DMPD: Macrophage-stimulating protein and RON receptor tyrosine kinase: potentialregulators of macrophage inflammatory activities. [Dynamic Macrophage Pathway CSML Database

Lifescience Database Archive (English)

Full Text Available 12472665 Macrophage-stimulating protein and RON receptor tyrosine kinase: potential...:545-53. (.png) (.svg) (.html) (.csml) Show Macrophage-stimulating protein and RON receptor tyrosine kinase:...le Macrophage-stimulating protein and RON receptor tyrosine kinase: potentialregulators of macrophage inflam

Analysis of genetic variation of inducible nitric oxide synthase and ...

African Journals Online (AJOL)

The genetic diversity of 100 Malaysian native chickens was investigated using polymerase chain reaction-restriction fragment polymorphism (PCR-RFLP) for two candidate genes: inducible nitric oxide synthase (INOS) and natural resistance-associated macrophage protein 1 (NRAMP1). The two genes were selected ...

Anti-inflammatory effect of garlic 14-kDa protein on LPS-stimulated-J774A.1 macrophages.

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Rabe, Shahrzad Zamani Taghizadeh; Ghazanfari, Tooba; Siadat, Zahra; Rastin, Maryam; Rabe, Shahin Zamani Taghizadeh; Mahmoudi, Mahmoud

2015-04-01

Garlic 14-kDa protein is purified from garlic (Allium sativum L.) which is used in traditional medicine and exerts various immunomodulatory activities. The present study investigated the suppressive effect of garlic 14-kDa protein on LPS-induced expression of pro-inflammatory mediators and underlying mechanism in inflammatory macrophages. J774A.1 macrophages were treated with 14-kDa protein (5-30 μg/ml) with/without LPS (1 μg/ml) and the production of inflammatory mediators such as prostaglandin E2 (PGE2), TNF-α, and IL-1β released were measured using ELISA. Nitric oxide (NO) production was determined using the Griess method. The anti-inflammatory activity of 14-kDa protein was examined by measuring inducible nitric oxide synthase and cyclooxygenase-2 proteins using western blot. The expression of nuclear NF-κB p65 subunit was assessed by western blot. Garlic 14-kDa protein significantly inhibited the excessive production of NO, PGE, TNF-α, and IL-1β in lipopolysaccharide (LPS)-activated J774A.1 macrophages in a concentration-related manner without cytotoxic effect. Western blot analysis demonstrated that garlic 14-kDa protein suppressed corresponding inducible NO synthase expression and activated cyclooxygenase-2 protein expression. The inhibitory effect was mediated partly by a reduction in the activity and expression of transcription factor NF-κB protein. Our results suggested, for the first time, garlic 14-kDa protein exhibits anti-inflammatory properties in macrophages possibly by suppressing the inflammatory mediators via the inhibition of transcription factor NF-κB signaling pathway. The traditional use of garlic as anti-inflammatory remedy could be ascribed partly to 14-kDa protein content. This protein might be a useful candidate for controlling inflammatory diseases and further investigations in vivo.

Immunotherapy for Prostate Cancer with Gc Protein-Derived Macrophage-Activating Factor, GcMAF1

OpenAIRE

Yamamoto, Nobuto; Suyama, Hirofumi; Yamamoto, Nobuyuki

2008-01-01

Serum Gc protein (known as vitamin D3-binding protein) is the precursor for the principal macrophage-activating factor (MAF). The MAF precursor activity of serum Gc protein of prostate cancer patients was lost or reduced because Gc protein was deglycosylated by serum α-N-acetylgalactosaminidase (Nagalase) secreted from cancerous cells. Therefore, macrophages of prostate cancer patients having deglycosylated Gc protein cannot be activated, leading to immunosuppression. Stepwise treatment of pu...

Triglyceride-induced macrophage cell death is triggered by caspase-1.

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Son, Sin Jee; Rhee, Ki-Jong; Lim, Jaewon; Kim, Tae Ue; Kim, Tack-Joong; Kim, Yoon Suk

2013-01-01

Triglyceride (TG) induces macrophage cell death which contributes to the development of atherosclerosis. We confirmed that exogenous TG accumulates in human THP-1 macrophages and causes cell death. TG treated THP-1 macrophages exhibited no change in tumor necrosis factor (TNF)-α, interleukin (IL)-18, macrophage inflammatory protein (MIP)-1α, and IL-1R1 receptor mRNA expression. However, there was a marked decrease in IL-1β mRNA expression but an increase in IL-1β protein secretion. Decreased expression of IL-1β mRNA and increased secretion of IL-1β protein was not the direct cause of cell death. Until now, TG was assumed to induce necrotic cell death in macrophages. Since caspase-1 is known to be involved in activation and secretion of IL-1β protein and pyroptotic cell death, next we determined whether caspase-1 is associated with TG-induced macrophage cell death. We found an increase in caspase-1 activity in TG-treated THP-1 macrophages and inhibition of caspase-1 activity using a specific inhibitor partially rescued cell death. These results suggest activation of the pyroptotic pathway by TG. This is the first report implicating the activation of caspase-1 and the triggering of the pyroptosis pathway in TG-induced macrophage cell death.

Activation of M1 macrophages in sepsis-induced acute kidney injury in response to heparin-binding protein.

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Li Xing

Full Text Available In the early stage of sepsis, M1 macrophages result in the production of inflammatory mediators and AKI. Heparin-binding protein (HBP have been shown to play important roles in sepsis-induced AKI. In this study, we investigate the association of HBP with M1 macrophages in sepsis-induced AKI.Male C57BL6 mice were subjected to cecal ligation and puncture (CLP or sham surgery. Biochemical and histological renal damage was assessed. Macrophage infiltration was assessed by immunohistochemistry. RT-PCR was used to investigate the expression of heparin-binding protein (HBP, the inducible nitric oxide synthase (iNOS and arginase 1 (Arg-1 mRNAs. Western blots were performed to assay the tissue levels of HBP, tumor necrosis factor alpha (TNF-α and interleukin-6 (IL-6.High levels of HBP were obviously detected 24 h after sepsis-induced AKI. Heparin inhibited HBP expression during sepsis-induced AKI. The suppression of HBP expression by heparin injection after the establishment of sepsis-induced AKI resulted in a reduction in renal injury severity accompanied with a significant repression of M1 macrophage activation and expression of TNF-α and IL-6.HBP plays an important role in the initial inflammatory reaction associated with sepsis-induced AKI, presumably by activating M1 macrophages and suppressing TNF-α and IL-6 secretion.

Role of CC chemokines (macrophage inflammatory protein-1 beta, monocyte chemoattractant protein-1, RANTES) in acute lung injury in rats

DEFF Research Database (Denmark)

Bless, N M; Huber-Lang, M; Guo, R F

2000-01-01

The role of the CC chemokines, macrophage inflammatory protein-1 beta (MIP-1 beta), monocyte chemotactic peptide-1 (MCP-1), and RANTES, in acute lung inflammatory injury induced by intrapulmonary deposition of IgG immune complexes injury in rats was determined. Rat MIP-1 beta, MCP-1, and RANTES...... were cloned, the proteins were expressed, and neutralizing Abs were developed. mRNA and protein expression for MIP-1 beta and MCP-1 were up-regulated during the inflammatory response, while mRNA and protein expression for RANTES were constitutive and unchanged during the inflammatory response....... Treatment of rats with anti-MIP-1 beta Ab significantly decreased vascular permeability by 37% (p = 0.012), reduced neutrophil recruitment into lung by 65% (p = 0.047), and suppressed levels of TNF-alpha in bronchoalveolar lavage fluids by 61% (p = 0.008). Treatment of rats with anti-rat MCP-1 or anti...

Transport of Magnesium by a Bacterial Nramp-Related Gene

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Rodionov, Dmitry A.; Freedman, Benjamin G.; Senger, Ryan S.; Winkler, Wade C.

2014-01-01

Magnesium is an essential divalent metal that serves many cellular functions. While most divalent cations are maintained at relatively low intracellular concentrations, magnesium is maintained at a higher level (∼0.5–2.0 mM). Three families of transport proteins were previously identified for magnesium import: CorA, MgtE, and MgtA/MgtB P-type ATPases. In the current study, we find that expression of a bacterial protein unrelated to these transporters can fully restore growth to a bacterial mutant that lacks known magnesium transporters, suggesting it is a new importer for magnesium. We demonstrate that this transport activity is likely to be specific rather than resulting from substrate promiscuity because the proteins are incapable of manganese import. This magnesium transport protein is distantly related to the Nramp family of proteins, which have been shown to transport divalent cations but have never been shown to recognize magnesium. We also find gene expression of the new magnesium transporter to be controlled by a magnesium-sensing riboswitch. Importantly, we find additional examples of riboswitch-regulated homologues, suggesting that they are a frequent occurrence in bacteria. Therefore, our aggregate data discover a new and perhaps broadly important path for magnesium import and highlight how identification of riboswitch RNAs can help shed light on new, and sometimes unexpected, functions of their downstream genes. PMID:24968120

M1 Macrophages but Not M2 Macrophages Are Characterized by Upregulation of CRP Expression via Activation of NFκB: a Possible Role for Ox-LDL in Macrophage Polarization.

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Kaplan, Marielle; Shur, Anna; Tendler, Yvgeny

2018-04-23

Arterial macrophages comprise a heterogeneous population: pro-inflammatory (M1) and anti-inflammatory (M2). Since C-reactive protein (CRP) is produced by macrophages in atherosclerotic lesions, understanding of CRP regulation in macrophages could be crucial to decipher inflammatory patterns in atherogenesis. We aimed to analyze CRP expression in M1/M2 macrophages and to question whether it involves NFκB signaling pathway. Furthermore, we questioned whether oxidative stress affect macrophage phenotype and modulate macrophage CRP expression. M1/M2 macrophage polarization was validated using THP-1 macrophages. CRP mRNA and protein expression were determined using real-time PCR and immunohistochemistry. Involvement of NFκB was determined by nuclear translocation of p50 subunit and the use of NFκB inhibitor. Involvement of oxidative stress in macrophage phenotypes induction was studied using oxidized-LDL (Ox-LDL) and antioxidants. M1 macrophages were characterized by elevated CRP mRNA expression (by 67%), CRP protein levels (by 108%), and upregulation of NFκB activation compared to control, but these features were not shared by M2 macrophages. Macrophages incubation with Ox-LDL led to a moderate M1 phenotype combined with a M2 phenotype, correlated with increased CRP mRNA expression. Antioxidants inhibited by up to 86% IL6 expression but did not significantly affect IL10 secretion. Antioxidants significantly inhibited CRP expression in M1 macrophages, but not in M2 macrophages. Elevated expression of CRP was characteristic of M1 macrophages rather than M2 through NFκB activation. Oxidative stress could be one of the endogenous triggers for macrophage activation to a mixed M1 and M2 phenotype, in association with increased expression of CRP.

Phagocytosis by macrophages mediated by receptors for denatured proteins - dependence on tyrosine protein kinases

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M.R. Hespanhol

2002-03-01

Full Text Available Previous studies have demonstrated that some components of the leukocyte cell membrane, CR3 (Mac-1, CD11b/CD18 and p150/95, are able to bind to denatured proteins. Thus, it is of interest to know which effector functions of these cells can be triggered by these receptors when they interact with particles or surfaces covered with denatured proteins. In the present study we analyzed their possible role as mediators of phagocytosis of red cells covered with denatured bovine serum albumin (BSA by mouse peritoneal macrophages. We observed that a macrophages are able to recognize (bind to these red cells, b this interaction can be inhibited by denatured BSA in the fluid phase, c there is no phagocytosis of these particles by normal macrophages, d phagocytosis mediated by denatured BSA can be, however, effectively triggered in inflammatory macrophages induced by glycogen or in macrophages activated in vivo with LPS, and e this phagocytic capacity is strongly dependent on the activity of tyrosine protein kinases in its signal transduction pathway, as demonstrated by using three kinds of enzyme inhibitors (genistein, quercetin and herbimycin A.

BET protein function is required for inflammation: Brd2 genetic disruption and BET inhibitor JQ1 impair mouse macrophage inflammatory responses1

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Belkina, Anna C.; Nikolajczyk, Barbara S.; Denis, Gerald V.

2013-01-01

Histone acetylation regulates activation and repression of multiple inflammatory genes known to play critical roles in chronic inflammatory diseases. However, proteins responsible for translating the histone acetylation code into an orchestrated pro-inflammatory cytokine response remain poorly characterized. Bromodomain extra terminal (BET) proteins are “readers” of histone acetylation marks with demonstrated roles in gene transcription, but the ability of BET proteins to coordinate the response of inflammatory cytokine genes through translation of histone marks is unknown. We hypothesize that members of the BET family of dual bromodomain-containing transcriptional regulators directly control inflammatory genes. We examined the genetic model of brd2 lo mice, a BET protein hypomorph, to show that Brd2 is essential for pro-inflammatory cytokine production in macrophages. Studies that utilize siRNA knockdown and a small molecule inhibitor of BET protein binding, JQ1, independently demonstrate BET proteins are critical for macrophage inflammatory responses. Furthermore, we show that Brd2 and Brd4 physically associate with the promoters of inflammatory cytokine genes in macrophages. This association is absent in the presence of BET inhibition by JQ1. Finally, we demonstrate that JQ1 ablates cytokine production in vitro and blunts the “cytokine storm” in endotoxemic mice by reducing levels of IL-6 and TNF-α while rescuing mice from LPS-induced death. We propose that targeting BET proteins with small molecule inhibitors will benefit hyper-inflammatory conditions associated with high levels of cytokine production. PMID:23420887

Wip1-dependent modulation of macrophage migration and phagocytosis

DEFF Research Database (Denmark)

Tang, Yiting; Pan, Bing; Zhou, Xin

2017-01-01

Macrophage accumulation within the vascular wall is a hallmark of atherosclerosis. Controlling macrophage conversion into foam cells remains a major challenge for treatment of atherosclerotic diseases. Here, we show that Wip1, a member of the PP2C family of Ser/Thr protein phosphatases, modulates...... macrophage migration and phagocytosis associated with atherosclerotic plaque formation. Wip1 deficiency increases migratory and phagocytic activities of the macrophage under stress conditions. Enhanced migration of Wip1-/- macrophages is mediated by Rac1-GTPase and PI3K/AKT signalling pathways. Elevated...... phagocytic ability of Wip1-/- macrophages is linked to CD36 plasma membrane recruitment that is regulated by AMPK activity. Our study identifies Wip1 as an intrinsic negative regulator of macrophage chemotaxis. We propose that Wip1-dependent control of macrophage function may provide avenues for preventing...

Immunotherapy for Prostate Cancer with Gc Protein-Derived Macrophage-Activating Factor, GcMAF1

Science.gov (United States)

Yamamoto, Nobuto; Suyama, Hirofumi; Yamamoto, Nobuyuki

2008-01-01

Serum Gc protein (known as vitamin D3-binding protein) is the precursor for the principal macrophage-activating factor (MAF). The MAF precursor activity of serum Gc protein of prostate cancer patients was lost or reduced because Gc protein was deglycosylated by serum α-N-acetylgalactosaminidase (Nagalase) secreted from cancerous cells. Therefore, macrophages of prostate cancer patients having deglycosylated Gc protein cannot be activated, leading to immunosuppression. Stepwise treatment of purified Gc protein with immobilized β-galactosidase and sialidase generated the most potent MAF (termed GcMAF) ever discovered, which produces no adverse effect in humans. Macrophages activated by GcMAF develop a considerable variation of receptors that recognize the abnormality in malignant cell surface and are highly tumoricidal. Sixteen nonanemic prostate cancer patients received weekly administration of 100 ng of GcMAF. As the MAF precursor activity increased, their serum Nagalase activity decreased. Because serum Nagalase activity is proportional to tumor burden, the entire time course analysis for GcMAF therapy was monitored by measuring the serum Nagalase activity. After 14 to 25 weekly administrations of GcMAF (100 ng/week), all 16 patients had very low serum Nagalase levels equivalent to those of healthy control values, indicating that these patients are tumor-free. No recurrence occurred for 7 years. PMID:18633461

BIGH3 protein and macrophages in retinal endothelial cell apoptosis.

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Mondragon, Albert A; Betts-Obregon, Brandi S; Moritz, Robert J; Parvathaneni, Kalpana; Navarro, Mary M; Kim, Hong Seok; Lee, Chi Fung; LeBaron, Richard G; Asmis, Reto; Tsin, Andrew T

2015-01-01

Diabetes is a pandemic disease with a higher occurrence in minority populations. The molecular mechanism to initiate diabetes-associated retinal angiogenesis remains largely unknown. We propose an inflammatory pathway of diabetic retinopathy in which macrophages in the diabetic eye provide TGFβ to retinal endothelial cells (REC) in the retinal microvasculature. In response to TGFβ, REC synthesize and secrete a pro-apoptotic BIGH3 (TGFβ-Induced Gene Human Clone 3) protein, which acts in an autocrine loop to induce REC apoptosis. Rhesus monkey retinal endothelial cells (RhREC) were treated with dMCM (cell media of macrophages treated with high glucose and LDL) and assayed for apoptosis (TUNEL), BIGH3 mRNA (qPCR), and protein (Western blots) expressions. Cells were also treated with ΤGFβ1 and 2 for BIGH3 mRNA and protein expression. Inhibition assays were carried out using antibodies for TGFβ1 and for BIGH3 to block apoptosis and mRNA expression. BIGH3 in cultured RhREC cells were identified by immunohistochemistry (IHC). Distribution of BIGH3 and macrophages in the diabetic mouse retina was examined with IHC. RhRECs treated with dMCM or TGFβ showed a significant increase in apoptosis and BIGH3 protein expression. Recombinant BIGH3 added to RhREC culture medium led to a dose-dependent increase in apoptosis. Antibodies (Ab) directed against BIGH3 and TGFβ, as well as TGFβ receptor blocker resulted in a significant reduction in apoptosis induced by either dMCM, TGFβ or BIGH3. IHC showed that cultured RhREC constitutively expressed BIGH3. Macrophage and BIGH3 protein were co-localized to the inner retina of the diabetic mouse eye. Our results support a novel inflammatory pathway for diabetic retinopathy. This pathway is initiated by TGFβ released from macrophages, which promotes synthesis and release of BIGH3 protein by REC and REC apoptosis.

Azurophil granule proteins constitute the major mycobactericidal proteins in human neutrophils and enhance the killing of mycobacteria in macrophages.

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Prajna Jena

Full Text Available Pathogenic mycobacteria reside in, and are in turn controlled by, macrophages. However, emerging data suggest that neutrophils also play a critical role in innate immunity to tuberculosis, presumably by their different antibacterial granule proteins. In this study, we purified neutrophil azurophil and specific granules and systematically analyzed the antimycobacterial activity of some purified azurophil and specific granule proteins against M. smegmatis, M. bovis-BCG and M. tuberculosis H37Rv. Using gel overlay and colony forming unit assays we showed that the defensin-depleted azurophil granule proteins (AZP were more active against mycobacteria compared to other granule proteins and cytosolic proteins. The proteins showing antimycobacterial activity were identified by MALDI-TOF mass spectrometry. Electron microscopic studies demonstrate that the AZP disintegrate bacterial cell membrane resulting in killing of mycobacteria. Exogenous addition of AZP to murine macrophage RAW 264.7, THP-1 and peripheral blood monocyte-derived macrophages significantly reduced the intracellular survival of mycobacteria without exhibiting cytotoxic activity on macrophages. Immunofluorescence studies showed that macrophages actively endocytose neutrophil granular proteins. Treatment with AZP resulted in increase in co-localization of BCG containing phagosomes with lysosomes but not in increase of autophagy. These data demonstrate that neutrophil azurophil proteins may play an important role in controlling intracellular survival of mycobacteria in macrophages.

Proteomic Analysis of ABCA1-Null Macrophages Reveals a Role for Stomatin-Like Protein-2 in Raft Composition and Toll-Like Receptor Signaling.

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Chowdhury, Saiful M; Zhu, Xuewei; Aloor, Jim J; Azzam, Kathleen M; Gabor, Kristin A; Ge, William; Addo, Kezia A; Tomer, Kenneth B; Parks, John S; Fessler, Michael B

2015-07-01

Lipid raft membrane microdomains organize signaling by many prototypical receptors, including the Toll-like receptors (TLRs) of the innate immune system. Raft-localization of proteins is widely thought to be regulated by raft cholesterol levels, but this is largely on the basis of studies that have manipulated cell cholesterol using crude and poorly specific chemical tools, such as β-cyclodextrins. To date, there has been no proteome-scale investigation of whether endogenous regulators of intracellular cholesterol trafficking, such as the ATP binding cassette (ABC)A1 lipid efflux transporter, regulate targeting of proteins to rafts. Abca1(-/-) macrophages have cholesterol-laden rafts that have been reported to contain increased levels of select proteins, including TLR4, the lipopolysaccharide receptor. Here, using quantitative proteomic profiling, we identified 383 proteins in raft isolates from Abca1(+/+) and Abca1(-/-) macrophages. ABCA1 deletion induced wide-ranging changes to the raft proteome. Remarkably, many of these changes were similar to those seen in Abca1(+/+) macrophages after lipopolysaccharide exposure. Stomatin-like protein (SLP)-2, a member of the stomatin-prohibitin-flotillin-HflK/C family of membrane scaffolding proteins, was robustly and specifically increased in Abca1(-/-) rafts. Pursuing SLP-2 function, we found that rafts of SLP-2-silenced macrophages had markedly abnormal composition. SLP-2 silencing did not compromise ABCA1-dependent cholesterol efflux but reduced macrophage responsiveness to multiple TLR ligands. This was associated with reduced raft levels of the TLR co-receptor, CD14, and defective lipopolysaccharide-induced recruitment of the common TLR adaptor, MyD88, to rafts. Taken together, we show that the lipid transporter ABCA1 regulates the protein repertoire of rafts and identify SLP-2 as an ABCA1-dependent regulator of raft composition and of the innate immune response. © 2015 by The American Society for Biochemistry and

Proteomic Analysis of ABCA1-Null Macrophages Reveals a Role for Stomatin-Like Protein-2 in Raft Composition and Toll-Like Receptor Signaling*

Science.gov (United States)

Chowdhury, Saiful M.; Zhu, Xuewei; Aloor, Jim J.; Azzam, Kathleen M.; Gabor, Kristin A.; Ge, William; Addo, Kezia A.; Tomer, Kenneth B.; Parks, John S.; Fessler, Michael B.

2015-01-01

Lipid raft membrane microdomains organize signaling by many prototypical receptors, including the Toll-like receptors (TLRs) of the innate immune system. Raft-localization of proteins is widely thought to be regulated by raft cholesterol levels, but this is largely on the basis of studies that have manipulated cell cholesterol using crude and poorly specific chemical tools, such as β-cyclodextrins. To date, there has been no proteome-scale investigation of whether endogenous regulators of intracellular cholesterol trafficking, such as the ATP binding cassette (ABC)A1 lipid efflux transporter, regulate targeting of proteins to rafts. Abca1−/− macrophages have cholesterol-laden rafts that have been reported to contain increased levels of select proteins, including TLR4, the lipopolysaccharide receptor. Here, using quantitative proteomic profiling, we identified 383 proteins in raft isolates from Abca1+/+ and Abca1−/− macrophages. ABCA1 deletion induced wide-ranging changes to the raft proteome. Remarkably, many of these changes were similar to those seen in Abca1+/+ macrophages after lipopolysaccharide exposure. Stomatin-like protein (SLP)-2, a member of the stomatin-prohibitin-flotillin-HflK/C family of membrane scaffolding proteins, was robustly and specifically increased in Abca1−/− rafts. Pursuing SLP-2 function, we found that rafts of SLP-2-silenced macrophages had markedly abnormal composition. SLP-2 silencing did not compromise ABCA1-dependent cholesterol efflux but reduced macrophage responsiveness to multiple TLR ligands. This was associated with reduced raft levels of the TLR co-receptor, CD14, and defective lipopolysaccharide-induced recruitment of the common TLR adaptor, MyD88, to rafts. Taken together, we show that the lipid transporter ABCA1 regulates the protein repertoire of rafts and identify SLP-2 as an ABCA1-dependent regulator of raft composition and of the innate immune response. PMID:25910759

Alternatively Activated (M2) Macrophage Phenotype Is Inducible by Endothelin-1 in Cultured Human Macrophages.

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Soldano, Stefano; Pizzorni, Carmen; Paolino, Sabrina; Trombetta, Amelia Chiara; Montagna, Paola; Brizzolara, Renata; Ruaro, Barbara; Sulli, Alberto; Cutolo, Maurizio

2016-01-01

Alternatively activated (M2) macrophages are phenotypically characterized by the expression of specific markers, mainly macrophage scavenger receptors (CD204 and CD163) and mannose receptor-1 (CD206), and participate in the fibrotic process by over-producing pro-fibrotic molecules, such as transforming growth factor-beta1 (TGFbeta1) and metalloproteinase (MMP)-9. Endothelin-1 (ET-1) is implicated in the fibrotic process, exerting its pro-fibrotic effects through the interaction with its receptors (ETA and ETB). The study investigated the possible role of ET-1 in inducing the transition from cultured human macrophages into M2 cells. Cultured human monocytes (THP-1 cell line) were activated into macrophages (M0 macrophages) with phorbol myristate acetate and subsequently maintained in growth medium (M0-controls) or treated with either ET-1 (100nM) or interleukin-4 (IL-4, 10ng/mL, M2 inducer) for 72 hours. Similarly, primary cultures of human peripheral blood monocyte (PBM)-derived macrophages obtained from healthy subjects, were maintained in growth medium (untreated cells) or treated with ET-1 or IL-4 for 6 days. Both M0 and PBM-derived macrophages were pre-treated with ET receptor antagonist (ETA/BRA, bosentan 10-5M) for 1 hour before ET-1 stimulation. Protein and gene expression of CD204, CD206, CD163, TGFbeta1 were analysed by immunocytochemistry, Western blotting and quantitative real time polymerase chain reaction (qRT-PCR). Gene expression of interleukin(IL)-10 and macrophage derived chemokine (CCL-22) was evaluated by qRT-PCR. MMP-9 production was investigated by gel zymography. ET-1 significantly increased the expression of M2 phenotype markers CD204, CD206, CD163, IL-10 and CCL-22, and the production of MMP-9 in both cultures of M0 and PBM-derived macrophages compared to M0-controls and untreated cells. In cultured PBM-derived macrophages, ET-1 increased TGFbeta1 protein and gene expression compared to untreated cells. The ET-1-mediated effects were

Differential S1P Receptor Profiles on M1- and M2-Polarized Macrophages Affect Macrophage Cytokine Production and Migration.

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Müller, Jan; von Bernstorff, Wolfram; Heidecke, Claus-Dieter; Schulze, Tobias

2017-01-01

Introduction . Macrophages are key players in complex biological processes. In response to environmental signals, macrophages undergo polarization towards a proinflammatory (M1) or anti-inflammatory (M2) phenotype. Sphingosine 1-phosphate (S1P) is a bioactive lysophospholipid that acts via 5 G-protein coupled receptors (S1P 1-5 ) in order to influence a broad spectrum of biological processes. This study assesses S1P receptor expression on macrophages before and after M1 and M2 polarization and performs a comparative analysis of S1P signalling in the two activational states of macrophages. Methods . Bone marrow derived macrophages (BMDM) from C57 BL/6 mice were cultured under either M1- or M2-polarizing conditions. S1P-receptor expression was determined by quantitative RT-PCR. Influence of S1P on macrophage activation, migration, phagocytosis, and cytokine secretion was assessed in vitro. Results . All 5 S1P receptor subclasses were expressed in macrophages. Culture under both M1- and M2-polarizing conditions led to significant downregulation of S1P 1 . In contrast, M1-polarized macrophages significantly downregulated S1P 4 . The expression of the remaining three S1P receptors did not change. S1P increased expression of iNOS under M2-polarizing conditions. Furthermore, S1P induced chemotaxis in M1 macrophages and changed cytokine production in M2 macrophages. Phagocytosis was not affected by S1P-signalling. Discussion . The expression of different specific S1P receptor profiles may provide a possibility to selectively influence M1- or M2-polarized macrophages.

Spliced XBP1 promotes macrophage survival and autophagy by interacting with Beclin-1

Energy Technology Data Exchange (ETDEWEB)

Tian, Ping-Ge [Southern Medical University, Guangzhou, Guangdong 510515 (China); Jiang, Zhi-Xin [Centre Laboratory, The 305th Hospital of the People' s Liberation Army, Beijing 100017 (China); Li, Jian-Hua [Department of Geriatric Cardiology, Chinese PLA General Hosptial, Beijing 100853 (China); Zhou, Zhe, E-mail: zhouzhe76@126.com [Laboratory of Biotechnology, Beijing Institute of Radiation Medicine, Beijing 100850 (China); Zhang, Qing-Hua, E-mail: 1056055170@qq.com [Department of Cardiology, The 305th Hospital of the People' s Liberation Army, Beijing 100017 (China)

2015-08-07

Macrophage autophagy plays an important role in the development of atherosclerosis, but the precise mechanism mediating this process is unclear. The potential role of the X-box binding protein 1 (XBP1), a crucial transduction factor that is involved in endoplasmic reticulum stress and the unfolded protein response, in bone marrow-derived macrophage autophagy is unknown. This study mainly explores the roles of XBP1 mRNA splicing in bone marrow-derived macrophage autophagy. The present study shows that the transient overexpression of spliced XBP1 via adenovirus-mediated gene transfer induces autophagy and promotes proliferation in bone marrow-derived macrophages via the down-regulation of Beclin-1, but that the sustained overexpression of spliced XBP1 leads to apoptosis. When XBP1 is down-regulated in bone marrow-derived macrophages using siRNA, rapamycin-induced autophagosome formation is ablated. Furthermore, we have detected the overexpression of XBP1 in areas of atherosclerotic plaques in the arteries of ApoE−/− mice. These results demonstrate that XBP1 mRNA splicing plays an important role in maintaining the function of bone marrow-derived macrophages and provide new insight into the study and treatment of atherosclerosis. - Highlights: • XBP1 was up-regulated in atherosclerotic plaques of ApoE−/− mice. • Transient spliced XBP1 overexpression induced macrophages autophagy via Beclin-1. • Sustained spliced XBP1 overexpression triggered macrophages apoptosis. • Spliced XBP1 plays a key role in maintaining the macrophages survival.

Gc protein-derived macrophage activating factor (GcMAF): isoelectric focusing pattern and tumoricidal activity.

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Mohamad, Saharuddin Bin; Nagasawa, Hideko; Sasaki, Hideyuki; Uto, Yoshihiro; Nakagawa, Yoshinori; Kawashima, Ken; Hori, Hitoshi

2003-01-01

Gc protein is the precursor for Gc protein-derived macrophage activating factor (GcMAF), with three phenotypes: Gc1f, Gc1s and Gc2, based on its electrophoretic mobility. The difference in electrophoretic mobility is because of the difference in its posttranslational sugar moiety composition. We compared the difference between Gc protein and GcMAF electrophoretic mobility using the isoelectric focusing (IEF) method. The tumoricidal activity of GcMAF-treated macrophage was evaluated after coculture with L-929 cell. The tumoricidal mechanism was investigated using TNF bioassay and nitric oxide (NO) release. The difference in Gc protein and GcMAF electrophoretic mobility was detected. The tumoricidal activity of GcMAF-treated macrophage was detected, but no release of TNF and NO was detected. The difference of isoelectric focusing mobility in Gc protein and GcMAF would be useful to develop a GcMAF detection method. GcMAF increased macrophage tumoricidal activity but TNF and NO release were not involved in the mechanism.

Role of macrophage inflammatory protein-1alpha in T-cell-mediated immunity to viral infection

DEFF Research Database (Denmark)

Madsen, Andreas N; Nansen, Anneline; Christensen, Jan P

2003-01-01

The immune response to lymphocytic choriomeningitis virus in mice lacking macrophage inflammatory protein-1alpha (MIP-1alpha) was evaluated. Generation of virus-specific effector T cells is unimpaired in MIP-1alpha-deficient mice. Furthermore, MIP-1alpha is not required for T-cell-mediated virus...... control or virus-induced T-cell-dependent inflammation. Thus, MIP-1alpha is not mandatory for T-cell-mediated antiviral immunity....

CD163-L1 is an endocytic macrophage protein strongly regulated by mediators in the inflammatory response

DEFF Research Database (Denmark)

Moeller, Jesper B; Nielsen, Marianne J; Reichhardt, Martin P

2012-01-01

CD163-L1 belongs to the group B scavenger receptor cysteine-rich family of proteins, where the CD163-L1 gene arose by duplication of the gene encoding the hemoglobin scavenger receptor CD163 in late evolution. The current data demonstrate that CD163-L1 is highly expressed and colocalizes with CD163...... on large subsets of macrophages, but in contrast to CD163 the expression is low or absent in monocytes and in alveolar macrophages, glia, and Kupffer cells. The expression of CD163-L1 increases when cultured monocytes are M-CSF stimulated to macrophages, and the expression is further increased by the acute......-phase mediator IL-6 and the anti-inflammatory mediator IL-10 but is suppressed by the proinflammatory mediators IL-4, IL-13, TNF-α, and LPS/IFN-γ. Furthermore, we show that CD163-L1 is an endocytic receptor, which internalizes independently of cross-linking through a clathrin-mediated pathway. Two cytoplasmic...

Protein energy malnutrition increases arginase activity in monocytes and macrophages.

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Corware, Karina; Yardley, Vanessa; Mack, Christopher; Schuster, Steffen; Al-Hassi, Hafid; Herath, Shanthi; Bergin, Philip; Modolell, Manuel; Munder, Markus; Müller, Ingrid; Kropf, Pascale

2014-01-01

Protein energy malnutrition is commonly associated with immune dysfunctions and is a major factor in susceptibility to infectious diseases. In this study, we evaluated the impact of protein energy malnutrition on the capacity of monocytes and macrophages to upregulate arginase, an enzyme associated with immunosuppression and increased pathogen replication. Our results show that monocytes and macrophages are significantly increased in the bone marrow and blood of mice fed on a protein low diet. No alteration in the capacity of bone marrow derived macrophages isolated from malnourished mice to phagocytose particles, to produce the microbicidal molecule nitric oxide and to kill intracellular Leishmania parasites was detected. However, macrophages and monocytes from malnourished mice express significantly more arginase both in vitro and in vivo. Using an experimental model of visceral leishmaniasis, we show that following protein energy malnutrition, the increased parasite burden measured in the spleen of these mice coincided with increased arginase activity and that macrophages provide a more permissive environment for parasite growth. Taken together, these results identify a novel mechanism in protein energy malnutrition that might contributes to increased susceptibility to infectious diseases by upregulating arginase activity in myeloid cells.

C-reactive protein interaction with macrophages: in vitro induction of tumor cytotoxicity, and characterization of C-reactive protein binding to macrophages

International Nuclear Information System (INIS)

Zahedi, K.A.

1987-01-01

The ability of C-reactive protein (CRP) to activate macrophages to tumoricidal state was examined. CRP was able to activate macrophages to kill tumor cells. The activation was shown to be due to CRP and not to low levels of other activators present in the CRP preparations, since specific removal of CRP led to abrogation of the CRP mediated activation of macrophages. The role of lipopolysaccharide (LPS) as a contaminating activator was eliminated by showing the ability of CRP preparations to activate macrophages from LPS non-responsive strains of mice, and to activate macrophages under conditions which specifically inactivated or removed the contaminating LPS. In order to exclude the possibility of indirect activation of macrophages by other cells present in the peritoneal exudate cell population, effect of CRP on pure macrophages was examined. Bone marrow derived macrophages as well as well as macrophage cell lines exhibited a significant increase in their capacity to kill tumor cells after treatment with CRP. The nature of CRP and macrophage interaction was examined using radioiodinated CRP. Labelled CRP bound specifically to macrophages and macrophage cell lines

Mitogen-activated protein kinase phosphatase 1 (MKP-1) in macrophage biology and cardiovascular disease. A redox-regulated master controller of monocyte function and macrophage phenotype.

Science.gov (United States)

Kim, Hong Seok; Asmis, Reto

2017-08-01

MAPK pathways play a critical role in the activation of monocytes and macrophages by pathogens, signaling molecules and environmental cues and in the regulation of macrophage function and plasticity. MAPK phosphatase 1 (MKP-1) has emerged as the main counter-regulator of MAPK signaling in monocytes and macrophages. Loss of MKP-1 in monocytes and macrophages in response to metabolic stress leads to dysregulation of monocyte adhesion and migration, and gives rise to dysfunctional, proatherogenic monocyte-derived macrophages. Here we review the properties of this redox-regulated dual-specificity MAPK phosphatase and the role of MKP-1 in monocyte and macrophage biology and cardiovascular diseases. Copyright © 2017 Elsevier Inc. All rights reserved.

Solute carrier protein family 11 member 1 (Slc11a1) activation efficiently inhibits Leishmania donovani survival in host macrophages.

Science.gov (United States)

Singh, Nisha; Gedda, Mallikarjuna Rao; Tiwari, Neeraj; Singh, Suya P; Bajpai, Surabhi; Singh, Rakesh K

2017-09-01

Visceral leishmaniasis (kala-azar), a life threatening disease caused by L. donovani , is a latent threat to more than 147 million people living in disease endemic South East Asia region of the Indian subcontinent. The therapeutic option to control leishmanial infections are very limited, and at present comprise only two drugs, an antifungal amphotericin B and an antitumor miltefosine, which are also highly vulnerable for parasitic resistance. Therefore, identification and development of alternate control measures is an exigent requirement to control leishmanial infections. In this study, we report that functionally induced expression of solute carrier protein family 11 member 1 ( Slc11a1), a transmembrane divalent cationic transporter recruited on the surface of phagolysosomes after phagocytosis of parasites, effectively inhibits Leishmania donovani growth in host macrophages. Further, the increased Slc11a1 functionality also resulted in increased production of NOx, TNF-α and IL-12 by activated macrophages. The findings of this study signify the importance of interplay between Slc11a1 expression and macrophages activation that can be effectively used to control of Leishmania growth and survival.

Metformin reduces lipid accumulation in macrophages by inhibiting FOXO1-mediated transcription of fatty acid-binding protein 4

International Nuclear Information System (INIS)

Song, Jun; Ren, Pingping; Zhang, Lin; Wang, Xing Li; Chen, Li; Shen, Ying H.

2010-01-01

Objective: The accumulation of lipids in macrophages contributes to the development of atherosclerosis. Strategies to reduce lipid accumulation in macrophages may have therapeutic potential for preventing and treating atherosclerosis and cardiovascular complications. The antidiabetic drug metformin has been reported to reduce lipid accumulation in adipocytes. In this study, we examined the effects of metformin on lipid accumulation in macrophages and investigated the mechanisms involved. Methods and results: We observed that metformin significantly reduced palmitic acid (PA)-induced intracellular lipid accumulation in macrophages. Metformin promoted the expression of carnitine palmitoyltransferase I (CPT-1), while reduced the expression of fatty acid-binding protein 4 (FABP4) which was involved in PA-induced lipid accumulation. Quantitative real-time PCR showed that metformin regulates FABP4 expression at the transcriptional level. We identified forkhead transcription factor FOXO1 as a positive regulator of FABP4 expression. Inhibiting FOXO1 expression with FOXO1 siRNA significantly reduced basal and PA-induced FABP4 expression. Overexpression of wild-type FOXO1 and constitutively active FOXO1 significantly increased FABP4 expression, whereas dominant negative FOXO1 dramatically decreased FABP4 expression. Metformin reduced FABP4 expression by promoting FOXO1 nuclear exclusion and subsequently inhibiting its activity. Conclusions: Taken together, these results suggest that metformin reduces lipid accumulation in macrophages by repressing FOXO1-mediated FABP4 transcription. Thus, metformin may have a protective effect against lipid accumulation in macrophages and may serve as a therapeutic agent for preventing and treating atherosclerosis in metabolic syndrome.

Metformin reduces lipid accumulation in macrophages by inhibiting FOXO1-mediated transcription of fatty acid-binding protein 4

Energy Technology Data Exchange (ETDEWEB)

Song, Jun [Qilu Hospital, Shandong University, Jinan, Shandong (China); Division of Cardiothoracic Surgery, Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston, TX (United States); Texas Heart Institute at St. Luke' s Episcopal Hospital, Houston, TX (United States); Ren, Pingping; Zhang, Lin [Division of Cardiothoracic Surgery, Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston, TX (United States); Texas Heart Institute at St. Luke' s Episcopal Hospital, Houston, TX (United States); Wang, Xing Li [Qilu Hospital, Shandong University, Jinan, Shandong (China); Division of Cardiothoracic Surgery, Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston, TX (United States); Texas Heart Institute at St. Luke' s Episcopal Hospital, Houston, TX (United States); Chen, Li [Qilu Hospital, Shandong University, Jinan, Shandong (China); Shen, Ying H., E-mail: hyshen@bcm.edu [Division of Cardiothoracic Surgery, Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston, TX (United States); Texas Heart Institute at St. Luke' s Episcopal Hospital, Houston, TX (United States)

2010-02-26

Objective: The accumulation of lipids in macrophages contributes to the development of atherosclerosis. Strategies to reduce lipid accumulation in macrophages may have therapeutic potential for preventing and treating atherosclerosis and cardiovascular complications. The antidiabetic drug metformin has been reported to reduce lipid accumulation in adipocytes. In this study, we examined the effects of metformin on lipid accumulation in macrophages and investigated the mechanisms involved. Methods and results: We observed that metformin significantly reduced palmitic acid (PA)-induced intracellular lipid accumulation in macrophages. Metformin promoted the expression of carnitine palmitoyltransferase I (CPT-1), while reduced the expression of fatty acid-binding protein 4 (FABP4) which was involved in PA-induced lipid accumulation. Quantitative real-time PCR showed that metformin regulates FABP4 expression at the transcriptional level. We identified forkhead transcription factor FOXO1 as a positive regulator of FABP4 expression. Inhibiting FOXO1 expression with FOXO1 siRNA significantly reduced basal and PA-induced FABP4 expression. Overexpression of wild-type FOXO1 and constitutively active FOXO1 significantly increased FABP4 expression, whereas dominant negative FOXO1 dramatically decreased FABP4 expression. Metformin reduced FABP4 expression by promoting FOXO1 nuclear exclusion and subsequently inhibiting its activity. Conclusions: Taken together, these results suggest that metformin reduces lipid accumulation in macrophages by repressing FOXO1-mediated FABP4 transcription. Thus, metformin may have a protective effect against lipid accumulation in macrophages and may serve as a therapeutic agent for preventing and treating atherosclerosis in metabolic syndrome.

Metabolic reprogramming through fatty acid transport protein 1 (FATP1 regulates macrophage inflammatory potential and adipose inflammation

Directory of Open Access Journals (Sweden)

Amy R. Johnson

2016-07-01

Full Text Available Objective: A novel approach to regulate obesity-associated adipose inflammation may be through metabolic reprogramming of macrophages (MΦs. Broadly speaking, MΦs dependent on glucose are pro-inflammatory, classically activated MΦs (CAM, which contribute to adipose inflammation and insulin resistance. In contrast, MΦs that primarily metabolize fatty acids are alternatively activated MΦs (AAM and maintain tissue insulin sensitivity. In actuality, there is much flexibility and overlap in the CAM-AAM spectrum in vivo dependent upon various stimuli in the microenvironment. We hypothesized that specific lipid trafficking proteins, e.g. fatty acid transport protein 1 (FATP1, would direct MΦ fatty acid transport and metabolism to limit inflammation and contribute to the maintenance of adipose tissue homeostasis. Methods: Bone marrow derived MΦs (BMDMs from Fatp1−/− and Fatp1+/+ mice were used to investigate FATP1-dependent substrate metabolism, bioenergetics, metabolomics, and inflammatory responses. We also generated C57BL/6J chimeric mice by bone marrow transplant specifically lacking hematopoetic FATP1 (Fatp1B−/− and controls Fatp1B+/+. Mice were challenged by high fat diet (HFD or low fat diet (LFD and analyses including MRI, glucose and insulin tolerance tests, flow cytometric, histologic, and protein quantification assays were conducted. Finally, an FATP1-overexpressing RAW 264.7 MΦ cell line (FATP1-OE and empty vector control (FATP1-EV were developed as a gain of function model to test effects on substrate metabolism, bioenergetics, metabolomics, and inflammatory responses. Results: Fatp1 is downregulated with pro-inflammatory stimulation of MΦs. Fatp1−/− BMDMs and FATP1-OE RAW 264.7 MΦs demonstrated that FATP1 reciprocally controled metabolic flexibility, i.e. lipid and glucose metabolism, which was associated with inflammatory response. Supporting our previous work demonstrating the positive relationship between glucose

HIV-1 Nef in Macrophage-Mediated Disease Pathogenesis

Science.gov (United States)

Lamers, Susanna L.; Fogel, Gary B.; Singer, Elyse J.; Salemi, Marco; Nolan, David J.; Huysentruyt, Leanne C.; McGrath, Michael S.

2013-01-01

Combined anti-retroviral therapy (cART) has significantly reduced the number of AIDS-associated illnesses and changed the course of HIV-1 disease in developed countries. Despite the ability of cART to maintain high CD4+ T-cell counts, a number of macrophage-mediated diseases can still occur in HIV-infected subjects. These diseases include lymphoma, metabolic diseases, and HIV-associated neurological disorders. Within macrophages, the HIV-1 regulatory protein “Nef” can modulate surface receptors, interact with signaling pathways, and promote specific environments that contribute to each of these pathologies. Moreover, genetic variation in Nef may also guide the macrophage response. Herein, we review findings relating to the Nef–macrophage interaction and how this relationship contributes to disease pathogenesis. PMID:23215766

BET protein function is required for inflammation: Brd2 genetic disruption and BET inhibitor JQ1 impair mouse macrophage inflammatory responses.

Science.gov (United States)

Belkina, Anna C; Nikolajczyk, Barbara S; Denis, Gerald V

2013-04-01

Histone acetylation regulates activation and repression of multiple inflammatory genes known to play critical roles in chronic inflammatory diseases. However, proteins responsible for translating the histone acetylation code into an orchestrated proinflammatory cytokine response remain poorly characterized. Bromodomain and extraterminal (BET) proteins are "readers" of histone acetylation marks, with demonstrated roles in gene transcription, but the ability of BET proteins to coordinate the response of inflammatory cytokine genes through translation of histone marks is unknown. We hypothesize that members of the BET family of dual bromodomain-containing transcriptional regulators directly control inflammatory genes. We examined the genetic model of brd2 lo mice, a BET protein hypomorph, to show that Brd2 is essential for proinflammatory cytokine production in macrophages. Studies that use small interfering RNA knockdown and a small-molecule inhibitor of BET protein binding, JQ1, independently demonstrate BET proteins are critical for macrophage inflammatory responses. Furthermore, we show that Brd2 and Brd4 physically associate with the promoters of inflammatory cytokine genes in macrophages. This association is absent in the presence of BET inhibition by JQ1. Finally, we demonstrate that JQ1 ablates cytokine production in vitro and blunts the "cytokine storm" in endotoxemic mice by reducing levels of IL-6 and TNF-α while rescuing mice from LPS-induced death. We propose that targeting BET proteins with small-molecule inhibitors will benefit hyperinflammatory conditions associated with high levels of cytokine production.

Loss of monocyte chemoattractant protein-1 alters macrophage polarization and reduces NFκB activation in the foreign body response.

Science.gov (United States)

Moore, Laura Beth; Sawyer, Andrew J; Charokopos, Antonios; Skokos, Eleni A; Kyriakides, Themis R

2015-01-01

Implantation of biomaterials elicits a foreign body response characterized by fusion of macrophages to form foreign body giant cells and fibrotic encapsulation. Studies of the macrophage polarization involved in this response have suggested that alternative (M2) activation is associated with more favorable outcomes. Here we investigated this process in vivo by implanting mixed cellulose ester filters or polydimethylsiloxane disks in the peritoneal cavity of wild-type (WT) and monocyte chemoattractant protein-1 (MCP-1) knockout mice. We analyzed classical (M1) and alternative (M2) gene expression via quantitative polymerase chain reaction, immunohistochemistry and enzyme-linked immunosorbent assay in both non-adherent cells isolated by lavage and implant-adherent cells. Our results show that macrophages undergo unique activation that displays features of both M1 and M2 polarization including induction of tumor necrosis factor α (TNF), which induces the expression and nuclear translocation of p50 and RelA determined by immunofluorescence and Western blot. Both processes were compromised in fusion-deficient MCP-1 KO macrophages in vitro and in vivo. Furthermore, inclusion of BAY 11-7028, an inhibitor of NFκB activation, reduced nuclear translocation of RelA and fusion in WT macrophages. Our studies suggest that peritoneal implants elicit a unique macrophage polarization phenotype leading to induction of TNF and activation of the NFκB pathway. Published by Elsevier Ltd.

Expression analysis of G Protein-Coupled Receptors in mouse macrophages.

Science.gov (United States)

Lattin, Jane E; Schroder, Kate; Su, Andrew I; Walker, John R; Zhang, Jie; Wiltshire, Tim; Saijo, Kaoru; Glass, Christopher K; Hume, David A; Kellie, Stuart; Sweet, Matthew J

2008-04-29

Monocytes and macrophages express an extensive repertoire of G Protein-Coupled Receptors (GPCRs) that regulate inflammation and immunity. In this study we performed a systematic micro-array analysis of GPCR expression in primary mouse macrophages to identify family members that are either enriched in macrophages compared to a panel of other cell types, or are regulated by an inflammatory stimulus, the bacterial product lipopolysaccharide (LPS). Several members of the P2RY family had striking expression patterns in macrophages; P2ry6 mRNA was essentially expressed in a macrophage-specific fashion, whilst P2ry1 and P2ry5 mRNA levels were strongly down-regulated by LPS. Expression of several other GPCRs was either restricted to macrophages (e.g. Gpr84) or to both macrophages and neural tissues (e.g. P2ry12, Gpr85). The GPCR repertoire expressed by bone marrow-derived macrophages and thioglycollate-elicited peritoneal macrophages had some commonality, but there were also several GPCRs preferentially expressed by either cell population. The constitutive or regulated expression in macrophages of several GPCRs identified in this study has not previously been described. Future studies on such GPCRs and their agonists are likely to provide important insights into macrophage biology, as well as novel inflammatory pathways that could be future targets for drug discovery.

Phosphorylation of the adaptor protein SH2B1β regulates its ability to enhance growth hormone-dependent macrophage motility

OpenAIRE

Su, Hsiao-Wen; Lanning, Nathan J.; Morris, David L.; Argetsinger, Lawrence S.; Lumeng, Carey N.; Carter-Su, Christin

2013-01-01

Previous studies have shown that growth hormone (GH) recruits the adapter protein SH2B1β to the GH-activated, GH receptor-associated tyrosine kinase JAK2, implicating SH2B1β in GH-dependent actin cytoskeleton remodeling, and suggesting that phosphorylation at serines 161 and 165 in SH2B1β releases SH2B1β from the plasma membrane. Here, we examined the role of SH2B1β in GH regulation of macrophage migration. We show that GH stimulates migration of cultured RAW264.7 macrophages, and primary cul...

Association of the solute carrier family 11 member 1 gene polymorphisms with susceptibility to leprosy in a Brazilian sample

Directory of Open Access Journals (Sweden)

Maria José Franco Brochado

2016-02-01

Full Text Available Natural resistance-associated macrophage protein 1/solute carrier family 11 member 1 gene (Nramp1/Slc11a1 is a gene that controls the susceptibility of inbred mice to intracellular pathogens. Polymorphisms in the human Slc11a1/Nramp1 gene have been associated with host susceptibility to leprosy. This study has evaluated nine polymorphisms of the Slc11a1/Nramp1 gene [(GTn, 274C/T, 469+14G/C, 577-18G/A, 823C/T, 1029 C/T, 1465-85G/A, 1703G/A, and 1729+55del4] in 86 leprosy patients (67 and 19 patients had the multibacillary and the paucibacillary clinical forms of the disease, respectively, and 239 healthy controls matched by age, gender, and ethnicity. The frequency of allele 2 of the (GTn polymorphism was higher in leprosy patients [p = 0.04, odds ratio (OR = 1.49], whereas the frequency of allele 3 was higher in the control group (p = 0.03; OR = 0.66. Patients carrying the 274T allele (p = 0.04; OR = 1.49 and TT homozygosis (p = 0.02; OR = 2.46, such as the 469+14C allele (p = 0.03; OR = 1.53 of the 274C/T and 469+14G/C polymorphisms, respectively, were more frequent in the leprosy group. The leprosy and control groups had similar frequency of the 577-18G/A, 823C/T, 1029C/T, 1465-85G/A, 1703G/A, and 1729+55del4 polymorphisms. The 274C/T polymorphism in exon 3 and the 469+14G/C polymorphism in intron 4 were associated with susceptibility to leprosy, while the allele 2 and 3 of the (GTn polymorphism in the promoter region were associated with susceptibility and protection to leprosy, respectively.

Postprandial triglyceride-rich lipoproteins regulate perilipin-2 and perilipin-3 lipid-droplet-associated proteins in macrophages.

Science.gov (United States)

Varela, Lourdes M; López, Sergio; Ortega-Gómez, Almudena; Bermúdez, Beatriz; Buers, Insa; Robenek, Horst; Muriana, Francisco J G; Abia, Rocío

2015-04-01

Lipid accumulation in macrophages contributes to atherosclerosis. Within macrophages, lipids are stored in lipid droplets (LDs); perilipin-2 and perilipin-3 are the main LD-associated proteins. Postprandial triglyceride (TG)-rich lipoproteins induce LD accumulation in macrophages. The role of postprandial lipoproteins in perilipin-2 and perilipin-3 regulation was studied. TG-rich lipoproteins (TRLs) induced the levels of intracellular TGs, LDs and perilipin-2 protein expression in THP-1 macrophages and in Apoe(-/-) mice bone-marrow-derived macrophages with low and high basal levels of TGs. Perilipin-3 was only synthesized in mice macrophages with low basal levels of TGs. The regulation was dependent on the fatty acid composition of the lipoproteins; monounsaturated and polyunsaturated fatty acids (PUFAs) more strongly attenuated these effects compared with saturated fatty acids. In THP-1 macrophages, immunofluorescence microscopy and freeze-fracture immunogold labeling indicated that the lipoproteins translocated perilipin-3 from the cytoplasm to the LD surface; only the lipoproteins that were rich in PUFAs suppressed this effect. Chemical inhibition showed that lipoproteins induced perilipin-2 protein expression through the peroxisome proliferator-activated nuclear receptor (PPAR) PPARα and PPARγ pathways. Overall, our data indicate that postprandial TRLs may be involved in atherosclerotic plaque formation through the regulation of perilipin-2 and perilipin-3 proteins in macrophages. Because the fatty acid composition of the lipoproteins is dependent on the type of fat consumed, the ingestion of olive oil, which is rich in monounsaturated fatty acids, and fish oil, which is rich in omega-3 fatty acids, can be considered a good nutritional strategy to reduce the risk of atherosclerosis by LD-associated proteins decrease. Copyright © 2015 Elsevier Inc. All rights reserved.

Interaction of the heterotrimeric G protein alpha subunit SSG-1 of Sporothrix schenckii with proteins related to stress response and fungal pathogenicity using a yeast two-hybrid assay

Directory of Open Access Journals (Sweden)

González-Méndez Ricardo

2010-12-01

Full Text Available Abstract Background Important biological processes require selective and orderly protein-protein interactions at every level of the signalling cascades. G proteins are a family of heterotrimeric GTPases that effect eukaryotic signal transduction through the coupling of cell surface receptors to cytoplasmic effector proteins. They have been associated with growth and pathogenicity in many fungi through gene knock-out studies. In Sporothrix schenckii, a pathogenic, dimorphic fungus, we previously identified a pertussis sensitive G alpha subunit, SSG-1. In this work we inquire into its interactions with other proteins. Results Using the yeast two-hybrid technique, we identified protein-protein interactions between SSG-1 and other important cellular proteins. The interactions were corroborated using co-immuneprecipitation. Using these techniques we identified a Fe/Mn superoxide dismutase (SOD, a glyceraldehyde-3-P dehydrogenase (GAPDH and two ion transport proteins, a siderophore-iron transporter belonging to the Major Facilitator Superfamily (MFS and a divalent-cation transporter of the Nramp (natural resistance-associated macrophage protein family as interacting with SSG-1. The cDNA's encoding these proteins were sequenced and bioinformatic macromolecular sequence analyses were used for the correct classification and functional assignment. Conclusions This study constitutes the first report of the interaction of a fungal G alpha inhibitory subunit with SOD, GAPDH, and two metal ion transporters. The identification of such important proteins as partners of a G alpha subunit in this fungus suggests possible mechanisms through which this G protein can affect pathogenicity and survival under conditions of environmental stress or inside the human host. The two ion transporters identified in this work are the first to be reported in S. schenckii and the first time they are identified as interacting with fungal G protein alpha subunits. The association

Macrophage inflammatory protein-1alpha: a link between innate immunity and familial Mediterranean fever?

Science.gov (United States)

Dizdar, Omer; Kalyoncu, Umut; Karadag, Omer; Akdogan, Ali; Kiraz, Sedat; Ertenli, Ihsan; Barista, Ibrahim; Calguneri, Meral

2007-01-01

The aim of this study is to investigate the relationship between chemokines and the inflammation in Familial Mediterranean Fever (FMF). Forty-nine patients with FMF (41 in remission and 8 in acute attack period) and 20 healthy controls were included in the study. Serum levels of macrophage inflammatory protein-1alpha (MIP-1alpha) were assessed in the patients and the controls, along with other parameters of disease activity, i.e., fibrinogen, C-reactive protein and erythrocyte sedimentation rate. Serum MIP-1alpha levels of the patients with FMF in acute attack period were significantly higher than the patients in remission and healthy controls (p=0.02 and p=0.038, respectively). MIP-1alpha levels were weakly correlated with CRP (r=0.32, p=0.032) levels. MIP-1alpha may have a role in the pathogenesis of FMF attacks. MIP-1alpha and other chemokines may constitute a link between the innate immune system and FMF.

Soluble ICAM-1 activates lung macrophages and enhances lung injury

DEFF Research Database (Denmark)

Schmal, H; Czermak, B J; Lentsch, A B

1998-01-01

production of TNF-alpha and the CXC chemokine, macrophage inflammatory protein-2 (MIP-2). Alveolar macrophages exhibited cytokine responses to both sICAM-1 and immobilized sICAM-1, while rat PBMCs failed to demonstrate similar responses. Exposure of alveolar macrophages to sICAM-1 resulted in NFkappa......B activation (which was blocked by the presence of the aldehyde peptide inhibitor of 28S proteosome and by genistein, a tyrosine kinase inhibitor). As expected, cross-linking of CD18 on macrophages with Ab resulted in generation of TNF-alpha and MIP-2. This response was also inhibited in the presence...... of TNF-alpha and MIP-2 and increased neutrophil recruitment. Therefore, through engagement of beta2 integrins, sICAM-1 enhances alveolar macrophage production of MIP-2 and TNF-alpha, the result of which is intensified lung injury after intrapulmonary disposition of immune complexes....

Identification and comparison of macrophage-induced proteins and proteins induced under various stress conditions in Brucella abortus.

OpenAIRE

Rafie-Kolpin, M; Essenberg, R C; Wyckoff, J H

1996-01-01

Brucella abortus is a facultative intracellular pathogen of cattle and humans that is capable of survival inside macrophages. In order to understand how B. abortus copes with the conditions during intracellular growth in macrophages, the protein synthesis pattern of the bacteria grown inside bovine macrophages has been compared with that of bacteria grown in the cell culture medium by two-dimensional polyacrylamide gel electrophoresis. Approximately 24 new proteins that are not detected in th...

DMPD: G-protein-coupled receptor expression, function, and signaling in macrophages. [Dynamic Macrophage Pathway CSML Database

Lifescience Database Archive (English)

Full Text Available 17456803 G-protein-coupled receptor expression, function, and signaling in macropha...2007 Apr 24. (.png) (.svg) (.html) (.csml) Show G-protein-coupled receptor expression, function, and signali...ng in macrophages. PubmedID 17456803 Title G-protein-coupled receptor expression, function

The Orosomucoid 1 protein is involved in the vitamin D – mediated macrophage de-activation process

Energy Technology Data Exchange (ETDEWEB)

Gemelli, Claudia, E-mail: claudia.gemelli@unimore.it [Department of Life Sciences, University of Modena and Reggio Emilia, Via Campi 287, 41125 Modena (Italy); Center for Regenerative Medicine, University of Modena and Reggio Emilia, Via Gottardi 100, 41125 Modena (Italy); Martello, Andrea; Montanari, Monica; Zanocco Marani, Tommaso; Salsi, Valentina; Zappavigna, Vincenzo; Parenti, Sandra; Vignudelli, Tatiana; Selmi, Tommaso; Ferrari, Sergio; Grande, Alexis [Department of Life Sciences, University of Modena and Reggio Emilia, Via Campi 287, 41125 Modena (Italy)

2013-12-10

Orosomucoid 1 (ORM1), also named Alpha 1 acid glycoprotein A (AGP-A), is an abundant plasma protein characterized by anti-inflammatory and immune-modulating properties. The present study was designed to identify a possible correlation between ORM1 and Vitamin D3 (1,25(OH)2D3), a hormone exerting a widespread effect on cell proliferation, differentiation and regulation of the immune system. In particular, the data described here indicated that ORM1 is a 1,25(OH)2D3 primary response gene, characterized by the presence of a VDRE element inside the 1 kb sequence of its proximal promoter region. This finding was demonstrated with gene expression studies, Chromatin Immunoprecipitation and luciferase transactivation experiments and confirmed by VDR full length and dominant negative over-expression. In addition, several experiments carried out in human normal monocytes demonstrated that the 1,25(OH)2D3 – VDR – ORM1 pathway plays a functional role inside the macrophage de-activation process and that ORM1 may be considered as a signaling molecule involved in the maintenance of tissue homeostasis and remodeling. - Highlights: • ORM1 is a Vitamin D primary response gene. • VD and its receptor VDR are involved in the de-activation process mediated by human resident macrophages. • The signaling pathway VD-VDR-ORM1 plays an important role in the control of macrophage de-activation process. • ORM1 may be defined as a signaling molecule implicated in the maintenance of tissue homeostasis and remodeling.

The Orosomucoid 1 protein is involved in the vitamin D – mediated macrophage de-activation process

International Nuclear Information System (INIS)

Gemelli, Claudia; Martello, Andrea; Montanari, Monica; Zanocco Marani, Tommaso; Salsi, Valentina; Zappavigna, Vincenzo; Parenti, Sandra; Vignudelli, Tatiana; Selmi, Tommaso; Ferrari, Sergio; Grande, Alexis

2013-01-01

Orosomucoid 1 (ORM1), also named Alpha 1 acid glycoprotein A (AGP-A), is an abundant plasma protein characterized by anti-inflammatory and immune-modulating properties. The present study was designed to identify a possible correlation between ORM1 and Vitamin D3 (1,25(OH)2D3), a hormone exerting a widespread effect on cell proliferation, differentiation and regulation of the immune system. In particular, the data described here indicated that ORM1 is a 1,25(OH)2D3 primary response gene, characterized by the presence of a VDRE element inside the 1 kb sequence of its proximal promoter region. This finding was demonstrated with gene expression studies, Chromatin Immunoprecipitation and luciferase transactivation experiments and confirmed by VDR full length and dominant negative over-expression. In addition, several experiments carried out in human normal monocytes demonstrated that the 1,25(OH)2D3 – VDR – ORM1 pathway plays a functional role inside the macrophage de-activation process and that ORM1 may be considered as a signaling molecule involved in the maintenance of tissue homeostasis and remodeling. - Highlights: • ORM1 is a Vitamin D primary response gene. • VD and its receptor VDR are involved in the de-activation process mediated by human resident macrophages. • The signaling pathway VD-VDR-ORM1 plays an important role in the control of macrophage de-activation process. • ORM1 may be defined as a signaling molecule implicated in the maintenance of tissue homeostasis and remodeling

Notch signaling regulates expression of Mcl-1 and apoptosis in PPD-treated macrophages.

Science.gov (United States)

Palaga, Tanapat; Ratanabunyong, Siriluk; Pattarakankul, Thitiporn; Sangphech, Naunpun; Wongchana, Wipawee; Hadae, Yukihiro; Kueanjinda, Patipark

2013-09-01

Macrophages are cellular targets for infection by bacteria and viruses. The fate of infected macrophages plays a key role in determining the outcome of the host immune response. Apoptotic cell death of macrophages is considered to be a protective host defense that eliminates pathogens and infected cells. In this study, we investigated the involvement of Notch signaling in regulating apoptosis in macrophages treated with tuberculin purified protein derivative (PPD). Murine bone marrow-derived macrophages (BMMs) treated with PPD or infected with Mycobacterium bovis Bacillus Calmette-Guérin (BCG) induced upregulation of Notch1. This upregulation correlated well with the upregulation of the anti-apoptotic gene mcl-1 both at the transcriptional and translational levels. Decreased levels of Notch1 and Mcl-1 were observed in BMM treated with PPD when a gamma secretase inhibitor (GSI), which inhibits the processing of Notch receptors, was used. Moreover, silencing Notch1 in the macrophage-like cell line RAW264.7 decreased Mcl-1 protein expression, suggesting that Notch1 is critical for Mcl-1 expression in macrophages. A significant increase in apoptotic cells was observed upon treatment of BMM with PPD in the presence of GSI compared to the vehicle-control treated cells. Finally, analysis of the mcl-1 promoter in humans and mice revealed a conserved potential CSL/RBP-Jκ binding site. The association of Notch1 with the mcl-1 promoter was confirmed by chromatin immunoprecipitation. Taken together, these results indicate that Notch1 inhibits apoptosis of macrophages stimulated with PPD by directly controlling the mcl-1 promoter.

Isolation of a macrophage receptor for proteins modified by advanced glycosylation end products

International Nuclear Information System (INIS)

Radoff, S.; Vlassara, H.; Cerami, A.

1987-01-01

The nonenzymatic reaction of glucose with protein amino groups leads to the formation of irreversible AGE, such as the recently characterized glucose-derived crosslink, [2-furoyl-4(5)-(2-furanyl)-1-H-imidazole] (FFI). These products accumulate with time in aging tissues and diabetes, and are implicated in irreversible tissue damage. The authors have recently shown that macrophages bind and degrade AGE-proteins via a specific surface receptor, which is thus selectively removing senescent macromolecules. Scatchard plot analysis of binding data has indicated 1.5 x 10 5 receptors/cell with a binding affinity (Ka) of 1.7 x 10 7 /M. They have now isolated this receptor from murine macrophage RAW 264.7 membranes, solubilized with octylglucoside/protease inhibitors, and using FFI-Sepharose affinity chromatography and FPLC. The purified receptor binds radioactive FFI-containing compounds competitively. SDS-PAGE gels under reducing conditions indicate the receptor to be composed of two polypeptides, 83 Kda and 36 Kda. Crosslinking experiments with 125 I-AGE-albumin as ligand, indicate the 83 Kda subunit to be the AGE-binding peptide. These studies further characterize a macrophage receptor which selectively recognizes time-dependent glucose-modified proteins associated with aging and diabetes

Mycobacterium avium MAV2052 protein induces apoptosis in murine macrophage cells through Toll-like receptor 4.

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Lee, Kang-In; Choi, Han-Gyu; Son, Yeo-Jin; Whang, Jake; Kim, Kwangwook; Jeon, Heat Sal; Park, Hye-Soo; Back, Yong Woo; Choi, Seunga; Kim, Seong-Woo; Choi, Chul Hee; Kim, Hwa-Jung

2016-04-01

Mycobacterium avium and its sonic extracts induce apoptosis in macrophages. However, little is known about the M. avium components regulating macrophage apoptosis. In this study, using multidimensional fractionation, we identified MAV2052 protein, which induced macrophage apoptosis in M. avium culture filtrates. The recombinant MAV2052 induced macrophage apoptosis in a caspase-dependent manner. The loss of mitochondrial transmembrane potential (ΔΨm), mitochondrial translocation of Bax, and release of cytochrome c from mitochondria were observed in macrophages treated with MAV2052. Further, reactive oxygen species (ROS) production was required for the apoptosis induced by MAV2052. In addition, ROS and mitogen-activated protein kinases were involved in MAV2052-mediated TNF-α and IL-6 production. ROS-mediated activation of apoptosis signal-regulating kinase 1 (ASK1)-JNK pathway was a major signaling pathway for MAV2052-induced apoptosis. Moreover, MAV2052 bound to Toll-like receptor (TLR) 4 molecule and MAV2052-induced ROS production, ΔΨm loss, and apoptosis were all significantly reduced in TLR4(-/-) macrophages. Altogether, our results suggest that MAV2052 induces apoptotic cell death through TLR4 dependent ROS production and JNK pathway in murine macrophages.

Trichinella spiralis infection enhances protein kinase C phosphorylation in guinea pig alveolar macrophages.

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Dzik, J M; Zieliński, Z; Cieśla, J; Wałajtys-Rode, E

2010-03-01

To learn more about the signalling pathways involved in superoxide anion production in guinea pig alveolar macrophages, triggered by Trichinella spiralis infection, protein level and phosphorylation of mitogen activated protein (MAP) kinases and protein kinase C (PKC) were investigated. Infection with T. spiralis, the nematode having 'lung phase' during colonization of the host, enhances PKC phosphorylation in guinea pig alveolar macrophages. Isoenzymes beta and delta of PKC have been found significantly phosphorylated, although their location was not changed as a consequence of T. spiralis infection. Neither in macrophages from T. spiralis-infected guinea pig nor in platelet-activating factor (PAF)-stimulated macrophages from uninfected animals, participation of MAP kinases in respiratory burst activation was statistically significant. The parasite antigens seem to act through macrophage PAF receptors, transducing a signal for enhanced NADPH oxidase activity, as stimulating effect of newborn larvae homogenate on respiratory burst was abolished by specific PAF receptor antagonist CV 6209. A suppressive action of T. spiralis larvae on host alveolar macrophage innate immunological response was reflected by diminished protein level of ERK2 kinase and suppressed superoxide anion production, in spite of high level of PKC phosphorylation.

Involvement of fractalkine and macrophage inflammatory protein-1 alpha in moderate-severe depression

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Rosaria Alba Merendino

2004-01-01

Full Text Available MODERATE-severe depression (MSD is linked to overexpression of proinflammatory cytokines and chemokines. Fractalkine (FKN and macrophage inflammatory protein-1 alpha (MIP-1α are, respectively, members of CX3C and C-C chemokines, and both are involved in recruiting and activating mononuclear phagocytes in the central nervous system. We analysed the presence of FKN and MIP-1α in sera of untreated MSD patients and healthy donors. High FKN levels were observed in all MSD patients as compared with values only detectable in 26% of healthy donors. MIP-1α was measurable in 20% of patients, while no healthy donors showed detectable chemokine levels. In conclusion, we describe a previously unknown involvement of FKN in the pathogenesis of MSD, suggesting that FKN may represent a target for a specific immune therapy of this disease.

Response of Mammalian Macrophages to Challenge with the Chlorovirus Acanthocystis turfacea Chlorella Virus 1.

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Petro, Thomas M; Agarkova, Irina V; Zhou, You; Yolken, Robert H; Van Etten, James L; Dunigan, David D

2015-12-01

It was recently reported that 44% of the oropharyngeal samples from the healthy humans in a study cohort had DNA sequences similar to that of the chlorovirus ATCV-1 (Acanthocystis turfacea chlorella virus 1, family Phycodnaviridae) and that these study subjects had decreases in visual processing and visual motor speed compared with individuals in whom no virus was detected. Moreover, mice inoculated orally with ATCV-1 developed immune responses to ATCV-1 proteins and had decreases in certain cognitive domains. Because heightened interleukin-6 (IL-6), nitric oxide (NO), and ERK mitogen-activated protein (MAP) kinase activation from macrophages are linked to cognitive impairments, we evaluated cellular responses and viral PFU counts in murine RAW264.7 cells and primary macrophages after exposure to ATCV-1 in vitro for up to 72 h after a virus challenge. Approximately 8% of the ATCV-1 inoculum was associated with macrophages after 1 h, and the percentage increased 2- to 3-fold over 72 h. Immunoblot assays with rabbit anti-ATCV-1 antibody detected a 55-kDa protein consistent with the viral capsid protein from 1 to 72 h and increasing de novo synthesis of a previously unidentified 17-kDa protein beginning at 24 h. Emergence of the 17-kDa protein did not occur and persistence of the 55-kDa protein declined over time when cells were exposed to heat-inactivated ATCV-1. Moreover, starting at 24 h, RAW264.7 cells exhibited cytopathic effects, annexin V staining, and cleaved caspase 3. Activation of ERK MAP kinases occurred in these cells by 30 min postchallenge, which preceded the expression of IL-6 and NO. Therefore, ATCV-1 persistence in and induction of inflammatory factors by these macrophages may contribute to declines in the cognitive abilities of mice and humans. Virus infections that persist in and stimulate inflammatory factors in macrophages contribute to pathologies in humans. A previous study showed that DNA sequences homologous to the chlorovirus ATCV-1 were

A macrophage inflammatory protein homolog encoded by guinea pig cytomegalovirus signals via CC chemokine receptor 1

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Penfold, Mark; Miao Zhenhua; Wang Yu; Haggerty, Shannon; Schleiss, Mark R.

2003-01-01

Cytomegaloviruses encode homologs of cellular immune effector proteins, including chemokines (CKs) and CK receptor-like G protein-coupled receptors (GPCRs). Sequence of the guinea pig cytomegalovirus (GPCMV) genome identified an open reading frame (ORF) which predicted a 101 amino acid (aa) protein with homology to the macrophage inflammatory protein (MIP) subfamily of CC (β) CKs, designated GPCMV-MIP. To assess functionality of this CK, recombinant GPCMV-MIP was expressed in HEK293 cells and assayed for its ability to bind to and functionally interact with a variety of GPCRs. Specific signaling was observed with the hCCR1 receptor, which could be blocked with hMIP -1α in competition experiments. Migration assays revealed that GPCMV-MIP was able to induce chemotaxis in hCCR1-L1.2 cells. Antisera raised against a GST-MIP fusion protein immunoprecipitated species of ∼12 and 10 kDa from GPCMV-inoculated tissue culture lysates, and convalescent antiserum from GPCMV-infected animals was immunoreactive with GST-MIP by ELISA assay. These results represent the first substantive in vitro characterization of a functional CC CK encoded by a cytomegalovirus

A recombinant fusion toxin based on enzymatic inactive C3bot1 selectively targets macrophages.

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Lydia Dmochewitz

Full Text Available BACKGROUND: The C3bot1 protein (~23 kDa from Clostridium botulinum ADP-ribosylates and thereby inactivates Rho. C3bot1 is selectively taken up into the cytosol of monocytes/macrophages but not of other cell types such as epithelial cells or fibroblasts. Most likely, the internalization occurs by a specific endocytotic pathway via acidified endosomes. METHODOLOGY/PRINCIPAL FINDINGS: Here, we tested whether enzymatic inactive C3bot1E174Q serves as a macrophage-selective transport system for delivery of enzymatic active proteins into the cytosol of such cells. Having confirmed that C3bot1E174Q does not induce macrophage activation, we used the actin ADP-ribosylating C2I (∼50 kDa from Clostridium botulinum as a reporter enzyme for C3bot1E174Q-mediated delivery into macrophages. The recombinant C3bot1E174Q-C2I fusion toxin was cloned and expressed as GST-protein in Escherichia coli. Purified C3bot1E174Q-C2I was recognized by antibodies against C2I and C3bot and showed C2I-specific enzyme activity in vitro. When applied to cultured cells C3bot1E174Q-C2I ADP-ribosylated actin in the cytosol of macrophages including J774A.1 and RAW264.7 cell lines as well as primary cultured human macrophages but not of epithelial cells. Together with confocal fluorescence microscopy experiments, the biochemical data indicate the selective uptake of a recombinant C3-fusion toxin into the cytosol of macrophages. CONCLUSIONS/SIGNIFICANCE: In summary, we demonstrated that C3bot1E174Q can be used as a delivery system for fast, selective and specific transport of enzymes into the cytosol of living macrophages. Therefore, C3-based fusion toxins can represent valuable molecular tools in experimental macrophage pharmacology and cell biology as well as attractive candidates to develop new therapeutic approaches against macrophage-associated diseases.

M1 and M2 macrophages derived from THP-1 cells differentially modulate the response of cancer cells to etoposide

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Genin, Marie; Clement, Francois; Fattaccioli, Antoine; Raes, Martine; Michiels, Carine

2015-01-01

Tumor associated macrophages (TAMs) are present in high density in solid tumors. TAMs share many characteristics with alternatively activated macrophages, also called M2. They have been shown to favor tumor development and a role in chemoresistance has also been suggested. Here, we investigated the effects of M2 in comparison to M1 macrophages on cancer cell sensitivity to etoposide. We set up a model of macrophage polarization, starting from THP-1 monocytes differentiated into macrophages using PMA (Phorbol 12-myristate 13-acetate). Once differentiated (M0 macrophages), they were incubated with IL-4 and IL-13 in order to obtain M2 polarized macrophages or with IFN-gamma and LPS for classical macrophage activation (M1). To mimic the communication between cancer cells and TAMs, M0, M1 or M2 macrophages and HepG2 or A549 cancer cells were co-cultured during respectively 16 (HepG2) or 24 (A549) hours, before etoposide exposure for 24 (HepG2) or 16 (A549) hours. After the incubation, the impact of etoposide on macrophage polarization was studied and cancer cell apoptosis was assessed by western-blot for cleaved caspase-3 and cleaved PARP-1 protein, caspase activity assay and FACS analysis of Annexin V and PI staining. mRNA and protein expression of M1 and M2 markers confirmed the polarization of THP-1-derived macrophages, which provide a new, easy and well-characterized model of polarized human macrophages. Etoposide-induced cancer cell apoptosis was markedly reduced in the presence of THP-1 M2 macrophages, while apoptosis was increased in cells co-cultured with M1 macrophages. On the other hand, etoposide did not influence M1 or M2 polarization. These results evidence for the first time a clear protective effect of M2 on the contrary to M1 macrophages on etoposide-induced cancer cell apoptosis

The role of autophagy in THP-1 macrophages resistance to HIV- vpr-induced apoptosis

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Zhou, Hua-ying, E-mail: zhouhuaying_2004@126.com; Zheng, Yu-huang; He, Yan; Chen, Zi; He, Bo

2017-02-01

Macrophages are resistant to cell death and are one of HIV reservoirs. HIV viral protein Vpr has the potential to promote infection of and survival of macrophages, which could be a highly significant factor in the development and/or maintenance of macrophage viral reservoirs. However, the impact of vpr on macrophages resistance to apoptosis is yet to be comprehended. Autophagy is a cell survival mechanism under stress state. In this study, we investigated whether autophagy is involved in macrophages resistant to vpr-induced apoptosis. Using the THP1 macrophages, we studied the interconnection between macrophages resistance to apoptosis and autophagy. We found that vpr is able to trigger autophagy in transfected THP-1 macrophages confirmed by electron microscopy (EM) and western blot analysis, and inhibition of autophagy with 3MA increased vpr-induced apoptosis. The results indicate that autophagy may be responsible for maintenance of macrophage HIV reservoirs. - Highlights: • HIV Vpr is able to trigger autophagy in transfected THP-1 macrophages. • Autophagy inhibition increases vpr-transfected THP1-macrophages apoptosis. • Autophagy is involved in THP-1 macrophages resistant to vpr-induced apoptosis.

Cytoskeleton-centric protein transportation by exosomes transforms tumor-favorable macrophages

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Cui, Yizhi; Zhou, Yanlong; Yin, Xingfeng; Guo, Jiahui; Zhang, Gong; Wang, Tong; He, Qing-Yu

2016-01-01

The exosome is a key initiator of pre-metastatic niche in numerous cancers, where macrophages serve as primary inducers of tumor microenvironment. However, the proteome that can be exosomally transported from cancer cells to macrophages has not been sufficiently characterized so far. Here, we used colorectal cancer (CRC) exosomes to educate tumor-favorable macrophages. With a SILAC-based mass spectrometry strategy, we successfully traced the proteome transported from CRC exosomes to macrophages. Such a proteome primarily focused on promoting cytoskeleton rearrangement, which was biologically validated with multiple cell lines. We reproduced the exosomal transportation of functional vimentin as a proof-of-concept example. In addition, we found that some CRC exosomes could be recognized by macrophages via Fc receptors. Therefore, we revealed the active and necessary role of exosomes secreted from CRC cells to transform cancer-favorable macrophages, with the cytoskeleton-centric proteins serving as the top functional unit. PMID:27602764

Cytoskeleton-centric protein transportation by exosomes transforms tumor-favorable macrophages.

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Chen, Zhipeng; Yang, Lijuan; Cui, Yizhi; Zhou, Yanlong; Yin, Xingfeng; Guo, Jiahui; Zhang, Gong; Wang, Tong; He, Qing-Yu

2016-10-11

The exosome is a key initiator of pre-metastatic niche in numerous cancers, where macrophages serve as primary inducers of tumor microenvironment. However, the proteome that can be exosomally transported from cancer cells to macrophages has not been sufficiently characterized so far. Here, we used colorectal cancer (CRC) exosomes to educate tumor-favorable macrophages. With a SILAC-based mass spectrometry strategy, we successfully traced the proteome transported from CRC exosomes to macrophages. Such a proteome primarily focused on promoting cytoskeleton rearrangement, which was biologically validated with multiple cell lines. We reproduced the exosomal transportation of functional vimentin as a proof-of-concept example. In addition, we found that some CRC exosomes could be recognized by macrophages via Fc receptors. Therefore, we revealed the active and necessary role of exosomes secreted from CRC cells to transform cancer-favorable macrophages, with the cytoskeleton-centric proteins serving as the top functional unit.

Functional relevance of protein glycosylation to the pro-inflammatory effects of extracellular matrix metalloproteinase inducer (EMMPRIN) on monocytes/macrophages.

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Ge, Heng; Yuan, Wei; Liu, Jidong; He, Qing; Ding, Song; Pu, Jun; He, Ben

2015-01-01

Extracellular matrix metalloproteinase inducer (EMMPRIN) is an important pro-inflammatory protein involved in the cellular functions of monocytes/macrophages. We have hypothesized that high-level heterogeneousness of protein glycosylation of EMMPRIN may have functional relevance to its biological effects and affect the inflammatory activity of monocytes/macrophages. The glycosylation patterns of EMMPRIN expressed by monocytes/macrophages (THP-1 cells) in response to different extracellular stimuli were observed, and the structures of different glycosylation forms were identified. After the purification of highly- and less-glycosylated proteins respectively, the impacts of different glycosylation forms on the pro-inflammatory effects of EMMPRIN were examined in various aspects, such as cell adhesion to endothelial cells, cell migrations, cytokine expression, and activation of inflammatory signalling pathway. 1) It was mainly the highly-glycosylated form of EMMPRIN (HG-EMMPRIN) that increased after being exposed to inflammatory signals (PMA and H2O2). 2) Glycosylation of EMMPRIN in monocytes/macrophages led to N-linked-glycans being added to the protein, with the HG form containing complex-type glycans and the less-glycosylated form (LG) the simple type. 3) Only the HG-EMMPRIN but not the LG-EMMPRIN exhibited pro-inflammatory effects and stimulated inflammatory activities of the monocytes/macrophages (i.e., activation of ERK1/2 and NF-κB pathway, enhanced monocyte-endothelium adhesion, cell migration and matrix metalloproteinase -9 expression). Post-transcriptional glycosylation represents an important mechanism that determines the biological effects of EMMPRIN in monocytes/macrophages. Glycosylation of EMMPRIN may serve as a potential target for regulating the inflammatory activities of monocytes/macrophages.

Functional relevance of protein glycosylation to the pro-inflammatory effects of extracellular matrix metalloproteinase inducer (EMMPRIN on monocytes/macrophages.

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Heng Ge

Full Text Available Extracellular matrix metalloproteinase inducer (EMMPRIN is an important pro-inflammatory protein involved in the cellular functions of monocytes/macrophages. We have hypothesized that high-level heterogeneousness of protein glycosylation of EMMPRIN may have functional relevance to its biological effects and affect the inflammatory activity of monocytes/macrophages.The glycosylation patterns of EMMPRIN expressed by monocytes/macrophages (THP-1 cells in response to different extracellular stimuli were observed, and the structures of different glycosylation forms were identified. After the purification of highly- and less-glycosylated proteins respectively, the impacts of different glycosylation forms on the pro-inflammatory effects of EMMPRIN were examined in various aspects, such as cell adhesion to endothelial cells, cell migrations, cytokine expression, and activation of inflammatory signalling pathway.1 It was mainly the highly-glycosylated form of EMMPRIN (HG-EMMPRIN that increased after being exposed to inflammatory signals (PMA and H2O2. 2 Glycosylation of EMMPRIN in monocytes/macrophages led to N-linked-glycans being added to the protein, with the HG form containing complex-type glycans and the less-glycosylated form (LG the simple type. 3 Only the HG-EMMPRIN but not the LG-EMMPRIN exhibited pro-inflammatory effects and stimulated inflammatory activities of the monocytes/macrophages (i.e., activation of ERK1/2 and NF-κB pathway, enhanced monocyte-endothelium adhesion, cell migration and matrix metalloproteinase -9 expression.Post-transcriptional glycosylation represents an important mechanism that determines the biological effects of EMMPRIN in monocytes/macrophages. Glycosylation of EMMPRIN may serve as a potential target for regulating the inflammatory activities of monocytes/macrophages.

Induction of heat-shock proteins and phagocytic function of chicken macrophage following in vitro heat exposure

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Miller, L.; Qureshi, M.A.

1992-01-01

The protein profiles and phagocytic ability of Sephadex-elicited chicken peritoneal macrophages were examined following heat-shock exposure. Macrophage cultures were exposed to various temperatures, time exposures and recovery periods. Densitometric analysis of SDS-PAGE autoradiographs revealed that heat-induced macrophages synthesized three major (23, 70 and 90 kD) heat-shock proteins (HSPs). The optimal temperature and time for induction of these HSPs was 45-46 degrees C for 1 h, with a variable recovery period for each HSP. Macrophages exposed to 45 degrees C for 30 and 60 min were significantly depressed in phagocytosis of uncoated sheep erythrocytes (SE) under 45 degrees C incubation conditions. However, phagocytosis of antibody-coated SE was not affected when compared to 41 degrees C control cultures. Macrophages allowed to recover at 41 degrees C following heat-shock exhibited no alterations in their phagocytic ability for either antibody-coated or uncoated SE. This study suggests that heat shock induces three major HSPs in chicken peritoneal macrophages in addition to maintaining their Fc-mediated phagocytic function while significantly depressing their nonspecific phagocytosis

Cathepsin D Specifically Cleaves the Chemokines Macrophage Inflammatory Protein-1α, Macrophage Inflammatory Protein-1β, and SLC That Are Expressed in Human Breast Cancer

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Wolf, Marlene; Clark-Lewis, Ian; Buri, Caroline; Langen, Hanno; Lis, Maddalena; Mazzucchelli, Luca

2003-01-01

Cathepsin D (Cath-D) expression in human primary breast cancer has been associated with a poor prognosis. In search of a better understanding of the Cath-D substrates possibly involved in cancer invasiveness and metastasis, we investigated the potential interactions between this protease and chemokines. Here we report that purified Cath-D, as well as culture supernatants from the human breast carcinoma cell lines MCF-7 and T47D, selectively degrade macrophage inflammatory protein (MIP)-1α (CCL3), MIP-1β (CCL4), and SLC (CCL21). Proteolysis was totally blocked by the protease inhibitor pepstatin A, and specificity of Cath-D cleavage was demonstrated using a large chemokine panel. Whereas MIP-1α and MIP-1β degradation was rapid and complete, cleavage of SLC was slow and not complete. Mass spectrometry analysis showed that Cath-D cleaves the Leu58 to Trp59 bond of SLC producing two functionally inactive fragments. Analysis of Cath-D proteolysis of a series of monocyte chemoattractant protein-3/MIP-1β hybrids indicated that processing of MIP-1β might start by cleaving off amino acids located in the C-terminal domain. In situ hybridization studies revealed MIP-1α, MIP-1β, and Cath-D gene expression mainly in the stromal compartment of breast cancers whereas SLC transcripts were found in endothelial cells of capillaries and venules within the neoplastic tissues. Cath-D production in the breast carcinoma cell lines MCF-7 and T47D, as assessed by enzyme-linked immunosorbent assay of culture supernatants and cell lysates, was not affected by stimulation with chemokines such as interleukin-8 (CXCL8), SDF-1 (CXCL12), and SLC. These data suggest that inactivation of chemokines by Cath-D possibly influences regulatory mechanisms in the tumoral extracellular microenvironment that in turn may affect the generation of the antitumoral immune response, the migration of cancer cells, or both processes. PMID:12651610

ACAT1 deletion in murine macrophages associated with cytotoxicity and decreased expression of collagen type 3A1

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Rodriguez, Annabelle; Ashen, M. Dominique; Chen, Edward S.

2005-01-01

In contrast to some published studies of murine macrophages, we previously showed that ACAT inhibitors appeared to be anti-atherogenic in primary human macrophages in that they decreased foam cell formation without inducing cytotoxicity. Herein, we examined foam cell formation and cytotoxicity in murine ACAT1 knockout (KO) macrophages in an attempt to resolve the discrepancies. Elicited peritoneal macrophages from normal C57BL6 and ACAT1 KO mice were incubated with DMEM containing acetylated LDL (acLDL, 100 μg protein/ml) for 48 h. Cells became cholesterol enriched and there were no differences in the total cholesterol mass. Esterified cholesterol mass was lower in ACAT1 KO foam cells compared to normal macrophages (p 14 C]adenine from macrophages, was approximately 2-fold greater in ACAT1 KO macrophages as compared to normal macrophages (p < 0.0001), and this was independent of cholesterol enrichment. cDNA microarray analysis showed that ACAT1 KO macrophages expressed substantially less collagen type 3A1 (26-fold), which was confirmed by RT-PCR. Total collagen content was also significantly reduced (57%) in lung homogenates isolated from ACAT1 KO mice (p < 0.02). Thus, ACAT1 KO macrophages show biochemical changes consistent with increased cytotoxicity and also a novel association with decreased expression of collagen type 3A1

Mucosa-Associated Lymphoid Tissue Lymphoma Translocation Protein 1 Positively Modulates Matrix Metalloproteinase-9 Production in Alveolar Macrophages upon Toll-Like Receptor 7 Signaling and Influenza Virus Infection

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Yu-Hsiang Lee

2017-09-01

Full Text Available Influenza A virus (IAV infection causes significant morbidity and mortality worldwide. Matrix metalloproteinase-9 (MMP-9 degrades extracellular matrix and is involved in the pathology of influenza. It has been reported that MMP-9 mediates neutrophil migration in IAV infection. Whether alveolar macrophages, the first immune cells that encounter IAV, produce MMP-9, and the mechanism of its regulation have never been investigated. As Toll-like receptor 7 (TLR7 is one of the receptors in innate immune cells that recognize IAV, we used TLR7 agonists and IAV to stimulate alveolar macrophage MH-S cells, primary macrophages, and bone marrow neutrophils. Results showed that MMP-9 expression in macrophages is inducible by TLR7 agonists and IAV, yet, MMP-9 production by neutrophils is not inducible by either one of them. We hypothesized that MMP-9 production in macrophages is mediated through TLR7-NF-κB pathway and used microarray to analyze TLR7 agonist-induced NF-κB-related genes. Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1, a positive regulator of NF-κB, is amongst the top highly induced genes. By use of MALT1 inhibitor (z-VRPR-fmk and alveolar macrophages from MALT1-deficient mice, we found that MMP-9 production is MALT1-dependent. While MALT1 can act as a paracaspase in lymphocytes through degrading various signaling proteins, we discovered that MALT1 functions to reduce a negative regulator of NF-κB, cylindromatosis (CYLD, in alveolar macrophages. IAV-induced MMP-9, TNF, and IL-6 in lungs of MALT1-deficient mice are significantly lower than in wild-type mice after intratracheal infection. MALT1-deficient mice also have less body weight loss and longer survival after infection. Taken together, we demonstrated a novel role of MALT1 in regulating alveolar macrophage MMP-9 production whose presence exacerbates the severity of influenza.

Rac2 controls tumor growth, metastasis and M1-M2 macrophage differentiation in vivo.

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Shweta Joshi

Full Text Available Although it is well-established that the macrophage M1 to M2 transition plays a role in tumor progression, the molecular basis for this process remains incompletely understood. Herein, we demonstrate that the small GTPase, Rac2 controls macrophage M1 to M2 differentiation and the metastatic phenotype in vivo. Using a genetic approach, combined with syngeneic and orthotopic tumor models we demonstrate that Rac2-/- mice display a marked defect in tumor growth, angiogenesis and metastasis. Microarray, RT-PCR and metabolomic analysis on bone marrow derived macrophages isolated from the Rac2-/- mice identify an important role for Rac2 in M2 macrophage differentiation. Furthermore, we define a novel molecular mechanism by which signals transmitted from the extracellular matrix via the α4β1 integrin and MCSF receptor lead to the activation of Rac2 and potentially regulate macrophage M2 differentiation. Collectively, our findings demonstrate a macrophage autonomous process by which the Rac2 GTPase is activated downstream of the α4β1 integrin and the MCSF receptor to control tumor growth, metastasis and macrophage differentiation into the M2 phenotype. Finally, using gene expression and metabolomic data from our Rac2-/- model, and information related to M1-M2 macrophage differentiation curated from the literature we executed a systems biologic analysis of hierarchical protein-protein interaction networks in an effort to develop an iterative interactome map which will predict additional mechanisms by which Rac2 may coordinately control macrophage M1 to M2 differentiation and metastasis.

Macrophage polarisation: an immunohistochemical approach for identifying M1 and M2 macrophages.

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Mário Henrique M Barros

Full Text Available Macrophage polarization is increasingly recognised as an important pathogenetic factor in inflammatory and neoplastic diseases. Proinflammatory M1 macrophages promote T helper (Th 1 responses and show tumoricidal activity. M2 macrophages contribute to tissue repair and promote Th2 responses. CD68 and CD163 are used to identify macrophages in tissue sections. However, characterisation of polarised macrophages in situ has remained difficult. Macrophage polarisation is regulated by transcription factors, pSTAT1 and RBP-J for M1, and CMAF for M2. We reasoned that double-labelling immunohistochemistry for the detection of macrophage markers together with transcription factors may be suitable to characterise macrophage polarisation in situ. To test this hypothesis, we have studied conditions associated with Th1- and Th2-predominant immune responses: infectious mononucleosis and Crohn's disease for Th1 and allergic nasal polyps, oxyuriasis, wound healing and foreign body granulomas for predominant Th2 response. In all situations, CD163+ cells usually outnumbered CD68+ cells. Moreover, CD163+ cells, usually considered as M2 macrophages, co-expressing pSTAT1 and RBP-J were found in all conditions examined. The numbers of putative M1 macrophages were higher in Th1- than in Th2-associated diseases, while more M2 macrophages were seen in Th2- than in Th1 related disorders. In most Th1-related diseases, the balance of M1 over M2 cells was shifted towards M1 cells, while the reverse was observed for Th2-related conditions. Hierarchical cluster analysis revealed two distinct clusters: cluster I included Th1 diseases together with cases with high numbers of CD163+pSTAT1+, CD68+pSTAT1+, CD163+RBP-J+ and CD68+RBP-J+ macrophages; cluster II comprised Th2 conditions together with cases displaying high numbers of CD163+CMAF+ and CD68+CMAF+ macrophages. These results suggest that the detection of pSTAT1, RBP-J, and CMAF in the context of CD68 or CD163 expression is a

Macrophage polarisation: an immunohistochemical approach for identifying M1 and M2 macrophages.

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Barros, Mário Henrique M; Hauck, Franziska; Dreyer, Johannes H; Kempkes, Bettina; Niedobitek, Gerald

2013-01-01

Macrophage polarization is increasingly recognised as an important pathogenetic factor in inflammatory and neoplastic diseases. Proinflammatory M1 macrophages promote T helper (Th) 1 responses and show tumoricidal activity. M2 macrophages contribute to tissue repair and promote Th2 responses. CD68 and CD163 are used to identify macrophages in tissue sections. However, characterisation of polarised macrophages in situ has remained difficult. Macrophage polarisation is regulated by transcription factors, pSTAT1 and RBP-J for M1, and CMAF for M2. We reasoned that double-labelling immunohistochemistry for the detection of macrophage markers together with transcription factors may be suitable to characterise macrophage polarisation in situ. To test this hypothesis, we have studied conditions associated with Th1- and Th2-predominant immune responses: infectious mononucleosis and Crohn's disease for Th1 and allergic nasal polyps, oxyuriasis, wound healing and foreign body granulomas for predominant Th2 response. In all situations, CD163+ cells usually outnumbered CD68+ cells. Moreover, CD163+ cells, usually considered as M2 macrophages, co-expressing pSTAT1 and RBP-J were found in all conditions examined. The numbers of putative M1 macrophages were higher in Th1- than in Th2-associated diseases, while more M2 macrophages were seen in Th2- than in Th1 related disorders. In most Th1-related diseases, the balance of M1 over M2 cells was shifted towards M1 cells, while the reverse was observed for Th2-related conditions. Hierarchical cluster analysis revealed two distinct clusters: cluster I included Th1 diseases together with cases with high numbers of CD163+pSTAT1+, CD68+pSTAT1+, CD163+RBP-J+ and CD68+RBP-J+ macrophages; cluster II comprised Th2 conditions together with cases displaying high numbers of CD163+CMAF+ and CD68+CMAF+ macrophages. These results suggest that the detection of pSTAT1, RBP-J, and CMAF in the context of CD68 or CD163 expression is a suitable tool for

Fibronectin induces macrophage migration through a SFK-FAK/CSF-1R pathway.

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Digiacomo, Graziana; Tusa, Ignazia; Bacci, Marina; Cipolleschi, Maria Grazia; Dello Sbarba, Persio; Rovida, Elisabetta

2017-07-04

Integrins, following binding to proteins of the extracellular matrix (ECM) including collagen, laminin and fibronectin (FN), are able to transduce molecular signals inside the cells and to regulate several biological functions such as migration, proliferation and differentiation. Besides activation of adaptor molecules and kinases, integrins transactivate Receptor Tyrosine Kinases (RTK). In particular, adhesion to the ECM may promote RTK activation in the absence of growth factors. The Colony-Stimulating Factor-1 Receptor (CSF-1R) is a RTK that supports the survival, proliferation, and motility of monocytes/macrophages, which are essential components of innate immunity and cancer development. Macrophage interaction with FN is recognized as an important aspect of host defense and wound repair. The aim of the present study was to investigate on a possible cross-talk between FN-elicited signals and CSF-1R in macrophages. FN induced migration in BAC1.2F5 and J774 murine macrophage cell lines and in human primary macrophages. Adhesion to FN determined phosphorylation of the Focal Adhesion Kinase (FAK) and Src Family Kinases (SFK) and activation of the SFK/FAK complex, as witnessed by paxillin phosphorylation. SFK activity was necessary for FAK activation and macrophage migration. Moreover, FN-induced migration was dependent on FAK in either murine macrophage cell lines or human primary macrophages. FN also induced FAK-dependent/ligand-independent CSF-1R phosphorylation, as well as the interaction between CSF-1R and β1. CSF-1R activity was necessary for FN-induced macrophage migration. Indeed, genetic or pharmacological inhibition of CSF-1R prevented FN-induced macrophage migration. Our results identified a new SFK-FAK/CSF-1R signaling pathway that mediates FN-induced migration of macrophages.

Macrophage Immune Response Suppression by Recombinant Mycobacterium tuberculosis Antigens, the ESAT-6, CFP-10, and ESAT-6/CFP-10 Fusion Proteins

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Seghatoleslam, Atefeh; Hemmati, Mina; Ebadat, Saeedeh; Movahedi, Bahram; Mostafavi-Pour, Zohreh

2016-01-01

Background: Macrophage immune responses are affected by the secretory proteins of Mycobacterium tuberculosis (Mtb). This study aimed to examine the immune responses of macrophages to Mtb secretory antigens, namely ESAT-6, CFP-10, and ESAT-6/CFP-10. Methods: THP-1 cells (a human monocytic cell line) were cultured and differentiated to macrophages by phorbol 12-myristate 13-acetate. The cytotoxicity of the recombinant Mtb proteins was assessed using the MTT assay. Two important immune responses of macrophages, namely NO and ROS production, were measured in response to the ESAT-6, CFP-10, and ESAT-6/CFP-10 antigens. The data were analyzed using one-way ANOVA with SPSS, version 16, and considered significant at Pproteins markedly reduced macrophage immune response. The treatment of the THP-1-differentiated cells with ESAT-6, CFP-10, and ESAT-6/CFP-10 reduced NO and ROS production. The treated THP-1-differentiated cells exhibited less inducible NO synthase activity than did the untreated cells. No toxic effect on macrophage viability was observed for the applied proteins at the different concentrations. Conclusion: It seems that the decline in macrophage immune response is due to the suppression of NO and ROS production pathways without any effect on cell viability. PMID:27365551

Inactivation of lipoprotein lipase occurs on the surface of THP-1 macrophages where oligomers of angiopoietin-like protein 4 are formed

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Makoveichuk, Elena; Sukonina, Valentina; Kroupa, Olessia; Thulin, Petra; Ehrenborg, Ewa; Olivecrona, Thomas; Olivecrona, Gunilla

2012-01-01

Highlights: ► Lipoprotein lipase (LPL) activity is controlled by ANGPTL4 in THP-1 macrophages. ► Both LPL and ANGPTL4 bind to THP-1 macrophages in a heparin-releasable fashion. ► Only monomers of ANGPTL4 are present within THP-1 macrophages. ► Covalent oligomers of ANGPTL4 appear on cell surface and in medium. ► Inactivation of LPL coincide with ANGPTL4 oligomer formation on cell surfaces. -- Abstract: Lipoprotein lipase (LPL) hydrolyzes triglycerides in plasma lipoproteins causing release of fatty acids for metabolic purposes in muscles and adipose tissue. LPL in macrophages in the artery wall may, however, promote foam cell formation and atherosclerosis. Angiopoietin-like protein (ANGPTL) 4 inactivates LPL and ANGPTL4 expression is controlled by peroxisome proliferator-activated receptors (PPAR). The mechanisms for inactivation of LPL by ANGPTL4 was studied in THP-1 macrophages where active LPL is associated with cell surfaces in a heparin-releasable form, while LPL in the culture medium is mostly inactive. The PPARδ agonist GW501516 had no effect on LPL mRNA, but increased ANGPTL4 mRNA and caused a marked reduction of the heparin-releasable LPL activity concomitantly with accumulation of inactive, monomeric LPL in the medium. Intracellular ANGPTL4 was monomeric, while dimers and tetramers of ANGPTL4 were present in the heparin-releasable fraction and medium. GW501516 caused an increase in the amount of ANGPTL4 oligomers on the cell surface that paralleled the decrease in LPL activity. Actinomycin D blocked the effects of GW501516 on ANGPTL4 oligomer formation and prevented the inactivation of LPL. Antibodies against ANGPTL4 interfered with the inactivation of LPL. We conclude that inactivation of LPL in THP-1 macrophages primarily occurs on the cell surface where oligomers of ANGPTL4 are formed.

Differential regulation of caspase-1 activation, pyroptosis, and autophagy via Ipaf and ASC in Shigella-infected macrophages.

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Toshihiko Suzuki

2007-08-01

Full Text Available Shigella infection, the cause of bacillary dysentery, induces caspase-1 activation and cell death in macrophages, but the precise mechanisms of this activation remain poorly understood. We demonstrate here that caspase-1 activation and IL-1beta processing induced by Shigella are mediated through Ipaf, a cytosolic pattern-recognition receptor of the nucleotide-binding oligomerization domain (NOD-like receptor (NLR family, and the adaptor protein apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC. We also show that Ipaf was critical for pyroptosis, a specialized form of caspase-1-dependent cell death induced in macrophages by bacterial infection, whereas ASC was dispensable. Unlike that observed in Salmonella and Legionella, caspase-1 activation induced by Shigella infection was independent of flagellin. Notably, infection of macrophages with Shigella induced autophagy, which was dramatically increased by the absence of caspase-1 or Ipaf, but not ASC. Autophagy induced by Shigella required an intact bacterial type III secretion system but not VirG protein, a bacterial factor required for autophagy in epithelial-infected cells. Treatment of macrophages with 3-methyladenine, an inhibitor of autophagy, enhanced pyroptosis induced by Shigella infection, suggesting that autophagy protects infected macrophages from pyroptosis. Thus, Ipaf plays a critical role in caspase-1 activation induced by Shigella independently of flagellin. Furthermore, the absence of Ipaf or caspase-1, but not ASC, regulates pyroptosis and the induction of autophagy in Shigella-infected macrophages, providing a novel function for NLR proteins in bacterial-host interactions.

Pharmacological inhibition of dynamin II reduces constitutive protein secretion from primary human macrophages.

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Maaike Kockx

Full Text Available Dynamins are fission proteins that mediate endocytic and exocytic membrane events and are pharmacological therapeutic targets. These studies investigate whether dynamin II regulates constitutive protein secretion and show for the first time that pharmacological inhibition of dynamin decreases secretion of apolipoprotein E (apoE and several other proteins constitutively secreted from primary human macrophages. Inhibitors that target recruitment of dynamin to membranes (MiTMABs or directly target the GTPase domain (Dyngo or Dynole series, dose- and time- dependently reduced the secretion of apoE. SiRNA oligo's targeting all isoforms of dynamin II confirmed the involvement of dynamin II in apoE secretion. Inhibition of secretion was not mediated via effects on mRNA or protein synthesis. 2D-gel electrophoresis showed that inhibition occurred after apoE was processed and glycosylated in the Golgi and live cell imaging showed that inhibited secretion was associated with reduced post-Golgi movement of apoE-GFP-containing vesicles. The effect was not restricted to macrophages, and was not mediated by the effects of the inhibitors on microtubules. Inhibition of dynamin also altered the constitutive secretion of other proteins, decreasing the secretion of fibronectin, matrix metalloproteinase 9, Chitinase-3-like protein 1 and lysozyme but unexpectedly increasing the secretion of the inflammatory mediator cyclophilin A. We conclude that pharmacological inhibitors of dynamin II modulate the constitutive secretion of macrophage apoE as a class effect, and that their capacity to modulate protein secretion may affect a range of biological processes.

MicroRNA 27a-3p Regulates Antimicrobial Responses of Murine Macrophages Infected by Mycobacterium avium subspecies paratuberculosis by Targeting Interleukin-10 and TGF-β-Activated Protein Kinase 1 Binding Protein 2

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Tariq Hussain

2018-01-01

Full Text Available Mycobacterium avium subspecies paratuberculosis (MAP persistently survive and replicate in mononuclear phagocytic cells by adopting various strategies to subvert host immune response. Interleukin-10 (IL-10 upregulation via inhibition of macrophage bactericidal activity is a critical step for MAP survival and pathogenesis within the host cell. Mitogen-activated protein kinase p38 signaling cascade plays a crucial role in the elevation of IL-10 and progression of MAP pathogenesis. The contribution of microRNAs (miRNAs and their influence on the activation of macrophages during MAP pathogenesis are still unclear. In the current study, we found that miRNA-27a-3p (miR-27a expression is downregulated during MAP infection both in vivo and in vitro. Moreover, miR-27a is also downregulated in toll-like receptor 2 (TLR2-stimulated murine macrophages (RAW264.7 and bone marrow-derived macrophage. ELISA and real-time qRT-PCR results confirm that overexpression of miR-27a inhibited MAP-induced IL-10 production in macrophages and upregulated pro-inflammatory cytokines, while miR-27a inhibitor counteracted these effects. Luciferase reporter assay results revealed that IL-10 and TGF-β-activated protein kinase 1 binding protein 2 (TAB 2 are potential targets of miR-27a. In addition, we demonstrated that miR-27a negatively regulates TAB 2 expression and diminishes TAB 2-dependent p38/JNK phosphorylation, ultimately downregulating IL-10 expression in MAP-infected macrophages. Furthermore, overexpression of miR-27a significantly inhibited the intracellular survival of MAP in infected macrophages. Our data show that miR-27a augments antimicrobial activities of macrophages and inhibits the expression of IL-10, demonstrating that miR-27a regulates protective innate immune responses during MAP infection and can be exploited as a novel therapeutic target in the control of intracellular pathogens, including paratuberculosis.

Viral infection of human lung macrophages increases PDL1 expression via IFNβ.

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Karl J Staples

Full Text Available Lung macrophages are an important defence against respiratory viral infection and recent work has demonstrated that influenza-induced macrophage PDL1 expression in the murine lung leads to rapid modulation of CD8+ T cell responses via the PD1 receptor. This PD1/PDL1 pathway may downregulate acute inflammatory responses to prevent tissue damage. The aim of this study was to investigate the mechanisms of PDL1 regulation by human macrophages in response to viral infection. Ex-vivo viral infection models using influenza and RSV were established in human lung explants, isolated lung macrophages and monocyte-derived macrophages (MDM and analysed by flow cytometry and RT-PCR. Incubation of lung explants, lung macrophages and MDM with X31 resulted in mean cellular infection rates of 18%, 18% and 29% respectively. Viral infection significantly increased cell surface expression of PDL1 on explant macrophages, lung macrophages and MDM but not explant epithelial cells. Infected MDM induced IFNγ release from autologous CD8+ T cells, an effect enhanced by PDL1 blockade. We observed increases in PDL1 mRNA and IFNβ mRNA and protein release by MDM in response to influenza infection. Knockdown of IFNβ by siRNA, resulted in a 37.5% reduction in IFNβ gene expression in response to infection, and a significant decrease in PDL1 mRNA. Furthermore, when MDM were incubated with IFNβ, this cytokine caused increased expression of PDL1 mRNA. These data indicate that human macrophage PDL1 expression modulates CD8+ cell IFNγ release in response to virus and that this expression is regulated by autologous IFNβ production.

Unfolded protein response (UPR) signaling regulates arsenic trioxide-mediated macrophage innate immune function disruption

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Srivastava, Ritesh K.; Li, Changzhao; Chaudhary, Sandeep C.; Ballestas, Mary E.; Elmets, Craig A.; Robbins, David J.; Matalon, Sadis; Deshane, Jessy S.; Afaq, Farrukh; Bickers, David R.; Athar, Mohammad

2013-01-01

Arsenic exposure is known to disrupt innate immune functions in humans and in experimental animals. In this study, we provide a mechanism by which arsenic trioxide (ATO) disrupts macrophage functions. ATO treatment of murine macrophage cells diminished internalization of FITC-labeled latex beads, impaired clearance of phagocytosed fluorescent bacteria and reduced secretion of pro-inflammatory cytokines. These impairments in macrophage functions are associated with ATO-induced unfolded protein response (UPR) signaling pathway characterized by the enhancement in proteins such as GRP78, p-PERK, p-eIF2α, ATF4 and CHOP. The expression of these proteins is altered both at transcriptional and translational levels. Pretreatment with chemical chaperon, 4-phenylbutyric acid (PBA) attenuated the ATO-induced activation in UPR signaling and afforded protection against ATO-induced disruption of macrophage functions. This treatment also reduced ATO-mediated reactive oxygen species (ROS) generation. Interestingly, treatment with antioxidant N-acetylcysteine (NAC) prior to ATO exposure, not only reduced ROS production and UPR signaling but also improved macrophage functions. These data demonstrate that UPR signaling and ROS generation are interdependent and are involved in the arsenic-induced pathobiology of macrophage. These data also provide a novel strategy to block the ATO-dependent impairment in innate immune responses. - Highlights: • Inorganic arsenic to humans and experimental animals disrupt innate immune responses. • The mechanism underlying arsenic impaired macrophage functions involves UPR signaling. • Chemical chaperon attenuates arsenic-mediated macrophage function impairment. • Antioxidant, NAC blocks impairment in arsenic-treated macrophage functions

Unfolded protein response (UPR) signaling regulates arsenic trioxide-mediated macrophage innate immune function disruption

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Srivastava, Ritesh K.; Li, Changzhao; Chaudhary, Sandeep C. [Department of Dermatology and Skin Diseases Research Center, University of Alabama at Birmingham, Birmingham, AL (United States); Ballestas, Mary E. [Department of Pediatrics Infectious Disease, Children' s of Alabama, School of Medicine, University of Alabama at Birmingham, AL (United States); Elmets, Craig A. [Department of Dermatology and Skin Diseases Research Center, University of Alabama at Birmingham, Birmingham, AL (United States); Robbins, David J. [Department of Surgery, Molecular Oncology Program, Miller School of Medicine, University of Miami, Miami (United States); Matalon, Sadis [Department of Anesthesiology, University of Alabama at Birmingham, Birmingham, AL (United States); Deshane, Jessy S. [Department of Medicine, Division of Pulmonary, Allergy and Critical Care Medicine, University of Alabama at Birmingham, Birmingham, AL (United States); Afaq, Farrukh [Department of Dermatology and Skin Diseases Research Center, University of Alabama at Birmingham, Birmingham, AL (United States); Bickers, David R. [Department of Dermatology, Columbia University Medical Center, New York (United States); Athar, Mohammad, E-mail: mathar@uab.edu [Department of Dermatology and Skin Diseases Research Center, University of Alabama at Birmingham, Birmingham, AL (United States)

2013-11-01

Arsenic exposure is known to disrupt innate immune functions in humans and in experimental animals. In this study, we provide a mechanism by which arsenic trioxide (ATO) disrupts macrophage functions. ATO treatment of murine macrophage cells diminished internalization of FITC-labeled latex beads, impaired clearance of phagocytosed fluorescent bacteria and reduced secretion of pro-inflammatory cytokines. These impairments in macrophage functions are associated with ATO-induced unfolded protein response (UPR) signaling pathway characterized by the enhancement in proteins such as GRP78, p-PERK, p-eIF2α, ATF4 and CHOP. The expression of these proteins is altered both at transcriptional and translational levels. Pretreatment with chemical chaperon, 4-phenylbutyric acid (PBA) attenuated the ATO-induced activation in UPR signaling and afforded protection against ATO-induced disruption of macrophage functions. This treatment also reduced ATO-mediated reactive oxygen species (ROS) generation. Interestingly, treatment with antioxidant N-acetylcysteine (NAC) prior to ATO exposure, not only reduced ROS production and UPR signaling but also improved macrophage functions. These data demonstrate that UPR signaling and ROS generation are interdependent and are involved in the arsenic-induced pathobiology of macrophage. These data also provide a novel strategy to block the ATO-dependent impairment in innate immune responses. - Highlights: • Inorganic arsenic to humans and experimental animals disrupt innate immune responses. • The mechanism underlying arsenic impaired macrophage functions involves UPR signaling. • Chemical chaperon attenuates arsenic-mediated macrophage function impairment. • Antioxidant, NAC blocks impairment in arsenic-treated macrophage functions.

Impact of glutathione peroxidase-1 deficiency on macrophage foam cell formation and proliferation: implications for atherogenesis.

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Fei Cheng

Full Text Available Clinical and experimental evidence suggests a protective role for the antioxidant enzyme glutathione peroxidase-1 (GPx-1 in the atherogenic process. GPx-1 deficiency accelerates atherosclerosis and increases lesion cellularity in ApoE(-/- mice. However, the distribution of GPx-1 within the atherosclerotic lesion as well as the mechanisms leading to increased macrophage numbers in lesions is still unknown. Accordingly, the aims of the present study were (1 to analyze which cells express GPx-1 within atherosclerotic lesions and (2 to determine whether a lack of GPx-1 affects macrophage foam cell formation and cellular proliferation. Both in situ-hybridization and immunohistochemistry of lesions of the aortic sinus of ApoE(-/- mice after 12 weeks on a Western type diet revealed that both macrophages and - even though to a less extent - smooth muscle cells contribute to GPx-1 expression within atherosclerotic lesions. In isolated mouse peritoneal macrophages differentiated for 3 days with macrophage-colony-stimulating factor (MCSF, GPx-1 deficiency increased oxidized low density-lipoprotein (oxLDL induced foam cell formation and led to increased proliferative activity of peritoneal macrophages. The MCSF- and oxLDL-induced proliferation of peritoneal macrophages from GPx-1(-/-ApoE(-/- mice was mediated by the p44/42 MAPK (p44/42 mitogen-activated protein kinase, namely ERK1/2 (extracellular-signal regulated kinase 1/2, signaling pathway as demonstrated by ERK1/2 signaling pathways inhibitors, Western blots on cell lysates with primary antibodies against total and phosphorylated ERK1/2, MEK1/2 (mitogen-activated protein kinase kinase 1/2, p90RSK (p90 ribosomal s6 kinase, p38 MAPK and SAPK/JNK (stress-activated protein kinase/c-Jun N-terminal kinase, and immunohistochemistry of mice atherosclerotic lesions with antibodies against phosphorylated ERK1/2, MEK1/2 and p90RSK. Representative effects of GPx-1 deficiency on both macrophage proliferation and

HIV-1 Latency in Monocytes/Macrophages

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Amit Kumar

2014-04-01

Full Text Available Human immunodeficiency virus type 1 (HIV-1 targets CD4+ T cells and cells of the monocyte/macrophage lineage. HIV pathogenesis is characterized by the depletion of T lymphocytes and by the presence of a population of cells in which latency has been established called the HIV-1 reservoir. Highly active antiretroviral therapy (HAART has significantly improved the life of HIV-1 infected patients. However, complete eradication of HIV-1 from infected individuals is not possible without targeting latent sources of infection. HIV-1 establishes latent infection in resting CD4+ T cells and findings indicate that latency can also be established in the cells of monocyte/macrophage lineage. Monocyte/macrophage lineage includes among others, monocytes, macrophages and brain resident macrophages. These cells are relatively more resistant to apoptosis induced by HIV-1, thus are important stable hideouts of the virus. Much effort has been made in the direction of eliminating HIV-1 resting CD4+ T-cell reservoirs. However, it is impossible to achieve a cure for HIV-1 without considering these neglected latent reservoirs, the cells of monocyte/macrophage lineage. In this review we will describe our current understanding of the mechanism of latency in monocyte/macrophage lineage and how such cells can be specifically eliminated from the infected host.

The Scavenger Protein Apoptosis Inhibitor of Macrophages (AIM) Potentiates the Antimicrobial Response against Mycobacterium tuberculosis by Enhancing Autophagy

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Sanjurjo, Lucía; Amézaga, Núria; Vilaplana, Cristina; Cáceres, Neus; Marzo, Elena; Valeri, Marta; Cardona, Pere-Joan; Sarrias, Maria-Rosa

2013-01-01

Apoptosis inhibitor of macrophages (AIM), a scavenger protein secreted by tissue macrophages, is transcriptionally regulated by the nuclear receptor Liver X Receptor (LXR) and Retinoid X Receptor (RXR) heterodimer. Given that LXR exerts a protective immune response against M. tuberculosis, here we analyzed whether AIM is involved in this response. In an experimental murine model of tuberculosis, AIM serum levels peaked dramatically early after infection with M. tuberculosis, providing an in vivo biological link to the disease. We therefore studied the participation of AIM in macrophage response to M. tuberculosis in vitro. For this purpose, we used the H37Rv strain to infect THP-1 macrophages transfected to stably express AIM, thereby increasing infected macrophage survival. Furthermore, the expression of this protein enlarged foam cell formation by enhancing intracellular lipid content. Phagocytosis assays with FITC-labeled M. tuberculosis bacilli indicated that this protein was not involved in bacterial uptake; however, AIM expression decreased the number of intracellular cfus by up to 70% in bacterial killing assays, suggesting that AIM enhances macrophage mycobactericidal activity. Accordingly, M. tuberculosis-infected AIM-expressing cells upregulated the production of reactive oxygen species. Moreover, real-time PCR analysis showed increased mRNA levels of the antimicrobial peptides cathelicidin and defensin 4B. These increases were concomitant with greater cellular concentrations of the autophagy-related molecules Beclin 1 and LC3II, as well as enhanced acidification of mycobacterial phagosomes and LC3 co-localization. In summary, our data support the notion that AIM contributes to key macrophage responses to M. tuberculosis. PMID:24223991

Immunotherapy of HIV-infected patients with Gc protein-derived macrophage activating factor (GcMAF).

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Yamamoto, Nobuto; Ushijima, Naofumi; Koga, Yoshihiko

2009-01-01

Serum Gc protein (known as vitamin D3-binding protein) is the precursor for the principal macrophage activating factor (MAF). The MAF precursor activity of serum Gc protein of HIV-infected patients was lost or reduced because Gc protein is deglycosylated by alpha-N-acetylgalactosaminidase (Nagalase) secreted from HIV-infected cells. Therefore, macrophages of HIV-infected patients having deglycosylated Gc protein cannot be activated, leading to immunosuppression. Since Nagalase is the intrinsic component of the envelope protein gp120, serum Nagalase activity is the sum of enzyme activities carried by both HIV virions and envelope proteins. These Nagalase carriers were already complexed with anti-HIV immunoglobulin G (IgG) but retained Nagalase activity that is required for infectivity. Stepwise treatment of purified Gc protein with immobilized beta-galactosidase and sialidase generated the most potent macrophage activating factor (termed GcMAF), which produces no side effects in humans. Macrophages activated by administration of 100 ng GcMAF develop a large amount of Fc-receptors as well as an enormous variation of receptors that recognize IgG-bound and unbound HIV virions. Since latently HIV-infected cells are unstable and constantly release HIV virions, the activated macrophages rapidly intercept the released HIV virions to prevent reinfection resulting in exhaustion of infected cells. After less than 18 weekly administrations of 100 ng GcMAF for nonanemic patients, they exhibited low serum Nagalase activities equivalent to healthy controls, indicating eradication of HIV-infection, which was also confirmed by no infectious center formation by provirus inducing agent-treated patient PBMCs. No recurrence occurred and their healthy CD + cell counts were maintained for 7 years.

Macrophage fusion is controlled by the cytoplasmic protein tyrosine phosphatase PTP-PEST/PTPN12.

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Rhee, Inmoo; Davidson, Dominique; Souza, Cleiton Martins; Vacher, Jean; Veillette, André

2013-06-01

Macrophages can undergo cell-cell fusion, leading to the formation of multinucleated giant cells and osteoclasts. This process is believed to promote the proteolytic activity of macrophages toward pathogens, foreign bodies, and extracellular matrices. Here, we examined the role of PTP-PEST (PTPN12), a cytoplasmic protein tyrosine phosphatase, in macrophage fusion. Using a macrophage-targeted PTP-PEST-deficient mouse, we determined that PTP-PEST was not needed for macrophage differentiation or cytokine production. However, it was necessary for interleukin-4-induced macrophage fusion into multinucleated giant cells in vitro. It was also needed for macrophage fusion following implantation of a foreign body in vivo. Moreover, in the RAW264.7 macrophage cell line, PTP-PEST was required for receptor activator of nuclear factor kappa-B ligand (RANKL)-triggered macrophage fusion into osteoclasts. PTP-PEST had no impact on expression of fusion mediators such as β-integrins, E-cadherin, and CD47, which enable macrophages to become fusion competent. However, it was needed for polarization of macrophages, migration induced by the chemokine CC chemokine ligand 2 (CCL2), and integrin-induced spreading, three key events in the fusion process. PTP-PEST deficiency resulted in specific hyperphosphorylation of the protein tyrosine kinase Pyk2 and the adaptor paxillin. Moreover, a fusion defect was induced upon treatment of normal macrophages with a Pyk2 inhibitor. Together, these data argue that macrophage fusion is critically dependent on PTP-PEST. This function is seemingly due to the ability of PTP-PEST to control phosphorylation of Pyk2 and paxillin, thereby regulating cell polarization, migration, and spreading.

Cyclic GMP-dependent protein kinase II is necessary for macrophage M1 polarization and phagocytosis via toll-like receptor 2.

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Liao, Wei-Ting; You, Huey-Ling; Li, Changgui; Chang, Jan-Gowth; Chang, Shun-Jen; Chen, Chung-Jen

2015-05-01

Cyclic GMP-dependent protein kinase II (cGKII; PRKG2) phosphorylates a variety of biological targets and has been identified as a gout-susceptible gene. However, the regulatory role of cGKII in triggering gout disease has yet to be clarified. Thus, we plan to explore the specific function of cGKII in macrophages related to gout disease. By using cGKII gene knockdown method, we detected macrophage M1/M2 polarization, phagocytosis, and their responses to stimulation by monosodium urate (MSU). cGKII was highly expressed in M1 phenotype, but not in M2, and cGKII knockdown significantly inhibited macrophage M1 polarization by decreasing M1 chemokine markers (CXCL10 and CCL2) and downregulating phagocytosis function. We further identified that cGKII-associated phagocytosis was mediated by upregulating toll-like receptor 2 (TLR2) expression, but not by TLR4. Mimicking gout condition by MSU treatments, we found that MSU alone induced cGKII and TLR2 expression with increased M1 polarization markers and phagocytosis activity. It means that cGKII knockdown significantly inhibited this MSU-induced cGKII-TLR2-phagocytosis axis. Our study showed that cGKII plays a key role in M1 polarization, especially in TLR2-mediated phagocytosis under MSU exposure. The findings provide evidence for the possible role of cGKII as an inflammation exciter in gout disease. Gout-susceptible gene cGKII is necessary for macrophage M1 polarization. cGKII regulates M1 phagocytosis function via TLR2. Monosodium urate treatments increase cGKII expression and related function. This study reveals the role of cGKII in enhancing gouty inflammatory responses.

Localization of CORO1A in the Macrophages Containing Mycobacterium leprae

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Suzuki, Koichi; Takeshita, Fumihiko; Nakata, Noboru; Ishii, Norihisa; Makino, Masahiko

2006-01-01

Mycobacteria have acquired an intracellular lifestyle within the macrophage, which is best exemplified by the enlarged infected histiocytes seen in lepromatous leprosy. To survive within the cell, mycobacteria must escape intracellular bactericidal mechanisms. In a study of Mycobacterium bovis Bacille Calmette-Guérin (M. bovis BCG) infection, it was shown that the host protein, CORO1A, also known as tryptophan aspartate-containing coat protein (TACO), accumulates on the phagosomal membrane, resulting in inhibition of phagosome-lysosome fusion, and thus augmenting intracellular survival. In this study, we show that CORO1A strongly localizes on the membrane of phagosomes that contain Mycobacterium leprae (M. leprae), where Toll-like receptor 2 was also visualized by immunostaining. When cultured macrophages were infected with M. leprae, CORO1A recruitment from the plasma membrane to the phagosomal membrane was observed. Moderate to strong CORO1A retention was observed in late lesions that contained foamy histiocytes, in which M. leprae were difficult to detect by acid-fast staining. These results suggest that components accumulating within the phagosome rather than viable bacilli are responsible for the retention of CORO1A, and that there is also a bactericidal mechanism in the macrophage that might counter the effects of CORO1A

Quantitative GPCR and ion channel transcriptomics in primary alveolar macrophages and macrophage surrogates

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Groot-Kormelink Paul J

2012-10-01

Full Text Available Abstract Background Alveolar macrophages are one of the first lines of defence against invading pathogens and play a central role in modulating both the innate and acquired immune systems. By responding to endogenous stimuli within the lung, alveolar macrophages contribute towards the regulation of the local inflammatory microenvironment, the initiation of wound healing and the pathogenesis of viral and bacterial infections. Despite the availability of protocols for isolating primary alveolar macrophages from the lung these cells remain recalcitrant to expansion in-vitro and therefore surrogate cell types, such as monocyte derived macrophages and phorbol ester-differentiated cell lines (e.g. U937, THP-1, HL60 are frequently used to model macrophage function. Methods The availability of high throughput gene expression technologies for accurate quantification of transcript levels enables the re-evaluation of these surrogate cell types for use as cellular models of the alveolar macrophage. Utilising high-throughput TaqMan arrays and focussing on dynamically regulated families of integral membrane proteins, we explore the similarities and differences in G-protein coupled receptor (GPCR and ion channel expression in alveolar macrophages and their widely used surrogates. Results The complete non-sensory GPCR and ion channel transcriptome is described for primary alveolar macrophages and macrophage surrogates. The expression of numerous GPCRs and ion channels whose expression were hitherto not described in human alveolar macrophages are compared across primary macrophages and commonly used macrophage cell models. Several membrane proteins known to have critical roles in regulating macrophage function, including CXCR6, CCR8 and TRPV4, were found to be highly expressed in macrophages but not expressed in PMA-differentiated surrogates. Conclusions The data described in this report provides insight into the appropriate choice of cell models for

Proprotein convertase 1/3 inhibited macrophages: A novel therapeutic based on drone macrophages.

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Duhamel, Marie; Rodet, Franck; Murgoci, Adriana; Wisztorski, Maxence; Day, Robert; Fournier, Isabelle; Salzet, Michel

2016-06-01

We demonstrated here thanks to proteomic, that proprotein convertase 1/3 knockdown macrophages present all the characteristic of activated pro-inflammatory macrophages. TLR4 and TLR9 signaling pathways can be enhanced leading to the secretion of pro-inflammatory factors and antitumor factors. We can control their activation by controlling one enzyme, PC1/3. In a tumor context, PC1/3 inhibition in macrophages may reactivate them and lead to a cytokine storm after stimulation "at distance" with a TLR ligand. Therefore, we name these proprotein convertase inhibited macrophages the "drone macrophages". They constitute an innovative cell therapy to treat efficiently tumors.

Inactivation of lipoprotein lipase occurs on the surface of THP-1 macrophages where oligomers of angiopoietin-like protein 4 are formed

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Makoveichuk, Elena; Sukonina, Valentina; Kroupa, Olessia [Department of Medical Biosciences, Physiological Chemistry Umea University, SE-901 87 Umea (Sweden); Thulin, Petra; Ehrenborg, Ewa [Atherosclerosis Research Unit, Department of Medicine, Karolinska Institutet, SE-171 76 Stockholm (Sweden); Olivecrona, Thomas [Department of Medical Biosciences, Physiological Chemistry Umea University, SE-901 87 Umea (Sweden); Olivecrona, Gunilla, E-mail: Gunilla.Olivecrona@medbio.umu.se [Department of Medical Biosciences, Physiological Chemistry Umea University, SE-901 87 Umea (Sweden)

2012-08-24

Highlights: Black-Right-Pointing-Pointer Lipoprotein lipase (LPL) activity is controlled by ANGPTL4 in THP-1 macrophages. Black-Right-Pointing-Pointer Both LPL and ANGPTL4 bind to THP-1 macrophages in a heparin-releasable fashion. Black-Right-Pointing-Pointer Only monomers of ANGPTL4 are present within THP-1 macrophages. Black-Right-Pointing-Pointer Covalent oligomers of ANGPTL4 appear on cell surface and in medium. Black-Right-Pointing-Pointer Inactivation of LPL coincide with ANGPTL4 oligomer formation on cell surfaces. -- Abstract: Lipoprotein lipase (LPL) hydrolyzes triglycerides in plasma lipoproteins causing release of fatty acids for metabolic purposes in muscles and adipose tissue. LPL in macrophages in the artery wall may, however, promote foam cell formation and atherosclerosis. Angiopoietin-like protein (ANGPTL) 4 inactivates LPL and ANGPTL4 expression is controlled by peroxisome proliferator-activated receptors (PPAR). The mechanisms for inactivation of LPL by ANGPTL4 was studied in THP-1 macrophages where active LPL is associated with cell surfaces in a heparin-releasable form, while LPL in the culture medium is mostly inactive. The PPAR{delta} agonist GW501516 had no effect on LPL mRNA, but increased ANGPTL4 mRNA and caused a marked reduction of the heparin-releasable LPL activity concomitantly with accumulation of inactive, monomeric LPL in the medium. Intracellular ANGPTL4 was monomeric, while dimers and tetramers of ANGPTL4 were present in the heparin-releasable fraction and medium. GW501516 caused an increase in the amount of ANGPTL4 oligomers on the cell surface that paralleled the decrease in LPL activity. Actinomycin D blocked the effects of GW501516 on ANGPTL4 oligomer formation and prevented the inactivation of LPL. Antibodies against ANGPTL4 interfered with the inactivation of LPL. We conclude that inactivation of LPL in THP-1 macrophages primarily occurs on the cell surface where oligomers of ANGPTL4 are formed.

circRNA Mediates Silica-Induced Macrophage Activation Via HECTD1/ZC3H12A-Dependent Ubiquitination

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Zhou, Zewei; Jiang, Rong; Yang, Xiyue; Guo, Huifang; Fang, Shencun; Zhang, Yingming; Cheng, Yusi; Wang, Jing; Yao, Honghong; Chao, Jie

2018-01-01

Rationale: Phagocytosis of silicon dioxide (SiO2) into lung cells causes an inflammatory cascade that results in fibroblast proliferation and migration, followed by fibrosis. Circular RNAs (circRNAs) are a subclass of non-coding RNAs detected within mammalian cells; however, researchers have not determined whether circRNAs are involved in the pathophysiological process of silicosis. The upstream molecular mechanisms and functional effects on cell apoptosis, proliferation and migration were investigated to elucidate the role of circRNAs in SiO2-induced inflammation in pulmonary macrophages. Methods: Primary cultures of alveolar macrophages from healthy donors and patients as well as the RAW264.7 macrophage cell line were used to explore the functions of circHECTD1 (HECT domain E3 ubiquitin protein ligase 1) in macrophage activation. Results: The results of the experiments indicated that 1) SiO2 concomitantly decreased circHECTD1 levels and increased HECTD1 protein expression; 2) circHECTD1 and HECTD1 were involved in SiO2-induced macrophage activation via ubiquitination; and 3) SiO2-activated macrophages promoted fibroblast proliferation and migration via the circHECTD1/HECTD1 pathway. Tissue samples from silicosis patients confirmed the upregulation of HECTD1. Conclusions: Our study elucidated a link between SiO2-induced macrophage activation and the circHECTD1/HECTD1 pathway, thereby providing new insight into the potential use of HECTD1 in the development of novel therapeutic strategies for treating silicosis. PMID:29290828

circRNA Mediates Silica-Induced Macrophage Activation Via HECTD1/ZC3H12A-Dependent Ubiquitination.

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Zhou, Zewei; Jiang, Rong; Yang, Xiyue; Guo, Huifang; Fang, Shencun; Zhang, Yingming; Cheng, Yusi; Wang, Jing; Yao, Honghong; Chao, Jie

2018-01-01

Rationale: Phagocytosis of silicon dioxide (SiO 2 ) into lung cells causes an inflammatory cascade that results in fibroblast proliferation and migration, followed by fibrosis. Circular RNAs (circRNAs) are a subclass of non-coding RNAs detected within mammalian cells; however, researchers have not determined whether circRNAs are involved in the pathophysiological process of silicosis. The upstream molecular mechanisms and functional effects on cell apoptosis, proliferation and migration were investigated to elucidate the role of circRNAs in SiO 2 -induced inflammation in pulmonary macrophages. Methods: Primary cultures of alveolar macrophages from healthy donors and patients as well as the RAW264.7 macrophage cell line were used to explore the functions of circHECTD1 (HECT domain E3 ubiquitin protein ligase 1) in macrophage activation. Results: The results of the experiments indicated that 1) SiO 2 concomitantly decreased circHECTD1 levels and increased HECTD1 protein expression; 2) circHECTD1 and HECTD1 were involved in SiO 2 -induced macrophage activation via ubiquitination; and 3) SiO 2 -activated macrophages promoted fibroblast proliferation and migration via the circHECTD1/HECTD1 pathway. Tissue samples from silicosis patients confirmed the upregulation of HECTD1. Conclusions: Our study elucidated a link between SiO 2 -induced macrophage activation and the circHECTD1/HECTD1 pathway, thereby providing new insight into the potential use of HECTD1 in the development of novel therapeutic strategies for treating silicosis.

The cellular prion protein negatively regulates phagocytosis and cytokine expression in murine bone marrow-derived macrophages.

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Min Wang

Full Text Available The cellular prion protein (PrP(C is a glycosylphosphatidylinositol (GPI-anchored glycoprotein on the cell surface. Previous studies have demonstrated contradictory roles for PrP(C in connection with the phagocytic ability of macrophages. In the present work, we investigated the function of PrP(C in phagocytosis and cytokine expression in bone marrow-derived macrophages infected with Escherichia coli. E. coli infection induced an increase in the PRNP mRNA level. Knockout of PrP(C promoted bacterial uptake; upregulated Rab5, Rab7, and Eea1 mRNA expression; and increased the recruitment of lysosomal-associated membrane protein-2 to phagosomes, suggesting enhanced microbicidal activity. Remarkably, knockout of PrP(C suppressed the proliferation of internalized bacteria and increased the expression of cytokines such as interleukin-1β. Collectively, our data reveal an important role of PrP(C as a negative regulator for phagocytosis, phagosome maturation, cytokine expression, and macrophage microbicidal activity.

Adipocyte-Macrophage Cross-Talk in Obesity.

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Engin, Ayse Basak

2017-01-01

Obesity is characterized by the chronic low-grade activation of the innate immune system. In this respect, macrophage-elicited metabolic inflammation and adipocyte-macrophage interaction has a primary importance in obesity. Large amounts of macrophages are accumulated by different mechanisms in obese adipose tissue. Hypertrophic adipocyte-derived chemotactic monocyte chemoattractant protein-1 (MCP-1)/C-C chemokine receptor 2 (CCR2) pathway also promotes more macrophage accumulation into the obese adipose tissue. However, increased local extracellular lipid concentrations is a final mechanism for adipose tissue macrophage accumulation. A paracrine loop involving free fatty acids and tumor necrosis factor-alpha (TNF-alpha) between adipocytes and macrophages establishes a vicious cycle that aggravates inflammatory changes in the adipose tissue. Adipocyte-specific caspase-1 and production of interleukin-1beta (IL-1beta) by macrophages; both adipocyte and macrophage induction by toll like receptor-4 (TLR4) through nuclear factor-kappaB (NF-kappaB) activation; free fatty acid-induced and TLR-mediated activation of c-Jun N-terminal kinase (JNK)-related pro-inflammatory pathways in CD11c+ immune cells; are effective in macrophage accumulation and in the development of adipose tissue inflammation. Old adipocytes are removed by macrophages through trogocytosis or sending an "eat me" signal. The obesity-induced changes in adipose tissue macrophage numbers are mainly due to increases in the triple-positive CD11b+ F4/80+ CD11c+ adipose tissue macrophage subpopulation. The ratio of M1-to-M2 macrophages is increased in obesity. Furthermore, hypoxia along with higher concentrations of free fatty acids exacerbates macrophage-mediated inflammation in obesity. The metabolic status of adipocytes is a major determinant of macrophage inflammatory output. Macrophage/adipocyte fatty-acid-binding proteins act at the interface of metabolic and inflammatory pathways. Both macrophages and

Macrophage heterogeneity and cholesterol homeostasis: classically-activated macrophages are associated with reduced cholesterol accumulation following treatment with oxidized LDL.

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Chu, Eugene M; Tai, Daven C; Beer, Jennifer L; Hill, John S

2013-02-01

Macrophages are centrally involved during atherosclerosis development and are the predominant cell type that accumulates cholesterol in the plaque. Macrophages however, are heterogeneous in nature reflecting a variety of microenvironments and different phenotypes may be more prone to contribute towards atherosclerosis progression. Using primary human monocyte-derived macrophages, we sought to evaluate one aspect of atherogenic potential of different macrophage phenotypes by determining their propensity to associate with and accumulate oxidized low density lipoprotein (oxLDL). Classically-activated macrophages treated simultaneously with interferon γ (IFNγ) and tumor necrosis factor α (TNFα) associated with less oxLDL and accumulated less cholesterol compared to untreated controls. The combined treatment of IFNγ and TNFα reduced the mRNA expression of CD36 and the expression of both cell surface CD36 and macrophage scavenger receptor 1 (MSR1) protein. Under oxLDL loaded conditions, IFNγ and TNFα did not reduce macrophage protein expression of the transcription factor peroxisome proliferator-actived receptor γ (PPARγ) which is known to positively regulate CD36 expression. However, macrophages treated with IFNγ attenuated the ability of the PPARγ-specific agonist rosiglitazone from upregulating cell surface CD36 protein expression. Our results demonstrate that the observed reduction of cholesterol accumulation in macrophages treated with IFNγ and TNFα following oxLDL treatment was due at least in part to reduced cell surface CD36 and MSR1 protein expression. Copyright © 2012 Elsevier B.V. All rights reserved.

[Macrophage activation in atherosclerosis. Message 1: Activation of macrophages normally and in atherosclerotic lesions].

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Nikiforov, N G; Kornienko, V Y; Karagodin, V P; Orekhov, A N

2015-01-01

Macrophages play important role in initiation and progression of inflammation in atherosclerosis. Plaque macrophages were shown to exhibit a phenotypic range that is intermediate between two extremes, M1 (proinflammatory) and M2 (anti-inflammatory). Indeed, in atherosclerosis, macrophages demonstrate phenotypic plasticity to rapidly adjust to changing microenvironmental conditions. In plaque macrophages demonstrate different phenotypes, and besides macrophage phenotypes could be changed. Phenotypes M1, M2, M4, Mhem, HA-mac, M(Hb) u Mox are described in the article. Ability of macrophages change their phenotype also considered.

The AP-1 Transcription Factor c-Jun Promotes Arthritis by Regulating Cyclooxygenase-2 and Arginase-1 Expression in Macrophages.

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Hannemann, Nicole; Jordan, Jutta; Paul, Sushmita; Reid, Stephen; Baenkler, Hanns-Wolf; Sonnewald, Sophia; Bäuerle, Tobias; Vera, Julio; Schett, Georg; Bozec, Aline

2017-05-01

Activation of proinflammatory macrophages is associated with the inflammatory state of rheumatoid arthritis. Their polarization and activation are controlled by transcription factors such as NF-κB and the AP-1 transcription factor member c-Fos. Surprisingly, little is known about the role of the AP-1 transcription factor c-Jun in macrophage activation. In this study, we show that mRNA and protein levels of c-Jun are increased in macrophages following pro- or anti-inflammatory stimulations. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment cluster analyses of microarray data using wild-type and c-Jun-deleted macrophages highlight the central function of c-Jun in macrophages, in particular for immune responses, IL production, and hypoxia pathways. Mice deficient for c-Jun in macrophages show an amelioration of inflammation and bone destruction in the serum-induced arthritis model. In vivo and in vitro gene profiling, together with chromatin immunoprecipitation analysis of macrophages, revealed direct activation of the proinflammatory factor cyclooxygenase-2 and indirect inhibition of the anti-inflammatory factor arginase-1 by c-Jun. Thus, c-Jun regulates the activation state of macrophages and promotes arthritis via differentially regulating cyclooxygenase-2 and arginase-1 levels. Copyright © 2017 by The American Association of Immunologists, Inc.

Increased cerebrospinal fluid levels of cytokines monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1β (MIP-1β) in patients with amyotrophic lateral sclerosis.

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Martínez, H R; Escamilla-Ocañas, C E; Camara-Lemarroy, C R; González-Garza, M T; Moreno-Cuevas, J; García Sarreón, M A

2017-10-10

Neuroinflammation has recently been described in amyotrophic lateral sclerosis (ALS). However, the precise role of such proinflammatory cytokines as monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1β (MIP-1β) in ALS has not yet been determined. In this study, we determined cerebrospinal fluid (CSF) MCP-1 and MIP-1β levels and assessed their association with the duration and severity of ALS. Concentrations of MCP-1 and MIP-1β were determined in the CSF of 77 patients diagnosed with ALS and 13 controls. Cytokine levels were analysed in relation to ALS duration (12months) and severity (30points on the ALS Functional Rating Scale administered at hospital admission). Higher CSF MIP-1β (10.68pg/mL vs. 4.69pg/mL, P<.0001) and MCP-1 (234.89pg/mL vs. 160.95pg/mL, P=.011) levels were found in the 77 patients with ALS compared to controls. There were no differences in levels of either cytokine in relation to disease duration or severity. However, we did observe a significant positive correlation between MIP-1β and MCP-1 in patients with ALS. The increase in MIP-1β and MCP-1 levels suggests that these cytokines may have a synergistic effect on ALS pathogenesis. However, in our cohort, no association was found with either the duration or the clinical severity of the disease. Copyright © 2017 Sociedad Española de Neurología. Publicado por Elsevier España, S.L.U. All rights reserved.

Effect of 14-kDa and 47-kDa protein molecules of age garlic extract on peritoneal macrophages.

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Daneshmandi, Saeed; Hajimoradi, Monire; Ahmadabad, Hasan Namdar; Hassan, Zuhair Mohammad; Roudbary, Maryam; Ghazanfari, Tooba

2011-03-01

Garlic (Allium sativum), traditionally being used as a spice worldwide, has different applications and is claimed to possess beneficial effects in several health ailments such as tumor and atherosclerosis. Garlic is also an immunomodulator and its different components are responsible for different properties. The present work aimed to assess the effect of protein fractions of garlic on peritoneal macrophages. 14-kDa and 47-kDa protein fractions of garlic were purified. Mice peritoneal macrophages were lavaged and cultured in a microtiter plate and exposed to different concentrations of garlic proteins. MTT assay was performed to evaluate the viability of macrophage. The amount of nitric oxide (NO) was detected in culture supernatants of macrophages by Griess reagent and furthermore, the cytotoxicity study of culture supernatants was carried out on WEHI-164 fibrosarcoma cell line as tumor necrosis factor-α bioassay. MTT assay results for both 14-kDa and 47-kDa protein fractions of stimulated macrophages were not significant (P > 0.05). Both 14-kDa and 47-kDa fractions significantly suppressed production of NO from macrophages (P = 0.007 and P = 0.003, respectively). Cytotoxicity of macrophages' supernatant on WEHI-164 fibrosarcoma cells was not affected by garlic protein fractions (P = 0.066 for 14-kDa and P = 0.085 for 47-kDa fractions). according to our finding, 14-kDa and 47-kDa fractions of aged garlic extract are able to suppress NO production from macrophages, which can be used as a biological advantage. These molecules had no cytotoxic effect on macrophages and do not increase tumoricidal property of macrophages.

Cytosolic Pellino-1-Mediated K63-Linked Ubiquitination of IRF5 in M1 Macrophages Regulates Glucose Intolerance in Obesity

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Donghyun Kim

2017-07-01

Full Text Available IRF5 is a signature transcription factor that induces M1 macrophage polarization. However, little is known regarding cytosolic proteins that induce IRF5 activation for M1 polarization. Here, we report the interaction between ubiquitin E3 ligase Pellino-1 and IRF5 in the cytoplasm, which increased nuclear translocation of IRF5 by K63-linked ubiquitination in human and mouse M1 macrophages. LPS and/or IFN-γ increased Pellino-1 expression, and M1 polarization was attenuated in Pellino-1-deficient macrophages in vitro and in vivo. Defective M1 polarization in Pellino-1-deficient macrophages improved glucose intolerance in mice fed a high-fat diet. Furthermore, macrophages in adipose tissues from obese humans exhibited increased Pellino-1 expression and IRF5 nuclear translocation compared with nonobese subjects, and these changes are associated with insulin resistance index. This study demonstrates that cytosolic Pellino-1-mediated K63-linked ubiquitination of IRF5 in M1 macrophages regulates glucose intolerance in obesity, suggesting a cytosolic mediator function of Pellino-1 in TLR4/IFN-γ receptor-IRF5 axis during M1 polarization.

The effect of Alcoholic garlic (Allium sativum extract on ABCA1 expression in human THP-1 macrophages

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Malekpour-Dehkordi Z

2011-06-01

Full Text Available "n Normal 0 false false false EN-US X-NONE AR-SA MicrosoftInternetExplorer4 /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-qformat:yes; mso-style-parent:""; mso-padding-alt:0in 5.4pt 0in 5.4pt; mso-para-margin:0in; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:11.0pt; font-family:"Calibri","sans-serif"; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:"Times New Roman"; mso-fareast-theme-font:minor-fareast; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:Arial; mso-bidi-theme-font:minor-bidi;} Background: ATP-binding cassette transporter A1 (ABCA1 is a key mediator of cholesterol efflux to apoA-I in lipid-laden macrophages, the first step of reverse cholesterol transport (RCT in vivo and a critical step in preventing atherosclerosis. Enhanced ABCA1 expression may inhibit foam cell formation and consequently reduce atherogenic risk. On the other hand, garlic, Allium sativum, and garlic extracts have been demonstrated to have potential cardiovascular benefits. Moreover, garlic has direct antiatherogenic and antiathersclerotic effects on artery walls. The aim of this study was to evaluate the effects of alcoholic garlic extract on the expression of ABCA1 in macrophages."n"nMethods: Cell viability assay was used in order to detect the cytotoxic dose of alcoholic garlic extract on macrophages. Real-time PCR and Western blotting were performed to study the effects of alcoholic garlic extract on the expression of ABCA1. Macrophage cells were treated by different concentrations of alcoholic garlic extract for 48 h. The total RNA of the treated macrophages were extracted and analyzed by real-time PCR. ABCA1 protein expression was also analyzed using the Western blotting technique."n"nResults: Alcoholic garlic extract

Immunotherapy for Prostate Cancer with Gc Protein-Derived Macrophage-Activating Factor, GcMAF.

Science.gov (United States)

Yamamoto, Nobuto; Suyama, Hirofumi; Yamamoto, Nobuyuki

2008-07-01

Serum Gc protein (known as vitamin D(3)-binding protein) is the precursor for the principal macrophage-activating factor (MAF). The MAF precursor activity of serum Gc protein of prostate cancer patients was lost or reduced because Gc protein was deglycosylated by serum alpha-N-acetylgalactosaminidase (Nagalase) secreted from cancerous cells. Therefore, macrophages of prostate cancer patients having deglycosylated Gc protein cannot be activated, leading to immunosuppression. Stepwise treatment of purified Gc protein with immobilized beta-galactosidase and sialidase generated the most potent MAF (termed GcMAF) ever discovered, which produces no adverse effect in humans. Macrophages activated by GcMAF develop a considerable variation of receptors that recognize the abnormality in malignant cell surface and are highly tumoricidal. Sixteen nonanemic prostate cancer patients received weekly administration of 100 ng of GcMAF. As the MAF precursor activity increased, their serum Nagalase activity decreased. Because serum Nagalase activity is proportional to tumor burden, the entire time course analysis for GcMAF therapy was monitored by measuring the serum Nagalase activity. After 14 to 25 weekly administrations of GcMAF (100 ng/week), all 16 patients had very low serum Nagalase levels equivalent to those of healthy control values, indicating that these patients are tumor-free. No recurrence occurred for 7 years.

Active spice-derived components can inhibit inflammatory responses of adipose tissue in obesity by suppressing inflammatory actions of macrophages and release of monocyte chemoattractant protein-1 from adipocytes.

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Woo, Hae-Mi; Kang, Ji-Hye; Kawada, Teruo; Yoo, Hoon; Sung, Mi-Kyung; Yu, Rina

2007-02-13

Inflammation plays a key role in obesity-related pathologies such as cardiovascular disease, type II diabetes, and several types of cancer. Obesity-induced inflammation entails the enhancement of the recruitment of macrophages into adipose tissue and the release of various proinflammatory proteins from fat tissue. Therefore, the modulation of inflammatory responses in obesity may be useful for preventing or ameliorating obesity-related pathologies. Some spice-derived components, which are naturally occurring phytochemicals, elicit antiobesity and antiinflammatory properties. In this study, we investigated whether active spice-derived components can be applied to the suppression of obesity-induced inflammatory responses. Mesenteric adipose tissue was isolated from obese mice fed a high-fat diet and cultured to prepare an adipose tissue-conditioned medium. Raw 264.7 macrophages were treated with the adipose tissue-conditioned medium with or without active spice-derived components (i.e., diallyl disulfide, allyl isothiocyanate, piperine, zingerone and curcumin). Chemotaxis assay was performed to measure the degree of macrophage migration. Macrophage activation was estimated by measuring tumor necrosis factor-alpha (TNF-alpha), nitric oxide, and monocyte chemoattractant protein-1 (MCP-1) concentrations. The active spice-derived components markedly suppressed the migration of macrophages induced by the mesenteric adipose tissue-conditioned medium in a dose-dependent manner. Among the active spice-derived components studied, allyl isothiocyanate, zingerone, and curcumin significantly inhibited the cellular production of proinflammatory mediators such as TNF-alpha and nitric oxide, and significantly inhibited the release of MCP-1 from 3T3-L1 adipocytes. Our findings suggest that the spice-derived components can suppress obesity-induced inflammatory responses by suppressing adipose tissue macrophage accumulation or activation and inhibiting MCP-1 release from adipocytes

DMPD: CSF-1 and cell cycle control in macrophages. [Dynamic Macrophage Pathway CSML Database

Lifescience Database Archive (English)

Full Text Available 8981359 CSF-1 and cell cycle control in macrophages. Hamilton JA. Mol Reprod Dev. 1...997 Jan;46(1):19-23. (.png) (.svg) (.html) (.csml) Show CSF-1 and cell cycle control in macrophages. PubmedI...D 8981359 Title CSF-1 and cell cycle control in macrophages. Authors Hamilton JA. Publication Mol Reprod Dev

Inhibition of macrophage oxidative stress prevents the reduction of ABCA-1 transporter induced by advanced glycated albumin.

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de Souza Pinto, Raphael; Castilho, Gabriela; Paim, Bruno Alves; Machado-Lima, Adriana; Inada, Natalia M; Nakandakare, Edna Regina; Vercesi, Aníbal Eugênio; Passarelli, Marisa

2012-05-01

We investigated the role of aminoguanidine and benfotiamine on the inhibition of reactive oxygen species (ROS) generation in macrophages induced by advanced glycated albumin (AGE-albumin) and its relationship with cell cholesterol homeostasis, emphasizing the expression of the ATP binding cassette transporter A-1 (ABCA-1). AGE-albumin was made by incubating fatty acid-free albumin with 10 mM glycolaldehyde. ROS production and ABCA-1 protein level were determined by flow cytometry in J774 macrophages treated along time with control (C) or AGE-albumin alone or in the presence of aminoguanidine or benfotiamine. Mitochondrial function was evaluated by oxygraphy. Compared to C-albumin, AGE-albumin increased ROS production in macrophages, which was ascribed to the activities of NADPH oxidase and of the mitochondrial system. Mitochondrial respiratory chain activity was reduced in cells incubated with AGE-albumin. ROS generation along time was associated with the reduction in macrophage ABCA-1 protein level. Aminoguanidine prevented ROS elevation and restored the ABCA-1 content in macrophages; on the other hand, benfotiamine that promoted a lesser reduction in ROS generation was not able to restore ABCA-1 levels. Inhibition of oxidative stress induced by AGE-albumin prevents disturbances in reverse cholesterol transport by curbing the reduction of ABCA-1 elicited by advanced glycation in macrophages and therefore may contribute to the prevention of atherosclerosis in diabetes mellitus.

Induction of experimental autoimmune encephalomyelitis in C57BL/6 mice deficient in either the chemokine macrophage inflammatory protein-1alpha or its CCR5 receptor

DEFF Research Database (Denmark)

Tran, E H; Kuziel, W A; Owens, T

2000-01-01

-type mice in Th1 cytokine gene expression, the kinetics and severity of disease, and infiltration of the central nervous system by lymphocytes, macrophages and granulocytes. RNase protection assays showed comparable accumulation of mRNA for the chemokines interferon-inducible protein-10, RANTES, macrophage...... and its CCR5 receptor in the induction of EAE by immunizing C57BL / 6 mice deficient in either MIP-1alpha or CCR5 with myelin oligodendrocyte glycoprotein (MOG). We found that MIP-1alpha-deficient mice were fully susceptible to MOG-induced EAE. These knockout animals were indistinguishable from wild...... chemoattractant protein-1, MIP-1beta, MIP-2, lymphotactin and T cell activation gene-3 during the course of the disease. CCR5-deficient mice were also susceptible to disease induction by MOG. The dispensability of MIP-1alpha and CCR5 for MOG-induced EAE in C57BL / 6 mice supports the idea that differential...

Disrupted epithelial/macrophage crosstalk via Spinster homologue 2-mediated S1P signaling may drive defective macrophage phagocytic function in COPD.

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Hai B Tran

Full Text Available We have previously established a link between impaired phagocytic capacity and deregulated S1P signaling in alveolar macrophages from COPD subjects. We hypothesize that this defect may include a disruption of epithelial-macrophage crosstalk via Spns2-mediated intercellular S1P signaling.Primary alveolar macrophages and bronchial epithelial cells from COPD subjects and controls, cell lines, and a mouse model of chronic cigarette smoke exposure were studied. Cells were exposed to 10% cigarette smoke extract, or vehicle control. Spns2 expression and subcellular localization was studied by immunofluorescence, confocal microscopy and RT-PCR. Phagocytosis was assessed by flow-cytometry. Levels of intra- and extracellular S1P were measured by S1P [3H]-labeling.Spns2 expression was significantly increased (p<0.05 in alveolar macrophages from current-smokers/COPD patients (n = 5 compared to healthy nonsmokers (n = 8 and non-smoker lung transplant patients (n = 4. Consistent with this finding, cigarette smoke induced a significant increase in Spns2 expression in both human alveolar and THP-1 macrophages. In contrast, a remarkable Spns2 down-regulation was noted in response to cigarette smoke in 16HBE14o- cell line (p<0.001 in 3 experiments, primary nasal epithelial cells (p<0.01 in 2 experiments, and in smoke-exposed mice (p<0.001, n = 6 animals per group. Spns2 was localized to cilia in primary bronchial epithelial cells. In both macrophage and epithelial cell types, Spns2 was also found localized to cytoplasm and the nucleus, in line with a predicted bipartile Nuclear Localization Signal at the position aa282 of the human Spns2 sequence. In smoke-exposed mice, alveolar macrophage phagocytic function positively correlated with Spns2 protein expression in bronchial epithelial cells.Our data suggest that the epithelium may be the major source for extracellular S1P in the airway and that there is a possible disruption of epithelial/macrophage cross talk via

Monosodium Urate Crystals Induce Upregulation of NK1.1-Dependent Killing by Macrophages and Support Tumor-Resident NK1.1+ Monocyte/Macrophage Populations in Antitumor Therapy.

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Steiger, Stefanie; Kuhn, Sabine; Ronchese, Franca; Harper, Jacquie L

2015-12-01

Macrophages display phenotypic and functional heterogeneity dependent on the changing inflammatory microenvironment. Under some conditions, macrophages can acquire effector functions commonly associated with NK cells. In the current study, we investigated how the endogenous danger signal monosodium urate (MSU) crystals can alter macrophage functions. We report that naive, primary peritoneal macrophages rapidly upregulate the expression of the NK cell-surface marker NK1.1 in response to MSU crystals but not in response to LPS or other urate crystals. NK1.1 upregulation by macrophages was associated with mechanisms including phagocytosis of crystals, NLRP3 inflammasome activation, and autocrine proinflammatory cytokine signaling. Further analysis demonstrated that MSU crystal-activated macrophages exhibited NK cell-like cytotoxic activity against target cells in a perforin/granzyme B-dependent manner. Furthermore, analysis of tumor hemopoietic cell populations showed that effective, MSU-mediated antitumor activity required coadministration with Mycobacterium smegmatis to induce IL-1β production and significant accumulation of monocytes and macrophages (but not granulocytes or dendritic cells) expressing elevated levels of NK1.1. Our findings provide evidence that MSU crystal-activated macrophages have the potential to develop tumoricidal NK cell-like functions that may be exploited to boost antitumor activity in vivo. Copyright © 2015 by The American Association of Immunologists, Inc.

Proprotein convertase 1/3 inhibited macrophages: A novel therapeutic based on drone macrophages

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Marie Duhamel

2016-06-01

Full Text Available We demonstrated here thanks to proteomic, that proprotein convertase 1/3 knockdown macrophages present all the characteristic of activated pro-inflammatory macrophages. TLR4 and TLR9 signaling pathways can be enhanced leading to the secretion of pro-inflammatory factors and antitumor factors. We can control their activation by controlling one enzyme, PC1/3. In a tumor context, PC1/3 inhibition in macrophages may reactivate them and lead to a cytokine storm after stimulation “at distance” with a TLR ligand. Therefore, we name these proprotein convertase inhibited macrophages the “drone macrophages”. They constitute an innovative cell therapy to treat efficiently tumors.

Functional Roles of p38 Mitogen-Activated Protein Kinase in Macrophage-Mediated Inflammatory Responses

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Yanyan Yang

2014-01-01

Full Text Available Inflammation is a natural host defensive process that is largely regulated by macrophages during the innate immune response. Mitogen-activated protein kinases (MAPKs are proline-directed serine and threonine protein kinases that regulate many physiological and pathophysiological cell responses. p38 MAPKs are key MAPKs involved in the production of inflammatory mediators, including tumor necrosis factor-α (TNF-α and cyclooxygenase-2 (COX-2. p38 MAPK signaling plays an essential role in regulating cellular processes, especially inflammation. In this paper, we summarize the characteristics of p38 signaling in macrophage-mediated inflammation. In addition, we discuss the potential of using inhibitors targeting p38 expression in macrophages to treat inflammatory diseases.

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of macrophage growth locus A (MglA) protein from Francisella tularensis

International Nuclear Information System (INIS)

Subburaman, Priadarsini; Austin, Brian P.; Shaw, Gary X.; Waugh, David S.; Ji, Xinhua

2010-01-01

The macrophage growth locus A (MglA) protein from F. tularensis crystallized in the hexagonal space group P6 1 or P6 5 , with unit-cell parameters a = b = 125, c = 54 Å. Francisella tularensis, a potential bioweapon, causes a rare infectious disease called tularemia in humans and animals. The macrophage growth locus A (MglA) protein from F. tularensis associates with RNA polymerase to positively regulate the expression of multiple virulence factors that are required for its survival and replication within macrophages. The MglA protein was overproduced in Escherichia coli, purified and crystallized. The crystals diffracted to 7.5 Å resolution at the Advanced Photon Source, Argonne National Laboratory and belonged to the hexagonal space group P6 1 or P6 5 , with unit-cell parameters a = b = 125, c = 54 Å

Mechanism of interleukin-13 production by granulocyte-macrophage colony-stimulating factor-dependent macrophages via protease-activated receptor-2.

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Yamaguchi, Rui; Yamamoto, Takatoshi; Sakamoto, Arisa; Ishimaru, Yasuji; Narahara, Shinji; Sugiuchi, Hiroyuki; Hirose, Eiji; Yamaguchi, Yasuo

2015-06-01

Granulocyte-macrophage colony-stimulating factor (GM-CSF) promotes classically activated M1 macrophages. GM-CSF upregulates protease-activated receptor-2 (PAR-2) protein expression and activation of PAR-2 by human neutrophil elastase (HNE) regulates cytokine production. This study investigated the mechanism of PAR-2-mediated interleukin (IL)-13 production by GM-CSF-dependent macrophages stimulated with HNE. Adherent macrophages were obtained from primary cultures of human mononuclear cells. After stimulation with HNE to activate the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling pathway, IL-13 mRNA and protein levels were assessed by the reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. PAR-2 protein was detected in GM-CSF-dependent macrophages by Western blotting. Unexpectedly, PD98059 (an ERK1 inhibitor) increased IL-13 production, even at higher concentrations. Interestingly, U0126 (an ERK1/2 inhibitor) reduced IL-13 production in a concentration-dependent manner. Neither SB203580 (a p38alpha/p38beta inhibitor) nor BIRB796 (a p38gamma/p38delta inhibitor) affected IL-13 production, while TMB-8 (a calcium chelator) diminished IL-13 production. Stimulation with HNE promoted the production of IL-13 (a Th2 cytokine) by GM-CSF-dependent M1 macrophages. PAR-2-mediated IL-13 production may be dependent on the Ca(2+)/ERK2 signaling pathway. Copyright © 2015 Elsevier Inc. All rights reserved.

Macrophage Stimulating Protein Enhances Hepatic Inflammation in a NASH Model

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Li, Jieyi; Chanda, Dipanjan; van Gorp, Patrick J.; Jeurissen, Mike L. J.; Houben, Tom; Walenbergh, Sofie M. A.; Debets, Jacques; Oligschlaeger, Yvonne; Gijbels, Marion J. J.; Neumann, Dietbert; Shiri-Sverdlov, Ronit

2016-01-01

Non-alcoholic steatohepatitis (NASH) is a common liver disease characterized by hepatic lipid accumulation (steatosis) and inflammation. Currently, therapeutic options are poor and the long-term burden to society is constantly increasing. Previously, macrophage stimulating protein (MSP)-a serum

BTB and CNC homolog 1 (Bach1) deficiency ameliorates TNBS colitis in mice: role of M2 macrophages and heme oxygenase-1.

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Harusato, Akihito; Naito, Yuji; Takagi, Tomohisa; Uchiyama, Kazuhiko; Mizushima, Katsura; Hirai, Yasuko; Higashimura, Yasuki; Katada, Kazuhiro; Handa, Osamu; Ishikawa, Takeshi; Yagi, Nobuaki; Kokura, Satoshi; Ichikawa, Hiroshi; Muto, Akihiko; Igarashi, Kazuhiko; Yoshikawa, Toshikazu

2013-01-01

BTB and CNC homolog 1 (Bach1) is a transcriptional repressor of heme oxygenase-1 (HO-1), which plays an important role in the protection of cells and tissues against acute and chronic inflammation. However, the role of Bach1 in the gastrointestinal mucosal defense system remains little understood. HO-1 supports the suppression of experimental colitis and localizes mainly in macrophages in colonic mucosa. This study was undertaken to elucidate the Bach1/HO-1 system's effects on the pathogenesis of experimental colitis. This study used C57BL/6 (wild-type) and homozygous Bach1-deficient C57BL/6 mice in which colonic damage was induced by the administration of an enema of 2,4,6-trinitrobenzene sulfonic acid (TNBS). Subsequently, they were evaluated macroscopically, histologically, and biochemically. Peritoneal macrophages from the respective mice were isolated and analyzed. Then, wild-type mice were injected with peritoneal macrophages from the respective mice. Acute colitis was induced similarly. TNBS-induced colitis was inhibited in Bach1-deficient mice. TNBS administration increased the expression of HO-1 messenger RNA and protein in colonic mucosa in Bach1-deficient mice. The expression of HO-1 mainly localized in F4/80-immunopositive and CD11b-immunopositive macrophages. Isolated peritoneal macrophages from Bach1-deficient mice highly expressed HO-1 and also manifested M2 macrophage markers, such as Arginase-1, Fizz-1, Ym1, and MRC1. Furthermore, TNBS-induced colitis was inhibited by the transfer of Bach1-deficient macrophages into wild-type mice. Deficiency of Bach1 ameliorated TNBS-induced colitis. Bach1-deficient macrophages played a key role in protection against colitis. Targeting of this mechanism is applicable to cell therapy for human inflammatory bowel disease.

MiR-146a modulates macrophage polarization by inhibiting Notch1 pathway in RAW264.7 macrophages.

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Huang, Cheng; Liu, Xue-Jiao; QunZhou; Xie, Juan; Ma, Tao-Tao; Meng, Xiao-Ming; Li, Jun

2016-03-01

Macrophages are heterogeneous and plastic cells which are able to undergo dynamic transition between M1 and M2 polarized phenotypes in response to the microenvironment signals. However, the underlying molecular mechanisms of macrophage polarization are still obscure. In the current study, it was revealed that miR-146a might play a pivotal role in macrophage polarization. As our results indicated, miR-146a was highly expressed in M2 macrophages rather than M1 macrophages. Over-expression of miR-146a resulted in significantly decreased production of pro-inflammatory cytokines including iNOS and TNF-α in M1 macrophages, while increased production of M2 marker genes such as Arg1 and CD206 in M2 macrophages. In contrast, knockdown of miR-146a promoted M1 macrophage polarization but diminished M2 macrophage polarization. Mechanistically, it was revealed that miR-146a modulated macrophage polarization by targeting Notch1. Of note, PPARγ was responsible as another target for miR-146a-mediated macrophage polarization. Taken together, it was suggested that miR-146a might serve as a molecular regulator in macrophage polarization and is a potential therapeutic target for inflammatory diseases. Copyright © 2016 Elsevier B.V. All rights reserved.

Role of 14-3-3η protein on cardiac fatty acid metabolism and macrophage polarization after high fat diet induced type 2 diabetes mellitus.

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Sreedhar, Remya; Arumugam, Somasundaram; Thandavarayan, Rajarajan A; Karuppagounder, Vengadeshprabhu; Koga, Yusuke; Nakamura, Takashi; Harima, Meilei; Watanabe, Kenichi

2017-07-01

Diabetic cardiomyopathy (DCM), a metabolic disorder, is one of the leading causes of mortality around the world and its pathogenesis involves cardiac inflammation and altered metabolic profile. Altered fatty acid metabolism during DCM can cause macrophage polarization in which inflammatory M1 phenotype dominates over the anti-inflammatory M2 phenotype. Hence, it is essential to identify a specific target, which could revert the metabolic profile and thereby reducing the M1 macrophage polarization. 14-3-3η protein has several cellular protective functions especially in the heart as plenty of reports available in various animal models of heart failure including diabetes mellitus. However, its role in the cardiac fatty acid metabolism and macrophage polarization remains unidentified. The present study has been designed to delineate the effect of cardiospecific dominant negative mutation of 14-3-3η protein (DN14-3-3) on various lipid metabolism related marker proteins expressions and cardiac macrophage phenotype in high fat diet (HFD) fed mice. Feeding HFD for 12 weeks has produced significant increase in body weight in the DN14-3-3 (TG) mice than C57BL6/J (WT) mice. Western blotting and immunohistochemical staining analysis of the heart tissue has revealed an increase in the expression of markers of cardiac fatty acid synthesis related proteins in addition to the reduced expression of fatty acid oxidation related proteins in TG mice fed HFD than WT mice fed HFD. Furthermore, the M1 macrophage marker proteins were increasingly expressed while M2 markers expressions were reduced in the hearts of TG mice fed HFD. In conclusion, our current study has identified that there is a definite role for the 14-3-3η protein against the pathogenesis of heart failure via regulation of cardiac fatty acid metabolism and macrophage polarization. Copyright © 2017. Published by Elsevier Ltd.

Heterochromatin protein 1 gamma and IκB kinase alpha interdependence during tumour necrosis factor gene transcription elongation in activated macrophages.

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Thorne, James L; Ouboussad, Lylia; Lefevre, Pascal F

2012-09-01

IκB kinase α (IKKα) is part of the cytoplasmic IKK complex regulating nuclear factor-κB (NF-κB) release and translocation into the nucleus in response to pro-inflammatory signals. IKKα can also be recruited directly to the promoter of NF-κB-dependent genes by NF-κB where it phosphorylates histone H3 at serine 10, triggering recruitment of the bromodomain-containing protein 4 and the positive transcription elongation factor b. Herein, we report that IKKα travels with the elongating form of ribonucleic acid polymerase II together with heterochromatin protein 1 gamma (HP1γ) at NF-κB-dependent genes in activated macrophages. IKKα binds to and phosphorylates HP1γ, which in turn controls IKKα binding to chromatin and phosphorylation of the histone variant H3.3 at serine 31 within transcribing regions. Downstream of transcription end sites, IKKα accumulates with its inhibitor the CUE-domain containing protein 2, suggesting a link between IKKα inactivation and transcription termination.

Virulent Mycobacterium bovis Beijing Strain Activates the NLRP7 Inflammasome in THP-1 Macrophages.

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Yang Zhou

Full Text Available Mycobacterium bovis is the causative agent of tuberculosis in a wide range of mammals, including humans. Macrophages are the first line of host defense. They secrete proinflammatory cytokines, such as interleukin-1 beta (IL-1β, in response to mycobacterial infection, but the underlying mechanisms by which human macrophages are activated and release IL-1β following M. bovis infection are poorly understood. Here we show that the 'nucleotide binding and oligomerization of domain-like receptor (NLR family pyrin domain containing 7 protein' (NLRP7 inflammasome is involved in IL-1β secretion and caspase-1 activation induced by M. bovis infection in THP-1 macrophages. NLRP7 inflammasome activation promotes the induction of pyroptosis as well as the expression of tumor necrosis factor alpha (TNF-α, Chemokine (C-C motif ligand 3 (CCL3 and IL-1β mRNAs. Thus, the NLRP7 inflammasome contributes to IL-1β secretion and induction of pyroptosis in response to M. bovis infection in THP-1 macrophages.

Post-transcriptional regulation of macrophage ABCA1, an early response gene to IFN-γ

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Alfaro Leon, Martha Leticia; Evans, Glenn F.; Farmen, Mark W.; Zuckerman, Steven H.

2005-01-01

Interferon-γ (IFN-γ) down-regulates receptors associated with reverse cholesterol transport including ABCA1. In the present study, the kinetics and mechanism of ABCA1 down-regulation were determined in mouse peritoneal macrophages. IFN-γ decreased ABCA1 mRNA 1 h following IFN-γ addition and was maximally reduced by 3 h. Down-regulation was protein synthesis dependent and involved post-transcriptional processes. ABCA1 message had a T 1/2 of 115 min in actinomycin treated cells that was reduced to a T 1/2 of 37 min by IFN-γ. The decrease in message stability was also associated with a rapid loss of ABCA1 protein, significant 3 h following IFN-γ addition. The kinetics of ABCA1 message and protein decrease was consistent with the early IFN-γ-induced changes in Stat1 phosphorylation and nuclear translocation observed in these cells. Therefore, ABCA1 can be considered as an early response gene to macrophage activation by IFN-γ with down-regulation occurring by message destabilization

The D543N polymorphism of the SLC11A1/NRAMP1 gene is associated with treatment failure in male patients with pulmonary tuberculosis.

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Salinas-Delgado, Yvain; Galaviz-Hernández, Carlos; Toral, René García; Ávila Rejón, Carmen A; Reyes-Lopez, Miguel A; Martínez, Antonio Rojas; Martínez-Aguilar, Gerardo; Sosa-Macías, Martha

2015-09-01

Polymorphisms in SLC11A1/NRAMP1 have shown an important association with susceptibility to tuberculosis and progression to active disease. However, whether there is an association of these polymorphisms with treatment failure is unknown. The aim of this study was to determine the association of SLC11A1 polymorphisms with treatment failure in Mexican subjects with pulmonary tuberculosis. Thirty-three subjects with treatment failure were paired by age and body mass index with 33 patients who successfully completed treatment and were considered cured. We assessed the polymorphisms of SLC11A1 in the regions of D543N and INT4 via polymerase chain reaction real-time TaqMan® single nucleotide polymorphism (SNP) genotyping. We found that D543N (G/A genotype) was associated with treatment failure in patients with pulmonary tuberculosis [odds ratio (OR) 11.61, 95% confidence interval (CI) 3.66-36.78]. When adjusted by gender, this association remained significant in males (OR 11.09, 95% CI 3.46-35.51). In our male population, the presence of the D543N polymorphism of SLC11A1 is a risk factor for treatment failure. This finding should be confirmed in other populations.

Pitavastatin Differentially Modulates MicroRNA-Associated Cholesterol Transport Proteins in Macrophages.

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Haijun Zhang

Full Text Available There is emerging evidence identifying microRNAs (miRNAs as mediators of statin-induced cholesterol efflux, notably through the ATP-binding cassette transporter A1 (ABCA1 in macrophages. The objective of this study was to assess the impact of an HMG-CoA reductase inhibitor, pitavastatin, on macrophage miRNAs in the presence and absence of oxidized-LDL, a hallmark of a pro-atherogenic milieu. Treatment of human THP-1 cells with pitavastatin prevented the oxLDL-mediated suppression of miR-33a, -33b and -758 mRNA in these cells, an effect which was not uniquely attributable to induction of SREBP2. Induction of ABCA1 mRNA and protein by oxLDL was inhibited (30% by pitavastatin, while oxLDL or pitavastatin alone significantly induced and repressed ABCA1 expression, respectively. These findings are consistent with previous reports in macrophages. miRNA profiling was also performed using a miRNA array. We identified specific miRNAs which were up-regulated (122 and down-regulated (107 in THP-1 cells treated with oxLDL plus pitavastatin versus oxLDL alone, indicating distinct regulatory networks in these cells. Moreover, several of the differentially expressed miRNAs identified are functionally associated with cholesterol trafficking (six miRNAs in cells treated with oxLDL versus oxLDL plus pitavastatin. Our findings indicate that pitavastatin can differentially modulate miRNA in the presence of oxLDL; and, our results provide evidence that the net effect on cholesterol homeostasis is mediated by a network of miRNAs.

Signaling via ITGB1/FAK and microfilament rearrangement mediates the internalization of Leptospira interrogans in mouse J774A.1 macrophages

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Zhao Xin

2015-01-01

Full Text Available Leptospirosis caused by pathogenic Leptospira species is a worldwide zoonotic 2 infectious disease, but the mechanisms of leptospiral internalization remain poorly understood. Here, we report that mouse J774A.1 macrophages expressed integrin-subfamily proteins (ITGB1, ITGB2 and ITGB3. Antibody blockage and siRNA-based knockdown of ITGB1 decreased the internalization of leptospires into mouse J774A.1 macrophage cells. The internalization required focal adhesion kinase (FAK activation in J774A.1 cells rather than phosphoinositide-3-kinase (PI3K, and microfilament rather than microtubule aggregation during infection. The data indicated that the ITGB1/FAK/microfilament signaling pathway is responsible for leptospiral internalization in mouse macrophages.

Real-time visualization of HIV-1 GAG trafficking in infected macrophages.

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Karine Gousset

2008-03-01

Full Text Available HIV-1 particle production is driven by the Gag precursor protein Pr55(Gag. Despite significant progress in defining both the viral and cellular determinants of HIV-1 assembly and release, the trafficking pathway used by Gag to reach its site of assembly in the infected cell remains to be elucidated. The Gag trafficking itinerary in primary monocyte-derived macrophages is especially poorly understood. To define the site of assembly and characterize the Gag trafficking pathway in this physiologically relevant cell type, we have made use of the biarsenical-tetracysteine system. A small tetracysteine tag was introduced near the C-terminus of the matrix domain of Gag. The insertion of the tag at this position did not interfere with Gag trafficking, virus assembly or release, particle infectivity, or the kinetics of virus replication. By using this in vivo detection system to visualize Gag trafficking in living macrophages, Gag was observed to accumulate both at the plasma membrane and in an apparently internal compartment that bears markers characteristic of late endosomes or multivesicular bodies. Significantly, the internal Gag rapidly translocated to the junction between the infected macrophages and uninfected T cells following macrophage/T-cell synapse formation. These data indicate that a population of Gag in infected macrophages remains sequestered internally and is presented to uninfected target cells at a virological synapse.

Effect of the Gc-derived macrophage-activating factor precursor (preGcMAF) on phagocytic activation of mouse peritoneal macrophages.

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Uto, Yoshihiro; Yamamoto, Syota; Takeuchi, Ryota; Nakagawa, Yoshinori; Hirota, Keiji; Terada, Hiroshi; Onizuka, Shinya; Nakata, Eiji; Hori, Hitoshi

2011-07-01

The 1f1f subtype of the Gc protein (Gc(1f1f) protein) was converted into Gc-derived macrophage-activating factor (GcMAF) by enzymatic processing in the presence of β-galactosidase of an activated B-cell and sialidase of a T-cell. We hypothesized that preGc(1f1f)MAF, the only Gc(1f1f) protein lacking galactose, can be converted to GcMAF in vivo because sialic acid is cleaved by residual sialidase. Hence, we investigated the effect of preGc(1f1f)MAF on the phagocytic activation of mouse peritoneal macrophages. We examined the sugar moiety of preGc(1f1f)MAF with a Western blot using peanut agglutinin (PNA) and Helix pomatia agglutinin (HPA) lectin. We also found that preGc(1f1f)MAF significantly enhanced phagocytic activity in mouse peritoneal macrophages but only in the presence of the mouse peritoneal fluid; the level of phagocytic activity was the same as that observed for GcMAF. PreGc(1f1f)MAF can be used as an effective macrophage activator in vivo.

Effects of protein-energy malnutrition on NF-kappaB signalling in murine peritoneal macrophages.

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Fock, Ricardo Ambrósio; Rogero, Marcelo Macedo; Vinolo, Marco Aurélio Ramirez; Curi, Rui; Borges, Maria Carolina; Borelli, Primavera

2010-04-01

Protein-energy malnutrition (PEM) is an important public health problem affecting millions of people worldwide. PEM decreases resistance to infection, impairing a number of physiological processes. In unstimulated cells, NF-kappaB is kept from binding to its consensus sequence by the inhibitor I kappaB alpha, which retains NF-kappaB in the cytoplasm. Upon various signals, such as lipopolysaccharide (LPS), I kappaB alpha is rapidly degraded and NF-kappaB is induced to translocate into the nucleus, where it activates expression of various genes that participate in the inflammatory response, including those involved in the synthesis of TNF-alpha. TRAF-6 is a cytoplasmic adapter protein that links the stimulatory signal from Toll like receptor-4 to NF-kappaB. The aim of this study was to evaluate the effect of malnutrition on induction of TNF-alpha by LPS in murine peritoneal macrophages. We evaluated peritoneal cellularity, the expression of MyD88, TRAF-6, IKK, I kappaB alpha and NF-kappaB, NF-kappaB activation and TNF-alpha mRNA and protein synthesis in macrophages. Two-month-old male BALB/C mice were submitted to PEM with a low-protein diet that contained 2% protein, compared to 12% protein in the control diet. When the experimental group had lost about 20% of the original body weight, it was used in the subsequent experiments. Malnourished animals presented anemia, leucopenia and severe reduction in peritoneal cavity cellularity. TNF-alpha mRNA and protein levels of macrophages stimulated with LPS were significantly lower in malnourished animals. PEM also decreased TRAF-6 expression and NF-kappaB activation after LPS stimulation. These results led us to conclude that PEM changes NF-kB signalling pathway in macrophages to LPS stimulus.

Biglycan- and Sphingosine Kinase-1 Signaling Crosstalk Regulates the Synthesis of Macrophage Chemoattractants

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Louise Tzung-Harn Hsieh

2017-03-01

Full Text Available In its soluble form, the extracellular matrix proteoglycan biglycan triggers the synthesis of the macrophage chemoattractants, chemokine (C-C motif ligand CCL2 and CCL5 through selective utilization of Toll-like receptors (TLRs and their adaptor molecules. However, the respective downstream signaling events resulting in biglycan-induced CCL2 and CCL5 production have not yet been defined. Here, we show that biglycan stimulates the production and activation of sphingosine kinase 1 (SphK1 in a TLR4- and Toll/interleukin (IL-1R domain-containing adaptor inducing interferon (IFN-β (TRIF-dependent manner in murine primary macrophages. We provide genetic and pharmacological proof that SphK1 is a crucial downstream mediator of biglycan-triggered CCL2 and CCL5 mRNA and protein expression. This is selectively driven by biglycan/SphK1-dependent phosphorylation of the nuclear factor NF-κB p65 subunit, extracellular signal-regulated kinase (Erk1/2 and p38 mitogen-activated protein kinases. Importantly, in vivo overexpression of soluble biglycan causes Sphk1-dependent enhancement of renal CCL2 and CCL5 and macrophage recruitment into the kidney. Our findings describe the crosstalk between biglycan- and SphK1-driven extracellular matrix- and lipid-signaling. Thus, SphK1 may represent a new target for therapeutic intervention in biglycan-evoked inflammatory conditions.

Low Levels of IGF-1 Contribute to Alveolar Macrophage Dysfunction in Cystic Fibrosis1

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Bessich, Jamie L.; Nymon, Amanda B.; Moulton, Lisa A; Dorman, Dana; Ashare, Alix

2013-01-01

Alveolar macrophages are major contributors to lung innate immunity. Although alveolar macrophages from CFTR−/− mice have impaired function, no study has investigated primary alveolar macrophages in adults with cystic fibrosis (CF). CF patients have low levels of insulin-like growth factor 1 (IGF-1), and our prior studies demonstrate a relationship between IGF-1 and macrophage function. We hypothesize that reduced IGF-1 in CF leads to impaired alveolar macrophage function and chronic infections. Serum and bronchoalveolar lavage (BAL) samples were obtained from 8 CF subjects and 8 healthy subjects. Macrophages were isolated from BAL fluid. We measured the ability of alveolar macrophages to kill Pseudomonas aeruginosa. Subsequently, macrophages were incubated with IGF-1 prior to inoculation with bacteria to determine the effect of IGF-1 on bacterial killing. We found a significant decrease in bacterial killing by CF alveolar macrophages compared to controls. CF subjects had lower serum and BAL IGF-1 levels compared to healthy controls. Exposure to IGF-1 enhanced alveolar macrophage macrophages in both groups. Finally, exposing healthy alveolar macrophages to CF BAL fluid decreased bacterial killing, and this was reversed by the addition of IGF-1, while IGF-1 blockade worsened bacterial killing. Our studies demonstrate that alveolar macrophage function is impaired in patients with CF. Reductions in IGF-1 levels in CF contribute to the impaired alveolar macrophage function. Exposure to IGF-1 ex vivo, results in improved function of CF alveolar macrophages. Further studies are needed to determine whether alveolar macrophage function can be enhanced in vivo with IGF-1 treatment. PMID:23698746

Granulocyte-macrophage colony-stimulating factor primes interleukin-13 production by macrophages via protease-activated receptor-2.

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Aoki, Manabu; Yamaguchi, Rui; Yamamoto, Takatoshi; Ishimaru, Yasuji; Ono, Tomomichi; Sakamoto, Arisa; Narahara, Shinji; Sugiuchi, Hiroyuki; Hirose, Eiji; Yamaguchi, Yasuo

2015-04-01

Chronic inflammation is often linked to the presence of type 2-polarized macrophages, which are induced by the T helper type 2 cytokines interleukin-4 and interleukin-13 (IL-13). IL-13 is a key mediator of tissue fibrosis caused by T helper type 2-based inflammation. Human neutrophil elastase (HNE) plays a pivotal role in the pathogenesis of pulmonary fibrosis. This study investigated the priming effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) on IL-13 expression by macrophages stimulated with HNE. Adherent macrophages were obtained from primary cultures of human mononuclear cells. Expression of IL-13 mRNA and protein by GM-CSF-dependent macrophages was investigated after stimulation with HNE, using the polymerase chain reaction and enzyme-linked immunosorbent assay. GM-CSF had a priming effect on IL-13 mRNA and protein expression by macrophages stimulated with HNE, while this effect was not observed for various other cytokines. GM-CSF-dependent macrophages showed a significant increase in the expression of protease activated receptor-2 (PAR-2) mRNA and protein. The response of IL-13 mRNA to HNE was significantly decreased by pretreatment with alpha1-antitrypsin, a PAR-2 antibody (SAM11), or a PAR-2 antagonist (ENMD-1068). These findings suggest that stimulation with HNE can induce IL-13 production by macrophages, especially GM-CSF-dependent macrophages. Accordingly, neutrophil elastase may have a key role in fibrosis associated with chronic inflammation. Copyright © 2015 Elsevier Inc. All rights reserved.

Modulation of the nuclear factor-kappa B (NF-κB) signalling pathway by glutamine in peritoneal macrophages of a murine model of protein malnutrition.

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da Silva Lima, Fabiana; Rogero, Marcelo Macedo; Ramos, Mayara Caldas; Borelli, Primavera; Fock, Ricardo Ambrósio

2013-06-01

Protein malnutrition affects resistance to infection by impairing the inflammatory response, modifying the function of effector cells, such as macrophages. Recent studies have revealed that glutamine-a non-essential amino acid, which could become conditionally essential in some situations like trauma, infection, post-surgery and sepsis-is able to modulate the synthesis of cytokines. The aim of this study was to evaluate the effect of glutamine on the expression of proteins involved in the nuclear factor-kappa B (NF-κB) signalling pathway of peritoneal macrophages from malnourished mice. Two-month-old male Balb/c mice were submitted to protein-energy malnutrition (n = 10) with a low-protein diet containing 2 % protein, whereas control mice (n = 10) were fed a 12 % protein-containing diet. The haemogram and analysis of plasma glutamine and corticosterone were evaluated. Peritoneal macrophages were pre-treated in vitro with glutamine (0, 0.6, 2 and 10 mmol/L) for 24 h and then stimulated with 1.25 μg LPS for 30 min, and the synthesis of TNF-α and IL-1α and the expression of proteins related to the NF-κB pathway were evaluated. Malnourished animals had anaemia, leucopoenia, lower plasma glutamine and increased corticosterone levels. TNF-α production of macrophages stimulated with LPS was significantly lower in cells from malnourished animals when cultivated in supraphysiological (2 and 10 mmol/L) concentrations of glutamine. Further, glutamine has a dose-dependent effect on the activation of macrophages, in both groups, when stimulated with LPS, inducing a decrease in TNF-α and IL-1α production and negatively modulating the NF-κB signalling pathway. These data lead us to infer that the protein malnutrition state interferes with the activation of macrophages and that higher glutamine concentrations, in vitro, have the capacity to act negatively in the NF-κB signalling pathway.

Signal regulatory protein α associated with the progression of oral leukoplakia and oral squamous cell carcinoma regulates phenotype switch of macrophages.

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Ye, Xiaojing; Zhang, Jing; Lu, Rui; Zhou, Gang

2016-12-06

Signal regulatory protein α (SIRPα) is a cell-surface protein expressed on macrophages that are regarded as an important component of the tumor microenvironment. The expression of SIRPα in oral leukoplakia (OLK) and oral squamous cell carcinoma (OSCC), and further explored the role of SIRPα on the phenotype, phagocytosis ability, migration, and invasion of macrophages in OSCC were investigated. The expression of SIRPα in OLK was higher than in OSCC, correlating with the expression of CD68 and CD163 on macrophages. After cultured with the conditioned media of oral cancer cells, the expression of SIRPα on THP-1 cells was decreased gradually. In co-culture system, macrophages were induced into M2 phenotype by oral cancer cells. Blockade of SIRPα inhibited phagocytosis ability and IL-6, TNF-α productions of macrophages. In addition, the proliferation, migration, and IL-10, TGF-β productions of macrophages were upregulated after blockade of SIRPα. Macrophages upregulated the expression of SIRPα and phagocytosis ability, and inhibited the migration and invasion when the activation of NF-κB was inhibited by pyrrolidine dithiocarbamate ammonium (PDTC). Hence, SIRPα might play an important role in the progression of OLK and oral cancer, and could be a pivotal therapeutic target in OSCC by regulating the phenotype of macrophages via targeting NF-κB.

Loss of Macrophage Low-Density Lipoprotein Receptor-Related Protein 1 Confers Resistance to the Antiatherogenic Effects of Tumor Necrosis Factor-α Inhibition.

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Zhu, Lin; Giunzioni, Ilaria; Tavori, Hagai; Covarrubias, Roman; Ding, Lei; Zhang, Youmin; Ormseth, Michelle; Major, Amy S; Stafford, John M; Linton, MacRae F; Fazio, Sergio

2016-08-01

Antiatherosclerotic effects of tumor necrosis factor-α (TNF-α) blockade in patients with systemic inflammatory states are not conclusively demonstrated, which suggests that effects depend on the cause of inflammation. Macrophage LRP1 (low-density lipoprotein receptor-related protein 1) and apoE contribute to inflammation through different pathways. We studied the antiatherosclerosis effects of TNF-α blockade in hyperlipidemic mice lacking either LRP1 (MΦLRP1(-/-)) or apoE from macrophages. Lethally irradiated low-density lipoprotein receptor (LDLR)(-/-) mice were reconstituted with bone marrow from either wild-type, MΦLRP1(-/-), apoE(-/-) or apoE(-/-)/MΦLRP1(-/-)(DKO) mice, and then treated with the TNF-α inhibitor adalimumab while fed a Western-type diet. Adalimumab reduced plasma TNF-α concentration, suppressed blood ly6C(hi) monocyte levels and their migration into the lesion, and reduced lesion cellularity and inflammation in both wild-type→LDLR(-/-) and apoE(-/-)→LDLR(-/-) mice. Overall, adalimumab reduced lesion burden by 52% to 57% in these mice. Adalimumab reduced TNF-α and blood ly6C(hi) monocyte levels in MΦLRP1(-/-)→LDLR(-/-) and DKO→LDLR(-/-) mice, but it did not suppress ly6C(hi) monocyte migration into the lesion or atherosclerosis progression. Our results show that TNF-α blockade exerts antiatherosclerotic effects that are dependent on the presence of macrophage LRP1. © 2016 American Heart Association, Inc.

Immunotherapy of metastatic colorectal cancer with vitamin D-binding protein-derived macrophage-activating factor, GcMAF.

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Yamamoto, Nobuto; Suyama, Hirofumi; Nakazato, Hiroaki; Yamamoto, Nobuyuki; Koga, Yoshihiko

2008-07-01

Serum vitamin D binding protein (Gc protein) is the precursor for the principal macrophage-activating factor (MAF). The MAF precursor activity of serum Gc protein of colorectal cancer patients was lost or reduced because Gc protein is deglycosylated by serum alpha-N-acetylgalactosaminidase (Nagalase) secreted from cancerous cells. Deglycosylated Gc protein cannot be converted to MAF, leading to immunosuppression. Stepwise treatment of purified Gc protein with immobilized beta-galactosidase and sialidase generated the most potent macrophage-activating factor (GcMAF) ever discovered, but it produces no side effect in humans. Macrophages treated with GcMAF (100 microg/ml) develop an enormous variation of receptors and are highly tumoricidal to a variety of cancers indiscriminately. Administration of 100 nanogram (ng)/ human maximally activates systemic macrophages that can kill cancerous cells. Since the half-life of the activated macrophages is approximately 6 days, 100 ng GcMAF was administered weekly to eight nonanemic colorectal cancer patients who had previously received tumor-resection but still carried significant amounts of metastatic tumor cells. As GcMAF therapy progressed, the MAF precursor activities of all patients increased and conversely their serum Nagalase activities decreased. Since serum Nagalase is proportional to tumor burden, serum Nagalase activity was used as a prognostic index for time course analysis of GcMAF therapy. After 32-50 weekly administrations of 100 ng GcMAF, all colorectal cancer patients exhibited healthy control levels of the serum Nagalase activity, indicating eradication of metastatic tumor cells. During 7 years after the completion of GcMAF therapy, their serum Nagalase activity did not increase, indicating no recurrence of cancer, which was also supported by the annual CT scans of these patients.

Ketamine inhibits tumor necrosis factor-α and interleukin-6 gene expressions in lipopolysaccharide-stimulated macrophages through suppression of toll-like receptor 4-mediated c-Jun N-terminal kinase phosphorylation and activator protein-1 activation

International Nuclear Information System (INIS)

Wu, G.-J.; Chen, T.-L.; Ueng, Y.-F.; Chen, R.-M.

2008-01-01

Our previous study showed that ketamine, an intravenous anesthetic agent, has anti-inflammatory effects. In this study, we further evaluated the effects of ketamine on the regulation of tumor necrosis factor-α (TNF-α) and interlukin-6 (IL-6) gene expressions and its possible signal-transducing mechanisms in lipopolysaccharide (LPS)-activated macrophages. Exposure of macrophages to 1, 10, and 100 μM ketamine, 100 ng/ml LPS, or a combination of ketamine and LPS for 1, 6, and 24 h was not cytotoxic to macrophages. A concentration of 1000 μM of ketamine alone or in combined treatment with LPS caused significant cell death. Administration of LPS increased cellular TNF-α and IL-6 protein levels in concentration- and time-dependent manners. Meanwhile, treatment with ketamine concentration- and time-dependently alleviated the enhanced effects. LPS induced TNF-α and IL-6 mRNA syntheses. Administration of ketamine at a therapeutic concentration (100 μM) significantly inhibited LPS-induced TNF-α and IL-6 mRNA expressions. Application of toll-like receptor 4 (TLR4) small interfering (si)RNA into macrophages decreased cellular TLR4 levels. Co-treatment of macrophages with ketamine and TLR4 siRNA decreased the LPS-induced TNF-α and IL-6 productions more than alone administration of TLR4 siRNA. LPS stimulated phosphorylation of c-Jun N-terminal kinase and translocation of c-Jun and c-Fos from the cytoplasm to nuclei. However, administration of ketamine significantly decreased LPS-induced activation of c-Jun N-terminal kinase and translocation of c-Jun and c-Fos. LPS increased the binding of nuclear extracts to activator protein-1 consensus DNA oligonucleotides. Administration of ketamine significantly ameliorated LPS-induced DNA binding activity of activator protein-1. Therefore, a clinically relevant concentration of ketamine can inhibit TNF-α and IL-6 gene expressions in LPS-activated macrophages. The suppressive mechanisms occur through suppression of TLR4-mediated

Macrophage Resistance to HIV-1 Infection Is Enhanced by the Neuropeptides VIP and PACAP

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Temerozo, Jairo R.; Joaquim, Rafael; Regis, Eduardo G.; Savino, Wilson; Bou-Habib, Dumith Chequer

2013-01-01

It is well established that host factors can modulate HIV-1 replication in macrophages, critical cells in the pathogenesis of HIV-1 infection due to their ability to continuously produce virus. The neuropeptides VIP and PACAP induce well-characterized effects on macrophages through binding to the G protein-coupled receptors VPAC1, VPAC2 and PAC1, but their influence on HIV-1 production by these cells has not been established. Here, we describe that VIP and PACAP reduce macrophage production of HIV-1, acting in a synergistic or additive manner to decrease viral growth. Using receptor antagonists, we detected that the HIV-1 inhibition promoted by VIP is dependent on its ligation to VPAC1/2, whereas PACAP decreases HIV-1 growth via activation of the VPAC1/2 and PAC1 receptors. Specific agonists of VPAC2 or PAC1 decrease macrophage production of HIV-1, whereas sole activation of VPAC1 enhances viral growth. However, the combination of specific agonists mimicking the receptor preference of the natural neuropeptides reproduces the ability of VIP and PACAP to increase macrophage resistance to HIV-1 replication. VIP and PACAP up-regulated macrophage secretion of the β-chemokines CCL3 and CCL5 and the cytokine IL-10, whose neutralization reversed the neuropeptide-induced inhibition of HIV-1 replication. Our results suggest that VIP and PACAP and the receptors VPAC2 and PAC1 could be used as targets for developing alternative therapeutic strategies for HIV-1 infection. PMID:23818986

High density lipoprotein (HDL)-associated sphingosine 1-phosphate (S1P) inhibits macrophage apoptosis by stimulating STAT3 activity and survivin expression.

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Feuerborn, Renata; Becker, Susen; Potì, Francesco; Nagel, Petra; Brodde, Martin; Schmidt, Harmut; Christoffersen, Christina; Ceglarek, Uta; Burkhardt, Ralph; Nofer, Jerzy-Roch

2017-02-01

Macrophage apoptosis is critically involved in atherosclerosis. We here examined the effect of anti-atherogenic high density lipoprotein (HDL) and its component sphingosine-1-phosphate (S1P) on apoptosis in RAW264.7 murine macrophages. Mitochondrial or endoplasmic reticulum-dependent apoptosis was induced by exposure of macrophages to etoposide or thapsigargin/fukoidan, respectively. Cell death induced by these compounds was inhibited by S1P as inferred from reduced annexin V binding, TUNEL staining, and caspase 3, 9 and 12 activities. S1P induced expression of the inhibitor of apoptosis protein (IAP) family proteins cIAP1, cIAP2 and survivin, but only the inhibitor of survivin expression YM155 and not the cIAP1/2 blocker GDC0152 reversed the inhibitory effect of S1P on apoptosis. Moreover, S1P activated signal transducer and activator of transcription 3 (STAT3) and Janus kinase 2 (JAK2) and the stimulatory effect of S1P on survivin expression and inhibitory effects on apoptosis were attenuated by STAT3 or JAK2 inhibitors, S3I-201 or AG490, respectively. The effects of S1P on STAT3 activation, survivin expression and macrophage apoptosis were emulated by HDL, HDL lipids, and apolipoprotein (apo) M-containing HDL, but not by apoA-I or HDL deprived of S1P or apoM. In addition, JTE013 and CAY10444, S1P receptor 2 and 3 antagonists, respectively, compromised the S1P and HDL capacities to stimulate STAT3 activation and survivin expression, and to inhibit apoptosis. HDL-associated S1P inhibits macrophage apoptosis by stimulating STAT3 activity and survivin expression. The suppression of macrophage apoptosis may represent a novel mechanism utilized by HDL to exert its anti-atherogenic effects. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Helicobacter pylori protein HP0986 (TieA) interacts with mouse TNFR1 and triggers proinflammatory and proapoptotic signaling pathways in cultured macrophage cells (RAW 264.7).

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Ansari, Suhail A; Devi, Savita; Tenguria, Shivendra; Kumar, Ashutosh; Ahmed, Niyaz

2014-08-01

HP0986 protein of Helicobacter pylori has been shown to trigger induction of proinflammatory cytokines (IL-8 and TNF-α) through the activation of NF-κB and also to induce Fas mediated apoptosis of human macrophage cells (THP-1). In this study, we unravel mechanistic details of the biological effects of this protein in a murine macrophage environment. Up regulation of MCP-1 and TNF-α in HP0986-induced RAW 264.7 cells occurred subsequent to the activation and translocation of NF-κB to the cell nucleus. Further, HP0986 induced apoptosis of RAW 264.7 cells through Fas activation and this was in agreement with previous observations made with THP-1 cells. Our studies indicated activation of TNFR1 through interaction with HP0986 and this elicited the aforementioned responses independent of TLR2, TLR4 or TNFR2. We found that mouse TNFR1 activation by HP0986 facilitates formation of a complex comprising of TNFR1, TRADD and TRAF2, and this occurs upstream of NF-κB activation. Furthermore, FADD also forms a second complex, at a later stage, together with TNFR1 and TRADD, resulting in caspase-8 activation and thereby the apoptosis of RAW 264.7 cells. In summary, our observations reveal finer details of the functional activity of HP0986 protein in relation to its behavior in a murine macrophage cell environment. These findings reconfirm the proinflammatory and apoptotic role of HP0986 signifying it to be an important trigger of innate responses. These observations form much needed baseline data entailing future in vivo studies of the functions of HP0986 in a murine model. Copyright © 2014 Elsevier Ltd. All rights reserved.

Macrophage mitochondrial oxidative stress promotes atherosclerosis and nuclear factor-κB-mediated inflammation in macrophages.

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Wang, Ying; Wang, Gary Z; Rabinovitch, Peter S; Tabas, Ira

2014-01-31

Mitochondrial oxidative stress (mitoOS) has been shown to correlate with the progression of human atherosclerosis. However, definitive cell type-specific causation studies in vivo are lacking, and the molecular mechanisms of potential proatherogenic effects remain to be determined. Our aims were to assess the importance of macrophage mitoOS in atherogenesis and to explore the underlying molecular mechanisms. We first validated Western diet-fed Ldlr(-/-) mice as a model of human mitoOS-atherosclerosis association by showing that non-nuclear oxidative DNA damage, a marker of mitoOS in lesional macrophages, correlates with aortic root lesion development. To investigate the importance of macrophage mitoOS, we used a genetic engineering strategy in which the OS suppressor catalase was ectopically expressed in mitochondria (mCAT) in macrophages. MitoOS in lesional macrophages was successfully suppressed in these mice, and this led to a significant reduction in aortic root lesional area. The mCAT lesions had less monocyte-derived cells, less Ly6c(hi) monocyte infiltration into lesions, and lower levels of monocyte chemotactic protein-1. The decrease in lesional monocyte chemotactic protein-1 was associated with the suppression of other markers of inflammation and with decreased phosphorylation of RelA (NF-κB p65), indicating decreased activation of the proinflammatory NF-κB pathway. Using models of mitoOS in cultured macrophages, we showed that mCAT suppressed monocyte chemotactic protein-1 expression by decreasing the activation of the IκB-kinase β-RelA NF-κB pathway. MitoOS in lesional macrophages amplifies atherosclerotic lesion development by promoting NF-κB-mediated entry of monocytes and other inflammatory processes. In view of the mitoOS-atherosclerosis link in human atheromata, these findings reveal a potentially new therapeutic target to prevent the progression of atherosclerosis.

Scavenger receptor B1 facilitates macrophage uptake of silver nanoparticles and cellular activation

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Aldossari, Abdullah A.; Shannahan, Jonathan H. [The University of Colorado Anschutz Medical Campus, Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences (United States); Podila, Ramakrishna [Clemson University, Department of Physics and Astronomy (United States); Brown, Jared M., E-mail: jared.brown@ucdenver.edu [The University of Colorado Anschutz Medical Campus, Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences (United States)

2015-07-15

Due to increased use of silver nanoparticles (AgNPs) for their antimicrobial activity, concerns have risen regarding potential adverse human health effects. Scavenger receptor B1 (SR-B1), a major receptor for high-density lipoprotein (HDL), is expressed by macrophages and has also been reported to play a role in recognition of negatively charged particles. We, therefore, hypothesized that SR-B1 mediates macrophage uptake of AgNPs and inflammatory activation. To test this hypothesis, we exposed a mouse macrophage cell line RAW264.7 (RAW) and bone marrow-derived macrophages (BMDM) to 20 nm citrate-suspended AgNPs. To verify the role of the SR-B1 receptor, we utilized a SR-B1 inhibitor (Blt2). In vitro studies demonstrated uptake of AgNPs and HDL-coated AgNPs by macrophages which were significantly reduced following pretreatment with Blt2. Inflammatory cytokine arrays revealed that macrophages exposed to AgNPs up-regulated expression of Tnf-α, Oncostatin m (OSM), Ccl4, Il17f, Ccl7, and Ccl2, whereas Il16 was found to be down-regulated. Macrophage activation was observed following AgNP and HDL-coated AgNP exposure as measured by OSM protein production and increased surface expression of CD86. These markers of activation were reduced with Blt2 pretreatment. The in vitro findings were confirmed in vivo through pulmonary instillation of AgNPs in mice. Pulmonary instillation of AgNPs resulted in a recruitment of inflammatory cells that were reduced in SR-B1-deficient mice or following Blt2 pretreatment. This study suggests that SR-B1 plays a major role in cellular recognition of AgNPs and the induction of cell responses that could contribute to inflammation caused by AgNP exposure.

Scavenger receptor B1 facilitates macrophage uptake of silver nanoparticles and cellular activation

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Aldossari, Abdullah A.; Shannahan, Jonathan H.; Podila, Ramakrishna; Brown, Jared M.

2015-07-01

Due to increased use of silver nanoparticles (AgNPs) for their antimicrobial activity, concerns have risen regarding potential adverse human health effects. Scavenger receptor B1 (SR-B1), a major receptor for high-density lipoprotein (HDL), is expressed by macrophages and has also been reported to play a role in recognition of negatively charged particles. We, therefore, hypothesized that SR-B1 mediates macrophage uptake of AgNPs and inflammatory activation. To test this hypothesis, we exposed a mouse macrophage cell line RAW264.7 (RAW) and bone marrow-derived macrophages (BMDM) to 20 nm citrate-suspended AgNPs. To verify the role of the SR-B1 receptor, we utilized a SR-B1 inhibitor (Blt2). In vitro studies demonstrated uptake of AgNPs and HDL-coated AgNPs by macrophages which were significantly reduced following pretreatment with Blt2. Inflammatory cytokine arrays revealed that macrophages exposed to AgNPs up-regulated expression of Tnf- α, Oncostatin m (OSM), Ccl4, Il17f, Ccl7, and Ccl2, whereas Il16 was found to be down-regulated. Macrophage activation was observed following AgNP and HDL-coated AgNP exposure as measured by OSM protein production and increased surface expression of CD86. These markers of activation were reduced with Blt2 pretreatment. The in vitro findings were confirmed in vivo through pulmonary instillation of AgNPs in mice. Pulmonary instillation of AgNPs resulted in a recruitment of inflammatory cells that were reduced in SR-B1-deficient mice or following Blt2 pretreatment. This study suggests that SR-B1 plays a major role in cellular recognition of AgNPs and the induction of cell responses that could contribute to inflammation caused by AgNP exposure.

Homocysteine elicits an M1 phenotype in murine macrophages through an EMMPRIN-mediated pathway.

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Winchester, Lee J; Veeranki, Sudhakar; Givvimani, Srikanth; Tyagi, Suresh C

2015-07-01

Hyperhomocysteinemia (HHcy) is associated with inflammatory diseases and is known to increase the production of reactive oxygen species (ROS), matrix metalloproteinase (MMP)-9, and inducible nitric oxide synthase, and to decrease endothelial nitric oxide production. However, the impact of HHcy on macrophage phenotype differentiation is not well-established. It has been documented that macrophages have 2 distinct phenotypes: the "classically activated/destructive" (M1), and the "alternatively activated/constructive" (M2) subtypes. We hypothesize that HHcy increases M1 macrophage differentiation through extracellular matrix metalloproteinase inducer (EMMPRIN), a known inducer of matrix metalloproteinases. murine J774A.1 and Raw 264.7 macrophages were treated with 100 and 500 μmol/L Hcy, respectively, for 24 h. Samples were analyzed using Western blotting and immunocytochemistry. Homocysteine treatment increased cluster of differentiation 40 (CD40; M1 marker) in J774A.1 and Raw 264.7 macrophages. MMP-9 was induced in both cell lines. EMMPRIN protein expression was also increased in both cell lines. Blocking EMMPRIN function by pre-treating cells with anti-EMMPRIN antibody, with or without Hcy, resulted in significantly lower expression of CD40 in both cell lines by comparison with the controls. A DCFDA assay demonstrated increased ROS production in both cell lines with Hcy treatment when compared with the controls. Our results suggest that HHcy results in an increase of the M1 macrophage phenotype. This effect seems to be at least partially mediated by EMMPRIN induction.

Uncaria rhynchophylla inhibits the production of nitric oxide and interleukin-1β through blocking nuclear factor κB, Akt, and mitogen-activated protein kinase activation in macrophages.

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Kim, Ji-Hee; Bae, Chang Hwan; Park, Sun Young; Lee, Sang Joon; Kim, YoungHee

2010-10-01

The stems with hook of Uncaria rhynchophylla have been used in traditional medicine as an antipyretic, antihypertensive, and anticonvulsant in China and Korea. In this study, we investigated the mechanism responsible for anti-inflammatory effects of U. rhynchophylla in RAW 264.7 macrophages. The aqueous extract of U. rhynchophylla inhibited lipopolysaccharide (LPS)-induced nitric oxide (NO) and interleukin (IL)-1β secretion as well as inducible NO synthase (iNOS) expression, without affecting cell viability. Furthermore, U. rhynchophylla suppressed LPS-induced nuclear factor κB (NF-κB) activation, phosphorylation, and degradation of inhibitory protein IκB (IκB)-α, phosphorylation of Akt, extracellular signal-regulated kinase 1/2, p38 kinase, and c-Jun N-terminal kinase. These results suggest that U. rhynchophylla has the inhibitory effects on LPS-induced NO and IL-1β production in macrophages through blockade in the phosphorylation of Akt and mitogen-activated protein kinases, following IκB-α degradation and NF-κB activation.

Regulation of ICAM-1 in Cells of the Monocyte/Macrophage System in Microgravity

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Katrin Paulsen

2015-01-01

Full Text Available Cells of the immune system are highly sensitive to altered gravity, and the monocyte as well as the macrophage function is proven to be impaired under microgravity conditions. In our study, we investigated the surface expression of ICAM-1 protein and expression of ICAM-1 mRNA in cells of the monocyte/macrophage system in microgravity during clinostat, parabolic flight, sounding rocket, and orbital experiments. In murine BV-2 microglial cells, we detected a downregulation of ICAM-1 expression in clinorotation experiments and a rapid and reversible downregulation in the microgravity phase of parabolic flight experiments. In contrast, ICAM-1 expression increased in macrophage-like differentiated human U937 cells during the microgravity phase of parabolic flights and in long-term microgravity provided by a 2D clinostat or during the orbital SIMBOX/Shenzhou-8 mission. In nondifferentiated U937 cells, no effect of microgravity on ICAM-1 expression could be observed during parabolic flight experiments. We conclude that disturbed immune function in microgravity could be a consequence of ICAM-1 modulation in the monocyte/macrophage system, which in turn could have a strong impact on the interaction with T lymphocytes and cell migration. Thus, ICAM-1 can be considered as a rapid-reacting and sustained gravity-regulated molecule in mammalian cells.

Macrophage recruitment, but not interleukin 1 beta activation, enhances noise-induced hearing damage.

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Mizushima, Yu; Fujimoto, Chisato; Kashio, Akinori; Kondo, Kenji; Yamasoba, Tatsuya

2017-11-18

It has been suggested that macrophages or inflammatory monocytes participate in the pathology of noise-induced hearing loss (NIHL), but it is unclear how extensively these cells contribute to the development of temporary and/or permanent NIHL. To address this question, we used clodronate liposomes to deplete macrophages and monocytes. After clodronate liposome injection, mice were exposed to 4-kHz octave band noise at 121 dB for 4 h. Compared to vehicle-injected controls, clodronate-treated mice exhibited significantly reduced permanent threshold shifts at 4 and 8 kHz and significantly smaller outer hair cell losses in the lower-apical cochlear turn. Following noise exposure, the stria vascularis had significantly more cells expressing the macrophage-specific protein F4/80, and this effect was significantly suppressed by clodronate treatment. These F4/80-positive cells expressed interleukin 1 beta (IL-1β), which noise exposure activated. However, IL-1β deficient mice did not exhibit significant resistance to intense noise when compared to wild-type mice. These findings suggest that macrophages that enter the cochlea after noise exposure are involved in NIHL, whereas IL-1β inhibition does not reverse this cochlear damage. Therefore, macrophages may be a promising therapeutic target in human sensorineural hearing losses such as NIHL. Copyright © 2017 Elsevier Inc. All rights reserved.

Preparation of Gc protein-derived macrophage activating factor (GcMAF) and its structural characterization and biological activities.

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Mohamad, Saharuddin Bin; Nagasawa, Hideko; Uto, Yoshihiro; Hori, Hitoshi

2002-01-01

Gc protein has been reported to be a precursor of Gc protein-derived macrophage activation factor (GcMAF) in the inflammation-primed macrophage activation cascade. An inducible beta-galactosidase of B cells and neuraminidase of T cells convert Gc protein to GcMAF. Gc protein from human serum was purified using 25(OH)D3 affinity column chromatography and modified to GcMAF using immobilized glycosidases (beta-galactosidase and neuraminidase) The sugar moiety structure of GcMAF was characterized by lectin blotting by Helix pomatia agglutinin. The biological activities of GcMAF were evaluated by a superoxide generation assay and a phagocytosis assay. We successfully purified Gc protein from human serum. GcMAF was detected by lectin blotting and showed a high biological activity. Our results support the importance of the terminal N-acetylgalactosamine moiety in the GcMAF-mediated macrophage activation cascade, and the existence of constitutive GcMAF in human serum. These preliminary data are important for designing small molecular GcMAF mimics.

Nucleotide-oligomerizing domain-1 (NOD1) receptor activation induces pro-inflammatory responses and autophagy in human alveolar macrophages.

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Juárez, Esmeralda; Carranza, Claudia; Hernández-Sánchez, Fernando; Loyola, Elva; Escobedo, Dante; León-Contreras, Juan Carlos; Hernández-Pando, Rogelio; Torres, Martha; Sada, Eduardo

2014-09-25

Nucleotide-binding oligomerizing domain-1 (NOD1) is a cytoplasmic receptor involved in recognizing bacterial peptidoglycan fragments that localize to the cytosol. NOD1 activation triggers inflammation, antimicrobial mechanisms and autophagy in both epithelial cells and murine macrophages. NOD1 mediates intracellular pathogen clearance in the lungs of mice; however, little is known about NOD1's role in human alveolar macrophages (AMs) or its involvement in Mycobacterium tuberculosis (Mtb) infection. AMs, monocytes (MNs), and monocyte-derived macrophages (MDMs) from healthy subjects were assayed for NOD1 expression. Cells were stimulated with the NOD1 ligand Tri-DAP and cytokine production and autophagy were assessed. Cells were infected with Mtb and treated with Tri-DAP post-infection. CFUs counting determined growth control, and autophagy protein recruitment to pathogen localization sites was analyzed by immunoelectron microscopy. NOD1 was expressed in AMs, MDMs and to a lesser extent MNs. Tri-DAP stimulation induced NOD1 up-regulation and a significant production of IL1β, IL6, IL8, and TNFα in AMs and MDMs; however, the level of NOD1-dependent response in MNs was limited. Autophagy activity determined by expression of proteins Atg9, LC3, IRGM and p62 degradation was induced in a NOD1-dependent manner in AMs and MDMs but not in MNs. Infected AMs could be activated by stimulation with Tri-DAP to control the intracellular growth of Mtb. In addition, recruitment of NOD1 and the autophagy proteins IRGM and LC3 to the Mtb localization site was observed in infected AMs after treatment with Tri-DAP. NOD1 is involved in AM and MDM innate responses, which include proinflammatory cytokines and autophagy, with potential implications in the killing of Mtb in humans.

Adrenomedullin Regulates IL-1β Gene Expression in F4/80+ Macrophages during Synovial Inflammation

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Takano, Shotaro; Miyagi, Masayuki; Inoue, Gen; Aikawa, Jun; Iwabuchi, Kazuya; Takaso, Masashi

2017-01-01

Adrenomedullin (AM) plays an important role in the regulation of inflammatory processes; however, the role and expression of AM in synovial inflammation have not been determined. To investigate the expression and role of AM in inflamed synovial tissue (ST), the gene expression profiles of AM in the ST, including synovial macrophages and fibroblasts, of a murine patellar surgical dislocation model were characterized. In addition, the effects of interleukin- (IL-) 1β and AM in cultured synovial cells were also examined. CD11c+ macrophages were found to be elevated in ST of the surgically dislocated patella. Higher gene expression of CD11c, IL-1β, AM, receptor activity-modifying proteins 2 (RAMP2), and 3 (RAMP3) was also observed in ST obtained from the dislocated side. AM expression was also significantly increased in synovial fibroblasts and macrophages in response to IL-1β treatment. Synovial macrophages also highly expressed RAMP3 compared to fibroblasts and this expression was further stimulated by exogenously added IL-1β. Further, the treatment of the F4/80-positive cell fraction obtained from ST with AM inhibited IL-1β expression. Taken together, these findings demonstrated that AM was produced by synovial fibroblasts and macrophages in inflamed ST and that increased levels of AM may exert anti-inflammatory effects on synovial macrophages. PMID:28299347

Activation of nucleotide-binding domain-like receptor containing protein 3 inflammasome in dendritic cells and macrophages by Streptococcus sanguinis.

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Saeki, Ayumi; Suzuki, Toshihiko; Hasebe, Akira; Kamezaki, Ryousuke; Fujita, Mari; Nakazawa, Futoshi; Shibata, Ken-Ichiro

2017-03-01

Streptococcus sanguinis is frequently isolated from the blood of patients with infective endocarditis and contributes to the pathology of this disease through induction of interleukin (IL)-1β responsible for the development of the disease. However, the mechanism of IL-1β induction remains unknown. In this study, S. sanguinis activated a murine dendritic cell (DC) to induce IL-1β and this activity was attenuated by silencing the mRNAs of nucleotide-binding domain-like receptor containing protein 3 (NLRP3) and caspase-1. S. sanguinis induced IL-1β production in murine bone marrow-derived macrophage, but this activity was significantly reduced in bone marrow-derived macrophages from NLRP3-, apoptosis-associated speck-like protein containing a caspase-recruitment domain-, and caspase-1-deficient mice. DC phagocytosed S. sanguinis cells, followed by the release of adenosine triphosphate (ATP). The ATP-degradating enzyme attenuated the release of ATP and IL-1β. The inhibitors for ATP receptor reduced IL-1β release in DC. These results strongly suggest that S. sanguinis has the activity to induce IL-1β through the NLRP3 inflammasome in macrophage and DC and interaction of purinergic receptors with ATP released is involved in expression of the activity. © 2016 John Wiley & Sons Ltd.

Production of macrophage inflammatory protein (MIP)-1alpha and MIP-1beta by human polymorphonuclear neutrophils stimulated with Porphyromonas endodontalis lipopolysaccharide.

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Ko, Hyun Jung; Lim, Sung Sam

2002-11-01

This study was undertaken to investigate the capacity of polymorphonuclear neutrophils (PMNs) to secrete Macrophage Inflammatory Protein (MIP)-1alpha and MIP-1beta after stimulation with Porphyromonas endodontalis lipopolysaccharide (LPS). Escherichia coli LPS was used as a positive control. Venous blood was collected and PMNs were isolated from healthy volunteers. Cells were cultured with various concentrations of LPS for different periods of time. Cell supernatants were assayed by enzyme-linked immunosorbent assay. The levels of chemokine secretion in PMNs stimulated with each LPS were found to be significantly higher than in the unstimulated control cells (p endodontalis LPS. These findings demonstrated that P. endodontalis LPS is capable of stimulating PMNs to produce chemotactic cytokines and suggested that PMNs stimulated with P. endodontalis LPS may play a crucial role in the inflammatory and immunopathological reactions of pulpal and periapical diseases.

Galectin-3 disruption impaired tumoral angiogenesis by reducing VEGF secretion from TGFβ1-induced macrophages

International Nuclear Information System (INIS)

Machado, Camila Maria Longo; Andrade, Luciana Nogueira Sousa; Teixeira, Verônica Rodrigues; Costa, Fabrício Falconi; Melo, Camila Morais; Santos, Sofia Nascimento dos; Nonogaki, Suely; Liu, Fu-Tong; Bernardes, Emerson Soares; Camargo, Anamaria Aranha; Chammas, Roger

2014-01-01

In order to study the role of galectin-3 in tumor angiogenesis associated with tumor-associated macrophages (TAM) and tumor parenchyma, the galectin-3 expression was reconstituted in Tm1 melanoma cell line that lacks this protein. Galectin-3-expressing cells (Tm1G3) and mock-vector transfected cells (Tm1N3) were injected into wild-type (WT) and galectin-3 knockout (KO) C57Bl/6 mice. Tumors originated from Tm1G3 were larger in tumor volume with enlarged functional vessels, decreased necrotic areas, and increased vascular endothelial growth factor (VEGF) protein levels. Galectin-3-nonexpressing-cells injected into WT and KO showed increased levels of transforming growth factor beta 1 (TGFβ1) and, in WT animals this feature was also accompanied by increased VEGFR2 expression and its phosphorylation. In KO animals, tumors derived from galectin-3-expressing cells were infiltrated by CD68 + -cells, whereas in tumors derived from galectin-3-nonexpressing-cells, CD68 + cells failed to infiltrate tumors and accumulated in the periphery of the tumor mass. In vitro studies showed that Tm1G3 secreted more VEGF than Tm1N3 cells. In the latter case, TGFβ1 induced VEGF production. Basal secretion of VEGF was higher in WT-bone marrow-derived macrophages (BMDM) than in KO-BMDM. TGFβ1 induced secretion of VEGF only in WT-BMDM. Tm1G3-induced tumors had the Arginase I mRNA increased, which upregulated alternative macrophage (M2)/TAM induction. M2 stimuli, such as interleukin-4 (IL4) and TGFβ1, increased Arginase I protein levels and galectin-3 expression in WT- BMDM, but not in cells from KO mice. Hence, we report that galectin-3 disruption in tumor stroma and parenchyma decreases angiogenesis through interfering with the responses of macrophages to the interdependent VEGF and TGFβ1 signaling pathways

Structural definition of a potent macrophage activating factor derived from vitamin D3-binding protein with adjuvant activity for antibody production.

Science.gov (United States)

Yamamoto, N

1996-10-01

Incubation of human vitamin D3-binding protein (Gc protein), with a mixture of immobilized beta-galactosidase and sialidase, efficiently generated a potent macrophage activating factor, a protein with N-acetylgalactosamine as the remaining sugar. Stepwise incubation of Gc protein with immobilized beta-galactosidase and sialidase, and isolation of the intermediates with immobilized lectins, revealed that either sequence of hydrolysis of Gc glycoprotein by these glycosidases yields the macrophage-activating factor, implying that Gc protein carries a trisaccharide composed of N-acetylgalactosamine and dibranched galactose and sialic acid termini. A 3 hr incubation of mouse peritoneal macrophages with picomolar amounts of the enzymatically generated macrophage-activating factor (GcMAF) resulted in a greatly enhanced phagocytic activity. Administration of a minute amount (10-50 pg/mouse) of GcMAF resulted in a seven- to nine-fold enhanced phagocytic activity of macrophages. Injection of sheep red blood cells (SRBC) along with GcMAF into mice produced a large number of anti-SRBC antibody secreting splenic cells in 2-4 days.

Low Levels of IGF-1 Contribute to Alveolar Macrophage Dysfunction in Cystic Fibrosis1

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Bessich, Jamie L.; Nymon, Amanda B.; Moulton, Lisa A; Dorman, Dana; Ashare, Alix

2013-01-01

Alveolar macrophages are major contributors to lung innate immunity. Although alveolar macrophages from CFTR−/− mice have impaired function, no study has investigated primary alveolar macrophages in adults with cystic fibrosis (CF). CF patients have low levels of insulin-like growth factor 1 (IGF-1), and our prior studies demonstrate a relationship between IGF-1 and macrophage function. We hypothesize that reduced IGF-1 in CF leads to impaired alveolar macrophage function and chronic infectio...

Effects of miR-33a-5P on ABCA1/G1-mediated cholesterol efflux under inflammatory stress in THP-1 macrophages.

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Min Mao

Full Text Available The present study is to investigate whether inflammatory cytokines inhibit ABCA1/ABCG1-mediated cholesterol efflux by regulating miR-33a-5P in THP-1 macrophages. We used interleukin-6 and tumor necrosis factor-alpha in the presence or absence of native low density lipoprotein (LDL to stimulate THP-1 macrophages. THP-1 macrophages were infected by either control lentivirus vectors or lentivirus encoding miR-33a-5P or antisense miR-33a-5P. The effects of inflammatory cytokines, miR-33a-5P and antisense miR-33a-5P on intracellular lipids accumulation and intracellular cholesterol contents were assessed by oil red O staining and quantitative intracellular cholesterol assay. ApoA-I-mediated cholesterol efflux was examined using the fluorescent sterol (BODIPY-cholesterol. The gene and protein expressions of the molecules involved in cholesterol trafficking were examined using quantitative real-time polymerase chain reaction and Western blotting. Inflammatory cytokines or miR-33a-5P increased intracellular lipid accumulation and decreased apoA-I-mediated cholesterol efflux via decreasing the expression of ABCA1 and ABCG1 in the absence or presence of LDL in THP-1 macrophages. However, antisense miR-33a-5P reversed the effects of inflammatory cytokines on intracellular lipid accumulation, cholesterol efflux, and the expression of miR-33a-5P, ABCA1 and ABCG1 in the absence or presence of LDL in THP-1 macrophages. This study indicated that inflammatory cytokines inhibited ABCA1/ABCG1-mediated cholesterol efflux by up-regulating miR-33a-5P in THP-1 macrophages.

Protection against gamma-radiation injury by protein tyrosine phosphatase 1B

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Marina Mojena

2018-07-01

Full Text Available Protein tyrosine phosphatase 1B (PTP1B is widely expressed in mammalian tissues, in particular in immune cells, and plays a pleiotropic role in dephosphorylating many substrates. Moreover, PTP1B expression is enhanced in response to pro-inflammatory stimuli and to different cell stressors. Taking advantage of the use of mice deficient in PTP1B we have investigated the effect of γ-radiation in these animals and found enhanced lethality and decreased respiratory exchange ratio vs. the corresponding wild type animals. Using bone-marrow derived macrophages and mouse embryonic fibroblasts (MEFs from wild-type and PTP1B-deficient mice, we observed a differential response to various cell stressors. PTP1B-deficient macrophages exhibited an enhanced response to γ-radiation, UV-light, LPS and S-nitroso-glutathione. Macrophages exposed to γ-radiation show DNA damage and fragmentation, increased ROS production, a lack in GSH elevation and enhanced acidic β-galactosidase activity. Interestingly, these differences were not observed in MEFs. Differential gene expression analysis of WT and KO macrophages revealed that the main pathways affected after irradiation were an up-regulation of protein secretion, TGF-β signaling and angiogenesis among other, and downregulation of Myc targets and Hedgehog signaling. These results demonstrate a key role for PTP1B in the protection against the cytotoxicity of irradiation in intact animal and in macrophages, which might be therapeutically relevant. Keywords: Protein tyrosine phosphatase, Cell viability, Irradiation sensitivity, Lethality, p53

Purple perilla extracts allay ER stress in lipid-laden macrophages.

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Sin-Hye Park

Full Text Available There is a growing body of evidence that excess lipids, hypoxic stress and other inflammatory signals can stimulate endoplasmic reticulum (ER stress in metabolic diseases. However, the pathophysiological importance and the underlying mechanisms of this phenomenon remain unknown. The current study investigated that 50 ng/ml oxidized LDL promoted unfolded protein response (UPR and ER stress in J774A1 murine macrophages, which was blocked by extracts (PPE of purple Perilla frutescens, a plant of the mint family Lamiaceae. The ER stressor tunicamycin was employed as a positive control. Treating 1-10 µg/ml oxidized LDL for 24 h elicited lipotoxic apoptosis in macrophages with obvious nuclear condensation and DNA fragmentation, which was inhibited by PPE. Tunicamycin and oxidized LDL activated and induced the UPR components of activating transcription factor 6 and ER resident chaperone BiP/Grp78 in temporal manners and such effects were blocked by ≥5 µg/ml PPE. In addition, PPE suppressed the enhanced mRNA transcription and splicing of X-box binding protein 1 (XBP1 by tunicamycin and oxidized LDL. The protein induction and nuclear translocation of XBP1 were deterred in PPE-treated macrophages under ER stress. The induction of ATP-binding cassette transporter A1 (ABCA1, scavenger receptor-B1 (SR-B1 and intracellular adhesion molecule-1 (ICAM-1 was abolished by the ER stressor in activated macrophages. The protein induction of ABCA1 and ICAM1 but not SR-B1 was retrieved by adding 10 µg/ml PPE to cells. These results demonstrate that PPE inhibited lipotoxic apoptosis and demoted the induction and activation of UPR components in macrophages. PPE restored normal proteostasis in activated macrophages oxidized LDL. Therefore, PPE was a potent agent antagonizing macrophage ER stress due to lipotoxic signals associated with atherosclerosis.

The influence of protein malnutrition on the production of GM-CSF and M-CSF by macrophages

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Dalila Cunha de Oliveira

Full Text Available ABSTRACT It is well established that protein malnutrition (PM impairs immune defenses and increases susceptibility to infection. Macrophages are cells that play a central role in innate immunity, constituting one of the first barriers against infections. Macrophages produce several soluble factors, including cytokines and growth factors, important to the immune response. Among those growth factors, granulocyte-macrophage colony-stimulating factor (GM-CSF and macrophage colony-stimulating factor (M-CSF. GM-CSF and M-CSF are important to monocyte and macrophage development and stimulation of the immune response process. Knowing the importance of GM-CSF and M-CSF, we sought to investigate the influence of PM on macrophage production of these growth factors. Two-month-old male BALB/c mice were subjected to PM with a low-protein diet (2% and compared to a control diet (12% mouse group. Nutritional status, hemogram and the number of peritoneal cells were evaluated. Additionally, peritoneal macrophages were cultured and the production of GM-CSF and M-CSF and mRNA expression were evaluated. To determine if PM altered macrophage production of GM-CSF and M-CSF, they were stimulated with TNF-α. The PM animals had anemia, leukopenia and a reduced number of peritoneal cells. The production of M-CSF was not different between groups; however, cells from PM animals, stimulated with or without TNF-α, presented reduced capability to produce GM-CSF. These data imply that PM interferes with the production of GM-CSF, and consequently would affect the production and maturation of hematopoietic cells and the immune response.

Immunotherapy of metastatic breast cancer patients with vitamin D-binding protein-derived macrophage activating factor (GcMAF).

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Yamamoto, Nobuto; Suyama, Hirofumi; Yamamoto, Nobuyuki; Ushijima, Naofumi

2008-01-15

Serum vitamin D3-binding protein (Gc protein) is the precursor for the principal macrophage activating factor (MAF). The MAF precursor activity of serum Gc protein of breast cancer patients was lost or reduced because Gc protein was deglycosylated by serum alpha-N-acetylgalactosaminidase (Nagalase) secreted from cancerous cells. Patient serum Nagalase activity is proportional to tumor burden. The deglycosylated Gc protein cannot be converted to MAF, resulting in no macrophage activation and immunosuppression. Stepwise incubation of purified Gc protein with immobilized beta-galactosidase and sialidase generated probably the most potent macrophage activating factor (termed GcMAF) ever discovered, which produces no adverse effect in humans. Macrophages treated in vitro with GcMAF (100 pg/ml) are highly tumoricidal to mammary adenocarcinomas. Efficacy of GcMAF for treatment of metastatic breast cancer was investigated with 16 nonanemic patients who received weekly administration of GcMAF (100 ng). As GcMAF therapy progresses, the MAF precursor activity of patient Gc protein increased with a concomitant decrease in serum Nagalase. Because of proportionality of serum Nagalase activity to tumor burden, the time course progress of GcMAF therapy was assessed by serum Nagalase activity as a prognostic index. These patients had the initial Nagalase activities ranging from 2.32 to 6.28 nmole/min/mg protein. After about 16-22 administrations (approximately 3.5-5 months) of GcMAF, these patients had insignificantly low serum enzyme levels equivalent to healthy control enzyme levels, ranging from 0.38 to 0.63 nmole/min/mg protein, indicating eradication of the tumors. This therapeutic procedure resulted in no recurrence for more than 4 years. Copyright 2007 Wiley-Liss, Inc.

A novel role for a major component of the vitamin D axis: vitamin D binding protein-derived macrophage activating factor induces human breast cancer cell apoptosis through stimulation of macrophages.

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Thyer, Lynda; Ward, Emma; Smith, Rodney; Fiore, Maria Giulia; Magherini, Stefano; Branca, Jacopo J V; Morucci, Gabriele; Gulisano, Massimo; Ruggiero, Marco; Pacini, Stefania

2013-07-08

The role of vitamin D in maintaining health appears greater than originally thought, and the concept of the vitamin D axis underlines the complexity of the biological events controlled by biologically active vitamin D (1,25(OH)(2)D3), its two binding proteins that are the vitamin D receptor (VDR) and the vitamin D-binding protein-derived macrophage activating factor (GcMAF). In this study we demonstrate that GcMAF stimulates macrophages, which in turn attack human breast cancer cells, induce their apoptosis and eventually phagocytize them. These results are consistent with the observation that macrophages infiltrated implanted tumors in mice after GcMAF injections. In addition, we hypothesize that the last 23 hydrophobic amino acids of VDR, located at the inner part of the plasma membrane, interact with the first 23 hydrophobic amino acids of the GcMAF located at the external part of the plasma membrane. This allows 1,25(OH)(2)D3 and oleic acid to become sandwiched between the two vitamin D-binding proteins, thus postulating a novel molecular mode of interaction between GcMAF and VDR. Taken together, these results support and reinforce the hypothesis that GcMAF has multiple biological activities that could be responsible for its anti-cancer effects, possibly through molecular interaction with the VDR that in turn is responsible for a multitude of non-genomic as well as genomic effects.

A Novel Role for a Major Component of the Vitamin D Axis: Vitamin D Binding Protein-Derived Macrophage Activating Factor Induces Human Breast Cancer Cell Apoptosis through Stimulation of Macrophages

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Marco Ruggiero

2013-07-01

Full Text Available The role of vitamin D in maintaining health appears greater than originally thought, and the concept of the vitamin D axis underlines the complexity of the biological events controlled by biologically active vitamin D (1,25(OH(2D3, its two binding proteins that are the vitamin D receptor (VDR and the vitamin D-binding protein-derived macrophage activating factor (GcMAF. In this study we demonstrate that GcMAF stimulates macrophages, which in turn attack human breast cancer cells, induce their apoptosis and eventually phagocytize them. These results are consistent with the observation that macrophages infiltrated implanted tumors in mice after GcMAF injections. In addition, we hypothesize that the last 23 hydrophobic amino acids of VDR, located at the inner part of the plasma membrane, interact with the first 23 hydrophobic amino acids of the GcMAF located at the external part of the plasma membrane. This al1ows 1,25(OH(2D3 and oleic acid to become sandwiched between the two vitamin D-binding proteins, thus postulating a novel molecular mode of interaction between GcMAF and VDR. Taken together, these results support and reinforce the hypothesis that GcMAF has multiple biological activities that could be responsible for its anti-cancer effects, possibly through molecular interaction with the VDR that in turn is responsible for a multitude of non-genomic as well as genomic effects.

Monocyte to macrophage differentiation-associated (MMD) positively regulates ERK and Akt activation and TNF-α and NO production in macrophages.

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Liu, Qiang; Zheng, Jin; Yin, Dan-Dan; Xiang, Jie; He, Fei; Wang, Yao-Chun; Liang, Liang; Qin, Hong-Yan; Liu, Li; Liang, Ying-Min; Han, Hua

2012-05-01

Macrophage activation is modulated by both environmental cues and endogenous programs. In the present study, we investigated the role of a PAQR family protein, monocyte to macrophage differentiation-associated (MMD), in macrophage activation and unveiled its underlying molecular mechanism. Our results showed that while MMD expression could be detected in all tissues examined, its expression level is significantly up-regulated upon monocyte differentiation. Within cells, EGFP-MMD fusion protein could be co-localized to endoplasmic reticulum, mitochondria, Golgi apparatus, but not lysosomes and cytoplasm. MMD expression is up-regulated in macrophages after LPS stimulation, and this might be modulated by RBP-J, the critical transcription factor of Notch signaling. Overexpression of MMD in macrophages increased the production of TNF-α and NO upon LPS stimulation. We found that MMD overexpression enhanced ERK1/2 and Akt phosphorylation in macrophages after LPS stimulation. Blocking Erk or Akt by pharmacological agent reduced TNF-α or NO production in MMD-overexpressing macrophages, respectively. These results suggested that MMD modulates TNF-α and NO production in macrophages, and this process might involves Erk or Akt.

Caspase-1-dependent and -independent cell death pathways in Burkholderia pseudomallei infection of macrophages.

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Antje Bast

2014-03-01

Full Text Available The cytosolic pathogen Burkholderia pseudomallei and causative agent of melioidosis has been shown to regulate IL-1β and IL-18 production through NOD-like receptor NLRP3 and pyroptosis via NLRC4. Downstream signalling pathways of those receptors and other cell death mechanisms induced during B. pseudomallei infection have not been addressed so far in detail. Furthermore, the role of B. pseudomallei factors in inflammasome activation is still ill defined. In the present study we show that caspase-1 processing and pyroptosis is exclusively dependent on NLRC4, but not on NLRP3 in the early phase of macrophage infection, whereas at later time points caspase-1 activation and cell death is NLRC4- independent. In the early phase we identified an activation pathway involving caspases-9, -7 and PARP downstream of NLRC4 and caspase-1. Analyses of caspase-1/11-deficient infected macrophages revealed a strong induction of apoptosis, which is dependent on activation of apoptotic initiator and effector caspases. The early activation pathway of caspase-1 in macrophages was markedly reduced or completely abolished after infection with a B. pseudomallei flagellin FliC or a T3SS3 BsaU mutant. Studies using cells transfected with the wild-type and mutated T3SS3 effector protein BopE indicated also a role of this protein in caspase-1 processing. A T3SS3 inner rod protein BsaK mutant failed to activate caspase-1, revealed higher intracellular counts, reduced cell death and IL-1β secretion during early but not during late macrophage infection compared to the wild-type. Intranasal infection of BALB/c mice with the BsaK mutant displayed a strongly decreased mortality, lower bacterial loads in organs, and reduced levels of IL-1β, myeloperoxidase and neutrophils in bronchoalveolar lavage fluid. In conclusion, our results indicate a major role for a functional T3SS3 in early NLRC4-mediated caspase-1 activation and pyroptosis and a contribution of late caspase-1

Neogambogic acid prevents silica-induced fibrosis via inhibition of high-mobility group box 1 and MCP-1-induced protein 1

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Zhang, Wei [Department of Physiology, School of Medicine, Southeast University, Nanjing, Jiangsu 210009 (China); Department of Pharmacology, School of Medicine, Southeast University, Nanjing, Jiangsu 210009 (China); Key Laboratory of Developmental Genes and Human Disease, Southeast University, Nanjing 210096 (China); Department of Respiration, Zhongda Hospital, School of Medicine, Southeast University, Nanjing, Jiangsu 210009 (China); Zhang, Mei, E-mail: meizhang1717@163.com [Department of Respiration, Zhongda Hospital, School of Medicine, Southeast University, Nanjing, Jiangsu 210009 (China); Wang, Zhongjiang [Department of Radiology, Zhongda Hospital, School of Medicine, Southeast University, Nanjing, Jiangsu 210009 (China); Cheng, Yusi; Liu, Haijun [Department of Physiology, School of Medicine, Southeast University, Nanjing, Jiangsu 210009 (China); Zhou, Zewei [Department of Physiology, School of Medicine, Southeast University, Nanjing, Jiangsu 210009 (China); Department of Pharmacology, School of Medicine, Southeast University, Nanjing, Jiangsu 210009 (China); Han, Bing [Department of Pharmacology, School of Medicine, Southeast University, Nanjing, Jiangsu 210009 (China); Chen, Baoan [Department of Hematology and Oncology, Zhongda Hospital, School of Medicine, Southeast University, Nanjing, Jiangsu 210009 (China); Yao, Honghong, E-mail: yaohh@seu.edu.cn [Department of Pharmacology, School of Medicine, Southeast University, Nanjing, Jiangsu 210009 (China); Key Laboratory of Developmental Genes and Human Disease, Southeast University, Nanjing 210096 (China); Chao, Jie, E-mail: chaojie@seu.edu.cn [Department of Physiology, School of Medicine, Southeast University, Nanjing, Jiangsu 210009 (China); Key Laboratory of Developmental Genes and Human Disease, Southeast University, Nanjing 210096 (China); Department of Respiration, Zhongda Hospital, School of Medicine, Southeast University, Nanjing, Jiangsu 210009 (China)

2016-10-15

Background: Silicosis is a systemic disease caused by inhaling silicon dioxide (SiO{sub 2}); early stages are characterized by alveolar inflammation, and later stages are characterized by progressive lung fibrosis. Mounting evidence indicates that high-mobility group box 1 (HMGB1) is involved in pulmonary fibrosis. Whether neogambogic acid (NGA) inhibits macrophage and fibroblast activation induced by SiO{sub 2} by targeting HMGB1 remains unclear. Methods and results: Experiments using cultured mouse macrophages (RAW264.7 cells) demonstrated that SiO{sub 2} treatment induces the expression of HMGB1 in a time- and dose-dependent manner via mitogen-activated protein kinases (MAPKs) and the phosphatidylinositol 3-kinase (PI3K)/Akt pathway; in turn, this expression causes macrophage apoptosis and fibroblast activation. Pretreating macrophages with NGA inhibited the HMGB1 expression induced by SiO{sub 2} and attenuated both macrophage apoptosis and fibroblast activation. Moreover, NGA directly inhibited MCP-1-induced protein 1 (MCPIP1) expression, as well as markers of fibroblast activation and migration induced by SiO{sub 2}. Furthermore, the effects of NGA on macrophages and fibroblasts were confirmed in vivo by exposing mice to SiO{sub 2}. Conclusion: NGA can prevent SiO{sub 2}-induced macrophage activation and apoptosis via HMGB1 inhibition and SiO{sub 2}-induced fibrosis via the MCPIP1 pathway. Targeting HMGB1 and MCPIP1 with NGA could provide insights into the potential development of a therapeutic approach for alleviating the inflammation and fibrosis induced by SiO{sub 2}. - Highlights: • The SiO{sub 2} induced HMGB1 in alveolar macrophage and MCPIP1 in fibroblast. • NGA rescued the SiO{sub 2}-induced apoptosis of alveolar macrophages via HMGB1 signaling. • NGA inhibited the fibroblast activation induced by SiO{sub 2} via MCPIP1 signaling. • NGA might represent a potential therapeutic approach for silicosis.

Burkholderia cenocepacia type VI secretion system mediates escape of type II secreted proteins into the cytoplasm of infected macrophages.

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Roberto Rosales-Reyes

Full Text Available Burkholderia cenocepacia is an opportunistic pathogen that survives intracellularly in macrophages and causes serious respiratory infections in patients with cystic fibrosis. We have previously shown that bacterial survival occurs in bacteria-containing membrane vacuoles (BcCVs resembling arrested autophagosomes. Intracellular bacteria stimulate IL-1β secretion in a caspase-1-dependent manner and induce dramatic changes to the actin cytoskeleton and the assembly of the NADPH oxidase complex onto the BcCV membrane. A Type 6 secretion system (T6SS is required for these phenotypes but surprisingly it is not required for the maturation arrest of the BcCV. Here, we show that macrophages infected with B. cenocepacia employ the NLRP3 inflammasome to induce IL-1β secretion and pyroptosis. Moreover, IL-1β secretion by B. cenocepacia-infected macrophages is suppressed in deletion mutants unable to produce functional Type VI, Type IV, and Type 2 secretion systems (SS. We provide evidence that the T6SS mediates the disruption of the BcCV membrane, which allows the escape of proteins secreted by the T2SS into the macrophage cytoplasm. This was demonstrated by the activity of fusion derivatives of the T2SS-secreted metalloproteases ZmpA and ZmpB with adenylcyclase. Supporting this notion, ZmpA and ZmpB are required for efficient IL-1β secretion in a T6SS dependent manner. ZmpA and ZmpB are also required for the maturation arrest of the BcCVs and bacterial intra-macrophage survival in a T6SS-independent fashion. Our results uncover a novel mechanism for inflammasome activation that involves cooperation between two bacterial secretory pathways, and an unanticipated role for T2SS-secreted proteins in intracellular bacterial survival.

Effects of propofol on lipopolysaccharide-induced expression and release of HMGB1 in macrophages

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Wang, T.; Wei, X.Y.; Liu, B.; Wang, L.J.; Jiang, L.H. [Department of Anesthesiology, the Third Affiliated Hospital, Zhengzhou University, Zhengzhou (China)

2015-02-24

This study aimed to determine the effects of different concentrations of propofol (2,6-diisopropylphenol) on lipopolysaccharide (LPS)-induced expression and release of high-mobility group box 1 protein (HMGB1) in mouse macrophages. Mouse macrophage cell line RAW264.7 cells were randomly divided into 5 treatment groups. Expression levels of HMGB1 mRNA were detected using RT-PCR, and cell culture supernatant HMGB1 protein levels were detected using enzyme-linked immunosorbent assay (ELISA). Translocation of HMGB1 from the nucleus to the cytoplasm in macrophages was observed by Western blotting and activity of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in the nucleus was detected using ELISA. HMGB1 mRNA expression levels increased significantly in the cell culture supernatant and in cells after 24 h of stimulating RAW264.7 cells with LPS (500 ng/mL). However, HMGB1 mRNA expression levels in the P2 and P3 groups, which received 500 ng/mL LPS with 25 or 50 μmol/mL propofol, respectively, were significantly lower than those in the group receiving LPS stimulation (P<0.05). After stimulation by LPS, HMGB1 protein levels were reduced significantly in the nucleus but were increased in the cytoplasm (P<0.05). Simultaneously, the activity of NF-κB was enhanced significantly (P<0.05). After propofol intervention, HMGB1 translocation from the nucleus to the cytoplasm and NF-κB activity were inhibited significantly (each P<0.05). Thus, propofol can inhibit the LPS-induced expression and release of HMGB1 by inhibiting HMGB1 translocation and NF-κB activity in RAW264.7 cells, suggesting propofol may be protective in patients with sepsis.

IQGAP1 is involved in post-ischemic neovascularization by regulating angiogenesis and macrophage infiltration.

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Norifumi Urao

2010-10-01

Full Text Available Neovascularization is an important repair mechanism in response to ischemic injury and is dependent on inflammation, angiogenesis and reactive oxygen species (ROS. IQGAP1, an actin-binding scaffold protein, is a key regulator for actin cytoskeleton and motility. We previously demonstrated that IQGAP1 mediates vascular endothelial growth factor (VEGF-induced ROS production and migration of cultured endothelial cells (ECs; however, its role in post-ischemic neovascularization is unknown.Ischemia was induced by left femoral artery ligation, which resulted in increased IQGAP1 expression in Mac3(+ macrophages and CD31(+ capillary-like ECs in ischemic legs. Mice lacking IQGAP1 exhibited a significant reduction in the post-ischemic neovascularization as evaluated by laser Doppler blood flow, capillary density and α-actin positive arterioles. Furthermore, IQGAP1(-/- mice showed a decrease in macrophage infiltration and ROS production in ischemic muscles, leading to impaired muscle regeneration and increased necrosis and fibrosis. The numbers of bone marrow (BM-derived cells in the peripheral blood were not affected in these knockout mice. BM transplantation revealed that IQGAP1 expressed in both BM-derived cells and tissue resident cells, such as ECs, is required for post-ischemic neovascularization. Moreover, thioglycollate-induced peritoneal macrophage recruitment and ROS production were inhibited in IQGAP1(-/- mice. In vitro, IQGAP1(-/- BM-derived macrophages showed inhibition of migration and adhesion capacity, which may explain the defective macrophage recruitment into the ischemic tissue in IQGAP1(-/- mice.IQGAP1 plays a key role in post-ischemic neovascularization by regulating, not only, ECs-mediated angiogenesis but also macrophage infiltration as well as ROS production. Thus, IQGAP1 is a potential therapeutic target for inflammation- and angiogenesis-dependent ischemic cardiovascular diseases.

Yersinia pestis and host macrophages: immunodeficiency of mouse macrophages induced by YscW.

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Bi, Yujing; Du, Zongmin; Han, Yanping; Guo, Zhaobiao; Tan, Yafang; Zhu, Ziwen; Yang, Ruifu

2009-09-01

The virulence of the pathogenic Yersinia species depends on a plasmid-encoded type III secretion system (T3SS) that transfers six Yersinia outer protein (Yop) effector proteins into the cytoplasm of eukaryotic cells, leading to disruption of host defence mechanisms. It is shown in this study that Yersinia pestis YscW, a protein of the T3SS injectisome, contributes to the induction of a deficiency in phagocytosis in host macrophages and a reduction in their antigen-presenting capacity. A Y. pestis strain lacking yscW had no effect on uptake by host macrophages. In mice infected with wild-type Y. pestis, the yscW mutant or a complement strain, immunodeficiency was observed in host macrophages compared with those from uninfected mice. However, the phagocytosis and antigen presenting capacities of macrophages infected by yscW mutant strain both in vivo and in vitro were significantly higher than those by wild type strain. Consistent with this finding, when YscW was expressed in the RAW264.7 macrophage cell line, phagocytosis and antigen-presenting capacities were significantly lower than those of the control groups. These results indicate that Y. pestis YscW may directly induce immunodeficiency in murine macrophages by crippling their phagocytosis and antigen-presenting capacities. These data provide evidences to Y. pestis pathogenesis that some proteins in T3SS injectisome, such as YscW protein, might play independent roles in disrupting host defense apart from their known functions.

Celecoxib sensitizes Staphylococcus aureus to antibiotics in macrophages by modulating SIRT1.

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Annamanedi, Madhavi; Kalle, Arunasree M

2014-01-01

We have previously shown that celecoxib in combination with an antibiotic, increase the bacterial sensitivity to antibiotics. However, the underlying molecular mechanism remained elusive. Efficacy of the combinatorial treatment of celecoxib and ampicillin in vitro was evaluated on macrophage-phagocytosed S. aureus. To elucidate the mechanism, signaling pathway of infection and inflammation involving TLR2, JNK, SIRT1 and NF-κB was studied by FACS, Western blot, ELISA and activity assays. Combinatorial treatment of ampicillin and celecoxib reduced the bacterial load in the macrophages. Further studies clearly suggested the activation of the master regulator of oxidative stress and inflammation SIRT1, by celecoxib when used alone and/or in combination with ampicillin. Also, the results indicated that celecoxib inhibited JNK phosphorylation thereby stabilizing and activating SIRT1 protein that inhibited the COX-2 gene transcription with a significant decrease in the levels of protein inflammatory cytokines like IL-6, MIP-1α and IL-1β via inhibition of NF-κB. SIRT1 activation by celecoxib also resulted in increase of catalase and peroxidase activity with a decrease in Nitric oxide levels. In conclusion, we demonstrate a novel role of celecoxib in controlling inflammation as an enhancer of antibiotic activity against bacteria by modulating SIRT1.

Tsc1 is a Critical Regulator of Macrophage Survival and Function

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Chunmin Fang

2015-07-01

Full Text Available Background/Aims: Tuberous sclerosis complex 1 (Tsc1 has been shown to regulate M1/M2 polarization of macrophages, but the precise roles of Tsc1 in the function and stability of macrophages are not fully understood. Here we show that Tsc1 is required for regulating the survival, migration and phagocytosis of macrophages. Methods: Mice with Tsc1 homozygous deletion in myeloid cells (LysMCreTsc1flox/flox; Tsc1 KO were obtained by crossing Tsc1flox/flox mice with mice expressing Cre recombinase under the control of Lysozyme promoter (LysMCre. The apoptosis and growth of macrophages were determined by flow cytometry and Real-time PCR (RT-PCR. The phagocytosis was determined using a Vybrant™ phagocytosis assay kit. The migration of macrophages was determined using transwell migration assay. Results: Peritoneal macrophages of Tsc1 KO mice exhibited increased apoptosis and enlarged cell size. Both M1 and M2 phenotypes in Tsc1-deficient macrophages were elevated in steady-state as well as in inflammatory conditions. Tsc1-deficient macrophages demonstrated impaired migration and reduced expression of chemokine receptors including CCR2 and CCR5. Phagocytosis activity and ROS production were enhanced in Tsc1-deficient macrophages. Furthermore, pharmacological inhibition of the mammalian target of rapamycin complex 1 (mTORC1 partially reversed the aberrance of Tsc1-deficient macrophages. Conclusion: Tsc1 plays a critical role in regulating macrophage survival, function and polarization via inhibition of mTORC1 activity.

IQGAP1 is important for activation of caspase-1 in macrophages and is targeted by Yersinia pestis type III effector YopM.

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Chung, Lawton K; Philip, Naomi H; Schmidt, Valentina A; Koller, Antonius; Strowig, Till; Flavell, Richard A; Brodsky, Igor E; Bliska, James B

2014-07-01

YopM is a leucine-rich repeat (LRR)-containing effector in several Yersinia species, including Yersinia pestis and Y. pseudotuberculosis. Different Yersinia strains encode distinct YopM isoforms with variable numbers of LRRs but conserved C-terminal tails. A 15-LRR isoform in Y. pseudotuberculosis YPIII was recently shown to bind and inhibit caspase-1 via a YLTD motif in LRR 10, and attenuation of YopM(-) YPIII was reversed in mice lacking caspase-1, indicating that caspase-1 inhibition is a major virulence function of YopM(YPIII). To determine if other YopM proteins inhibit caspase-1, we utilized Y. pseudotuberculosis strains natively expressing a 21-LRR isoform lacking the YLTD motif (YopM(32777)) or ectopically expressing a Y. pestis 15-LRR version with a functional (YopM(KIM)) or inactivated (YopM(KIM) D271A) YLTD motif. Results of mouse and macrophage infections with these strains showed that YopM(32777), YopM(KIM), and YopM(KIM) D271A inhibit caspase-1 activation, indicating that the YLTD motif is dispensable for this activity. Analysis of YopM(KIM) deletion variants revealed that LRRs 6 to 15 and the C-terminal tail are required to inhibit caspase-1 activation. YopM(32777), YopM(KIM), and YopM(KIM) deletion variants were purified, and binding partners in macrophage lysates were identified. Caspase-1 bound to YopM(KIM) but not YopM(32777). Additionally, YopM(KIM) bound IQGAP1 and the use of Iqgap1(-/-) macrophages revealed that this scaffolding protein is important for caspase-1 activation upon infection with YopM(-) Y. pseudotuberculosis. Thus, while multiple YopM isoforms inhibit caspase-1 activation, their variable LRR domains bind different host proteins to perform this function and the LRRs of YopM(KIM) target IQGAP1, a novel regulator of caspase-1, in macrophages. Importance: Activation of caspase-1, mediated by macromolecular complexes termed inflammasomes, is important for innate immune defense against pathogens. Pathogens can, in turn, subvert

HIV-1-infected macrophages induce astrogliosis by SDF-1α and matrix metalloproteinases

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Okamoto, Mika; Wang, Xin; Baba, Masanori

2005-01-01

Brain macrophages/microglia and astrocytes are known to be involved in the pathogenesis of HIV-1-associated dementia (HAD). To clarify their interaction and contribution to the pathogenesis, HIV-1-infected or uninfected macrophages were used as a model of brain macrophages/microglia, and their effects on human astrocytes in vitro were examined. The culture supernatants of HIV-1-infected or uninfected macrophages induced significant astrocyte proliferation, which was annihilated with a neutralizing antibody to stromal cell-derived factor (SDF)-1α or a matrix metalloproteinase (MMP) inhibitor. In these astrocytes, CXCR4, MMP, and tissue inhibitors of matrix metalloproteinase mRNA expression and SDF-1α production were significantly up-regulated. The supernatants of infected macrophages were always more effective than those of uninfected cells. Moreover, the enhanced production of SDF-1α was suppressed by the MMP inhibitor. These results indicate that the activated and HIV-1-infected macrophages can indirectly induce astrocyte proliferation through up-regulating SDF-1α and MMP production, which implies a mechanism of astrogliosis in HAD

Kharon1 null mutants of Leishmania mexicana are avirulent in mice and exhibit a cytokinesis defect within macrophages.

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Khoa D Tran

Full Text Available In a variety of eukaryotes, flagella play important roles both in motility and as sensory organelles that monitor the extracellular environment. In the parasitic protozoan Leishmania mexicana, one glucose transporter isoform, LmxGT1, is targeted selectively to the flagellar membrane where it appears to play a role in glucose sensing. Trafficking of LmxGT1 to the flagellar membrane is dependent upon interaction with the KHARON1 protein that is located at the base of the flagellar axoneme. Remarkably, while Δkharon1 null mutants are viable as insect stage promastigotes, they are unable to survive as amastigotes inside host macrophages. Although Δkharon1 promastigotes enter macrophages and transform into amastigotes, these intracellular parasites are unable to execute cytokinesis and form multinucleate cells before dying. Notably, extracellular axenic amastigotes of Δkharon1 mutants replicate and divide normally, indicating a defect in the mutants that is only exhibited in the intra-macrophage environment. Although the flagella of Δkharon1 amastigotes adhere to the phagolysomal membrane of host macrophages, the morphology of the mutant flagella is often distorted. Additionally, these null mutants are completely avirulent following injection into BALB/c mice, underscoring the critical role of the KHARON1 protein for viability of intracellular amastigotes and disease in the animal model of leishmaniasis.

Down regulation of macrophage IFNGR1 exacerbates systemic L. monocytogenes infection.

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Emily M Eshleman

2017-05-01

Full Text Available Interferons (IFNs target macrophages to regulate inflammation and resistance to microbial infections. The type II IFN (IFNγ acts on a cell surface receptor (IFNGR to promote gene expression that enhance macrophage inflammatory and anti-microbial activity. Type I IFNs can dampen macrophage responsiveness to IFNγ and are associated with increased susceptibility to numerous bacterial infections. The precise mechanisms responsible for these effects remain unclear. Type I IFNs silence macrophage ifngr1 transcription and thus reduce cell surface expression of IFNGR1. To test how these events might impact macrophage activation and host resistance during bacterial infection, we developed transgenic mice that express a functional FLAG-tagged IFNGR1 (fGR1 driven by a macrophage-specific promoter. Macrophages from fGR1 mice expressed physiologic levels of cell surface IFNGR1 at steady state and responded equivalently to WT C57Bl/6 macrophages when treated with IFNγ alone. However, fGR1 macrophages retained cell surface IFNGR1 and showed enhanced responsiveness to IFNγ in the presence of type I IFNs. When fGR1 mice were infected with the bacterium Listeria monocytogenes their resistance was significantly increased, despite normal type I and II IFN production. Enhanced resistance was dependent on IFNγ and associated with increased macrophage activation and antimicrobial function. These results argue that down regulation of myeloid cell IFNGR1 is an important mechanism by which type I IFNs suppress inflammatory and anti-bacterial functions of macrophages.

Early secreted antigen ESAT-6 of Mycobacterium Tuberculosis promotes apoptosis of macrophages via targeting the microRNA155-SOCS1 interaction.

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Yang, Shaojun; Li, Fake; Jia, Shuangrong; Zhang, Kejun; Jiang, Wenbing; Shang, Ya; Chang, Kai; Deng, Shaoli; Chen, Ming

2015-01-01

The early secreted antigenic target 6-kDa protein (ESAT-6) of Mycobacterium tuberculosis (Mtb) not only acts as a key player for virulence but also exhibits a strong immunotherapeutic potential against Mtb. However, little is known about the molecular basis for its potential in immunotherapy. The present study was designed to unravel the role of miRNA-155 in ESAT-6-mediated enhancement of host immunity and apoptosis in macrophages. Lentivirus-mediated miR-155 sponge and miR-155 and SOCS1 overexpression vectors were developed in macrophages. TLR2- or p65-specific siRNA knockdown was employed to silence TLR2 or p65. Quantitative polymerase chain reaction and western blotting analyses were performed to determine mRNA and protein expression levels, respectively. Macrophage apoptosis was analyzed by flow cytometry. ESAT-6 significantly increased miR-155 expression, which was dependent on TLR2/NF-κB activation in macrophages. Induced expression of miRNA-155 was required for the ESAT-6-mediated protective immune response and macrophage apoptosis. ESAT-6 promoted macrophage apoptosis by targeting the miR-155-SOCS1 pathway. The differential expression levels of TLR2, BIC, and SOCS1 were involved in regulating the immune response in human peripheral blood mononuclear cells of patients with active tuberculosis (TB) and latent TB (LTB). ESAT-6 promotes apoptosis of macrophages via targeting the miRNA155-SOCS1 interaction. © 2015 S. Karger AG, Basel.

Early Secreted Antigen ESAT-6 of Mycobacterium Tuberculosis Promotes Apoptosis of Macrophages via Targeting the MicroRNA155-SOCS1 Interaction

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Shaojun Yang

2015-02-01

Full Text Available Background: The early secreted antigenic target 6-kDa protein (ESAT-6 of Mycobacterium tuberculosis (Mtb not only acts as a key player for virulence but also exhibits a strong immunotherapeutic potential against Mtb. However, little is known about the molecular basis for its potential in immunotherapy. The present study was designed to unravel the role of miRNA-155 in ESAT-6-mediated enhancement of host immunity and apoptosis in macrophages. Methods: Lentivirus-mediated miR-155 sponge and miR-155 and SOCS1 overexpression vectors were developed in macrophages. TLR2- or p65-specific siRNA knockdown was employed to silence TLR2 or p65. Quantitative polymerase chain reaction and western blotting analyses were performed to determine mRNA and protein expression levels, respectively. Macrophage apoptosis was analyzed by flow cytometry. Results: ESAT-6 significantly increased miR-155 expression, which was dependent on TLR2/NF-κB activation in macrophages. Induced expression of miRNA-155 was required for the ESAT-6-mediated protective immune response and macrophage apoptosis. ESAT-6 promoted macrophage apoptosis by targeting the miR-155-SOCS1 pathway. The differential expression levels of TLR2, BIC, and SOCS1 were involved in regulating the immune response in human peripheral blood mononuclear cells of patients with active tuberculosis (TB and latent TB (LTB. Conclusion: ESAT-6 promotes apoptosis of macrophages via targeting the miRNA155-SOCS1 interaction.

Administration of Protein kinase D1 induce an immunomodulatory effect on lipopolysaccharide-induced intestinal inflammation in a co-culture model of intestinal epithelial Caco-2 cells and RAW 264.7 macrophage cells

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Nielsen, Ditte Søvsø Gundelund; Fredborg, Marlene; Andersen, Vibeke

2017-01-01

the effects of human PKD1 in relation to intestinal inflammation, using a co-culture model of intestinal epithelial Caco-2 cells and RAW264.7 macrophages. An inflammatory response was induced in the macrophages by lipopolysaccharide (LPS), upregulating the expression of tumour necrosis factor alpha (TNF......-α), interleukin- (IL-) 1β, and IL-6 besides increasing the secretion of TNF-α protein. The effect of administering PKD1 to Caco-2 was evaluated in relation to both amelioration of inflammation and the ability to suppress inflammation initiation. Administration of PKD1 (10–100 ng/ml) following induction...

Increased expression of interleukin-1β in triglyceride-induced macrophage cell death is mediated by p38 MAP kinase.

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Sung, Ho Joong; Son, Sin Jee; Yang, Seung-ju; Rhee, Ki-Jong; Kim, Yoon Suk

2012-07-01

Triglycerides (TG) are implicated in the development of atherosclerosis through formation of foam cells and induction of macrophage cell death. In this study, we report that addition of exogenous TG induced cell death in phorbol 12-myristate 13-acetate-differentiated THP-1 human macrophages. TG treatment induced a dramatic decrease in interleukin-1β (IL-1β) mRNA expression in a dose- and time-dependent manner. The expression of granulocyte macrophage colony-stimulating factor and platelet endothelial cell adhesion molecule remained unchanged. To identify signaling pathways involved in TG-induced downregulation of IL-1β, we added p38 MAPK, protein kinase C (PKC) or c-Raf1 specific inhibitors. We found that inhibition of p38 MAPK alleviated the TG-induced downregulation of IL-1β, whereas inhibition of PKC and c-Raf1 had no effect. This is the first report showing decreased IL-1β expression during TG-induced cell death in a human macrophage line. Our results suggest that downregulation of IL-1β expression by TG-treated macrophages may play a role during atherogenesis.

Macrophage replication screen identifies a novel Francisella hydroperoxide resistance protein involved in virulence.

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Anna C Llewellyn

Full Text Available Francisella tularensis is a gram-negative facultative intracellular pathogen and the causative agent of tularemia. Recently, genome-wide screens have identified Francisella genes required for virulence in mice. However, the mechanisms by which most of the corresponding proteins contribute to pathogenesis are still largely unknown. To further elucidate the roles of these virulence determinants in Francisella pathogenesis, we tested whether each gene was required for replication of the model pathogen F. novicida within macrophages, an important virulence trait. Fifty-three of the 224 genes tested were involved in intracellular replication, including many of those within the Francisella pathogenicity island (FPI, validating our results. Interestingly, over one third of the genes identified are annotated as hypothetical, indicating that F. novicida likely utilizes novel virulence factors for intracellular replication. To further characterize these virulence determinants, we selected two hypothetical genes to study in more detail. As predicted by our screen, deletion mutants of FTN_0096 and FTN_1133 were attenuated for replication in macrophages. The mutants displayed differing levels of attenuation in vivo, with the FTN_1133 mutant being the most attenuated. FTN_1133 has sequence similarity to the organic hydroperoxide resistance protein Ohr, an enzyme involved in the bacterial response to oxidative stress. We show that FTN_1133 is required for F. novicida resistance to, and degradation of, organic hydroperoxides as well as resistance to the action of the NADPH oxidase both in macrophages and mice. Furthermore, we demonstrate that F. holarctica LVS, a strain derived from a highly virulent human pathogenic species of Francisella, also requires this protein for organic hydroperoxide resistance as well as replication in macrophages and mice. This study expands our knowledge of Francisella's largely uncharacterized intracellular lifecycle and

Effects of vitamin D(3)-binding protein-derived macrophage activating factor (GcMAF) on angiogenesis.

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Kanda, Shigeru; Mochizuki, Yasushi; Miyata, Yasuyoshi; Kanetake, Hiroshi; Yamamoto, Nobuto

2002-09-04

The vitamin D(3)-binding protein (Gc protein)-derived macrophage activating factor (GcMAF) activates tumoricidal macrophages against a variety of cancers indiscriminately. We investigated whether GcMAF also acts as an antiangiogenic factor on endothelial cells. The effects of GcMAF on angiogenic growth factor-induced cell proliferation, chemotaxis, and tube formation were examined in vitro by using cultured endothelial cells (murine IBE cells, porcine PAE cells, and human umbilical vein endothelial cells [HUVECs]) and in vivo by using a mouse cornea micropocket assay. Blocking monoclonal antibodies to CD36, a receptor for the antiangiogenic factor thrombospondin-1, which is also a possible receptor for GcMAF, were used to investigate the mechanism of GcMAF action. GcMAF inhibited the endothelial cell proliferation, chemotaxis, and tube formation that were all stimulated by fibroblast growth factor-2 (FGF-2), vascular endothelial growth factor-A, or angiopoietin 2. FGF-2-induced neovascularization in murine cornea was also inhibited by GcMAF. Monoclonal antibodies against murine and human CD36 receptor blocked the antiangiogenic action of GcMAF on the angiogenic factor stimulation of endothelial cell chemotaxis. In addition to its ability to activate tumoricidal macrophages, GcMAF has direct antiangiogenic effects on endothelial cells independent of tissue origin. The antiangiogenic effects of GcMAF may be mediated through the CD36 receptor.

YC-1 potentiates cAMP-induced CREB activation and nitric oxide production in alveolar macrophages

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Hwang, Tsong-Long, E-mail: htl@mail.cgu.edu.tw [Graduate Institute of Natural Products, College of Medicine, Chang Gung University, Taoyuan, Taiwan (China); Chinese Herbal Medicine Research Team, Healthy Aging Research Center, Chang Gung University, Kweishan, Taoyuan, Taiwan (China); Tang, Ming-Chi [Graduate Institute of Natural Products, College of Medicine, Chang Gung University, Taoyuan, Taiwan (China); Kuo, Liang-Mou [Department of General Surgery, Chang Gung Memorial Hospital at Chia-Yi, Taiwan (China); Chang, Wen-De; Chung, Pei-Jen; Chang, Ya-Wen; Fang, Yao-Ching [Graduate Institute of Natural Products, College of Medicine, Chang Gung University, Taoyuan, Taiwan (China)

2012-04-15

Alveolar macrophages play significant roles in the pathogenesis of several inflammatory lung diseases. Increases in exhaled nitric oxide (NO) are well documented to reflect disease severity in the airway. In this study, we investigated the effect of 3-(5′-hydroxymethyl-2′-furyl)-1-benzyl indazole (YC-1), a known activator of soluble guanylyl cyclase, on prostaglandin (PG)E{sub 1} (a stable PGE{sub 2} analogue) and forskolin (a adenylate cyclase activator) induced NO production and inducible NO synthase (iNOS) expression in rat alveolar macrophages (NR8383). YC-1 did not directly cause NO production or iNOS expression, but drastically potentiated PGE{sub 1}- or forskolin-induced NO production and iNOS expression in NR8383 alveolar macrophages. Combination treatment with YC-1 and PGE{sub 1} significantly increased phosphorylation of the cAMP response element-binding protein (CREB), but not nuclear factor (NF)-κB activation. The combined effect on NO production, iNOS expression, and CREB phosphorylation was reversed by a protein kinase (PK)A inhibitor (H89), suggesting that the potentiating functions were mediated through a cAMP/PKA signaling pathway. Consistent with this, cAMP analogues, but not the cGMP analogue, caused NO release, iNOS expression, and CREB activation. YC-1 treatment induced an increase in PGE{sub 1}-induced cAMP formation, which occurred through the inhibition of cAMP-specific phosphodiesterase (PDE) activity. Furthermore, the combination of rolipram (an inhibitor of PDE4), but not milronone (an inhibitor of PDE3), and PGE{sub 1} also triggered NO production and iNOS expression. In summary, YC-1 potentiates PGE{sub 1}-induced NO production and iNOS expression in alveolar macrophages through inhibition of cAMP PDE activity and activation of the cAMP/PKA/CREB signaling pathway. Highlights: ► YC-1 potentiated PGE1-induced iNOS expression in alveolar macrophages. ► The combination of YC-1 and PGE1 increased CREB but not NFκB activation. �

Structurally well-defined macrophage activating factor derived from vitamin D3-binding protein has a potent adjuvant activity for immunization.

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Yamamoto, N; Naraparaju, V R

1998-06-01

Freund's adjuvant produced severe inflammation that augments development of antibodies. Thus, mixed administration of antigens with adjuvant was not required as long as inflammation was induced in the hosts. Since macrophage activation for phagocytosis and antigen processing is the first step of antibody development, inflammation-primed macrophage activation plays a major role in immune development. Therefore, macrophage activating factor should act as an adjuvant for immunization. The inflammation-primed macrophage activation process is the major macrophage activating cascade that requires participation of serum vitamin D3-binding protein (DBP; human DBP is known as Gc protein) and glycosidases of B and T lymphocytes. Stepwise incubation of Gc protein with immobilized beta-galactosidase and sialidase efficiently generated the most potent macrophage activating factor (designated GcMAF) we have ever encountered. Administration of GcMAF (20 or 100 pg/mouse) resulted in stimulation of the progenitor cells for extensive mitogenesis and activation of macrophages. Administration of GcMAF (100 pg/mouse) along with immunization of mice with sheep red blood cells (SRBC) produced a large number of anti-SRBC antibody secreting splenic cells in 2-4 days. Thus, GcMAF has a potent adjuvant activity for immunization. Although malignant tumours are poorly immunogenic, 4 days after GcMAF-primed immunization of mice with heat-killed Ehrlich ascites tumour cells, the ascites tumour was no longer transplantable in these mice.

Tumor Cells and Tumor-Associated Macrophages: Secreted Proteins as Potential Targets for Therapy

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Baay, Marc; Brouwer, Anja; Pauwels, Patrick; Peeters, Marc; Lardon, Filip

2011-01-01

Inflammatory pathways, meant to defend the organism against infection and injury, as a byproduct, can promote an environment which favors tumor growth and metastasis. Tumor-associated macrophages (TAMs), which constitute a significant part of the tumor-infiltrating immune cells, have been linked to the growth, angiogenesis, and metastasis of a variety of cancers, most likely through polarization of TAMs to the M2 (alternative) phenotype. The interaction between tumor cells and macrophages provides opportunities for therapy. This paper will discuss secreted proteins as targets for intervention. PMID:22162712

Tumor Cells and Tumor-Associated Macrophages: Secreted Proteins as Potential Targets for Therapy

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Marc Baay

2011-01-01

Full Text Available Inflammatory pathways, meant to defend the organism against infection and injury, as a byproduct, can promote an environment which favors tumor growth and metastasis. Tumor-associated macrophages (TAMs, which constitute a significant part of the tumor-infiltrating immune cells, have been linked to the growth, angiogenesis, and metastasis of a variety of cancers, most likely through polarization of TAMs to the M2 (alternative phenotype. The interaction between tumor cells and macrophages provides opportunities for therapy. This paper will discuss secreted proteins as targets for intervention.

Hsp27 promotes ABCA1 expression and cholesterol efflux through the PI3K/PKCζ/Sp1 pathway in THP-1 macrophages.

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Kuang, Hai-Jun; Zhao, Guo-Jun; Chen, Wu-Jun; Zhang, Min; Zeng, Gao-Feng; Zheng, Xi-Long; Tang, Chao-Ke

2017-09-05

Heat shock protein 27 (Hsp27) is a putative biomarker and therapeutic target in atherosclerosis. This study was to explore the potential mechanisms underlying Hsp27 effects on ATP-binding cassette transporter A1 (ABCA1) expression and cellular cholesterol efflux. THP-1 macrophage-derived foam cells were infected with adenovirus to express wild-type Hsp27, hyper-phosphorylated Hsp27 mimic (3D Hsp27), antisense Hsp27 or hypo-phosphorylated Hsp27 mimic (3A Hsp27). Wild-type and 3D Hsp27 were found to up-regulate ABCA1 mRNA and protein expression and increase cholesterol efflux from cells. Expression of antisense or 3A Hsp27 suppressed the expression of ABCA1 and cholesterol efflux. Furthermore, over-expression of wild-type and 3D Hsp27 significantly increased the levels of phosphorylated specificity protein 1 (Sp1), protein kinase C ζ (PKCζ) and phosphatidylinositol 3-kinase (PI3K). In addition, the up-regulation of ABCA1 expression and cholesterol efflux induced by 3D Hsp27 was suppressed by inhibition of Sp1, PKCζ and PI3K with specific kinase inhibitors. Taken together, our results revealed that Hsp27 may up-regulate the expression of ABCA1 and promotes cholesterol efflux through activation of the PI3K/PKCζ/Sp1 signal pathway in THP-1 macrophage-derived foam cells. Our findings may partly explain the mechanisms underlying the anti-atherogenic effect of Hsp27. Copyright © 2017 Elsevier B.V. All rights reserved.

Human macrophage hemoglobin-iron metabolism in vitro

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Custer, G.; Balcerzak, S.; Rinehart, J.

1982-01-01

An entirely in vitro technique was employed to characterize hemoglobin-iron metabolism by human macrophages obtained by culture of blood monocytes and pulmonary alveolar macrophages. Macrophages phagocytized about three times as many erythrocytes as monocytes and six times as many erythrocytes as pulmonary alveolar macrophages. The rate of subsequent release of 59 Fe to the extracellular transferrin pool was two- to fourfold greater for macrophages as compared to the other two cell types. The kinetics of 59 Fe-transferrin release were characterized by a relatively rapid early phase (hours 1-4) followed by a slow phase (hours 4-72) for all three cell types. Intracellular movement of iron was characterized by a rapid shift from hemoglobin to ferritin that was complete with the onset of the slow phase of extracellular release. A transient increase in 59 Fe associated with an intracellular protein eluting with transferrin was also observed within 1 hour after phagocytosis. The process of hemoglobin-iron release to extracellular transferrin was inhibited at 4 degrees C but was unaffected by inhibitory of protein synthesis, glycolysis, microtubule function, and microfilament function. These data emphasize the rapidity of macrophage hemoglobin iron metabolism, provide a model for characterization of this process in vitro, and in general confirm data obtained utilizing in vivo animal models

Characteristics of adipose tissue macrophages and macrophage-derived insulin-like growth factor-1 in virus-induced obesity.

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Park, S; Park, H-L; Lee, S-Y; Nam, J-H

2016-03-01

Various pathogens are implicated in the induction of obesity. Previous studies have confirmed that human adenovirus 36 (Ad36) is associated with increased adiposity, improved glycemic control and induction of inflammation. The Ad36-induced inflammation is reflected in the infiltration of macrophages into adipose tissue. However, the characteristics and role of adipose tissue macrophages (ATMs) and macrophage-secreted factors in virus-induced obesity (VIO) are unclear. Although insulin-like growth factor-1 (IGF-1) is involved in obesity metabolism, the contribution of IGF secreted by macrophages in VIO has not been studied. Four-week-old male mice were studied 1 week and 12 weeks after Ad36 infection for determining the characteristics of ATMs in VIO and diet-induced obesity (DIO). In addition, macrophage-specific IGF-1-deficient (MIKO) mice were used to study the involvement of IGF-1 in VIO. In the early stage of VIO (1 week after Ad36 infection), the M1 ATM sub-population increased, which increased the M1/M2 ratio, whereas DIO did not cause this change. In the late stage of VIO (12 weeks after Ad36 infection), the M1/M2 ratio did not change because the M1 and M2 ATM sub-populations increased to a similar extent, despite an increase in adiposity. By contrast, DIO increased the M1/M2 ratio. In addition, VIO in wild-type mice upregulated angiogenesis in adipose tissue and improved glycemic control. However, MIKO mice showed no increase in adiposity, angiogenesis, infiltration of macrophages into adipose tissue, or improvement in glycemic control after Ad36 infection. These data suggest that IGF-1 secreted by macrophages may contribute to hyperplasia and hypertrophy in adipose tissue by increasing angiogenesis, which helps to maintain the 'adipose tissue robustness'.

Proteomic Investigation of the Time Course Responses of RAW 264.7 Macrophages to Infection with Salmonella enterica

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Shi, Liang; Chowdhury, Saiful M.; Smallwood, Heather S.; Yoon, Hyunjin; Mottaz-Brewer, Heather M.; Norbeck, Angela D.; McDermott, Jason E.; Clauss, Therese RW; Heffron, Fred; Smith, Richard D.; Adkins, Joshua N.

2009-08-01

Macrophages plan important roles in controlling Salmonella-mediated systemic infection. To investigate the responses of macrophages to Salmonella infection, we infected RAW 264.7 macrophages with Salmonella enterica serovar Typhimurium (STM) and then performed a comparative liquid chromatography-tandem mass spectrometry [LC-MS(/MS)]-based proteomics analysis of the infected macrophages. A total of 1006 macrophage and 115 STM proteins were indentified from this study. Most of STM proteins were found at late stage of the time course of infection, consistent with the fact that STM proliferates inside RAW 264.7 macrophages. Majority of the identified macrophage proteins were house keeping-related, including cytoplasmic superoxide dismutase 1 (SOD1), whose peptide abundances were relatively constant during the time course of infection. Compared to those in no infection control, the peptide abundances of 244 macrophage proteins (or 24% of total indentified macrophage proteins) changed considerably after STM infection. The functions of these STM infection-affected macrophage proteins were diverse and ranged from production of antibacterial nitric oxide (i.e., inducible nitric oxide synthase or iNOS) or production of prostaglandin H2 (i.e., prostaglandin-endoperoxide synthase 2, also know as cyclooxygenase-2 or COX-2) to regulation of intracellular traffic (e.g., sorting nexin or SNX 5, 6 and 9), demonstrating a global impact of STM infection on macrophage proteome. Western-blot analysis not only confirmed the LC-MS(/MS) results of SOD1, COX-2 and iNOS, but also revealed that the protein abundances of mitochondrial SOD2 increased after STM infection, indicating an infection-induced oxidative stress in mitochondria.

Truncated thioredoxin (Trx-80) promotes pro-inflammatory macrophages of the M1 phenotype and enhances atherosclerosis.

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Mahmood, Dler Faieeq Darweesh; Abderrazak, Amna; Couchie, Dominique; Lunov, Oleg; Diderot, Vimala; Syrovets, Tatiana; Slimane, Mohamed-Naceur; Gosselet, Fabien; Simmet, Thomas; Rouis, Mustapha; El Hadri, Khadija

2013-07-01

Vascular cells are particularly susceptible to oxidative stress that is believed to play a key role in the pathogenesis of cardiovascular disorders. Thioredoxin-1 (Trx-1) is an oxidative stress-limiting protein with anti-inflammatory and anti-apoptotic properties. In contrast, its truncated form (Trx-80) exerts pro-inflammatory effects. Here we analyzed whether Trx-80 might exert atherogenic effects by promoting macrophage differentiation into the M1 pro-inflammatory phenotype. Trx-80 at 1 µg/ml significantly attenuated the polarization of anti-inflammatory M2 macrophages induced by exposure to either IL-4 at 15 ng/ml or IL-4/IL-13 (10 ng/ml each) in vitro, as evidenced by the expression of the characteristic markers, CD206 and IL-10. By contrast, in LPS-challenged macrophages, Trx-80 significantly potentiated the differentiation into inflammatory M1 macrophages as indicated by the expression of the M1 cytokines, TNF-α and MCP-1. When Trx-80 was administered to hyperlipoproteinemic ApoE2.Ki mice at 30 µg/g body weight (b.w.) challenged either with LPS at 30 µg/30 g (b.w.) or IL-4 at 500 ng/30 g (b.w.), it significantly induced the M1 phenotype but inhibited differentiation of M2 macrophages in thymus and liver. When ApoE2.Ki mice were challenged once weekly with LPS for 5 weeks, they showed severe atherosclerotic lesions enriched with macrophages expressing predominantly M1 over M2 markers. Such effect was potentiated when mice received daily, in addition to LPS, the Trx-80. Moreover, the Trx-80 treatment led to a significantly increased aortic lesion area. The ability of Trx-80 to promote differentiation of macrophages into the classical proinflammatory phenotype may explain its atherogenic effects in cardiovascular diseases. Copyright © 2013 Wiley Periodicals, Inc.

ADAR1 attenuates allogeneic graft rejection by suppressing miR-21 biogenesis in macrophages and promoting M2 polarization.

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Li, Junjie; Xie, Jiangang; Liu, Shanshou; Li, Xiao; Zhang, Dongliang; Wang, Xianqi; Jiang, Jinquan; Hu, Wei; Zhang, Yuan; Jin, Boquan; Zhuang, Ran; Yin, Wen

2018-04-25

ADAR1 (adenosine deaminase acting on double-stranded RNA 1) is an RNA-editing enzyme that mediates adenosine-to-inosine RNA editing events, an important post-transcriptional modification mechanism that can alter the coding properties of mRNA or regulate microRNA biogenesis. ADAR1 also regulates the innate immune response. Here, we have demonstrated that ADAR1 expression increased in LPS-stimulated macrophages. Silencing ADAR1 by using small interfering RNA in macrophages resulted in the pronounced polarization of macrophages to M1, whereas ADAR1 overexpression promoted M2 polarization, which indicated that ADAR1 can inhibit macrophage hyperpolarization and prevent immune hyperactivity. The RNA-RNP immunoprecipitation binding assay demonstrated a direct interaction between ADAR1 and miR-21 precursor. Significant up-regulation in IL-10 and down-regulation in miR-21 were observed in ADAR1-overexpressing macrophages. We evaluated miR-21 target mRNAs and macrophage polarization signaling pathways and found that forkhead box protein O1 (Foxo1) was up-regulated in cells that overexpressed ADAR1. In a mouse allogeneic skin transplantation model, grafts in the ADAR1-overexpressed group survived longer and suffered less immune cell infiltration. In ADAR1-overexpressed recipients, splenic macrophages were significantly polarized to M2, and levels of sera IL-10 were markedly higher than those in the control group. In summary, ADAR1 modulates macrophage M2 polarization via the ADAR1-miR-21-Foxo1-IL-10 axis, thereby suppressing allogeneic graft rejection.-Li, J., Xie, J., Liu, S., Li, X., Zhang, D., Wang, X., Jiang, J., Hu, W., Zhang, Y., Jin, B., Zhuang, R., Yin, W. ADAR1 attenuates allogeneic graft rejection by suppressing miR-21 biogenesis in macrophages and promoting M2 polarization.

Association of the macrophage activating factor (MAF) precursor activity with polymorphism in vitamin D-binding protein.

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Nagasawa, Hideko; Sasaki, Hideyuki; Uto, Yoshihiro; Kubo, Shinichi; Hori, Hitoshi

2004-01-01

Serum vitamin D-binding protein (Gc protein or DBP) is a highly expressed polymorphic protein, which is a precursor of the inflammation-primed macrophage activating factor, GcMAF, by a cascade of carbohydrate processing reactions. In order to elucidate the relationship between Gc polymorphism and GcMAF precursor activity, we estimated the phagocytic ability of three homotypes of Gc protein, Gc1F-1F, Gc1S-1S and Gc2-2, through processing of their carbohydrate moiety. We performed Gc typing of human serum samples by isoelectric focusing (IEF). Gc protein from human serum was purified by affinity chromatography with 25-hydroxyvitamin D3-sepharose. A phagocytosis assay of Gc proteins, modified using beta-glycosidase and sialidase, was carried out. The Gc1F-1F phenotype was revealed to possess Galbeta1-4GalNAc linkage by the analysis of GcMAF precursor activity using beta1-4 linkage-specific galactosidase from jack bean. The GcMAF precursor activity of the Gc1F-1F phenotype was highest among three Gc homotypes. The Gc polymorphism and carbohydrate diversity of Gc protein are significant for its pleiotropic effects.

The metabolic ER stress sensor IRE1α suppresses alternative activation of macrophages and impairs energy expenditure in obesity.

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Shan, Bo; Wang, Xiaoxia; Wu, Ying; Xu, Chi; Xia, Zhixiong; Dai, Jianli; Shao, Mengle; Zhao, Feng; He, Shengqi; Yang, Liu; Zhang, Mingliang; Nan, Fajun; Li, Jia; Liu, Jianmiao; Liu, Jianfeng; Jia, Weiping; Qiu, Yifu; Song, Baoliang; Han, Jing-Dong J; Rui, Liangyou; Duan, Sheng-Zhong; Liu, Yong

2017-05-01

Obesity is associated with metabolic inflammation and endoplasmic reticulum (ER) stress, both of which promote metabolic disease progression. Adipose tissue macrophages (ATMs) are key players orchestrating metabolic inflammation, and ER stress enhances macrophage activation. However, whether ER stress pathways underlie ATM regulation of energy homeostasis remains unclear. Here, we identified inositol-requiring enzyme 1α (IRE1α) as a critical switch governing M1-M2 macrophage polarization and energy balance. Myeloid-specific IRE1α abrogation in Ern1 f/f ; Lyz2-Cre mice largely reversed high-fat diet (HFD)-induced M1-M2 imbalance in white adipose tissue (WAT) and blocked HFD-induced obesity, insulin resistance, hyperlipidemia and hepatic steatosis. Brown adipose tissue (BAT) activity, WAT browning and energy expenditure were significantly higher in Ern1 f/f ; Lyz2-Cre mice. Furthermore, IRE1α ablation augmented M2 polarization of macrophages in a cell-autonomous manner. Thus, IRE1α senses protein unfolding and metabolic and immunological states, and consequently guides ATM polarization. The macrophage IRE1α pathway drives obesity and metabolic syndrome through impairing BAT activity and WAT browning.

Augmented macrophage differentiation and polarization of tumor-associated macrophages towards M1 subtype in listeria-administered tumor-bearing host.

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Rai, Rakesh K; Vishvakarma, Naveen K; Mohapatra, Tribhuban M; Singh, Sukh Mahendra

2012-09-01

This study investigates the effect of Listeria administration on differentiation of macrophages from precursor bone marrow cells and functional status of tumor-associated macrophages (TAM). Listeria administration not only resulted in an augmented infiltration of tumor by F4/80 macrophages but also repolarized the functional status of TAM displaying features of some M1 macrophage subtype with upregulated phagocytosis and tumoricidal activity accompanied by altered expression of monocarboxylate transporter-1, toll-like receptor-2, surface markers: CD11c, interleukin-2 receptor, CD62L, and secreted molecules: nitric oxide, interleukin (IL)-1, IL-6, tumor necrosis factor-α, and vascular endothelial growth factor. Declined tumor cell survival and modulated repertoire of cytokines: interferon-γ, IL-6, IL-10, and transforming growth factor-β in tumor microenvironment indicated their role in polarization of TAM towards proinflammatory state. Bone marrow cell of Listeria-administered tumor-bearing mice showed augmented survival, declined expression of p53 upregulated modulator of apoptosis with an upregulated differentiation into activation responsive bone marrow-derived macrophages along with altered expression of macrophage-colony stimulating factor, macrophage-colony stimulating factor receptor, and granulocyte macrophage-colony stimulating factor receptor. These findings indicate that Listeria infection is associated with an augmented differentiation of macrophages accompanied by tumoricidal activation of TAM.

miR-181a Induces Macrophage Polarized to M2 Phenotype and Promotes M2 Macrophage-mediated Tumor Cell Metastasis by Targeting KLF6 and C/EBPα

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Jia Bi

2016-01-01

Full Text Available Macrophages can acquire a variety of polarization status and functions: classically activated macrophages (M1 macrophages; alternatively activated macrophages (M2 macrophages. However, the molecular basis of the process is still unclear. Here, this study addresses that microRNA-181a (miR-181a is a key molecule controlling macrophage polarization. We found that miR-181a is overexpressed in M2 macrophages than in M1 macrophages. miR-181a expression was decreased when M2 phenotype converted to M1, whereas it increased when M1 phenotype converted to M2. Overexpression of miR-181a in M1 macrophages diminished M1 phenotype expression while promoting polarization to the M2 phenotype. In contrast, knockdown of miR-181a in M2 macrophages promoted M1 polarization and diminished M2 phenotype expression. Mechanistically, Bioinformatic analysis revealed that Kruppel-like factor 6 (KLF6 and CCAAT/enhancer binding protein-α (C/EBPα is a potential target of miR-181a and luciferase assay confirmed that KLF6 and C/EBPα translation is suppressed by miR-181a through interaction with the 3′UTR of KLF6 and C/EBPα mRNA. Further analysis showed that induction of miR-181a suppressed KLF6 and C/EBPα protein expression. Importantly, miR-181a also diminishes M2 macrophages-mediated migration and invasion capacity of tumor cells. Collectively, our results suggest that miR-181a plays a significant role in regulating macrophage polarization through directly target KLF6 and C/EBPα.

High density lipoprotein stimulated migration of macrophages depends on the scavenger receptor class B, type I, PDZK1 and Akt1 and is blocked by sphingosine 1 phosphate receptor antagonists.

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Aishah Al-Jarallah

Full Text Available HDL carries biologically active lipids such as sphingosine-1-phosphate (S1P and stimulates a variety of cell signaling pathways in diverse cell types, which may contribute to its ability to protect against atherosclerosis. HDL and sphingosine-1-phosphate receptor agonists, FTY720 and SEW2871 triggered macrophage migration. HDL-, but not FTY720-stimulated migration was inhibited by an antibody against the HDL receptor, SR-BI, and an inhibitor of SR-BI mediated lipid transfer. HDL and FTY720-stimulated migration was also inhibited in macrophages lacking either SR-BI or PDZK1, an adaptor protein that binds to SR-BI's C-terminal cytoplasmic tail. Migration in response to HDL and S1P receptor agonists was inhibited by treatment of macrophages with sphingosine-1-phosphate receptor type 1 (S1PR1 antagonists and by pertussis toxin. S1PR1 activates signaling pathways including PI3K-Akt, PKC, p38 MAPK, ERK1/2 and Rho kinases. Using selective inhibitors or macrophages from gene targeted mice, we demonstrated the involvement of each of these pathways in HDL-dependent macrophage migration. These data suggest that HDL stimulates the migration of macrophages in a manner that requires the activities of the HDL receptor SR-BI as well as S1PR1 activity.

Reliable and inexpensive expression of large, tagged, exogenous proteins in murine bone marrow-derived macrophages using a second generation lentiviral system

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Matthew R. Miller

2015-08-01

Full Text Available Over the past two decades, researchers have struggled to efficiently express foreign DNA in primary macrophages, impeding research progress. The applications of lipofection, electroporation, microinjection, and viral-mediated transfer typically result in disruptions in macrophage differentiation and function, low expression levels of exogenous proteins, limited efficiency and high cell mortality. In this report, after extensive optimization, we present a method of expressing large tagged proteins at high efficiency, consistency, and low cost using lentiviral infection. This method utilizes laboratory-propagated second generation plasmids to produce efficient virus that can be stored for later use. The expression of proteins up to 150 kDa in size is achieved in 30–70% of cells while maintaining normal macrophage differentiation and morphology as determined by fluorescence microscopy and Western blot analysis. This manuscript delineates the reagents and methods used to produce lentivirus to express exogenous DNA in murine bone marrow-derived macrophages sufficient for single cell microscopy as well as functional assays requiring large numbers of murine bone marrow-derived macrophages.

Antitumor effect of vitamin D-binding protein-derived macrophage activating factor on Ehrlich ascites tumor-bearing mice.

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Koga, Y; Naraparaju, V R; Yamamoto, N

1999-01-01

Cancerous cells secrete alpha-N-acetylgalactosaminidase (NaGalase) into the blood stream, resulting in deglycosylation of serum vitamin D3-binding protein (known as Gc protein), which is a precursor for macrophage activating factor (MAF). Incubation of Gc protein with immobilized beta-galactosidase and sialidase generates the most potent macrophage activating factor (designated GcMAF). Administration of GcMAF to cancer-bearing hosts can bypass the inactivated MAF precursor and act directly on macrophages for efficient activation. Therapeutic effects of GcMAF on Ehrlich ascites tumor-bearing mice were assessed by survival time and serum NaGalase activity, because serum NaGalase activity was proportional to tumor burden. A single administration of GcMAF (100 pg/mouse) to eight mice on the same day after transplantation of the tumor (5 x 10(5) cells) showed a mean survival time of 21 +/- 3 days for seven mice, with one mouse surviving more than 60 days, whereas tumor-bearing controls had a mean survival time of 13 +/- 2 days. Six of the eight mice that received two GcMAF administrations, at Day 0 and Day 4 after transplantation, survived up to 31 +/- 4 days whereas, the remaining two mice survived for more than 60 days. Further, six of the eight mice that received three GcMAF administrations with 4-day intervals showed an extended survival of at least 60 days, and serum NaGalase levels were as low as those of control mice throughout the survival period. The cure with subthreshold GcMAF-treatments (administered once or twice) of tumor-bearing mice appeared to be a consequence of sustained macrophage activation by inflammation resulting from the macrophage-mediated tumoricidal process. Therefore, a protracted macrophage activation induced by a few administrations of minute amounts of GcMAF eradicated the murine ascites tumor.

Suitability of macrophage inflammatory protein-1beta production by THP-1 cells in differentiating skin sensitizers from irritant chemicals.

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Lim, Yeon-Mi; Moon, Seong-Joon; An, Su-Sun; Lee, Soo-Jin; Kim, Seo-Young; Chang, Ih-Seop; Park, Kui-Lea; Kim, Hyoung-Ah; Heo, Yong

2008-04-01

Worldwide restrictions in animal use for research have driven efforts to develop alternative methods. The study aimed to test the efficacy of the macrophage inflammatory protein-1beta (MIP-1beta) assay for testing chemicals' skin-sensitizing capacity. The assay was performed using 9 chemicals judged to be sensitizing and 7 non-sensitizing by the standard in vivo assays. THP-1 cells were cultured in the presence or absence of 4 doses, 0.01x, 0.1x, 0.5x, or 1x IC(50) (50% inhibitory concentration for THP-1 cell proliferation) of these chemicals for 24 hr, and the MIP-1beta level in the supernatants was determined. Skin sensitization by the test chemicals was determined by MIP-1beta production rates. The MIP-1beta production rate was expressed as the relative increase in MIP-1beta production in response to chemical treatment compared with vehicle treatment. When the threshold MIP-1beta production rate used was 100% or 105% of dimethyl sulfoxide, all the sensitizing chemicals tested (dinitrochlorobenzene, hexyl cinnamic aldehyde, eugenol, hydroquinone, dinitrofluorobenzene, benzocaine, nickel, chromium, and 5-chloro-2-methyl-4-isothiazolin-3-one) were positive, and all the non-sensitizing chemicals (methyl salicylate, benzalkonium chloride, lactic acid, isopropanol, and salicylic acid), with the exception of sodium lauryl sulfate, were negative for MIP-1beta production. These results indicate that MIP-1beta could be a biomarker for classification of chemicals as sensitizers or non-sensitizers.

Cytokine expression of macrophages in HIV-1-associated vacuolar myelopathy.

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Tyor, W R; Glass, J D; Baumrind, N; McArthur, J C; Griffin, J W; Becker, P S; Griffin, D E

1993-05-01

Macrophages are frequently present within the periaxonal and intramyelinic vacuoles that are located primarily in the posterior and lateral funiculi of the thoracic spinal cord in HIV-associated vacuolar myelopathy. But the role of these macrophages in the formation of the vacuoles is unclear. One hypothesis is that cytokines, such as interleukin-1 (IL-1) and tumor necrosis factor (TNF)-alpha, are produced locally by macrophages and have toxic effects on myelin or oligodendrocytes. The resulting myelin damage eventually culminates in the removal of myelin by macrophages and vacuole formation. We studied thoracic spinal cord specimens taken at autopsy from HIV-positive (+) and HIV-negative individuals. The predominant mononuclear cells present in HIV+ spinal cords are macrophages. They are located primarily in the posterior and lateral funiculi regardless of the presence or absence of vacuolar myelopathy. Macrophages and microglia are more frequent in HIV+ than HIV-negative individuals and these cells frequently stain for class I and class II antigens, IL-1, and TNF-alpha. Activated macrophages positive for IL-1 and TNF-alpha are great increased in the posterior and lateral funiculi of HIV+ individuals with and without vacuolar myelopathy, suggesting they are present prior to the development of vacuoles. Cytokines, such as TNF-alpha, may be toxic for myelin or oligodendrocytes, leading to myelin damage and removal by macrophages and vacuole formation.

Essential Role of DAP12 Signaling in Macrophage Programming into a Fusion-Competent State

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Helming, Laura; Tomasello, Elena; Kyriakides, Themis R.; Martinez, Fernando O.; Takai, Toshiyuki; Gordon, Siamon; Vivier, Eric

2009-01-01

Multinucleated giant cells, formed by fusion of macrophages, are a hallmark of granulomatous inflammation. With a genetic approach, we show that signaling through the adaptor protein DAP12 (DNAX activating protein of 12 kD), its associated receptor triggering receptor expressed by myeloid cells 2 (TREM-2), and the downstream protein tyrosine kinase Syk is required for the cytokine-induced formation of giant cells and that overexpression of DAP12 potentiates macrophage fusion. We also present evidence that DAP12 is a general macrophage fusion regulator and is involved in modulating the expression of several macrophage-associated genes, including those encoding known mediators of macrophage fusion, such as DC-STAMP and Cadherin 1. Thus, DAP12 is involved in programming of macrophages through the regulation of gene and protein expression to induce a fusion-competent state. PMID:18957693

Xylitol, an anticaries agent, exhibits potent inhibition of inflammatory responses in human THP-1-derived macrophages infected with Porphyromonas gingivalis.

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Park, Eunjoo; Na, Hee Sam; Kim, Sheon Min; Wallet, Shannon; Cha, Seunghee; Chung, Jin

2014-06-01

Xylitol is a well-known anticaries agent and has been used for the prevention and treatment of dental caries. In this study, the anti-inflammatory effects of xylitol are evaluated for possible use in the prevention and treatment of periodontal infections. Cytokine expression was stimulated in THP-1 (human monocyte cell line)-derived macrophages by live Porphyromonas gingivalis, and enzyme-linked immunosorbent assay and a commercial multiplex assay kit were used to determine the effects of xylitol on live P. gingivalis-induced production of cytokine. The effects of xylitol on phagocytosis and the production of nitric oxide were determined using phagocytosis assay, viable cell count, and Griess reagent. The effects of xylitol on P. gingivalis adhesion were determined by immunostaining, and costimulatory molecule expression was examined by flow cytometry. Live P. gingivalis infection increased the production of representative proinflammatory cytokines, such as tumor necrosis factor-α and interleukin (IL)-1β, in a multiplicity of infection- and time-dependent manner. Live P. gingivalis also enhanced the release of cytokines and chemokines, such as IL-12 p40, eotaxin, interferon γ-induced protein 10, monocyte chemotactic protein-1, and macrophage inflammatory protein-1. The pretreatment of xylitol significantly inhibited the P. gingivalis-induced cytokines production and nitric oxide production. In addition, xylitol inhibited the attachment of live P. gingivalis on THP-1-derived macrophages. Furthermore, xylitol exerted antiphagocytic activity against both Escherichia coli and P. gingivalis. These findings suggest that xylitol acts as an anti-inflammatory agent in THP-1-derived macrophages infected with live P. gingivalis, which supports its use in periodontitis.

L-plastin nanobodies perturb matrix degradation, podosome formation, stability and lifetime in THP-1 macrophages.

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Sarah De Clercq

Full Text Available Podosomes are cellular structures acting as degradation 'hot-spots' in monocytic cells. They appear as dot-like structures at the ventral cell surface, enriched in F-actin and actin regulators, including gelsolin and L-plastin. Gelsolin is an ubiquitous severing and capping protein, whereas L-plastin is a leukocyte-specific actin bundling protein. The presence of the capping protein CapG in podosomes has not yet been investigated. We used an innovative approach to investigate the role of these proteins in macrophage podosomes by means of nanobodies or Camelid single domain antibodies. Nanobodies directed against distinct domains of gelsolin, L-plastin or CapG were stably expressed in macrophage-like THP-1 cells. CapG was not enriched in podosomes. Gelsolin nanobodies had no effect on podosome formation or function but proved very effective in tracing distinct gelsolin populations. One gelsolin nanobody specifically targets actin-bound gelsolin and was effectively enriched in podosomes. A gelsolin nanobody that blocks gelsolin-G-actin interaction was not enriched in podosomes demonstrating that the calcium-activated and actin-bound conformation of gelsolin is a constituent of podosomes. THP-1 cells expressing inhibitory L-plastin nanobodies were hampered in their ability to form stable podosomes. Nanobodies did not perturb Ser5 phosphorylation of L-plastin although phosphorylated L-plastin was highly enriched in podosomes. Furthermore, nanobody-induced inhibition of L-plastin function gave rise to an irregular and unstable actin turnover of podosomes, resulting in diminished degradation of the underlying matrix. Altogether these results indicate that L-plastin is indispensable for podosome formation and function in macrophages.

Inflammasome sensor NLRP1 controls rat macrophage susceptibility to Toxoplasma gondii.

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Kimberly M Cirelli

2014-03-01

Full Text Available Toxoplasma gondii is an intracellular parasite that infects a wide range of warm-blooded species. Rats vary in their susceptibility to this parasite. The Toxo1 locus conferring Toxoplasma resistance in rats was previously mapped to a region of chromosome 10 containing Nlrp1. This gene encodes an inflammasome sensor controlling macrophage sensitivity to anthrax lethal toxin (LT induced rapid cell death (pyroptosis. We show here that rat strain differences in Toxoplasma infected macrophage sensitivity to pyroptosis, IL-1β/IL-18 processing, and inhibition of parasite proliferation are perfectly correlated with NLRP1 sequence, while inversely correlated with sensitivity to anthrax LT-induced cell death. Using recombinant inbred rats, SNP analyses and whole transcriptome gene expression studies, we narrowed the candidate genes for control of Toxoplasma-mediated rat macrophage pyroptosis to four genes, one of which was Nlrp1. Knockdown of Nlrp1 in pyroptosis-sensitive macrophages resulted in higher parasite replication and protection from cell death. Reciprocally, overexpression of the NLRP1 variant from Toxoplasma-sensitive macrophages in pyroptosis-resistant cells led to sensitization of these resistant macrophages. Our findings reveal Toxoplasma as a novel activator of the NLRP1 inflammasome in rat macrophages.

Adipocyte fetuin-A contributes to macrophage migration into adipose tissue and polarization of macrophages.

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Chatterjee, Priyajit; Seal, Soma; Mukherjee, Sandip; Kundu, Rakesh; Mukherjee, Sutapa; Ray, Sukanta; Mukhopadhyay, Satinath; Majumdar, Subeer S; Bhattacharya, Samir

2013-09-27

Macrophage infiltration into adipose tissue during obesity and their phenotypic conversion from anti-inflammatory M2 to proinflammatory M1 subtype significantly contributes to develop a link between inflammation and insulin resistance; signaling molecule(s) for these events, however, remains poorly understood. We demonstrate here that excess lipid in the adipose tissue environment may trigger one such signal. Adipose tissue from obese diabetic db/db mice, high fat diet-fed mice, and obese diabetic patients showed significantly elevated fetuin-A (FetA) levels in respect to their controls; partially hepatectomized high fat diet mice did not show noticeable alteration, indicating adipose tissue to be the source of this alteration. In adipocytes, fatty acid induces FetA gene and protein expressions, resulting in its copious release. We found that FetA could act as a chemoattractant for macrophages. To simulate lipid-induced inflammatory conditions when proinflammatory adipose tissue and macrophages create a niche of an altered microenvironment, we set up a transculture system of macrophages and adipocytes; the addition of fatty acid to adipocytes released FetA into the medium, which polarized M2 macrophages to M1. This was further confirmed by direct FetA addition to macrophages. Taken together, lipid-induced FetA from adipocytes is an efficient chemokine for macrophage migration and polarization. These findings open a new dimension for understanding obesity-induced inflammation.

Alternative activation of macrophages and pulmonary fibrosis are modulated by scavenger receptor, macrophage receptor with collagenous structure.

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Murthy, Shubha; Larson-Casey, Jennifer L; Ryan, Alan J; He, Chao; Kobzik, Lester; Carter, A Brent

2015-08-01

Alternative activation of alveolar macrophages is linked to fibrosis following exposure to asbestos. The scavenger receptor, macrophage receptor with collagenous structure (MARCO), provides innate immune defense against inhaled particles and pathogens; however, a receptor for asbestos has not been identified. We hypothesized that MARCO acts as an initial signaling receptor for asbestos, polarizes macrophages to a profibrotic M2 phenotype, and is required for the development of asbestos-induced fibrosis. Compared with normal subjects, alveolar macrophages isolated from patients with asbestosis express higher amounts of MARCO and have greater profibrotic polarization. Arginase 1 (40-fold) and IL-10 (265-fold) were higher in patients. In vivo, the genetic deletion of MARCO attenuated the profibrotic environment and pulmonary fibrosis in mice exposed to chrysotile. Moreover, alveolar macrophages from MARCO(-/-) mice polarize to an M1 phenotype, whereas wild-type mice have higher Ym1 (>3.0-fold) and nearly 7-fold more active TGF-β1 in bronchoalveolar lavage (BAL) fluid (BALF). Arg(432) and Arg(434) in domain V of MARCO are required for the polarization of macrophages to a profibrotic phenotype as mutation of these residues reduced FIZZ1 expression (17-fold) compared with cells expressing MARCO. These observations demonstrate that a macrophage membrane protein regulates the fibrotic response to lung injury and suggest a novel target for therapeutic intervention. © FASEB.

The Effects of the Recombinant CCR5 T4 Lysozyme Fusion Protein on HIV-1 Infection.

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Qingwen Jin

Full Text Available Insertion of T4 lysozyme (T4L into the GPCR successfully enhanced GPCR protein stability and solubilization. However, the biological functions of the recombinant GPCR protein have not been analyzed.We engineered the CCR5-T4L mutant and expressed and purified the soluble recombinant protein using an E.coli expression system. The antiviral effects of this recombinant protein in THP-1 cell lines, primary human macrophages, and PBMCs from different donors were investigated. We also explored the possible mechanisms underlying the observed antiviral effects.Our data showed the biphasic inhibitory and promotion effects of different concentrations of soluble recombinant CCR5-T4L protein on R5 tropic human immunodeficiency virus-1 (HIV-1 infection in THP-1 cell lines, human macrophages, and PBMCs from clinical isolates. We demonstrated that soluble recombinant CCR5-T4L acts as a HIV-1 co-receptor, interacts with wild type CCR5, down-regulates the surface CCR5 expression in human macrophages, and interacts with CCL5 to inhibit macrophage migration. Using binding assays, we further determined that recombinant CCR5-T4L and [125I]-CCL5 compete for the same binding site on wild type CCR5.Our results suggest that recombinant CCR5-T4L protein marginally promotes HIV-1 infection at low concentrations and markedly inhibits infection at higher concentrations. This recombinant protein may be helpful in the future development of anti-HIV-1 therapeutic agents.

Transient degradation of NF-κB proteins in macrophages after interaction with mast cell granules

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Noriko Ito

1998-01-01

Full Text Available The exposure of the macrophage cell line, J774 to mast cell granules (MCG led to the form ation of altered nuclear transcription factor proteins (NFκBx, which had faster electrophoretic mobility than the p50 homodimer of NF-κB, but retained comparable DNA binding capacity. Antibodies to N-terminal peptides of p50, p52, p65 or c-Rel supershifted only a fraction of NF-κBx. Western blot analyses revealed that nuclear p65 and c-Rel were progressively degraded after exposure to MCG, whereas nuclear p50 appeared to be unaffected. In contrast, cytoplasmic p50, p65, c-Rel as well as IkBα remained intact after MCG treatment, although p52 was clearly degraded. In comparison to J774 cells, incubation of m ouse peritoneal macrophages with MCG resulted in more extensive alterations to NF-κB proteins. The alterations in NF-κB proteins did not affect the expression of inducible nitric oxide synthase (iNOS or TNF-α mRNA in J774 cells. These data indicate that exposure of J774 cells to MCG leads to generation of altered nuclear p52, p65 and c-Rel, which retain intact N-terminal peptides, specific oligonucleotide binding and transactivating activity. On the other hand, in peritoneal macrophages, MCG induce more extensive modifications to NF-κB proteins with associated inhibition of iNOS or TNF-α mRNA expression.

Normal autophagic activity in macrophages from mice lacking Gαi3, AGS3, or RGS19.

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Ali Vural

Full Text Available In macrophages autophagy assists antigen presentation, affects cytokine release, and promotes intracellular pathogen elimination. In some cells autophagy is modulated by a signaling pathway that employs Gαi3, Activator of G-protein Signaling-3 (AGS3/GPSM1, and Regulator of G-protein Signaling 19 (RGS19. As macrophages express each of these proteins, we tested their importance in regulating macrophage autophagy. We assessed LC3 processing and the formation of LC3 puncta in bone marrow derived macrophages prepared from wild type, Gnai3(-/-, Gpsm1(-/-, or Rgs19(-/- mice following amino acid starvation or Nigericin treatment. In addition, we evaluated rapamycin-induced autophagic proteolysis rates by long-lived protein degradation assays and anti-autophagic action after rapamycin induction in wild type, Gnai3(-/-, and Gpsm1(-/- macrophages. In similar assays we compared macrophages treated or not with pertussis toxin, an inhibitor of GPCR (G-protein couple receptor triggered Gαi nucleotide exchange. Despite previous findings, the level of basal autophagy, autophagic induction, autophagic flux, autophagic degradation and the anti-autophagic action in macrophages that lacked Gαi3, AGS3, or RGS19; or had been treated with pertussis toxin, were similar to controls. These results indicate that while Gαi signaling may impact autophagy in some cell types it does not in macrophages.

Neopterin negatively regulates expression of ABCA1 and ABCG1 by the LXRα signaling pathway in THP-1 macrophage-derived foam cells.

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Yan, Jin-quan; Tan, Chun-zhi; Wu, Jin-hua; Zhang, Dong-cui; Chen, Ji-ling; Zeng, Bin-yuan; Jiang, Yu-ping; Nie, Jin; Liu, Wei; Liu, Qin; Dai, Hao

2013-07-01

To investigate the effects of neopterin on ABCA1 expression and cholesterol efflux in human THP-1 macrophage-derived foam cells, and to explore the role of the liver X receptor alpha (LXRα) involved. In the present study, THP-1 cells were pre-incubated with ox-LDL to become foam cells. The protein and mRNA expression were examined by Western blot assays and real-time quantitative PCR, respectively. Liquid scintillation counting and high performance liquid chromatography assays were used to test cellular cholesterol efflux and cholesterol content. Neopterin decreased ABCA1 expression and cholesterol efflux in a time- and concentration-dependent manner in THP-1 macrophage-derived foam cells, and the LXRα siRNA can reverse the inhibitory effects induced by neopterin. Neoterin has a negative regulation on ABCA1 expression via the LXRα signaling pathway, which suggests the aggravated effects of neopterin on atherosclerosis.

Modulation of macrophage activation state protects tissue from necrosis during critical limb ischemia in thrombospondin-1-deficient mice.

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Nicolas Bréchot

Full Text Available BACKGROUND: Macrophages, key regulators of healing/regeneration processes, strongly infiltrate ischemic tissues from patients suffering from critical limb ischemia (CLI. However pro-inflammatory markers correlate with disease progression and risk of amputation, suggesting that modulating macrophage activation state might be beneficial. We previously reported that thrombospondin-1 (TSP-1 is highly expressed in ischemic tissues during CLI in humans. TSP-1 is a matricellular protein that displays well-known angiostatic properties in cancer, and regulates inflammation in vivo and macrophages properties in vitro. We therefore sought to investigate its function in a mouse model of CLI. METHODS AND FINDINGS: Using a genetic model of tsp-1(-/- mice subjected to femoral artery excision, we report that tsp-1(-/- mice were clinically and histologically protected from necrosis compared to controls. Tissue protection was associated with increased postischemic angiogenesis and muscle regeneration. We next showed that macrophages present in ischemic tissues exhibited distinct phenotypes in tsp-1(-/- and wt mice. A strong reduction of necrotic myofibers phagocytosis was observed in tsp-1(-/- mice. We next demonstrated that phagocytosis of muscle cell debris is a potent pro-inflammatory signal for macrophages in vitro. Consistently with these findings, macrophages that infiltrated ischemic tissues exhibited a reduced postischemic pro-inflammatory activation state in tsp-1(-/- mice, characterized by a reduced Ly-6C expression and a less pro-inflammatory cytokine expression profile. Finally, we showed that monocyte depletion reversed clinical and histological protection from necrosis observed in tsp-1(-/- mice, thereby demonstrating that macrophages mediated tissue protection in these mice. CONCLUSION: This study defines targeting postischemic macrophage activation state as a new potential therapeutic approach to protect tissues from necrosis and promote tissue

Immune Enhancing Activity of β-(1,3)-Glucan Isolated from Genus Agrobacterium in Bone-Marrow Derived Macrophages and Mice Splenocytes.

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Byun, Eui-Baek; Jang, Beom-Su; Byun, Eui-Hong; Sung, Nak-Yun

2016-01-01

An effective method for activating macrophages and deriving a Th1 immune response could be used to improve the defenses of hosts. In this study, we investigated the immunomodulation effect and the related signaling mechanism of [Formula: see text]-(1,3)-glucan, isolated from the Agrobacterium species. Here, we found that [Formula: see text]-(1,3)-glucan predominantly induced the tumor necrosis factor (TNF)-[Formula: see text], interleukin (IL)-1[Formula: see text], IL-6, IL-12p70, and nitric oxide, which was dependent on mitogen-activated protein kinases (MAPK) and nuclear factor (NF)-[Formula: see text]B signaling. Additionally, [Formula: see text]-(1,3)-glucan treatment significantly up-regulated the expression of the co-stimulatory molecules CD80 and CD86, and also significantly increased the expression of iNOS and Dectin-1, which is a transmembrane protein that binds [Formula: see text]-glucan and associates with macrophage activation. Importantly, the splenic T cells co-cultured with [Formula: see text]-(1,3)-glucan-treated macrophages produced the a Th1 cytokine profile that includes high levels of IFN-[Formula: see text], but not IL-4 (Th2 cytokine), indicating that [Formula: see text]-(1,3)-glucan contributes to Th1 polarization of the immune response. Taken together, our results suggest that [Formula: see text]-(1,3)-glucan isolated from Agrobacterium species can induce macrophage activation through the MAPK and NF-[Formula: see text]B signaling pathway, as well as Th1 polarization.

Anti-Inflammatory Effect of Myristicin on RAW 264.7 Macrophages Stimulated with Polyinosinic-Polycytidylic Acid

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Wansu Park

2011-08-01

Full Text Available Myristicin (1-allyl-5-methoxy-3,4-methylenedioxybenzene is an active aromatic compound found in nutmeg (the seed of Myristica fragrans, carrot, basil, cinnamon, and parsley. Myristicin has been known to have anti-cholinergic, antibacterial, and hepatoprotective effects, however, the effects of myristicin on virus-stimulated macrophages are not fully reported. In this study, the anti-inflammatory effect of myristicin on double-stranded RNA (dsRNA-stimulated macrophages was examined. Myristicin did not reduce the cell viability of RAW 264.7 mouse macrophages at concentrations of up to 50 µM. Myristicin significantly inhibited the production of calcium, nitric oxide (NO, interleukin (IL-6, IL-10, interferon inducible protein-10, monocyte chemotactic protein (MCP-1, MCP-3, granulocyte-macrophage colony-stimulating factor, macrophage inflammatory protein (MIP-1α, MIP-1β, and leukemia inhibitory factor in dsRNA [polyinosinic-polycytidylic acid]-induced RAW 264.7 cells (P < 0.05. In conclusion, myristicin has anti-inflammatory properties related with its inhibition of NO, cytokines, chemokines, and growth factors in dsRNA-stimulated macrophages via the calcium pathway.

Drosophila divalent metal ion transporter Malvolio is required in dopaminergic neurons for feeding decisions.

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Søvik, E; LaMora, A; Seehra, G; Barron, A B; Duncan, J G; Ben-Shahar, Y

2017-06-01

Members of the natural resistance-associated macrophage protein (NRAMP) family are evolutionarily conserved metal ion transporters that play an essential role in regulating intracellular divalent cation homeostasis in both prokaryotes and eukaryotes. Malvolio (Mvl), the sole NRAMP family member in insects, plays a role in food choice behaviors in Drosophila and other species. However, the specific physiological and cellular processes that require the action of Mvl for appropriate feeding decisions remain elusive. Here, we show that normal food choice requires Mvl function specifically in the dopaminergic system, and can be rescued by supplementing food with manganese. Collectively, our data indicate that the action of the Mvl transporter affects food choice behavior via the regulation of dopaminergic innervation of the mushroom bodies, a principle brain region associated with decision-making in insects. Our studies suggest that the homeostatic regulation of the intraneuronal levels of divalent cations plays an important role in the development and function of the dopaminergic system and associated behaviors. © 2017 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.

Controlled release of cytokines using silk-biomaterials for macrophage polarization.

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Reeves, Andrew R D; Spiller, Kara L; Freytes, Donald O; Vunjak-Novakovic, Gordana; Kaplan, David L

2015-12-01

Polarization of macrophages into an inflammatory (M1) or anti-inflammatory (M2) phenotype is important for clearing pathogens and wound repair, however chronic activation of either type of macrophage has been implicated in several diseases. Methods to locally control the polarization of macrophages is of great interest for biomedical implants and tissue engineering. To that end, silk protein was used to form biopolymer films that release either IFN-γ or IL-4 to control the polarization of macrophages. Modulation of the solubility of the silk films through regulation of β-sheet (crystalline) content enabled a short-term release (4-8 h) of either cytokine, with smaller amounts released out to 24 h. Altering the solubility of the films was accomplished by varying the time that the films were exposed to water vapor. The released IFN-γ or IL-4 induced polarization of THP-1 derived macrophages into the M1 or M2 phenotypes, respectively. The silk biomaterials were able to release enough IFN-γ or IL-4 to repolarize the macrophage from M1 to M2 and vice versa, demonstrating the well-established plasticity of macrophages. High β-sheet content films that are not soluble and do not release the trapped cytokines were also able to polarize macrophages that adhered to the surface through degradation of the silk protein. Chemically conjugating IFN-γ to silk films through disulfide bonds allowed for longer-term release to 10 days. The release of covalently attached IFN-γ from the films was also able to polarize M1 macrophages in vitro. Thus, the strategy described here offers new approaches to utilizing biomaterials for directing the polarization of macrophages. Copyright © 2015 Elsevier Ltd. All rights reserved.

Inactivation of p27kip1 Promoted Nonspecific Inflammation by Enhancing Macrophage Proliferation in Islet Transplantation.

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Li, Yang; Ding, Xiaoming; Fan, Ping; Guo, Jian; Tian, Xiaohui; Feng, Xinshun; Zheng, Jin; Tian, Puxun; Ding, Chenguang; Xue, Wujun

2016-11-01

Islet transplantation suffers from low efficiency caused by nonspecific inflammation-induced graft loss after transplantation. This study reports increased islet loss and enhanced inflammatory response in p27-deficient mice (p27-/-) and proposes a possible mechanism. Compared with wild type, p27-/- mice showed more severe functional injury of islet, with increased serum levels of inflammatory cytokines IL-1 and TNF-α, inducing macrophage proliferation. Furthermore, the increased number, proapoptotic proteins, and nuclear factor-kappa b (NF-κB) phosphorylation status of the infiltrating macrophages were accompanied by increased TNF-α mRNA level of islet graft site in p27-/- mice. Moreover, in vitro, we found that macrophages were still activated and cocultured with islet and promoted islet loss even blocking the direct effect of TNF-α on islets. Malondialdehyde (MDA, an end product of lipid peroxidation) in islet and media were increased after cocultured with macrophages. p27 deficiency also increased macrophage proliferation and islet injury. Therefore, p27 inactivation promotes injury islet graft loss via the elevation of proliferation and inflammatory cytokines secretion in infiltrating macrophages which induced nonspecific inflammation independent of TNF-α/nuclear factor-kappa b pathway. This potentially represents a promising therapeutic target in improving islet graft survival.

The pro-atherogenic effects of macrophages are reduced upon formation of a complex between C-reactive protein and lysophosphatidylcholine

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Chang Mi-Kyung

2012-10-01

Full Text Available Abstract Rationale C-reactive protein (CRP and lysophosphatidylcholine (LPC are phosphorylcholine-(PC-containing oxidized phospholipids (oxPLs found in oxidized LDL (oxLDL, which trigger pro-atherogenic activities of macrophages during the process of atherosclerosis. It has been previously reported that CRP binds to the PC head group of oxLDL in a calcium-dependent manner. The aim of this study was to investigate the importance of binding between CRP and LPC to the pro-atherogenic activities of macrophages. Objectives and findings A chemiluminescent immunoassay and HPLC showed that human recombinant CRP formed a stable complex with LPC in the presence of calcium. The Kd value of the binding of the CRP-LPC complex to the receptors FcγRIA or FcγRIIA was 3–5 fold lower than that of CRP alone. The CRP-LPC complex triggered less potent generation of reactive oxygen species and less activation of the transcription factors AP-1 and NF-kB by human monocyte-derived macrophages in comparison to CRP or LPC alone. However, CRP did not affect activities driven by components of oxLDL lacking PC, such as upregulation of PPRE, ABCA1, CD36 and PPARγ and the enhancement of cholesterol efflux by human macrophages. The presence of CRP inhibited the association of Dil-labelled oxLDL to human macrophages. Conclusions The formation of complexes between CRP and PC-containing oxPLs, such as LPC, suppresses the pro-atherogenic effects of CRP and LPC on macrophages. This effect may in part retard the progression of atherosclerosis.

CRISPR/Cas9-Mediated Gene Editing in Human iPSC-Derived Macrophage Reveals Lysosomal Acid Lipase Function in Human Macrophages-Brief Report.

Science.gov (United States)

Zhang, Hanrui; Shi, Jianting; Hachet, Melanie A; Xue, Chenyi; Bauer, Robert C; Jiang, Hongfeng; Li, Wenjun; Tohyama, Junichiro; Millar, John; Billheimer, Jeffrey; Phillips, Michael C; Razani, Babak; Rader, Daniel J; Reilly, Muredach P

2017-11-01

To gain mechanistic insights into the role of LIPA (lipase A), the gene encoding LAL (lysosomal acid lipase) protein, in human macrophages. We used CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) technology to knock out LIPA in human induced pluripotent stem cells and then differentiate to macrophage (human-induced pluripotent stem cells-derived macrophage [IPSDM]) to explore the human macrophage LIPA loss-of-function phenotypes. LIPA was abundantly expressed in monocyte-derived macrophages and was markedly induced on IPSDM differentiation to comparable levels as in human monocyte-derived macrophage. IPSDM with knockout of LIPA ( LIPA -/- ) had barely detectable LAL enzymatic activity. Control and LIPA -/- IPSDM were loaded with [ 3 H]-cholesteryl oleate-labeled AcLDL (acetylated low-density lipoprotein) followed by efflux to apolipoprotein A-I. Efflux of liberated [ 3 H]-cholesterol to apolipoprotein A-I was abolished in LIPA -/- IPSDM, indicating deficiency in LAL-mediated lysosomal cholesteryl ester hydrolysis. In cells loaded with [ 3 H]-cholesterol-labeled AcLDL, [ 3 H]-cholesterol efflux was, however, not different between control and LIPA -/- IPSDM. ABCA1 (ATP-binding cassette, subfamily A, member 1) expression was upregulated by AcLDL loading but to a similar extent between control and LIPA -/- IPSDM. In nonlipid loaded state, LIPA -/- IPSDM had high levels of cholesteryl ester mass compared with minute amounts in control IPSDM. Yet, with AcLDL loading, overall cholesteryl ester mass was increased to similar levels in both control and LIPA -/- IPSDM. LIPA -/- did not impact lysosomal apolipoprotein-B degradation or expression of IL1B , IL6 , and CCL5. CONCLUSIONS: LIPA -/- IPSDM reveals macrophage-specific hallmarks of LIPA deficiency. CRISPR/Cas9 and IPSDM provide important tools to study human macrophage biology and more broadly for future studies of disease-associated LIPA genetic variation in human

Recombinant Protein Truncation Strategy for Inducing Bactericidal Antibodies to the Macrophage Infectivity Potentiator Protein of Neisseria meningitidis and Circumventing Potential Cross-Reactivity with Human FK506-Binding Proteins

OpenAIRE

Bielecka, Magdalena K.; Devos, Nathalie; Gilbert, Mélanie; Hung, Miao-Chiu; Weynants, Vincent; Heckels, John E.; Christodoulides, Myron

2014-01-01

A recombinant macrophage infectivity potentiator (rMIP) protein of Neisseria meningitidis induces significant serum bactericidal antibody production in mice and is a candidate meningococcal vaccine antigen. However, bioinformatics analysis of MIP showed some amino acid sequence similarity to human FK506-binding proteins (FKBPs) in residues 166 to 252 located in the globular domain of the protein. To circumvent the potential concern over generating antibodies that could recognize human protein...

Arctigenin promotes cholesterol efflux from THP-1 macrophages through PPAR-γ/LXR-α signaling pathway.

Science.gov (United States)

Xu, Xiaolin; Li, Qian; Pang, Liewen; Huang, Guoqian; Huang, Jiechun; Shi, Meng; Sun, Xiaotian; Wang, Yiqing

2013-11-15

Cholesterol efflux from macrophages is a critical mechanism to prevent the development of atherosclerosis. Here, we sought to investigate the effects of arctigenin, a bioactive component of Arctium lappa, on the cholesterol efflux in oxidized low-density lipoprotein (oxLDL)-loaded THP-1 macrophages. Our data showed that arctigenin significantly accelerated apolipoprotein A-I- and high-density lipoprotein-induced cholesterol efflux in both dose- and time-dependent manners. Moreover, arctigenin treatment enhanced the expression of ATP binding cassette transporter A1 (ABCA1), ABCG1, and apoE, all of which are key molecules in the initial step of cholesterol efflux, at both mRNA and protein levels. Arctigenin also caused a concentration-dependent elevation in the expression of peroxisome proliferator-activated receptor-gamma (PPAR-γ) and liver X receptor-alpha (LXR-α). The arctigenin-mediated induction of ABCA1, ABCG1, and apoE was abolished by specific inhibition of PPAR-γ or LXR-α using small interfering RNA technology. Our results collectively indicate that arctigenin promotes cholesterol efflux in oxLDL-loaded THP-1 macrophages through upregulation of ABCA1, ABCG1 and apoE, which is dependent on the enhanced expression of PPAR-γ and LXR-α. Copyright © 2013 Elsevier Inc. All rights reserved.

HIV-1 and the macrophage

NARCIS (Netherlands)

Bol, Sebastiaan M.; Cobos-Jimenez, Viviana; Kootstra, Neeltje A.; van 't Wout, Angelique B.

2011-01-01

Macrophages and CD4(+) T cells are natural target cells for HIV-1, and both cell types contribute to the establishment of the viral reservoir that is responsible for continuous residual virus replication during antiretroviral therapy and viral load rebound upon treatment interruption. Scientific

Nuclear proteins interacting with the promoter region of the human granulocyte/macrophage colony-stimulating factor gene

International Nuclear Information System (INIS)

Shannon, M.F.; Gamble, J.R.; Vadas, M.A.

1988-01-01

The gene for human granulocyte/macrophage colony-stimulating factor (GM-CSF) is expressed in a tissue-specific as well as an activation-dependent manner. The interaction of nuclear proteins with the promoter region of the GM-CSF gene that is likely to be responsible for this pattern of GM-CSF expression was investigated. The authors show that nuclear proteins interact with DNA fragments from the GM-CSF promoter in a cell-specific manner. A region spanning two cytokine-specific sequences, cytokine 1 (CK-1, 5', GAGATTCCAC 3') and cytokine 2 (CK-2, 5' TCAGGTA 3') bound two nuclear proteins from GM-CSF-expressing cells in gel retardation assays. NF-GMb was inducible with phorbol 12-myristate 13-acetate and accompanied induction of GM-CSF message. NF-GMb was absent in cell lines not producing GM-CSF, some of which had other distinct binding proteins. NF-GMa and NF-GMb eluted from a heparin-Sepharose column at 0.3 and 0.6 M KCl, respectively. They hypothesize that the sequences CK-1 and CK-2 bind specific proteins and regulate GM-CSF transcription

Urotensin II increases foam cell formation by repressing ABCA1 expression through the ERK/NF-κB pathway in THP-1 macrophages

Energy Technology Data Exchange (ETDEWEB)

Wang, Yan [Department of Anesthesiology, The Second Affiliated Hospital of University of South China, Hengyang 421001, Hunan (China); Wu, Jian-Feng [Department of Cardiovascular Medicine, The Second Affiliated Hospital of University of South China, Hengyang 421001, Hunan (China); Tang, Yan-Yan; Zhang, Min; Li, Yuan [Institute of Cardiovascular Research, Key Laboratory for Atherosclerology of Hunan Province, University of South China, Hengyang 421001, Hunan (China); Chen, Kong; Zeng, Meng-Ya [Department of Cardiovascular Medicine, The Second Affiliated Hospital of University of South China, Hengyang 421001, Hunan (China); Yao, Feng; Xie, Wei [Institute of Cardiovascular Research, Key Laboratory for Atherosclerology of Hunan Province, University of South China, Hengyang 421001, Hunan (China); Zheng, Xi-Long [Department of Biochemistry and Molecular Biology, Libin Cardiovascular Institute of Alberta, University of Calgary, Health Sciences Center, 3330 Hospital Dr NW, Calgary, Alberta T2N 4N1 (Canada); Zeng, Gao-Feng, E-mail: qichingnudou@tom.com [Department of Cardiovascular Medicine, The Second Affiliated Hospital of University of South China, Hengyang 421001, Hunan (China); Tang, Chao-Ke, E-mail: tangchaoke@qq.com [Institute of Cardiovascular Research, Key Laboratory for Atherosclerology of Hunan Province, University of South China, Hengyang 421001, Hunan (China)

2014-10-03

Highlights: • U II reduces cholesterol efflux in THP-1 macrophages. • U II decreases the expression of ABCA1. • Inhibition of the ERK/NF-κB pathway reduces U II effects on ABCA1 expression and cholesterol efflux. - Abstract: Objective: Foam cell formation in the arterial wall plays a key role in the development of atherosclerosis. Recent studies showed that Urotensin II (U II) is involved in the pathogenesis of atherosclerosis. Here we examined the effects of human U II on ATP-binding cassette transporter A1 (ABCA1) expression and the underlying mechanism in THP-1 macrophages. Methods and results: Cultured THP-1 macrophages were treated with U II, followed by measuring the intracellular lipid contents, cholesterol efflux and ABCA1 levels. The results showed that U II dramatically decreased ABCA1 levels and impaired cholesterol efflux. However, the effects of U II on ABCA1 protein expression and cellular cholesterol efflux were partially reversed by inhibition of extracellular signal regulated kinase 1/2 (ERK1/2) and nuclear factor kappa B (NF-κB) activity, suggesting the potential roles of ERK1/2 and NF-κB in ABCA1 expression, respectively. Conclusion: Our current data indicate that U II may have promoting effects on the progression of atherosclerosis, likely through suppressing ABCA1 expression via activation of the ERK/NF-κB pathway and reducing cholesterol efflux to promote macrophage foam cell formation.

Multiple Pseudomonas species secrete exolysin-like toxins and provoke Caspase-1-dependent macrophage death.

Science.gov (United States)

Basso, Pauline; Wallet, Pierre; Elsen, Sylvie; Soleilhac, Emmanuelle; Henry, Thomas; Faudry, Eric; Attrée, Ina

2017-10-01

Pathogenic bacteria secrete protein toxins that provoke apoptosis or necrosis of eukaryotic cells. Here, we developed a live-imaging method, based on incorporation of a DNA-intercalating dye into membrane-damaged host cells, to study the kinetics of primary bone marrow-derived macrophages (BMDMs) mortality induced by opportunistic pathogen Pseudomonas aeruginosa expressing either Type III Secretion System (T3SS) toxins or the pore-forming toxin, Exolysin (ExlA). We found that ExlA promotes the activation of Caspase-1 and maturation of interleukin-1β. BMDMs deficient for Caspase-1 and Caspase-11 were resistant to ExlA-induced death. Furthermore, by using KO BMDMs, we determined that the upstream NLRP3/ASC complex leads to the Caspase-1 activation. We also demonstrated that Pseudomonas putida and Pseudomonas protegens and the Drosophila pathogen Pseudomonas entomophila, which naturally express ExlA-like toxins, are cytotoxic toward macrophages and provoke the same type of pro-inflammatory death as does ExlA + P. aeruginosa. These results demonstrate that ExlA-like toxins of two-partner secretion systems from diverse Pseudomonas species activate the NLRP3 inflammasome and provoke inflammatory pyroptotic death of macrophages. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

Caprine arthritis encephalitis virus dysregulates the expression of cytokines in macrophages.

Science.gov (United States)

Lechner, F; Machado, J; Bertoni, G; Seow, H F; Dobbelaere, D A; Peterhans, E

1997-01-01

Caprine arthritis encephalitis virus (CAEV) is a lentivirus of goats that leads to chronic mononuclear infiltration of various tissues, in particular, the radiocarpal joints. Cells of the monocyte/macrophage lineage are the major host cells of CAEV in vivo. We have shown that infection of cultured goat macrophages with CAEV results in an alteration of cytokine expression in vitro. Constitutive expression of interleukin 8 (IL-8) and monocyte chemoattractant protein 1 (MCP-1) was increased in infected macrophages, whereas transforming growth factor beta1 (TGF-beta1) mRNA was down-regulated. When macrophages were infected with a CAEV clone lacking the trans-acting nuclear regulatory gene tat, IL-8 and MCP-1 were also increased. No significant differences from cells infected with the wild-type clone were observed, suggesting that Tat is not required for the increased expression of IL-8 and MCP-1 in infected macrophages. Furthermore, infection with CAEV led to an altered pattern of cytokine expression in response to lipopolysaccharide (LPS), heat-killed Listeria monocytogenes plus gamma interferon, or fixed cells of Staphylococcus aureus Cowan I. In infected macrophages, tumor necrosis factor alpha, IL-1beta, IL-6, and IL-12 p40 mRNA expression was reduced in response to all stimuli tested whereas changes in expression of granulocyte-macrophage colony-stimulating factor depended on the stimulating agent. Electrophoretic mobility shift assays demonstrated that, in contrast to effects of human immunodeficiency virus infection of macrophages, CAEV infection had no effect on the level of constitutive nuclear factor-kappaB (NF-kappaB) activity or on the level of LPS-stimulated NF-kappaB activity, suggesting that NF-kappaB is not involved in altered regulation of cytokine expression in CAEV-infected cells. In contrast, activator protein 1 (AP-1) binding activity was decreased in infected macrophages. These data show that CAEV infection may result in a dysregulation of

Hypoxia-Inducible Factor-1α Expression in Macrophages Promotes Development of Atherosclerosis

DEFF Research Database (Denmark)

Pedersen, Annemarie Aarup; Pedersen, Tanja X; Junker, Nanna

2016-01-01

transplanted with bone marrow from mice with HIF-1α deficiency in the myeloid cells or control bone marrow. The HIF-1α deficiency in myeloid cells reduced atherosclerosis in aorta of the Ldlr(-/-) recipient mice by ≈72% (P=0.006).In vitro, HIF-1α-deficient macrophages displayed decreased differentiation...... to proinflammatory M1 macrophages and reduced expression of inflammatory genes. HIF-1α deficiency also affected glucose uptake, apoptosis, and migratory abilities of the macrophages. CONCLUSIONS: HIF-1α expression in macrophages affects their intrinsic inflammatory profile and promotes development of atherosclerosis....

Keap1 silencing boosts lipopolysaccharide-induced transcription of interleukin 6 via activation of nuclear factor κB in macrophages

International Nuclear Information System (INIS)

Lv, Peng; Xue, Peng; Dong, Jian; Peng, Hui; Clewell, Rebecca; Wang, Aiping; Wang, Yue; Peng, Shuangqing; Qu, Weidong; Zhang, Qiang; Andersen, Melvin E.; Pi, Jingbo

2013-01-01

Interleukin-6 (IL6) is a multifunctional cytokine that regulates immune and inflammatory responses. Multiple transcription factors, including nuclear factor κB (NF-κB) and nuclear factor E2-related factor 2 (Nrf2), regulate IL6 transcription. Kelch-like ECH-associated protein 1 (Keap1) is a substrate adaptor protein for the Cullin 3-dependent E3 ubiquitin ligase complex, which regulates the degradation of many proteins, including Nrf2 and IκB kinase β (IKKβ). Here, we found that stable knockdown of Keap1 (Keap1-KD) in RAW 264.7 (RAW) mouse macrophages and human monocyte THP-1 cells significantly increased expression of Il6, and Nrf2-target genes, under basal and lipopolysaccharide (LPS, 0.001–0.1 μg/ml)-challenged conditions. However, Nrf2 activation alone, by tert-butylhydroquinone treatment of RAW cells, did not increase expression of Il6. Compared to cells transduced with scrambled non-target negative control shRNA, Keap1-KD RAW cells showed enhanced protein levels of IKKβ and increased expression and phosphorylation of NF-κB p65 under non-stressed and LPS-treated conditions. Because the expression of Il6 in Keap1-KD RAW cells was significantly attenuated by silencing of Ikkβ, but not Nrf2, it appears that stabilized IKKβ is responsible for the enhanced transactivation of Il6 in Keap1-KD cells. This study demonstrated that silencing of Keap1 in macrophages boosts LPS-induced transcription of Il6 via NF-κB activation. Given the importance of IL6 in the inflammatory response, the Keap1–IKKβ–NF-κB pathway may be a novel target for treatment and prevention of inflammation and associated disorders. - Highlights: • Knockdown of Keap1 increases expression of Il6 in macrophages. • Silencing of Keap1 results in protein accumulation of IKKβ and NF-κB p65. • Induction of Il6 resulting from Keap1 silencing is attributed to NF-κB activation

Keap1 silencing boosts lipopolysaccharide-induced transcription of interleukin 6 via activation of nuclear factor κB in macrophages

Energy Technology Data Exchange (ETDEWEB)

Lv, Peng [Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing (China); Institute for Chemical Safety Sciences, The Hamner Institutes for Health Sciences, 6 Davis Drive, Research Triangle Park, NC 27709 (United States); Xue, Peng; Dong, Jian [Institute for Chemical Safety Sciences, The Hamner Institutes for Health Sciences, 6 Davis Drive, Research Triangle Park, NC 27709 (United States); Peng, Hui [Institute for Chemical Safety Sciences, The Hamner Institutes for Health Sciences, 6 Davis Drive, Research Triangle Park, NC 27709 (United States); Evaluation and Research Center for Toxicology, Institute of Disease Control and Prevention, Academy of Military Medical Sciences (China); Clewell, Rebecca [Institute for Chemical Safety Sciences, The Hamner Institutes for Health Sciences, 6 Davis Drive, Research Triangle Park, NC 27709 (United States); Wang, Aiping [Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing (China); Wang, Yue [Institute for Medical Device Standardization Administration, National Institutes for Food and Drug Control, Beijing (China); Peng, Shuangqing [Evaluation and Research Center for Toxicology, Institute of Disease Control and Prevention, Academy of Military Medical Sciences (China); Qu, Weidong [Key Laboratory of the Public Health Safety, Ministry of Education, School of Public Health, Fudan University, Shanghai (China); Zhang, Qiang; Andersen, Melvin E. [Institute for Chemical Safety Sciences, The Hamner Institutes for Health Sciences, 6 Davis Drive, Research Triangle Park, NC 27709 (United States); Pi, Jingbo, E-mail: jpi@thehamner.org [Institute for Chemical Safety Sciences, The Hamner Institutes for Health Sciences, 6 Davis Drive, Research Triangle Park, NC 27709 (United States)

2013-11-01

Interleukin-6 (IL6) is a multifunctional cytokine that regulates immune and inflammatory responses. Multiple transcription factors, including nuclear factor κB (NF-κB) and nuclear factor E2-related factor 2 (Nrf2), regulate IL6 transcription. Kelch-like ECH-associated protein 1 (Keap1) is a substrate adaptor protein for the Cullin 3-dependent E3 ubiquitin ligase complex, which regulates the degradation of many proteins, including Nrf2 and IκB kinase β (IKKβ). Here, we found that stable knockdown of Keap1 (Keap1-KD) in RAW 264.7 (RAW) mouse macrophages and human monocyte THP-1 cells significantly increased expression of Il6, and Nrf2-target genes, under basal and lipopolysaccharide (LPS, 0.001–0.1 μg/ml)-challenged conditions. However, Nrf2 activation alone, by tert-butylhydroquinone treatment of RAW cells, did not increase expression of Il6. Compared to cells transduced with scrambled non-target negative control shRNA, Keap1-KD RAW cells showed enhanced protein levels of IKKβ and increased expression and phosphorylation of NF-κB p65 under non-stressed and LPS-treated conditions. Because the expression of Il6 in Keap1-KD RAW cells was significantly attenuated by silencing of Ikkβ, but not Nrf2, it appears that stabilized IKKβ is responsible for the enhanced transactivation of Il6 in Keap1-KD cells. This study demonstrated that silencing of Keap1 in macrophages boosts LPS-induced transcription of Il6 via NF-κB activation. Given the importance of IL6 in the inflammatory response, the Keap1–IKKβ–NF-κB pathway may be a novel target for treatment and prevention of inflammation and associated disorders. - Highlights: • Knockdown of Keap1 increases expression of Il6 in macrophages. • Silencing of Keap1 results in protein accumulation of IKKβ and NF-κB p65. • Induction of Il6 resulting from Keap1 silencing is attributed to NF-κB activation.

Azurophil granule proteins constitute the major mycobactericidal proteins in human neutrophils and enhance the killing of mycobacteria in macrophages

DEFF Research Database (Denmark)

Jena, Prajna; Mohanty, Soumitra; Mohanty, Tirthankar

2012-01-01

Pathogenic mycobacteria reside in, and are in turn controlled by, macrophages. However, emerging data suggest that neutrophils also play a critical role in innate immunity to tuberculosis, presumably by their different antibacterial granule proteins. In this study, we purified neutrophil azurophil...... and specific granules and systematically analyzed the antimycobacterial activity of some purified azurophil and specific granule proteins against M. smegmatis, M. bovis-BCG and M. tuberculosis H37Rv. Using gel overlay and colony forming unit assays we showed that the defensin-depleted azurophil granule...... proteins (AZP) were more active against mycobacteria compared to other granule proteins and cytosolic proteins. The proteins showing antimycobacterial activity were identified by MALDI-TOF mass spectrometry. Electron microscopic studies demonstrate that the AZP disintegrate bacterial cell membrane...

Effect of Bothrops alternatus snake venom on macrophage phagocytosis and superoxide production: participation of protein kinase C

Directory of Open Access Journals (Sweden)

SS Setubal

2011-01-01

Full Text Available Envenomations caused by different species of Bothrops snakes result in severe local tissue damage, hemorrhage, pain, myonecrosis, and inflammation with a significant leukocyte accumulation at the bite site. However, the activation state of leukocytes is still unclear. According to clinical cases and experimental work, the local effects observed in envenenomation by Bothrops alternatus are mainly the appearance of edema, hemorrhage, and necrosis. In this study we investigated the ability of Bothrops alternatus crude venom to induce macrophage activation. At 6 to 100 ¼g/mL, BaV is not toxic to thioglycollate-elicited macrophages; at 3 and 6 ¼g/mL, it did not interfere in macrophage adhesion or detachment. Moreover, at concentrations of 1.5, 3, and 6 ¼g/mL the venom induced an increase in phagocytosis via complement receptor one hour after incubation. Pharmacological treatment of thioglycollate-elicited macrophages with staurosporine, a protein kinase (PKC inhibitor, abolished phagocytosis, suggesting that PKC may be involved in the increase of serum-opsonized zymosan phagocytosis induced by BaV. Moreover, BaV also induced the production of anion superoxide (O2_ by thioglycollate-elicited macrophages. This BaV stimulated superoxide production was abolished after treating the cells with staurosporine, indicating that PKC is an important signaling pathway for the production of this radical. Based on these results, we suggest that phagocytosis and reactive oxygen species are involved in the pathogenesis of local tissue damage characteristic of Bothrops spp. envenomations.

Blood-flow restricted training leads to myocelullar macrophage infiltration and upregulation of heat-shock proteins, but no apparent muscle damage

DEFF Research Database (Denmark)

Nielsen, Jakob L; Aagaard, Per; Prokhorova, Tatyana A

2017-01-01

Previous studies indicate that low-load muscle contractions performed under local blood-flow restriction (BFR) may initially induce muscle damage and stress. However, whether these factors are evoked with longitudinal BFR training remains unexplored at the myocellular level. Two distinct study...... into the intervention (Mid8) and 3 and 10 days after training cessation (Post3,Post10) to examine macrophage (M1/M2) content as well as heat-shock protein (HSP27/70) and tenascin-C expression. Blood samples (1 wk) were collected before and after (0.1-24 h) the first and last training session to examine markers...... of muscle damage (CK), oxidative stress (TAC,GSH) and inflammation (MCP1,IL-6,TNFa). M1-macrophage content increased 108-165% with BFRE and LLE at Post3 (P

Up-regulation of TLR2 and TLR4 in high mobility group Box1-stimulated macrophages in pulpitis patients

Science.gov (United States)

Mahmoudi, Javad; Sabermarouf, Babak; Baradaran, Behzad; Sadat-Hatamnezhad, Leila; Shotorbani, Siamak Sandoghchian

2017-01-01

Objective(s): High Mobility Group Box1 (HMGB1) is a nonhistone, DNA-binding protein that serves a crucial role in regulating gene transcription and is involved in a variety of proinflammatory, extracellular activities. The aim of this study was to explore whether HMGB1 stimulation can up-regulate the expression of Toll-like Receptor 2 (TLR2) and Toll-like Receptor 4 (TLR4) on macrophages from pulpitis and to clarify the subsequent events involving Th17 cells and Th17 cell-associated cytokine changes. Materials and Methods: Having prepared dental pulp tissues of pulpitis and healthy controls, macrophage were isolated and cultured. Macrophages were thereafter stimulated by HMGB1 time course. RT-QPCR, flowcytometer, immunofluorescence, Western blotting, and ELISA techniques were used in the present research. Results: Our results showed that the expression of TLR2 and TLR4 on macrophages stimulated with HMGB1 increased in pulpitis compared with controls (macrophages without HMGB1 stimulation) with a statistical significance (Ppulpitis increased, and NF-kB, the downstream target of TLR2 and TLR4, also showed a marked elevation after macrophages’ stimulation by HMGB1. Conclusion: The evidence from the present study suggests that the enhanced TLR2 and TLR4 pathways and Th17 cell polarization may be due to HMGB1 stimulation in pulpitis. PMID:28293399

Macrophage proinflammatory response to the titanium alloy equipment in dental implantation.

Science.gov (United States)

Chen, X; Li, H S; Yin, Y; Feng, Y; Tan, X W

2015-08-07

Titanium alloy and stainless steel (SS) had been widely used as dental implant materials because of their affinity with epithelial tissue and connective tissue, and good physical, chemical, biological, mechanical properties and processability. We compared the effects of titanium alloy and SS on macrophage cytokine expression as well as their biocompatibility. Mouse macrophage RAW264.7 cells were cultured on titanium alloy and SS surfaces. Cells were counted by scanning electron microscopy. A nitride oxide kit was used to detect released nitric oxide by macrophages on the different materials. An enzyme linked immunosorbent assay was used to detect monocyte chemoattractant protein-1 levels. Scanning electron microscopy revealed fewer macrophages on the surface of titanium alloy (48.2 ± 6.4 x 10(3) cells/cm(2)) than on SS (135 ± 7.3 x 10(3) cells/cm(2)). The nitric oxide content stimulated by titanium alloy was 22.5 mM, which was lower than that stimulated by SS (26.8 mM), but the difference was not statistically significant (P = 0.07). The level of monocyte chemoattractant protein-1 released was significantly higher in the SS group (OD value = 0.128) than in the titanium alloy group (OD value = 0.081) (P = 0.024). The transforming growth factor-b1 mRNA expression levels in macrophages after stimulation by titanium alloy for 12 and 36 h were significantly higher than that after stimulation by SS (P = 0.31 and 0.25, respectively). Macrophages participate in the inflammatory response by regulating cytokines such as nitric oxide, monocyte chemoattractant protein-1, and transforming growth factor-b1. There were fewer macrophages and lower inflammation on the titanium alloy surface than on the SS surface. Titanium alloy materials exhibited better biological compatibility than did SS.

Fcγ receptor I alpha chain (CD64) expression in macrophages is critical for the onset of meningitis by Escherichia coli K1.

Science.gov (United States)

Mittal, Rahul; Sukumaran, Sunil K; Selvaraj, Suresh K; Wooster, David G; Babu, M Madan; Schreiber, Alan D; Verbeek, J Sjef; Prasadarao, Nemani V

2010-11-18

Neonatal meningitis due to Escherichia coli K1 is a serious illness with unchanged morbidity and mortality rates for the last few decades. The lack of a comprehensive understanding of the mechanisms involved in the development of meningitis contributes to this poor outcome. Here, we demonstrate that depletion of macrophages in newborn mice renders the animals resistant to E. coli K1 induced meningitis. The entry of E. coli K1 into macrophages requires the interaction of outer membrane protein A (OmpA) of E. coli K1 with the alpha chain of Fcγ receptor I (FcγRIa, CD64) for which IgG opsonization is not necessary. Overexpression of full-length but not C-terminal truncated FcγRIa in COS-1 cells permits E. coli K1 to enter the cells. Moreover, OmpA binding to FcγRIa prevents the recruitment of the γ-chain and induces a different pattern of tyrosine phosphorylation of macrophage proteins compared to IgG2a induced phosphorylation. Of note, FcγRIa(-/-) mice are resistant to E. coli infection due to accelerated clearance of bacteria from circulation, which in turn was the result of increased expression of CR3 on macrophages. Reintroduction of human FcγRIa in mouse FcγRIa(-/-) macrophages in vitro increased bacterial survival by suppressing the expression of CR3. Adoptive transfer of wild type macrophages into FcγRIa(-/-) mice restored susceptibility to E. coli infection. Together, these results show that the interaction of FcγRI alpha chain with OmpA plays a key role in the development of neonatal meningitis by E. coli K1.

Regulation and control of nitric oxide (NO) in macrophages

DEFF Research Database (Denmark)

Kovacevic, Zaklina; Sahni, Sumit; Lok, K.H.

2017-01-01

We recently demonstrated that a novel storage and transport mechanism for nitric oxide (NO) mediated by glutathione-S-transferase P1 (GSTP1) and multidrug resistance protein 1 (MRP1/ABCC1), protects M1-macrophage (M1-MØ) models from large quantities of endogenous NO. This system stores and transp......We recently demonstrated that a novel storage and transport mechanism for nitric oxide (NO) mediated by glutathione-S-transferase P1 (GSTP1) and multidrug resistance protein 1 (MRP1/ABCC1), protects M1-macrophage (M1-MØ) models from large quantities of endogenous NO. This system stores...... be responsible for delivering cytotoxic NO as DNICs via MRP1 from M1-MØs, to tumor cell targets....

Arctigenin promotes cholesterol efflux from THP-1 macrophages through PPAR-γ/LXR-α signaling pathway

Energy Technology Data Exchange (ETDEWEB)

Xu, Xiaolin [Department of Cardiothoracic Surgery, Huashan Hospital, Fudan University, Shanghai 200032 (China); Li, Qian [Department of Integrative Medicine and Neurobiology, School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai (China); Pang, Liewen; Huang, Guoqian; Huang, Jiechun; Shi, Meng; Sun, Xiaotian [Department of Cardiothoracic Surgery, Huashan Hospital, Fudan University, Shanghai 200032 (China); Wang, Yiqing [Department of Cardiothoracic Surgery, Huashan Hospital, Fudan University, Shanghai 200032 (China)

2013-11-15

Highlights: •Arctigenin enhanced cholesterol efflux in oxLDL-loaded THP-1 macrophages. •The expression of ABCA1, ABCG1 and apoE was upregulated in arctigenin-treated cells. •Arctigenin promoted the expression of PPAR-γ and LXR-α. •Inhibition of PPAR-γ or LXR-α reversed arctigenin-mediated biological effects. •Arctigenin promotes cholesterol efflux via activation of PPAR-γ/LXR-α/ABCA1 pathway. -- Abstract: Cholesterol efflux from macrophages is a critical mechanism to prevent the development of atherosclerosis. Here, we sought to investigate the effects of arctigenin, a bioactive component of Arctium lappa, on the cholesterol efflux in oxidized low-density lipoprotein (oxLDL)-loaded THP-1 macrophages. Our data showed that arctigenin significantly accelerated apolipoprotein A-I- and high-density lipoprotein-induced cholesterol efflux in both dose- and time-dependent manners. Moreover, arctigenin treatment enhanced the expression of ATP binding cassette transporter A1 (ABCA1), ABCG1, and apoE, all of which are key molecules in the initial step of cholesterol efflux, at both mRNA and protein levels. Arctigenin also caused a concentration-dependent elevation in the expression of peroxisome proliferator-activated receptor-gamma (PPAR-γ) and liver X receptor-alpha (LXR-α). The arctigenin-mediated induction of ABCA1, ABCG1, and apoE was abolished by specific inhibition of PPAR-γ or LXR-α using small interfering RNA technology. Our results collectively indicate that arctigenin promotes cholesterol efflux in oxLDL-loaded THP-1 macrophages through upregulation of ABCA1, ABCG1 and apoE, which is dependent on the enhanced expression of PPAR-γ and LXR-α.

Arctigenin promotes cholesterol efflux from THP-1 macrophages through PPAR-γ/LXR-α signaling pathway

International Nuclear Information System (INIS)

Xu, Xiaolin; Li, Qian; Pang, Liewen; Huang, Guoqian; Huang, Jiechun; Shi, Meng; Sun, Xiaotian; Wang, Yiqing

2013-01-01

Highlights: •Arctigenin enhanced cholesterol efflux in oxLDL-loaded THP-1 macrophages. •The expression of ABCA1, ABCG1 and apoE was upregulated in arctigenin-treated cells. •Arctigenin promoted the expression of PPAR-γ and LXR-α. •Inhibition of PPAR-γ or LXR-α reversed arctigenin-mediated biological effects. •Arctigenin promotes cholesterol efflux via activation of PPAR-γ/LXR-α/ABCA1 pathway. -- Abstract: Cholesterol efflux from macrophages is a critical mechanism to prevent the development of atherosclerosis. Here, we sought to investigate the effects of arctigenin, a bioactive component of Arctium lappa, on the cholesterol efflux in oxidized low-density lipoprotein (oxLDL)-loaded THP-1 macrophages. Our data showed that arctigenin significantly accelerated apolipoprotein A-I- and high-density lipoprotein-induced cholesterol efflux in both dose- and time-dependent manners. Moreover, arctigenin treatment enhanced the expression of ATP binding cassette transporter A1 (ABCA1), ABCG1, and apoE, all of which are key molecules in the initial step of cholesterol efflux, at both mRNA and protein levels. Arctigenin also caused a concentration-dependent elevation in the expression of peroxisome proliferator-activated receptor-gamma (PPAR-γ) and liver X receptor-alpha (LXR-α). The arctigenin-mediated induction of ABCA1, ABCG1, and apoE was abolished by specific inhibition of PPAR-γ or LXR-α using small interfering RNA technology. Our results collectively indicate that arctigenin promotes cholesterol efflux in oxLDL-loaded THP-1 macrophages through upregulation of ABCA1, ABCG1 and apoE, which is dependent on the enhanced expression of PPAR-γ and LXR-α

Deficiency of ABCA1 and ABCG1 in Macrophages Increases Inflammation and Accelerates Atherosclerosis in Mice

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Westerterp, Marit; Murphy, Andrew J.; Wang, Mi; Pagler, Tamara A.; Vengrenyuk, Yuliya; Kappus, Mojdeh S.; Gorman, Darren J.; Nagareddy, Prabhakara R.; Zhu, Xuewei; Abramowicz, Sandra; Parks, John S.; Welch, Carrie; Fisher, Edward A.; Wang, Nan; Yvan-Charvet, Laurent; Tall, Alan R.

2013-01-01

Rationale Plasma HDL levels are inversely correlated with atherosclerosis. Although it is widely assumed that this is due to the ability of HDL to promote cholesterol efflux from macrophage foam cells, direct experimental support for this hypothesis is lacking. Objective To assess the role of macrophage cholesterol efflux pathways in atherogenesis. Methods and Results We developed MAC-ABCDKO mice with efficient deletion of the ATP Binding Cassette Transporters A1 and G1 (ABCA1 and ABCG1) in macrophages but not in hematopoietic stem or progenitor populations. MAC-ABCDKO bone marrow (BM) was transplanted into Ldlr-/- recipients. On the chow diet, these mice had similar plasma cholesterol and blood monocyte levels but increased atherosclerosis compared to controls. On the Western type diet (WTD), MAC-ABCDKO BM transplanted Ldlr-/- mice had disproportionate atherosclerosis, considering they also had lower VLDL/LDL cholesterol levels than controls. ABCA1/G1 deficient macrophages in lesions showed increased inflammatory gene expression. Unexpectedly, WTD-fed MAC-ABCDKO BM transplanted Ldlr-/- mice displayed monocytosis and neutrophilia in the absence of HSPC proliferation. Mechanistic studies revealed increased expression of M-CSF and G-CSF in splenic macrophage foam cells, driving BM monocyte and neutrophil production. Conclusion These studies 1) show that macrophage deficiency of ABCA1/G1 is pro-atherogenic likely by promoting plaque inflammation and 2) uncover a novel positive feedback loop in which cholesterol-laden splenic macrophages signal BM progenitors to produce monocytes, with suppression by macrophage cholesterol efflux pathways. PMID:23572498

Preparation, characterization, and safety evaluation of poly(lactide-co-glycolide nanoparticles for protein delivery into macrophages

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Guedj AS

2015-09-01

Full Text Available Anne-Sophie Guedj,1 Arnold J Kell,2 Michael Barnes,2 Sandra Stals,1 David Gonçalves,3 Denis Girard,3 Carole Lavigne11National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, MB, 2National Research Council of Canada, Ottawa, ON, 3Laboratoire de recherche en inflammation et physiologie des granulocytes, Université du Québec, INRS-Institut Armand-Frappier, Laval, QC, CanadaAbstract: Following infection, HIV establishes reservoirs within tissues that are inaccessible to optimal levels of antiviral drugs or within cells where HIV lies latent, thus escaping the action of anti-HIV drugs. Macrophages are a persistent reservoir for HIV and may contribute to the rebound viremia observed after antiretroviral treatment is stopped. In this study, we further investigate the potential of poly(lactic-co-glycolic acid (PLGA-based nanocarriers as a new strategy to enhance penetration of therapeutic molecules into macrophages. We have prepared stable PLGA nanoparticles (NPs and evaluated their capacity to transport an active molecule into the human monocyte/macrophage cell line THP-1 using bovine serum albumin (BSA as a proof-of-concept compound. Intracellular localization of fluorescent BSA molecules encapsulated into PLGA NPs was monitored in live cells using confocal microscopy, and cellular uptake was quantified by flow cytometry. In vitro and in vivo toxicological studies were performed to further determine the safety profile of PLGA NPs including inflammatory effects. The size of the PLGA NPs carrying BSA (PLGA-BSA in culture medium containing 10% serum was ~126 nm in diameter, and they were negatively charged at their surface (zeta potential =-5.6 mV. Our confocal microscopy studies and flow cytometry data showed that these PLGA-BSA NPs are rapidly and efficiently taken up by THP-1 monocyte-derived macrophages (MDMs at low doses. We found that PLGA-BSA NPs increased cellular uptake and internalization of the protein in vitro. PLGA

Evaluation of a nanotechnology-based approach to induce gene-expression in human THP-1 macrophages under inflammatory conditions.

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Bernal, Laura; Alvarado-Vázquez, Abigail; Ferreira, David Wilson; Paige, Candler A; Ulecia-Morón, Cristina; Hill, Bailey; Caesar, Marina; Romero-Sandoval, E Alfonso

2017-02-01

Macrophages orchestrate the initiation and resolution of inflammation by producing pro- and anti-inflammatory products. An imbalance in these mediators may originate from a deficient or excessive immune response. Therefore, macrophages are valid therapeutic targets to restore homeostasis under inflammatory conditions. We hypothesize that a specific mannosylated nanoparticle effectively induces gene expression in human macrophages under inflammatory conditions without undesirable immunogenic responses. THP-1 macrophages were challenged with lipopolysaccharide (LPS, 5μg/mL). Polyethylenimine (PEI) nanoparticles grafted with a mannose receptor ligand (Man-PEI) were used as a gene delivery method. Nanoparticle toxicity, Man-PEI cellular uptake rate and gene induction efficiency (GFP, CD14 or CD68) were studied. Potential immunogenic responses were evaluated by measuring the production of tumor necrosis factor-alpha (TNF-α), Interleukin (IL)-6 and IL-10. Man-PEI did not produce cytotoxicity, and it was effectively up-taken by THP-1 macrophages (69%). This approach produced a significant expression of GFP (mRNA and protein), CD14 and CD68 (mRNA), and transiently and mildly reduced IL-6 and IL-10 levels in LPS-challenged macrophages. Our results indicate that Man-PEI is suitable for inducing an efficient gene overexpression in human macrophages under inflammatory conditions with limited immunogenic responses. Our promising results set the foundation to test this technology to induce functional anti-inflammatory genes. Copyright © 2016 Elsevier GmbH. All rights reserved.

Glycan structure of Gc Protein-derived Macrophage Activating Factor as revealed by mass spectrometry.

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Borges, Chad R; Rehder, Douglas S

2016-09-15

Disagreement exists regarding the O-glycan structure attached to human vitamin D binding protein (DBP). Previously reported evidence indicated that the O-glycan of the Gc1S allele product is the linear core 1 NeuNAc-Gal-GalNAc-Thr trisaccharide. Here, glycan structural evidence is provided from glycan linkage analysis and over 30 serial glycosidase-digestion experiments which were followed by analysis of the intact protein by electrospray ionization mass spectrometry (ESI-MS). Results demonstrate that the O-glycan from the Gc1F protein is the same linear trisaccharide found on the Gc1S protein and that the hexose residue is galactose. In addition, the putative anti-cancer derivative of DBP known as Gc Protein-derived Macrophage Activating Factor (GcMAF, which is formed by the combined action of β-galactosidase and neuraminidase upon DBP) was analyzed intact by ESI-MS, revealing that the activating E. coli β-galactosidase cleaves nothing from the protein-leaving the glycan structure of active GcMAF as a Gal-GalNAc-Thr disaccharide, regardless of the order in which β-galactosidase and neuraminidase are applied. Moreover, glycosidase digestion results show that α-N-Acetylgalactosamindase (nagalase) lacks endoglycosidic function and only cleaves the DBP O-glycan once it has been trimmed down to a GalNAc-Thr monosaccharide-precluding the possibility of this enzyme removing the O-glycan trisaccharide from cancer-patient DBP in vivo. Copyright © 2016 Elsevier Inc. All rights reserved.

Inhibition of ecto-ATPase activities impairs HIV-1 infection of macrophages.

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Schachter, Julieta; Delgado, Kelly Valcárcel; Barreto-de-Souza, Victor; Bou-Habib, Dumith Chequer; Persechini, Pedro Muanis; Meyer-Fernandes, José Roberto

2015-05-01

Nucleotides and nucleosides are secreted into extracellular media at different concentrations as a consequence of different physiologic and pathological conditions. Ecto-nucleotidases, enzymes present on the surface of most cells, hydrolyze these extracellular nucleotides and reduce the concentration of them, thus affecting the activation of different nucleotide and nucleoside receptors. Also, ecto-nucleotidases are present in a number of microorganisms and play important roles in host-pathogen interactions. Here, we characterized the ecto-ATPase activities present on the surface of HIV-1 particle and human macrophages as well. We found that the kinetic properties of HIV-1 and macrophage ecto-ATPases are similar, suggesting that the enzyme is the same. This ecto-ATPase activity was increased in macrophages infected in vitro with HIV-1. Using three different non-related ecto-ATPase inhibitors-POM-1, ARL67156 and BG0-we showed that the inhibition of these macrophage and viral ecto-ATPase activities impairs HIV-1 infection. In addition, we also found that elevated extracellular concentrations of ATP inhibit HIV-1 production by infected macrophages. Copyright © 2014 Elsevier GmbH. All rights reserved.

Topology of Legionella pneumophila DotA: an inner membrane protein required for replication in macrophages.

OpenAIRE

Roy, C R; Isberg, R R

1997-01-01

The Legionella pneumophila dotA gene is required for intracellular growth of the bacterium in macrophages. In this study, a structure-function analysis of the DotA protein was conducted to elucidate the role of this protein in L. pneumophila pathogenesis. Translational fusions of dotA to the Escherichia coli phoA and lacZ genes indicated that DotA is an integral cytoplasmic membrane protein with eight membrane-spanning domains. DotA contains two large periplasmic domains of approximately 503 ...

Improved gene expression in resting macrophages using an oligopeptide derived from Vpr of human immunodeficiency virus type-1

International Nuclear Information System (INIS)

Mizoguchi, Izuru; Ooe, Yoshihiro; Hoshino, Shigeki; Shimura, Mari; Kasahara, Tadashi; Kano, Shigeyuki; Ohta, Toshiko; Takaku, Fumimaro; Nakayama, Yasuhide; Ishizaka, Yukihito

2005-01-01

Vpr, an accessory gene product of human immunodeficiency virus type-1, is thought to transport a viral DNA from the cytoplasm to the nucleus in resting macrophages. Previously, we reported that a peptide encompassing amino acids 52-78 of Vpr (C45D18) promotes the nuclear trafficking of recombinant proteins that are conjugated with C45D18. Here, we present evidence that C45D18, when conjugated with a six-branched cationic polymer of poly(N,N-dimethylaminopropylacrylamide)-block-oligo(4-aminostyrene) (SV: star vector), facilitates gene expression in resting macrophages. Although there was no difference between SV alone and C45D18-SV with respect to gene transduction into growing cells, C45D18-SV resulted in more than 40-fold greater expression of the exogenous gene upon transduction into chemically differentiated macrophages and human quiescent monocyte-derived macrophages. The data suggest that C45D18 contributes to improving the ability of a non-viral vector to transduce macrophages with exogenous genes and we discuss its further application

Macrophages support splenic erythropoiesis in 4T1 tumor-bearing mice.

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Min Liu

Full Text Available Anemia is a common complication of cancer; a role of spleen in tumor-stress erythropoiesis has been suggested. However, the molecular mechanisms involved in the splenic erythropoiesis following tumor maintenance remain poorly understood. Here we show that tumor development blocks medullar erythropoiesis by granulocyte colony-stimulating factor (G-CSF and then causes anemia in murine 4T1 breast tumor-bearing mice. Meanwhile, tumor-stress promotes splenic erythropoiesis. Splenectomy worsened tumor-induced anemia, and reduced tumor volume and tumor weight, indicating the essential role of spleen in tumor-stress erythropoiesis and tumor growth. Tumor progression of these mice led to increased amounts of bone morphogenetic protein 4 (BMP4 in spleen. The in vivo role of macrophages in splenic erythropoiesis under tumor-stress conditions was investigated. Macrophage depletion by injecting liposomal clodronate decreased the expression of BMP4, inhibited splenic erythropoiesis, aggravated the tumor-induced anemia and suppressed tumor growth. Our results provide insight that macrophages and BMP4 are positive regulators of splenic erythropoiesis in tumor pathological situations. These findings reveal that during the tumor-stress period, the microenvironment of the spleen is undergoing changes, which contributes to adopt a stress erythropoietic fate and supports the expansion and differentiation of stress erythroid progenitors, thereby replenishing red blood cells and promoting tumor growth.

Activated human mast cells induce LOX-1-specific scavenger receptor expression in human monocyte-derived macrophages.

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Mervi Alanne-Kinnunen

Full Text Available Activated mast cells in atherosclerotic lesions degranulate and release bioactive compounds capable of regulating atherogenesis. Here we examined the ability of activated human primary mast cells to regulate the expression of the major scavenger receptors in cultured human primary monocyte-derived macrophages (HMDMs.Components released by immunologically activated human primary mast cells induced a transient expression of lectin-like oxidized LDL receptor (LOX-1 mRNA in HMDMs, while the expression of two other scavenger receptors, MSR1 and CD36, remained unaffected. The LOX-1-inducing secretory components were identified as histamine, tumor necrosis factor alpha (TNF-α, and transforming growth factor beta (TGF-β1, which exhibited a synergistic effect on LOX-1 mRNA expression. Histamine induced a transient expression of LOX-1 protein. Mast cell -induced increase in LOX-1 expression was not associated with increased uptake of oxidized LDL by the macrophages.Mast cell-derived histamine, TNF-α, and TGF-β1 act in concert to induce a transient increase in LOX-1 expression in human primary monocyte-derived macrophages. The LOX-1-inducing activity potentially endows mast cells a hitherto unrecognized role in the regulation of innate immune reactions in atherogenesis.

Silica-coated Gd(DOTA)-loaded protein nanoparticles enable magnetic resonance imaging of macrophages

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Bruckman, Michael A.; Randolph, Lauren N.; Gulati, Neetu M.; Stewart, Phoebe L.; Steinmetz, Nicole F.

2015-01-01

The molecular imaging of in vivo targets allows non-invasive disease diagnosis. Nanoparticles offer a promising platform for molecular imaging because they can deliver large payloads of imaging reagents to the site of disease. Magnetic resonance imaging (MRI) is often preferred for clinical diagnosis because it uses non-ionizing radiation and offers both high spatial resolution and excellent penetration. We have explored the use of plant viruses as the basis of for MRI contrast reagents, specifically Tobacco mosaic virus (TMV), which can assemble to form either stiff rods or spheres. We loaded TMV particles with paramagnetic Gd ions, increasing the ionic relaxivity compared to free Gd ions. The loaded TMV particles were then coated with silica maintaining high relaxivities. Interestingly, we found that when Gd(DOTA) was loaded into the interior channel of TMV and the exterior was coated with silica, the T1 relaxivities increased by three-fold from 10.9 mM−1 s−1 to 29.7 mM−1s−1 at 60 MHz compared to uncoated Gd-loaded TMV. To test the performance of the contrast agents in a biological setting, we focused on interactions with macrophages because the active or passive targeting of immune cells is a popular strategy to investigate the cellular components involved in disease progression associated with inflammation. In vitro assays and phantom MRI experiments indicate efficient targeting and imaging of macrophages, enhanced contrast-to-noise ratio was observed by shape-engineering (SNP > TMV) and silica-coating (Si-TMV/SNP > TMV/SNP). Because plant viruses are in the food chain, antibodies may be prevalent in the population. Therefore we investigated whether the silica-coating could prevent antibody recognition; indeed our data indicate that mineralization can be used as a stealth coating option to reduce clearance. Therefore, we conclude that the silica-coated protein-based contrast agent may provide an interesting candidate material for further investigation

Visualisation of chicken macrophages using transgenic reporter genes: insights into the development of the avian macrophage lineage.

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Balic, Adam; Garcia-Morales, Carla; Vervelde, Lonneke; Gilhooley, Hazel; Sherman, Adrian; Garceau, Valerie; Gutowska, Maria W; Burt, David W; Kaiser, Pete; Hume, David A; Sang, Helen M

2014-08-01

We have generated the first transgenic chickens in which reporter genes are expressed in a specific immune cell lineage, based upon control elements of the colony stimulating factor 1 receptor (CSF1R) locus. The Fms intronic regulatory element (FIRE) within CSF1R is shown to be highly conserved in amniotes and absolutely required for myeloid-restricted expression of fluorescent reporter genes. As in mammals, CSF1R-reporter genes were specifically expressed at high levels in cells of the macrophage lineage and at a much lower level in granulocytes. The cell lineage specificity of reporter gene expression was confirmed by demonstration of coincident expression with the endogenous CSF1R protein. In transgenic birds, expression of the reporter gene provided a defined marker for macrophage-lineage cells, identifying the earliest stages in the yolk sac, throughout embryonic development and in all adult tissues. The reporter genes permit detailed and dynamic visualisation of embryonic chicken macrophages. Chicken embryonic macrophages are not recruited to incisional wounds, but are able to recognise and phagocytose microbial antigens. © 2014. Published by The Company of Biologists Ltd.

Induction of ER stress in macrophages of tuberculosis granulomas.

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Tracie A Seimon

2010-09-01

Full Text Available The endoplasmic reticulum (ER stress pathway known as the Unfolded Protein Response (UPR is an adaptive survival pathway that protects cells from the buildup of misfolded proteins, but under certain circumstances it can lead to apoptosis. ER stress has been causally associated with macrophage apoptosis in advanced atherosclerosis of mice and humans. Because atherosclerosis shares certain features with tuberculosis (TB with regard to lesional macrophage accumulation, foam cell formation, and apoptosis, we investigated if the ER stress pathway is activated during TB infection.Here we show that ER stress markers such as C/EBP homologous protein (CHOP; also known as GADD153, phosphorylated inositol-requiring enzyme 1 alpha (Ire1α and eukaryotic initiation factor 2 alpha (eIF2α, and activating transcription factor 3 (ATF3 are expressed in macrophage-rich areas of granulomas in lungs of mice infected with virulent Mycobacterium tuberculosis (Mtb. These areas were also positive for numerous apoptotic cells as assayed by TUNEL. Microarray analysis of human caseous TB granulomas isolated by laser capture microdissection reveal that 73% of genes involved in the UPR are upregulated at the mRNA transcript level. The expression of two ER stress markers, ATF3 and CHOP, were also increased in macrophages of human TB granulomas when assayed by immunohistochemistry. CHOP has been causally associated with ER stress-induced macrophage apoptosis. We found that apoptosis was more abundant in granulomas as compared to non-granulomatous tissue isolated from patients with pulmonary TB, and apoptosis correlated with CHOP expression in areas surrounding the centralized areas of caseation.In summary, ER stress is induced in macrophages of TB granulomas in areas where apoptotic cells accumulate in mice and humans. Although macrophage apoptosis is generally thought to be beneficial in initially protecting the host from Mtb infection, death of infected macrophages in

Extratumoral Heme Oxygenase-1 (HO-1 Expressing Macrophages Likely Promote Primary and Metastatic Prostate Tumor Growth.

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Sofia Halin Bergström

Full Text Available Aggressive tumors induce tumor-supporting changes in the benign parts of the prostate. One factor that has increased expression outside prostate tumors is hemoxygenase-1 (HO-1. To investigate HO-1 expression in more detail, we analyzed samples of tumor tissue and peritumoral normal prostate tissue from rats carrying cancers with different metastatic capacity, and human prostate cancer tissue samples from primary tumors and bone metastases. In rat prostate tumor samples, immunohistochemistry and quantitative RT-PCR showed that the main site of HO-1 synthesis was HO-1+ macrophages that accumulated in the tumor-bearing organ, and at the tumor-invasive front. Small metastatic tumors were considerably more effective in attracting HO-1+ macrophages than larger non-metastatic ones. In clinical samples, accumulation of HO-1+ macrophages was seen at the tumor invasive front, almost exclusively in high-grade tumors, and it correlated with the presence of bone metastases. HO-1+ macrophages, located at the tumor invasive front, were more abundant in bone metastases than in primary tumors. HO-1 expression in bone metastases was variable, and positively correlated with the expression of macrophage markers but negatively correlated with androgen receptor expression, suggesting that elevated HO-1 could be a marker for a subgroup of bone metastases. Together with another recent observation showing that selective knockout of HO-1 in macrophages reduced prostate tumor growth and metastatic capacity in animals, the results of this study suggest that extratumoral HO-1+ macrophages may have an important role in prostate cancer.

Clofazimine modulates the expression of lipid metabolism proteins in Mycobacterium leprae-infected macrophages.

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Degang, Yang; Akama, Takeshi; Hara, Takeshi; Tanigawa, Kazunari; Ishido, Yuko; Gidoh, Masaichi; Makino, Masahiko; Ishii, Norihisa; Suzuki, Koichi

2012-01-01

Mycobacterium leprae (M. leprae) lives and replicates within macrophages in a foamy, lipid-laden phagosome. The lipids provide essential nutrition for the mycobacteria, and M. leprae infection modulates expression of important host proteins related to lipid metabolism. Thus, M. leprae infection increases the expression of adipophilin/adipose differentiation-related protein (ADRP) and decreases hormone-sensitive lipase (HSL), facilitating the accumulation and maintenance of lipid-rich environments suitable for the intracellular survival of M. leprae. HSL levels are not detectable in skin smear specimens taken from leprosy patients, but re-appear shortly after multidrug therapy (MDT). This study examined the effect of MDT components on host lipid metabolism in vitro, and the outcome of rifampicin, dapsone and clofazimine treatment on ADRP and HSL expression in THP-1 cells. Clofazimine attenuated the mRNA and protein levels of ADRP in M. leprae-infected cells, while those of HSL were increased. Rifampicin and dapsone did not show any significant effects on ADRP and HSL expression levels. A transient increase of interferon (IFN)-β and IFN-γ mRNA was also observed in cells infected with M. leprae and treated with clofazimine. Lipid droplets accumulated by M. leprae-infection were significantly decreased 48 h after clofazimine treatment. Such effects were not evident in cells without M. leprae infection. In clinical samples, ADRP expression was decreased and HSL expression was increased after treatment. These results suggest that clofazimine modulates lipid metabolism in M. leprae-infected macrophages by modulating the expression of ADRP and HSL. It also induces IFN production in M. leprae-infected cells. The resultant decrease in lipid accumulation, increase in lipolysis, and activation of innate immunity may be some of the key actions of clofazimine.

Accumulation of M1-like macrophages in type 2 diabetic islets is followed by a systemic shift in macrophage polarization.

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Cucak, Helena; Grunnet, Lars Groth; Rosendahl, Alexander

2014-01-01

Human T2D is characterized by a low-grade systemic inflammation, loss of β-cells, and diminished insulin production. Local islet immunity is still poorly understood, and hence, we evaluated macrophage subpopulations in pancreatic islets in the well-established murine model of T2D, the db/db mouse. Already at 8 weeks of disease, on average, 12 macrophages were observed in the diabetic islets, whereas only two were recorded in the nondiabetic littermates. On a detailed level, the islet resident macrophages increased fourfold compared with nondiabetic littermates, whereas a pronounced recruitment (eightfold) of a novel subset of macrophages (CD68+F4/80-) was observed. The majority of the CD68+F4/80+ but only 40% of the CD68+F4/80- islet macrophages expressed CD11b. Both islet-derived macrophage subsets expressed moderate MHC-II, high galectin-3, and low CD80/CD86 levels, suggesting the cells to be macrophages rather than DCs. On a functional level, the vast majority of the macrophages in the diabetic islets was of the proinflammatory, M1-like phenotype. The systemic immunity in diabetic animals was characterized by a low-grade inflammation with elevated cytokine levels and increase of splenic cytokine, producing CD68+F4/80- macrophages. In late-stage diabetes, the cytokine signature changed toward a TGF-β-dominated profile, coinciding with a significant increase of galectin-3-positive macrophages in the spleen. In summary, our results show that proinflammatory M1-like galectin-3+ CD80/CD86(low) macrophages invade diabetic islets. Moreover, the innate immunity matures in a diabetes-dependent manner from an initial proinflammatory toward a profibrotic phenotype, supporting the concept that T2D is an inflammatory disease.

Soluble human leukocyte antigen G5 polarizes differentiation of macrophages toward a decidual macrophage-like phenotype.

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Lee, Cheuk-Lun; Guo, YiFan; So, Kam-Hei; Vijayan, Madhavi; Guo, Yue; Wong, Vera H H; Yao, YuanQing; Lee, Kai-Fai; Chiu, Philip C N; Yeung, William S B

2015-10-01

What are the actions of soluble human leukocyte antigen G5 (sHLAG5) on macrophage differentiation? sHLAG5 polarizes the differentiation of macrophages toward a decidual macrophage-like phenotype, which could regulate fetomaternal tolerance and placental development. sHLAG5 is a full-length soluble isoform of human leukocyte antigen implicated in immune tolerance during pregnancy. Low or undetectable circulating level of sHLAG5 in first trimester of pregnancy is associated with pregnancy complications such as pre-eclampsia and spontaneous abortion. Decidual macrophages are located in close proximity to invasive trophoblasts, and are involved in regulating fetomaternal tolerance and placental development. Human peripheral blood monocytes were differentiated into macrophages by treatment with granulocyte macrophage colony-stimulating factor in the presence or absence of recombinant sHLAG5 during the differentiation process. The phenotypes and the biological activities of the resulting macrophages were compared. Recombinant sHLAG5 was produced in Escherichia coli BL21 and the protein identity was verified by tandem mass spectrometry. The expression of macrophage markers were analyzed by flow cytometry and quantitative PCR. Phagocytosis was determined by flow cytometry. Indoleamine 2,3-dioxygenase 1 expression and activity were measured by western blot analysis and kynurenine assay, respectively. Cell proliferation and cell cycling were determined by fluorometric cell proliferation assay and flow cytometry, respectively. Cytokine secretion was determined by cytokine array and ELISA kits. Intracellular cytokine expression was measured by flow cytometry. Cell invasion and migration were determined by trans-well invasion and migration assay, respectively. sHLAG5 drove the differentiation of macrophages with 'immuno-modulatory' characteristics, including reduced expression of M1 macrophage marker CD86 and increased expression of M2 macrophage marker CD163. sHLAG5-polarized

HIV-1 infection induces changes in expression of cellular splicing factors that regulate alternative viral splicing and virus production in macrophages

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Purcell Damian FJ

2008-02-01

Full Text Available Abstract Background Macrophages are important targets and long-lived reservoirs of HIV-1, which are not cleared of infection by currently available treatments. In the primary monocyte-derived macrophage model of infection, replication is initially productive followed by a decline in virion output over ensuing weeks, coincident with a decrease in the levels of the essential viral transactivator protein Tat. We investigated two possible mechanisms in macrophages for regulation of viral replication, which appears to be primarily regulated at the level of tat mRNA: 1 differential mRNA stability, used by cells and some viruses for the rapid regulation of gene expression and 2 control of HIV-1 alternative splicing, which is essential for optimal viral replication. Results Following termination of transcription at increasing times after infection in macrophages, we found that tat mRNA did indeed decay more rapidly than rev or nef mRNA, but with similar kinetics throughout infection. In addition, tat mRNA decayed at least as rapidly in peripheral blood lymphocytes. Expression of cellular splicing factors in uninfected and infected macrophage cultures from the same donor showed an inverse pattern over time between enhancing factors (members of the SR family of RNA binding proteins and inhibitory factors (members of the hnRNP family. While levels of the SR protein SC35 were greatly up-regulated in the first week or two after infection, hnRNPs of the A/B and H groups were down-regulated. Around the peak of virus production in each culture, SC35 expression declined to levels in uninfected cells or lower, while the hnRNPs increased to control levels or above. We also found evidence for increased cytoplasmic expression of SC35 following long-term infection. Conclusion While no evidence of differential regulation of tat mRNA decay was found in macrophages following HIV-1 infection, changes in the balance of cellular splicing factors which regulate alternative

A defect in the inflammation-primed macrophage-activation cascade in osteopetrotic rats.

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Yamamoto, N; Lindsay, D D; Naraparaju, V R; Ireland, R A; Popoff, S N

1994-05-15

Macrophages were activated by administration of lysophosphatidylcholine (lyso-Pc) or dodecylglycerol (DDG) to wild-type rats but not in osteopetrotic (op) mutant rats. In vitro treatment of wild-type rat peritoneal cells with lyso-Pc or DDG efficiently activated macrophages whereas treatment of op mutant rat peritoneal cells with lyso-Pc or DDG did not activate macrophages. The inflammation-primed macrophage activation cascade in rats requires participation of B lymphocytes and vitamin D binding protein (DBP). Lyso-Pc-inducible beta-galactosidase of wild-type rat B lymphocytes can convert DBP to the macrophage-activating factor (MAF), whereas B lymphocytes of the op mutant rats were shown to be deficient in lyso-Pc-inducible beta-galactosidase. DBP is conserved among mammalian species. Treatment of human DBP (Gc1 protein) with commercial glycosidases yields an extremely high titrated MAF as assayed on mouse and rat macrophages. Because the enzymatically generated MAF (GcMAF) bypasses the role of lymphocytes in macrophage activation, the op mutant rat macrophages were efficiently activated by administration of a small quantity (100 pg/rat) of GcMAF. Likewise, in vitro treatment of op rat peritoneal cells with as little as 40 pg GcMAF/ml activated macrophages.

Cathepsin E deficiency impairs autophagic proteolysis in macrophages.

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Takayuki Tsukuba

Full Text Available Cathepsin E is an endosomal aspartic proteinase that is predominantly expressed in immune-related cells. Recently, we showed that macrophages derived from cathepsin E-deficient (CatE(-/- mice display accumulation of lysosomal membrane proteins and abnormal membrane trafficking. In this study, we demonstrated that CatE(-/- macrophages exhibit abnormalities in autophagy, a bulk degradation system for aggregated proteins and damaged organelles. CatE(-/- macrophages showed increased accumulation of autophagy marker proteins such as LC3 and p62, and polyubiquitinated proteins. Cathepsin E deficiency also altered autophagy-related signaling pathways such as those mediated by the mammalian target of rapamycin (mTOR, Akt, and extracellular signal-related kinase (ERK. Furthermore, immunofluorescence microscopy analyses showed that LC3-positive vesicles were merged with acidic compartments in wild-type macrophages, but not in CatE(-/- macrophages, indicating inhibition of fusion of autophagosome with lysosomes in CatE(-/- cells. Delayed degradation of LC3 protein was also observed under starvation-induced conditions. Since the autophagy system is involved in the degradation of damaged mitochondria, we examined the accumulation of damaged mitochondria in CatE(-/- macrophages. Several mitochondrial abnormalities such as decreased intracellular ATP levels, depolarized mitochondrial membrane potential, and decreased mitochondrial oxygen consumption were observed. Such mitochondrial dysfunction likely led to the accompanying oxidative stress. In fact, CatE(-/- macrophages showed increased reactive oxygen species (ROS production and up-regulation of oxidized peroxiredoxin-6, but decreased antioxidant glutathione. These results indicate that cathepsin E deficiency causes autophagy impairment concomitantly with increased aberrant mitochondria as well as increased oxidative stress.

Effects of as-cast and wrought Cobalt-Chrome-Molybdenum and Titanium-Aluminium-Vanadium alloys on cytokine gene expression and protein secretion in J774A.1 macrophages

DEFF Research Database (Denmark)

Jakobsen, Stig Storgaard; Larsen, Agnete; Stoltenberg, Meredin

2007-01-01

the cell viability. Surface properties of the discs were characterised with a profilometer and with energy dispersive X-ray spectroscopy. We here report, for the first time, that the prosthetic material surface (non-phagocytable) of as-cast high carbon CoCrMo reduces the pro-inflammatory cytokine IL-6......Insertion of metal implants is associated with a possible change in the delicate balance between pro- and anti-inflammatory proteins, probably leading to an unfavourable predominantly pro-inflammatory milieu. The most likely cause is an inappropriate activation of macrophages in close relation...... to the metal implant and wear-products. The aim of the present study was to compare surfaces of as-cast and wrought Cobalt-Chrome-Molybdenum (CoCrMo) alloys and Titanium-Aluminium-Vanadium (TiAlV) alloy when incubated with mouse macrophage J774A.1 cell cultures. Changes in pro- and anti-inflammatory cytokines...

Major Vault Protein Regulates Class A Scavenger Receptor-mediated Tumor Necrosis Factor-α Synthesis and Apoptosis in Macrophages*

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Ben, Jingjing; Zhang, Yan; Zhou, Rongmei; Zhang, Haiyang; Zhu, Xudong; Li, Xiaoyu; Zhang, Hanwen; Li, Nan; Zhou, Xiaodan; Bai, Hui; Yang, Qing; Li, Donghai; Xu, Yong; Chen, Qi

2013-01-01

Atherosclerosis is considered a disease of chronic inflammation largely initiated and perpetuated by macrophage-dependent synthesis and release of pro-inflammatory mediators. Class A scavenger receptor (SR-A) expressed on macrophages plays a key role in this process. However, how SR-A-mediated pro-inflammatory response is modulated in macrophages remains ill defined. Here through immunoprecipitation coupled with mass spectrometry, we reported major vault protein (MVP) as a novel binding partner for SR-A. The interaction between SR-A and MVP was confirmed by immunofluorescence staining and chemical cross-linking assay. Treatment of macrophages with fucoidan, a SR-A ligand, led to a marked increase in TNF-α production, which was attenuated by MVP depletion. Further analysis revealed that SR-A stimulated TNF-α synthesis in macrophages via the caveolin- instead of clathrin-mediated endocytic pathway linked to p38 and JNK, but not ERK, signaling pathways. Importantly, fucoidan invoked an enrichment of MVP in lipid raft, a caveolin-reliant membrane structure, and enhanced the interaction among SR-A, caveolin, and MVP. Finally, we demonstrated that MVP elimination ameliorated SR-A-mediated apoptosis in macrophages. As such, MVP may fine-tune SR-A activity in macrophages which contributes to the development of atherosclerosis. PMID:23703615

Herpes Simplex Virus Type 1 Infects Enteric Neurons and Triggers Gut Dysfunction via Macrophage Recruitment.

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Brun, Paola; Qesari, Marsela; Marconi, Peggy C; Kotsafti, Andromachi; Porzionato, Andrea; Macchi, Veronica; Schwendener, Reto A; Scarpa, Marco; Giron, Maria C; Palù, Giorgio; Calistri, Arianna; Castagliuolo, Ignazio

2018-01-01

Herpes Simplex Virus type 1 (HSV-1), a neurotropic pathogen widespread in human population, infects the enteric nervous system (ENS) in humans and rodents and causes intestinal neuromuscular dysfunction in rats. Although infiltration of inflammatory cells in the myenteric plexus and neurodegeneration of enteric nerves are common features of patients suffering from functional intestinal disorders, the proof of a pathogenic link with HSV-1 is still unsettled mainly because the underlying mechanisms are largely unknown. In this study we demonstrated that following intragastrical administration HSV-1 infects neurons within the myenteric plexus resulting in functional and structural alterations of the ENS. By infecting mice with HSV-1 replication-defective strain we revealed that gastrointestinal neuromuscular anomalies were however independent of viral replication. Indeed, enteric neurons exposed to UV-inactivated HSV-1 produced monocyte chemoattractant protein-1 (MCP-1/CCL2) to recruit activated macrophages in the longitudinal muscle myenteric plexus. Infiltrating macrophages produced reactive oxygen and nitrogen species and directly harmed enteric neurons resulting in gastrointestinal dysmotility. In HSV-1 infected mice intestinal neuromuscular dysfunctions were ameliorated by in vivo administration of (i) liposomes containing dichloromethylene bisphosphonic acid (clodronate) to deplete tissue macrophages, (ii) CCR2 chemokine receptor antagonist RS504393 to block the CCL2/CCR2 pathway, (iii) Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME) and AR-C 102222 to quench production of nitrogen reactive species produced via iNOS. Overall these data demonstrate that HSV-1 infection makes enteric neurons recruit macrophages via production of a specific chemoattractant factor. The resulting inflammatory reaction is mandatory for intestinal dysmotility. These findings provide insights into the neuro-immune communication that occurs in the ENS following HSV-1 infection

Herpes Simplex Virus Type 1 Infects Enteric Neurons and Triggers Gut Dysfunction via Macrophage Recruitment

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Paola Brun

2018-03-01

Full Text Available Herpes Simplex Virus type 1 (HSV-1, a neurotropic pathogen widespread in human population, infects the enteric nervous system (ENS in humans and rodents and causes intestinal neuromuscular dysfunction in rats. Although infiltration of inflammatory cells in the myenteric plexus and neurodegeneration of enteric nerves are common features of patients suffering from functional intestinal disorders, the proof of a pathogenic link with HSV-1 is still unsettled mainly because the underlying mechanisms are largely unknown. In this study we demonstrated that following intragastrical administration HSV-1 infects neurons within the myenteric plexus resulting in functional and structural alterations of the ENS. By infecting mice with HSV-1 replication-defective strain we revealed that gastrointestinal neuromuscular anomalies were however independent of viral replication. Indeed, enteric neurons exposed to UV-inactivated HSV-1 produced monocyte chemoattractant protein-1 (MCP-1/CCL2 to recruit activated macrophages in the longitudinal muscle myenteric plexus. Infiltrating macrophages produced reactive oxygen and nitrogen species and directly harmed enteric neurons resulting in gastrointestinal dysmotility. In HSV-1 infected mice intestinal neuromuscular dysfunctions were ameliorated by in vivo administration of (i liposomes containing dichloromethylene bisphosphonic acid (clodronate to deplete tissue macrophages, (ii CCR2 chemokine receptor antagonist RS504393 to block the CCL2/CCR2 pathway, (iii Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME and AR-C 102222 to quench production of nitrogen reactive species produced via iNOS. Overall these data demonstrate that HSV-1 infection makes enteric neurons recruit macrophages via production of a specific chemoattractant factor. The resulting inflammatory reaction is mandatory for intestinal dysmotility. These findings provide insights into the neuro-immune communication that occurs in the ENS following HSV-1

Postprandial phase time influences the uptake of TAG from postprandial TAG-rich lipoproteins by THP-1 macrophages.

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Cabello-Moruno, Rosana; Sinausia, Laura; Botham, Kathleen M; Montero, Emilio; Avella, Michael; Perona, Javier S

2014-11-14

Postprandial TAG-rich lipoproteins (TRL) can be taken up by macrophages, leading to the formation of foam cells, probably via receptor-mediated pathways. The present study was conducted to investigate whether the postprandial time point at which TRL are collected modulates this process. A meal containing refined olive oil was given to nine healthy young men and TRL were isolated from their serum at 2, 4 and 6 h postprandially. The lipid class and apoB compositions of TRL were determined by HPLC and SDS-PAGE, respectively. The accumulation of lipids in macrophages was determined after the incubation of THP-1 macrophages with TRL. The gene expression of candidate receptors was measured by real-time PCR. The highest concentrations of TAG, apoB48 and apoB100 in TRL were observed at 2 h after the consumption of the test meal. However, excessive intracellular TAG accumulation in THP-1 macrophages was observed in response to incubation with TRL isolated at 4 h, when their particle size (estimated as the TAG:apoB ratio) was intermediate. The abundance of mRNA transcripts in macrophages in response to incubation with TRL was down-regulated for LDL receptor (LDLR), slightly up-regulated for VLDL receptor and remained unaltered for LDLR-related protein, but no effect of the postprandial time point was observed. In contrast, the mRNA expression of scavenger receptors SRB1, SRA2 and CD36 was higher when cells were incubated with TRL isolated at 4 h after the consumption of the test meal. In conclusion, TRL led to excessive intracellular TAG accumulation in THP-1 macrophages, which was greater when cells were incubated with intermediate-sized postprandial TRL isolated at 4 h and was associated with a significant increase in the mRNA expression of scavenger receptors.

Surfactant protein A (SP-A) inhibits agglomeration and macrophage uptake of toxic amine modified nanoparticles.

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McKenzie, Zofi; Kendall, Michaela; Mackay, Rose-Marie; Whitwell, Harry; Elgy, Christine; Ding, Ping; Mahajan, Sumeet; Morgan, Cliff; Griffiths, Mark; Clark, Howard; Madsen, Jens

2015-01-01

The lung provides the main route for nanomaterial exposure. Surfactant protein A (SP-A) is an important respiratory innate immune molecule with the ability to bind or opsonise pathogens to enhance phagocytic removal from the airways. We hypothesised that SP-A, like surfactant protein D, may interact with inhaled nanoparticulates, and that this interaction will be affected by nanoparticle (NP) surface characteristics. In this study, we characterise the interaction of SP-A with unmodified (U-PS) and amine-modified (A-PS) polystyrene particles of varying size and zeta potential using dynamic light scatter analysis. SP-A associated with both 100 nm U-PS and A-PS in a calcium-independent manner. SP-A induced significant calcium-dependent agglomeration of 100 nm U-PS NPs but resulted in calcium-independent inhibition of A-PS self agglomeration. SP-A enhanced uptake of 100 nm U-PS into macrophage-like RAW264.7 cells in a dose-dependent manner but in contrast inhibited A-PS uptake. Reduced association of A-PS particles in RAW264.7 cells following pre-incubation of SP-A was also observed with coherent anti-Stokes Raman spectroscopy. Consistent with these findings, alveolar macrophages (AMs) from SP-A(-/-) mice were more efficient at uptake of 100 nm A-PS compared with wild type C57Bl/6 macrophages. No difference in uptake was observed with 500 nm U-PS or A-PS particles. Pre-incubation with SP-A resulted in a significant decrease in uptake of 100 nm A-PS in macrophages isolated from both groups of mice. In contrast, increased uptake by AMs of U-PS was observed after pre-incubation with SP-A. Thus we have demonstrated that SP-A promotes uptake of non-toxic U-PS particles but inhibits the clearance of potentially toxic A-PS particles by blocking uptake into macrophages.

IAP survivin regulates atherosclerotic macrophage survival

NARCIS (Netherlands)

Blanc-Brude, Olivier P.; Teissier, Elisabeth; Castier, Yves; Lesèche, Guy; Bijnens, Ann-Pascal; Daemen, Mat; Staels, Bart; Mallat, Ziad; Tedgui, Alain

2007-01-01

Inflammatory macrophage apoptosis is critical to atherosclerotic plaque formation, but its mechanisms remain enigmatic. We hypothesized that inhibitor of apoptosis protein (IAP) survivin regulates macrophage death in atherosclerosis. Western blot analysis revealed discrete survivin expression in

Translocator protein as an imaging marker of macrophage and stromal activation in RA pannus.

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Narayan, Nehal; Owen, David; Mandhair, Harpreet; Smyth, Erica; Carlucci, Francesco; Saleem, Azeem; Gunn, Roger; Rabiner, Eugenii Ilan A; Wells, Lisa; Dakin, Stephanie; Sabokbar, Afsie; Taylor, Peter

2018-01-04

Positron Emission Tomography (PET) radioligands targeted to Translocator protein (TSPO), offer a highly sensitive and specific means of imaging joint inflammation in rheumatoid arthritis (RA). Through high expression of TSPO on activated macrophages, TSPO PET has been widely reported in several studies of RA as a means of imaging synovial macrophages in vivo. However, this premise does not take into account the ubiquitous expression of TSPO. This study aimed to investigate TSPO expression in major cellular constituents of RA pannus; monocytes, macrophages, fibroblast-like synoviocytes (FLS) and CD4+ T lymphocytes, to more accurately interpret TSPO PET signal from RA synovium. Methods: 3 RA patients and 3 healthy volunteers underwent PET both knees using the TSPO radioligand 11 C-PBR28. Through synovial tissue 3H-PBR28 autoradiography and immunostaining of 6 RA patients and 6 healthy volunteers, cellular expression of TSPO in synovial tissue was evaluated. TSPO mRNA expression and 3H-PBR28 radioligand binding was assessed using in vitro monocytes, macrophages, FLS and CD4+ T-lymphocytes. Results: 11 C-PBR28 PET signal was significantly higher in RA compared to healthy joints (average SUV 0.82± 0.12 compared to 0.03± 0.004 respectively, p<0.01). Further, 3H-PBR28 specific binding in synovial tissue was approximately 10-fold higher in RA compared to healthy controls. Immunofluorescence revealed TSPO expression on macrophages, FLS and CD4+ T cells. In vitro study demonstrated highest TSPO mRNA expression and 3H-PBR28 specific binding, in activated FLS, non-activated and activated 'M2' reparative macrophages, with least TSPO expression in activated and non-activated CD4+ T lymphocytes. Conclusion: This study is the first evaluation of cellular TSPO expression in synovium, finding highest TSPO expression and PBR28 binding on activated synovial FLS and M2 phenotype macrophages. TSPO targeted PET may therefore have unique sensitivity to detect FLS and macrophage

Effect of Enzymatic Digestion of Protein Derivatives Obtained from Mucuna pruriens L. on Production of Proinflammatory Mediators by BALB/c Mouse Macrophages.

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Martínez Leo, Edwin E; Arana Argáez, Victor E; Acevedo Fernández, Juan J; Puc, Rosa Moo; Segura Campos, Maira R

2018-04-25

Inflammation is considered to be a major risk factor for the pathogenesis of chronic non-communicable diseases. Macrophages are important immune cells, which regulate inflammation and host defense by secretion of proinflammatory mediators. Obtaining biopeptides by enzymatic hydrolysis adds value to proteins of vegetative origin, such as Mucuna pruriens L. The present study evaluated the effect of enzymatic digestion of protein derivatives obtained from M. pruriens L. on the production of proinflammatory mediators by BALB/c mouse macrophages. Five different molecular weight peptide fractions were obtained (F > 10, 5-10, 3-5, 1-3, and < 1 kDa, respectively). At 300 μg/mL, F5-10 kDa inhibited 50.26 and 61.00% NO and H 2 O 2 production, respectively. Moreover, F5-10 kDa reduced the IL-6 and TNFα levels to 60.25 and 69.54%, respectively. After enzymatic digestive simulation, F5-10 kDa decreased the inflammatory mediators.

Role of macrophage CCAAT/enhancer binding protein delta in the pathogenesis of rheumatoid arthritis in collagen-induced arthritic mice.

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Ling-Hua Chang

Full Text Available BACKGROUND: The up-regulation of CCAAT/enhancer binding protein delta (CEBPD has frequently been observed in macrophages in age-associated disorders, including rheumatoid arthritis (RA. However, the role of macrophage CEBPD in the pathogenesis of RA is unclear. METHODOLOGY AND PRINCIPAL FINDINGS: We found that the collagen-induced arthritis (CIA score and the number of affected paws in Cebpd(-/- mice were significantly decreased compared with the wild-type (WT mice. The histological analysis revealed an attenuated CIA in Cebpd(-/- mice, as shown by reduced pannus formation and greater integrity of joint architecture in affected paws of Cebpd(-/- mice compared with WT mice. In addition, immunohistochemistry analysis revealed decreased pannus proliferation and angiogenesis in Cebpd(-/- mice compared with WT mice. CEBPD activated in macrophages played a functional role in promoting the tube formation of endothelial cells and the migration and proliferation of synoviocytes. In vivo DNA binding assays and reporter assays showed that CEBPD up-regulated CCL20, CXCL1, IL23A and TNFAIP6 transcripts through direct binding to their promoter regions. CCL20, IL23A, CXCL1 and TNFAIP6 contributed to the migration and proliferation of synoviocytes, and the latter two proteins were involved in tube formation of endothelial cells. Finally, two anti-inflammatory chemicals, inotilone and rosmanol, reduced the expression of CEBPD and its downstream targets and mitigated the above phenomena. CONCLUSIONS AND SIGNIFICANCE: Collectively, our findings suggest that CEBPD and its downstream effectors could be biomarkers for the diagnosis of RA and potentially serve as therapeutic targets for RA therapy.

Inhibition of the NLRP3 inflammasome attenuates foam cell formation of THP-1 macrophages by suppressing ox-LDL uptake and promoting cholesterol efflux.

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Chen, Liang; Yao, Qiying; Xu, Siwei; Wang, Hongyan; Qu, Peng

2018-01-01

The NOD-like receptor family, pyrin domain-containing protein 3 (NLRP3) inflammasome plays an important role in the development of atherosclerosis. The activated NLRP3 inflammasome has been reported to promote macrophage foam cell formation, but not all studies have obtained the same result, and how NLRP3 inflammasome is involved in the formation of foam cells remains elusive. We used selective NLRP3 inflammasome inhibitors and NLRP3-deficient THP-1 cells to assess the effect of NLRP3 inflammasome inhibition on macrophage foam cell formation, oxidized low-density lipoprotein (ox-LDL) uptake, esterification, and cholesterol efflux, as well as the expression of associated proteins. Inhibition of the NLRP3 inflammasome attenuated foam cell formation, diminished ox-LDL uptake, and promoted cholesterol efflux from THP-1 macrophages. Moreover, it downregulated CD36, acyl coenzyme A: cholesterol acyltransferase-1 and neutral cholesterol ester hydrolase expression; upregulated ATP-binding cassette transporter A1 (ABCA1) and scavenger receptor class B type I (SR-BI) expression; but had no effect on the expression of scavenger receptor class A and ATP-binding cassette transporter G1. Collectively, our findings show that inhibition of the NLRP3 inflammasome decreases foam cell formation of THP-1 macrophages via suppression of ox-LDL uptake and enhancement of cholesterol efflux, which may be due to downregulation of CD36 expression and upregulation of ABCA1 and SR-BI expression, respectively. Copyright © 2017 Elsevier Inc. All rights reserved.

Macrophage-specific nanotechnology-driven CD163 overexpression in human macrophages results in an M2 phenotype under inflammatory conditions.

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Alvarado-Vazquez, Perla Abigail; Bernal, Laura; Paige, Candler A; Grosick, Rachel L; Moracho Vilrriales, Carolina; Ferreira, David Wilson; Ulecia-Morón, Cristina; Romero-Sandoval, E Alfonso

2017-08-01

M1 macrophages release proinflammatory factors during inflammation. They transit to an M2 phenotype and release anti-inflammatory factors to resolve inflammation. An imbalance in the transition from M1 to M2 phenotype in macrophages contributes to the development of persistent inflammation. CD163, a member of the scavenger receptor cysteine-rich family, is an M2 macrophage marker. The functional role of CD163 during the resolution of inflammation is not completely known. We postulate that CD163 contributes to the transition from M1 to M2 phenotype in macrophages. We induced CD163 gene in THP-1 and primary human macrophages using polyethylenimine nanoparticles grafted with a mannose ligand (Man-PEI). This nanoparticle specifically targets cells of monocytic origin via mannose receptors. Cells were challenged with a single or a double stimulation of lipopolysaccharide (LPS). A CD163 or empty plasmid was complexed with Man-PEI nanoparticles for cell transfections. Quantitative RT-PCR, immunocytochemistry, and ELISAs were used for molecular assessments. CD163-overexpressing macrophages displayed reduced levels of tumor necrosis factor-alpha (TNF)-α and monocytes chemoattractant protein (MCP)-1 after a single stimulation with LPS. Following a double stimulation paradigm, CD163-overexpressing macrophages showed an increase of interleukin (IL)-10 and IL-1ra and a reduction of MCP-1. This anti-inflammatory phenotype was partially blocked by an anti-CD163 antibody (effects on IL-10 and IL-1ra). A decrease in the release of TNF-α, IL-1β, and IL-6 was observed in CD163-overexpressing human primary macrophages. The release of IL-6 was blocked by an anti-CD163 antibody in the CD163-overexpressing group. Our data show that the induction of the CD163 gene in human macrophages under inflammatory conditions produces changes in cytokine secretion in favor of an anti-inflammatory phenotype. Targeting macrophages to induce CD163 using cell-directed nanotechnology is an attractive

Generation of reactive oxygen species (ROS) is a key factor for stimulation of macrophage proliferation by ceramide 1-phosphate

International Nuclear Information System (INIS)

Arana, Lide; Gangoiti, Patricia; Ouro, Alberto; Rivera, Io-Guané; Ordoñez, Marta; Trueba, Miguel; Lankalapalli, Ravi S.; Bittman, Robert; Gomez-Muñoz, Antonio

2012-01-01

We previously demonstrated that ceramide 1-phosphate (C1P) is mitogenic for fibroblasts and macrophages. However, the mechanisms involved in this action were only partially described. Here, we demonstrate that C1P stimulates reactive oxygen species (ROS) formation in primary bone marrow-derived macrophages, and that ROS are required for the mitogenic effect of C1P. ROS production was dependent upon prior activation of NADPH oxidase by C1P, which was determined by measuring phosphorylation of the p40phox subunit and translocation of p47phox from the cytosol to the plasma membrane. In addition, C1P activated cytosolic calcium-dependent phospholipase A 2 and protein kinase C-α, and NADPH oxidase activation was blocked by selective inhibitors of these enzymes. These inhibitors, and inhibitors of ROS production, blocked the mitogenic effect of C1P. By using BHNB-C1P (a photolabile caged-C1P analog), we demonstrate that all of these C1P actions are caused by intracellular C1P. It can be concluded that the enzyme responsible for C1P-stimulated ROS generation in bone marrow-derived macrophages is NADPH oxidase, and that this enzyme is downstream of PKC-α and cPLA 2 -α in this pathway. -- Highlights: ► Ceramide 1-phosphate (C1P) stimulates reactive oxygen species (ROS) formation. ► The enzyme responsible for ROS generation by C1P in macrophages is NADPH oxidase. ► NADPH oxidase lies downstream of cPLA 2 -α and PKC-α in this pathway. ► ROS generation is essential for the stimulation of macrophage proliferation by C1P.

A role for the JAK-STAT1 pathway in blocking replication of HSV-1 in dendritic cells and macrophages

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Town Terrence

2009-05-01

Full Text Available Abstract Background Macrophages and dendritic cells (DCs play key roles in host defense against HSV-1 infection. Although macrophages and DCs can be infected by herpes simplex virus type 1 (HSV-1, both cell types are resistant to HSV-1 replication. The aim of our study was to determine factor (s that are involved in the resistance of DCs and macrophages to productive HSV-1 infection. Results We report here that, in contrast to bone marrow-derived DCs and macrophages from wild type mice, DCs and macrophages isolated from signal transducers and activators of transcription-1 deficient (STAT1-/- mice were susceptible to HSV-1 replication and the production of viral mRNAs and DNA. There were differences in expression of immediate early, early, and late gene transcripts between STAT1+/+ and STAT1-/- infected APCs. Conclusion These results suggest for the first time that the JAK-STAT1 pathway is involved in blocking replication of HSV-1 in DCs and macrophages.

A role for the JAK-STAT1 pathway in blocking replication of HSV-1 in dendritic cells and macrophages

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Mott, Kevin R; UnderHill, David; Wechsler, Steven L; Town, Terrence; Ghiasi, Homayon

2009-01-01

Background Macrophages and dendritic cells (DCs) play key roles in host defense against HSV-1 infection. Although macrophages and DCs can be infected by herpes simplex virus type 1 (HSV-1), both cell types are resistant to HSV-1 replication. The aim of our study was to determine factor (s) that are involved in the resistance of DCs and macrophages to productive HSV-1 infection. Results We report here that, in contrast to bone marrow-derived DCs and macrophages from wild type mice, DCs and macrophages isolated from signal transducers and activators of transcription-1 deficient (STAT1-/-) mice were susceptible to HSV-1 replication and the production of viral mRNAs and DNA. There were differences in expression of immediate early, early, and late gene transcripts between STAT1+/+ and STAT1-/- infected APCs. Conclusion These results suggest for the first time that the JAK-STAT1 pathway is involved in blocking replication of HSV-1 in DCs and macrophages. PMID:19439086

In vitro methods of assessing ocular biocompatibility using THP-1-derived macrophages.

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McCanna, David Joseph; Barthod-Malat, Aurore V; Gorbet, Maud B

2015-01-01

Macrophages play an important role in the elimination of infections, the removal of debris and in tissue repair after infection and trauma. In vitro models that assess ocular biomaterials for toxicity typically focus on the effects of these materials on epithelial or fibroblast cells. This investigation evaluated known ocular toxins deposited on model materials for their effects on the viability and activation of macrophages. THP-1-derived macrophages were cultured onto silicone films (used as a base biomaterial) deposited with chemical toxins (benzalkonium chloride (BAK), zinc diethyldithiocarbamate (ZDEC) and lipopolysaccharide (LPS)). Utilizing three fluorescent dyes calcein, ethidium homodimer-1 (EthD-1) and annexin V, the viability of macrophages attached to the biomaterial was determined using confocal microscopy. Propidium iodide (PI) staining and alamarBlue® (resazurin) reduction were used to assess cell death and metabolic activity. CD14, CD16, CD33, CD45, and CD54 expression of adherent macrophages, were also evaluated to detect LPS activation of macrophages using flow cytometry. The sensitivity of this test battery was demonstrated as significant toxicity from treated surfaces with ZDEC (0.001-0.01%), and BAK (0.001%-0.1%) was detected. Also, macrophage activation could be detected by measuring CD54 expression after exposure to adsorbed LPS. These in vitro methods will be helpful in determining the toxicity potential of new ocular biomaterials.

Leishmania eukaryotic initiation factor (LeIF inhibits parasite growth in murine macrophages.

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Olga Koutsoni

Full Text Available The leishmaniases constitute neglected global public health problems that require adequate control measures, prophylactic clinical vaccines and effective and non-toxic drug treatments. In this study, we explored the potential of Leishmania infantum eukaryotic initiation factor (LieIF, an exosomal protein, as a novel anti-infective therapeutic molecule. More specifically, we assessed the efficacy of recombinant LieIF, in combination with recombinant IFN-γ, in eliminating intracellular L. donovani parasites in an in vitro macrophage model. J774A.1 macrophages were initially treated with LieIF/IFN-γ prior to in vitro infection with L. donovani stationary phase promastigotes (pre-infection treatment, and resistance to infection was observed 72 h after infection. J774A.1 macrophages were also treated with LieIF/IFN-γ after L. donovani infection (post-infection treatment, and resistance to infection was also observed at both time points tested (19 h and 72 h after infection. To elucidate the LieIF/IFN-γ-induced mechanism(s that mediate the reduction of intracellular parasite growth, we examined the generation of potent microbicidal molecules, such as nitric oxide (NO and reactive oxygen species (ROS, within infected macrophages. Furthermore, macrophages pre-treated with LieIF/IFN-γ showed a clear up-regulation in macrophage inflammatory protein 1α (MIP-1α as well as tumor necrosis factor alpha (TNF-α expression. However, significant different protein levels were not detected. In addition, macrophages pre-treated with LieIF/IFN-γ combined with anti-TNF-α monoclonal antibody produced significantly lower amounts of ROS. These data suggest that during the pre-treatment state, LieIF induces intramacrophage parasite growth inhibition through the production of TNF-α, which induces microbicidal activity by stimulating NO and ROS production. The mechanisms of NO and ROS production when macrophages are treated with LieIF after infection are probably

Andrographolide Inhibits Oxidized LDL-Induced Cholesterol Accumulation and Foam Cell Formation in Macrophages.

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Lin, Hung-Chih; Lii, Chong-Kuei; Chen, Hui-Chun; Lin, Ai-Hsuan; Yang, Ya-Chen; Chen, Haw-Wen

2018-01-01

oxLDL is involved in the pathogenesis of atherosclerotic lesions through cholesterol accumulation in macrophage foam cells. Andrographolide, the bioactive component of Andrographis paniculata, possesses several biological activities such as anti-inflammatory, anti-oxidant, and anticancer functions. Scavenger receptors (SRs), including class A SR (SR-A) and CD36, are responsible for the internalization of oxLDL. In contrast, receptors for reverse cholesterol transport, including ABCA1 and ABCG1, mediate the efflux of cholesterol from macrophage foam cells. Transcription factor liver X receptor [Formula: see text] (LXR[Formula: see text] plays a key role in lipid metabolism and inflammation as well as in the regulation of ABCA1 and ABCG1 expression. Because of the contribution of inflammation to macrophage foam cell formation and the potent anti-inflammatory activity of andrographolide, we hypothesized that andrographolide might inhibit oxLDL-induced macrophage foam cell formation. The results showed that andrographolide reduced oxLDL-induced lipid accumulation in macrophage foam cells. Andrographolide decreased the mRNA and protein expression of CD36 by inducing the degradation of CD36 mRNA; however, andrographolide had no effect on SR-A expression. In contrast, andrographolide increased the mRNA and protein expression of ABCA1 and ABCG1, which were dependent on LXR[Formula: see text]. Andrographolide enhanced LXR[Formula: see text] nuclear translocation and DNA binding activity. Treatment with the LXR[Formula: see text] antagonist GGPP and transfection with LXR[Formula: see text] siRNA reversed the ability of andrographolide to stimulate ABCA1 and ABCG1 protein expression. In conclusion, inhibition of CD36-mediated oxLDL uptake and induction of ABCA1- and ABCG1-dependent cholesterol efflux are two working mechanisms by which andrographolide inhibits macrophage foam cell formation, which suggests that andrographolide could be a potential candidate to prevent

Genotypic variations in the dynamics of metal concentrations in poplar leaves: A field study with a perspective on phytoremediation

International Nuclear Information System (INIS)

Pottier, Mathieu; García de la Torre, Vanesa S.; Victor, Cindy; David, Laure C.; Chalot, Michel; Thomine, Sébastien

2015-01-01

Poplar is commonly used for phytoremediation of metal polluted soils. However, the high concentrations of trace elements present in leaves may return to soil upon leaf abscission. To investigate the mechanisms controlling leaf metal content, metal concentrations and expression levels of genes involved in metal transport were monitored at different developmental stages on leaves from different poplar genotypes growing on a contaminated field. Large differences in leaf metal concentrations were observed among genotypes. Whereas Mg was remobilized during senescence, Zn and Cd accumulation continued until leaf abscission in all genotypes. A positive correlation between Natural Resistance Associated Macrophage Protein 1 (NRAMP1) expression levels and Zn bio-concentration factors was observed. Principal component analyses of metal concentrations and gene expression levels clearly discriminated poplar genotypes. This study highlights a general absence of trace element remobilization from poplar leaves despite genotype specificities in the control of leaf metal homeostasis. - Highlights: • Poplar genotypes display large variations in leaf metal concentrations. • Trace elements are not remobilized during poplar leaf senescence. • Distinct transporter genes control metal homeostasis at different leaf stages. • Poplar genotypes use distinct mechanisms to control leaf metal homeostasis. • NRAMP1 metal transporter could contribute to Zn and Cd accumulation in poplar leaves. - In order to improve metal phytoextraction using poplars, this work provides new insights on the control of leaf metal concentrations through principal component and correlative analyses

Mouse adenovirus type 1 infection of macrophages

NARCIS (Netherlands)

Ashley, S.L.; Welton, A.R.; Harwood, K.M.; Rooijen, van N.; Spindler, K.R.

2009-01-01

Mouse adenovirus type 1 (MAV-1) causes acute and persistent infections in mice, with high levels of virus found in the brain, spinal cord and spleen in acute infections. MAV-1 infects endothelial cells throughout the mouse, and monocytes/macrophages have also been implicated as targets of the virus.

Disrupted epithelial/macrophage crosstalk via Spinster homologue 2-mediated S1P signaling may drive defective macrophage phagocytic function in COPD.

Science.gov (United States)

Tran, Hai B; Jersmann, Hubertus; Truong, Tung Thanh; Hamon, Rhys; Roscioli, Eugene; Ween, Miranda; Pitman, Melissa R; Pitson, Stuart M; Hodge, Greg; Reynolds, Paul N; Hodge, Sandra

2017-01-01

We have previously established a link between impaired phagocytic capacity and deregulated S1P signaling in alveolar macrophages from COPD subjects. We hypothesize that this defect may include a disruption of epithelial-macrophage crosstalk via Spns2-mediated intercellular S1P signaling. Primary alveolar macrophages and bronchial epithelial cells from COPD subjects and controls, cell lines, and a mouse model of chronic cigarette smoke exposure were studied. Cells were exposed to 10% cigarette smoke extract, or vehicle control. Spns2 expression and subcellular localization was studied by immunofluorescence, confocal microscopy and RT-PCR. Phagocytosis was assessed by flow-cytometry. Levels of intra- and extracellular S1P were measured by S1P [3H]-labeling. Spns2 expression was significantly increased (pS1P in the airway and that there is a possible disruption of epithelial/macrophage cross talk via Spns2-mediated S1P signaling in COPD and in response to cigarette smoke exposure.

Yersinia pestis Requires Host Rab1b for Survival in Macrophages.

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Michael G Connor

2015-10-01

Full Text Available Yersinia pestis is a facultative intracellular pathogen that causes the disease known as plague. During infection of macrophages Y. pestis actively evades the normal phagosomal maturation pathway to establish a replicative niche within the cell. However, the mechanisms used by Y. pestis to subvert killing by the macrophage are unknown. Host Rab GTPases are central mediators of vesicular trafficking and are commonly targeted by bacterial pathogens to alter phagosome maturation and killing by macrophages. Here we demonstrate for the first time that host Rab1b is required for Y. pestis to effectively evade killing by macrophages. We also show that Rab1b is specifically recruited to the Yersinia containing vacuole (YCV and that Y. pestis is unable to subvert YCV acidification when Rab1b expression is knocked down in macrophages. Furthermore, Rab1b knockdown also altered the frequency of association between the YCV with the lysosomal marker Lamp1, suggesting that Rab1b recruitment to the YCV directly inhibits phagosome maturation. Finally, we show that Rab1b knockdown also impacts the pH of the Legionella pneumophila containing vacuole, another pathogen that recruits Rab1b to its vacuole. Together these data identify a novel role for Rab1b in the subversion of phagosome maturation by intracellular pathogens and suggest that recruitment of Rab1b to the pathogen containing vacuole may be a conserved mechanism to control vacuole pH.

B-1 cells modulate the murine macrophage response to Leishmania major infection.

Science.gov (United States)

Arcanjo, Angelica F; Nunes, Marise P; Silva-Junior, Elias B; Leandro, Monique; da Rocha, Juliana Dutra Barbosa; Morrot, Alexandre; Decote-Ricardo, Debora; Freire-de-Lima, Celio Geraldo

2017-05-26

To investigate the modulatory effect of B-1 cells on murine peritoneal macrophages infected with Leishmania major ( L. major ) in vitro . Peritoneal macrophages obtained from BALB/c and BALB/c XID mice were infected with L. major and cultured in the presence or absence of B-1 cells obtained from wild-type BALB/c mice. Intracellular amastigotes were counted, and interleukin-10 (IL-10) production was quantified in the cellular supernatants using an enzyme-linked immunosorbent assay. The levels of the lipid mediator prostaglandin E2 (PGE 2 ) were determined using a PGE 2 enzyme immunoassay kit (Cayman Chemical, Ann Arbor, MI), and the number of lipid bodies was quantified in the cytoplasm of infected macrophages in the presence and absence of B-1 cells. Culturing the cells with selective PGE 2 -neutralizing drugs inhibited PGE 2 production and confirmed the role of this lipid mediator in IL-10 production. In contrast, we demonstrated that B-1 cells derived from IL-10 KO mice did not favor the intracellular growth of L. major . We report that B-1 cells promote the growth of L. major amastigotes inside peritoneal murine macrophages. We demonstrated that the modulatory effect was independent of physical contact between the cells, suggesting that soluble factor(s) were released into the cultures. We demonstrated in our co-culture system that B-1 cells trigger IL-10 production by L. major -infected macrophages. Furthermore, the increased secretion of IL-10 was attributed to the presence of the lipid mediator PGE 2 in supernatants of L. major -infected macrophages. The presence of B-1 cells also favors the production of lipid bodies by infected macrophages. In contrast, we failed to obtain the same effect on parasite replication inside L. major -infected macrophages when the B-1 cells were isolated from IL-10 knockout mice. Our results show that elevated levels of PGE 2 and IL-10 produced by B-1 cells increase L. major growth, as indicated by the number of parasites in cell

4T1 Murine Mammary Carcinoma Cells Enhance Macrophage-Mediated Innate Inflammatory Responses.

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Laurence Madera

Full Text Available Tumor progression and the immune response are intricately linked. While it is known that cancers alter macrophage inflammatory responses to promote tumor progression, little is known regarding how cancers affect macrophage-dependent innate host defense. In this study, murine bone-marrow-derived macrophages (BMDM were exposed to murine carcinoma-conditioned media prior to assessment of the macrophage inflammatory response. BMDMs exposed to 4T1 mammary carcinoma-conditioned medium demonstrated enhanced production of pro-inflammatory cytokines tumor necrosis factor α, interleukin-6, and CCL2 in response to lipopolysaccharide (LPS while production of interleukin-10 remained unchanged. The increased LPS-induced production of pro-inflammatory cytokines was transient and correlated with enhanced cytokine production in response to other Toll-like receptor agonists, including peptidoglycan and flagellin. In addition, 4T1-conditioned BMDMs exhibited strengthened LPS-induced nitric oxide production and enhanced phagocytosis of Escherichia coli. 4T1-mediated augmentation of macrophage responses to LPS was partially dependent on the NFκB pathway, macrophage-colony stimulating factor, and actin polymerization, as well as the presence of 4T1-secreted extracellular vesicles. Furthermore, peritoneal macrophages obtained from 4T1 tumor-bearing mice displayed enhanced pro-inflammatory cytokine production in response to LPS. These results suggest that uptake of 4T1-secreted factors and actin-mediated ingestion of 4T1-secreted exosomes by macrophages cause a transient enhancement of innate inflammatory responses. Mammary carcinoma-mediated regulation of innate immunity may have significant implications for our understanding of host defense and cancer progression.

Zinc and zinc transporters in macrophages and their roles in efferocytosis in COPD.

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Rhys Hamon

Full Text Available Our previous studies have shown that nutritional zinc restriction exacerbates airway inflammation accompanied by an increase in caspase-3 activation and an accumulation of apoptotic epithelial cells in the bronchioles of the mice. Normally, apoptotic cells are rapidly cleared by macrophage efferocytosis, limiting any secondary necrosis and inflammation. We therefore hypothesized that zinc deficiency is not only pro-apoptotic but also impairs macrophage efferocytosis. Impaired efferocytic clearance of apoptotic epithelial cells by alveolar macrophages occurs in chronic obstructive pulmonary disease (COPD, cigarette-smoking and other lung inflammatory diseases. We now show that zinc is a factor in impaired macrophage efferocytosis in COPD. Concentrations of zinc were significantly reduced in the supernatant of bronchoalveolar lavage fluid of patients with COPD who were current smokers, compared to healthy controls, smokers or COPD patients not actively smoking. Lavage zinc was positively correlated with AM efferocytosis and there was decreased efferocytosis in macrophages depleted of Zn in vitro by treatment with the membrane-permeable zinc chelator TPEN. Organ and cell Zn homeostasis are mediated by two families of membrane ZIP and ZnT proteins. Macrophages of mice null for ZIP1 had significantly lower intracellular zinc and efferocytosis capability, suggesting ZIP1 may play an important role. We investigated further using the human THP-1 derived macrophage cell line, with and without zinc chelation by TPEN to mimic zinc deficiency. There was no change in ZIP1 mRNA levels by TPEN but a significant 3-fold increase in expression of another influx transporter ZIP2, consistent with a role for ZIP2 in maintaining macrophage Zn levels. Both ZIP1 and ZIP2 proteins were localized to the plasma membrane and cytoplasm in normal human lung alveolar macrophages. We propose that zinc homeostasis in macrophages involves the coordinated action of ZIP1 and ZIP2

Dihydro-CDDO-trifluoroethyl amide suppresses inflammatory responses in macrophages via activation of Nrf2

International Nuclear Information System (INIS)

Li, Bin; Abdalrahman, Akram; Lai, Yimu; Janicki, Joseph S.; Ward, Keith W.; Meyer, Colin J.; Wang, Xing Li; Tang, Dongqi; Cui, Taixing

2014-01-01

Highlights: • Dh404 suppresses the expression of a selected set of pro-inflammatory cytokines in inflamed macrophages via activating Nrf2. • Dh404 activates Nrf2 while keeping Keap1 function intact in macrophages. • Dh404 minimally regulates NF-κB pathway in macrophages. - Abstract: Nuclear factor erythroid 2-related factor (Nrf2) is the major regulator of cellular defenses against various pathological stresses in a variety of organ systems, thus Nrf2 has evolved to be an attractive drug target for the treatment and/or prevention of human disease. Several synthetic oleanolic triterpenoids including dihydro-CDDO-trifluoroethyl amide (dh404) appear to be potent activators of Nrf2 and exhibit chemopreventive promises in multiple disease models. While the pharmacological efficacy of Nrf2 activators may be dependent on the nature of Nrf2 activation in specific cell types of target organs, the precise role of Nrf2 in mediating biological effects of Nrf2 activating compounds in various cell types remains to be further explored. Herein we report a unique and Nrf2-dependent anti-inflammatory profile of dh404 in inflamed macrophages. In lipopolysaccharide (LPS)-inflamed RAW264.7 macrophages, dh404 dramatically suppressed the expression of pro-inflammatory cytokines including inducible nitric oxide synthase (iNOS), monocyte chemotactic protein-1 (MCP-1), and macrophage inflammatory protein-1 beta (MIP-1β), while minimally regulating the expression of interleulin-6 (IL-6), IL-1β, and tumor necrosis factor alpha (TNFα). Dh404 potently activated Nrf2 signaling; however, it did not affect LPS-induced NF-κB activity. Dh404 did not interrupt the interaction of Nrf2 with its endogenous inhibitor Kelch-like ECH associating protein 1 (Keap1) in macrophages. Moreover, knockout of Nrf2 blocked the dh404-induced anti-inflammatory responses in LPS-inflamed macrophages. These results demonstrated that dh404 suppresses pro-inflammatory responses in macrophages via an activation

Dihydro-CDDO-trifluoroethyl amide suppresses inflammatory responses in macrophages via activation of Nrf2

Energy Technology Data Exchange (ETDEWEB)

Li, Bin [Shandong University Qilu Hospital Research Center for Cell Therapy, Key Laboratory of Cardiovascular Remodeling and Function Research, Qilu Hospital of Shandong University, Jinan 250012 (China); Department of Cell Biology and Anatomy, University of South Carolina School of Medicine, Columbia, SC 29208 (United States); Abdalrahman, Akram; Lai, Yimu; Janicki, Joseph S. [Department of Cell Biology and Anatomy, University of South Carolina School of Medicine, Columbia, SC 29208 (United States); Ward, Keith W.; Meyer, Colin J. [Department of Pharmacology, Reata Pharmaceuticals, Inc., Irving, TX 75063 (United States); Wang, Xing Li [Shandong University Qilu Hospital Research Center for Cell Therapy, Key Laboratory of Cardiovascular Remodeling and Function Research, Qilu Hospital of Shandong University, Jinan 250012 (China); Tang, Dongqi, E-mail: Dongqi.Tang@uscmed.sc.edu [Shandong University Qilu Hospital Research Center for Cell Therapy, Key Laboratory of Cardiovascular Remodeling and Function Research, Qilu Hospital of Shandong University, Jinan 250012 (China); Department of Cell Biology and Anatomy, University of South Carolina School of Medicine, Columbia, SC 29208 (United States); Cui, Taixing, E-mail: taixing.cui@uscmed.sc.edu [Shandong University Qilu Hospital Research Center for Cell Therapy, Key Laboratory of Cardiovascular Remodeling and Function Research, Qilu Hospital of Shandong University, Jinan 250012 (China); Department of Cell Biology and Anatomy, University of South Carolina School of Medicine, Columbia, SC 29208 (United States)

2014-02-21

Highlights: • Dh404 suppresses the expression of a selected set of pro-inflammatory cytokines in inflamed macrophages via activating Nrf2. • Dh404 activates Nrf2 while keeping Keap1 function intact in macrophages. • Dh404 minimally regulates NF-κB pathway in macrophages. - Abstract: Nuclear factor erythroid 2-related factor (Nrf2) is the major regulator of cellular defenses against various pathological stresses in a variety of organ systems, thus Nrf2 has evolved to be an attractive drug target for the treatment and/or prevention of human disease. Several synthetic oleanolic triterpenoids including dihydro-CDDO-trifluoroethyl amide (dh404) appear to be potent activators of Nrf2 and exhibit chemopreventive promises in multiple disease models. While the pharmacological efficacy of Nrf2 activators may be dependent on the nature of Nrf2 activation in specific cell types of target organs, the precise role of Nrf2 in mediating biological effects of Nrf2 activating compounds in various cell types remains to be further explored. Herein we report a unique and Nrf2-dependent anti-inflammatory profile of dh404 in inflamed macrophages. In lipopolysaccharide (LPS)-inflamed RAW264.7 macrophages, dh404 dramatically suppressed the expression of pro-inflammatory cytokines including inducible nitric oxide synthase (iNOS), monocyte chemotactic protein-1 (MCP-1), and macrophage inflammatory protein-1 beta (MIP-1β), while minimally regulating the expression of interleulin-6 (IL-6), IL-1β, and tumor necrosis factor alpha (TNFα). Dh404 potently activated Nrf2 signaling; however, it did not affect LPS-induced NF-κB activity. Dh404 did not interrupt the interaction of Nrf2 with its endogenous inhibitor Kelch-like ECH associating protein 1 (Keap1) in macrophages. Moreover, knockout of Nrf2 blocked the dh404-induced anti-inflammatory responses in LPS-inflamed macrophages. These results demonstrated that dh404 suppresses pro-inflammatory responses in macrophages via an activation

Chemokines, macrophage inflammatory protein-2 and stromal cell-derived factor-1{alpha}, suppress amyloid {beta}-induced neurotoxicity