FOXP3 renders activated human regulatory T cells resistant to restimulation-induced cell death by suppressing SAP expression.

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Katz, Gil; Voss, Kelsey; Yan, Toria F; Kim, Yong Chan; Kortum, Robert L; Scott, David W; Snow, Andrew L

2018-05-01

Restimulation-induced cell death (RICD) is an apoptotic program that regulates effector T cell expansion, triggered by repeated stimulation through the T cell receptor (TCR) in the presence of interleukin-2 (IL-2). Although CD4 + regulatory T cells (Tregs) consume IL-2 and experience frequent TCR stimulation, they are highly resistant to RICD. Resistance in Tregs is dependent on the forkhead box P3 (FOXP3) transcription factor, although the mechanism remains unclear. T cells from patients with X-linked lymphoproliferative disease (XLP-1), that lack the adaptor molecule SLAM-associated protein (SAP), are also resistant to RICD. Here we demonstrate that normal Tregs express very low levels of SAP compared to conventional T cells. FOXP3 reduces SAP expression by directly binding to and repressing the SH2D1A (SAP) promoter. Indeed, ectopic SAP expression restores RICD sensitivity in human FOXP3 + Tregs. Our findings illuminate the mechanism behind FOXP3-mediated RICD resistance in Tregs, providing new insight into their long-term persistence. Published by Elsevier Inc.

Ability of γδ T cells to modulate the Foxp3 T cell response is dependent on adenosine.

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Dongchun Liang

Full Text Available Whether γδ T cells inhibit or enhance the Foxp3 T cell response depends upon their activation status. The critical enhancing effector in the supernatant is adenosine. Activated γδ T cells express adenosine receptors at high levels, which enables them to deprive Foxp3+ T cells of adenosine, and to inhibit their expansion. Meanwhile, cell-free supernatants of γδ T cell cultures enhance Foxp3 T cell expansion. Thus, inhibition and enhancement by γδ T cells of Foxp3 T cell response are a reflection of the balance between adenosine production and absorption by γδ T cells. Non-activated γδ T cells produce adenosine but bind little, and thus enhance the Foxp3 T cell response. Activated γδ T cells express high density of adenosine receptors and have a greatly increased ability to bind adenosine. Extracellular adenosine metabolism and expression of adenosine receptor A2ARs by γδ T cells played a major role in the outcome of γδ and Foxp3 T cell interactions. A better understanding of the functional conversion of γδ T cells could lead to γδ T cell-targeted immunotherapies for related diseases.

Foxp3-dependent transformation of human primary CD4+ T lymphocytes by the retroviral protein tax.

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Chen, Li; Liu, Dan; Zhang, Yang; Zhang, Huan; Cheng, Hua

2015-10-23

The retroviral Tax proteins of human T cell leukemia virus type 1 and 2 (HTLV-1 and -2) are highly homologous viral transactivators. Both viral proteins can immortalize human primary CD4+ memory T cells, but when expressed alone they rarely transform T cells. In the present study, we found that the Tax proteins displayed a differential ability to immortalize human CD4+Foxp3+ T cells with characteristic expression of CTLA-4 and GITR. Because epidermal growth factor receptor (EGFR) was reportedly expressed and activated in a subset of CD4+Foxp3+ T cells, we introduced an activated EGFR into Tax-immortalized CD4+Foxp3+ T cells. We observed that these modified cells were grown independently of exogenous IL-2, correlating with a T cell transformation phenotype. In Tax-immortalized CD4+Foxp3- T cells, ectopic expression of Foxp3 was a prerequisite for Tax transformation of T cells. Accordingly, treatment of the transformed T cells with erlotinib, a selective inhibitor of EGFR, induced degradation of EGFR in lysosome, consequently causing T cell growth inhibition. Further, we identified autophagy as a crucial cellular survival pathway for the transformed T cells. Silencing key autophagy molecules including Beclin1, Atg5 and PI3 kinase class III (PI3KC3) resulted in drastic impairment of T cell growth. Our data, therefore, unveiled a previously unidentified role of Foxp3 in T cell transformation, providing a molecular basis for HTLV-1 transformation of CD4+Foxp3+ T cells. Copyright © 2015 Elsevier Inc. All rights reserved.

All-trans retinoic acid promotes TGF-β-induced Tregs via histone modification but not DNA demethylation on Foxp3 gene locus.

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Ling Lu

Full Text Available It has been documented all-trans retinoic acid (atRA promotes the development of TGF-β-induced CD4(+Foxp3(+ regulatory T cells (iTreg that play a vital role in the prevention of autoimmune responses, however, molecular mechanisms involved remain elusive. Our objective, therefore, was to determine how atRA promotes the differentiation of iTregs.Addition of atRA to naïve CD4(+CD25(- cells stimulated with anti-CD3/CD28 antibodies in the presence of TGF-β not only increased Foxp3(+ iTreg differentiation, but maintained Foxp3 expression through apoptosis inhibition. atRA/TGF-β-treated CD4(+ cells developed complete anergy and displayed increased suppressive activity. Infusion of atRA/TGF-β-treated CD4(+ cells resulted in the greater effects on suppressing symptoms and protecting the survival of chronic GVHD mice with typical lupus-like syndromes than did CD4(+ cells treated with TGF-β alone. atRA did not significantly affect the phosphorylation levels of Smad2/3 and still promoted iTreg differentiation in CD4(+ cells isolated from Smad3 KO and Smad2 conditional KO mice. Conversely, atRA markedly increased ERK1/2 activation, and blockade of ERK1/2 signaling completely abolished the enhanced effects of atRA on Foxp3 expression. Moreover, atRA significantly increased histone methylation and acetylation within the promoter and conserved non-coding DNA sequence (CNS elements at the Foxp3 gene locus and the recruitment of phosphor-RNA polymerase II, while DNA methylation in the CNS3 was not significantly altered.We have identified the cellular and molecular mechanism(s by which atRA promotes the development and maintenance of iTregs. These results will help to enhance the quantity and quality of development of iTregs and may provide novel insights into clinical cell therapy for patients with autoimmune diseases and those needing organ transplantation.

SA-4-1BBL costimulation inhibits conversion of conventional CD4+ T cells into CD4+ FoxP3+ T regulatory cells by production of IFN-γ.

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Shravan Madireddi

Full Text Available Tumors convert conventional CD4(+ T cells into induced CD4(+CD25(+FoxP3(+ T regulatory (iTreg cells that serve as an effective means of immune evasion. Therefore, the blockade of conventional CD4(+ T cell conversion into iTreg cells represents an attractive target for improving the efficacy of various immunotherapeutic approaches. Using a novel form of 4-1BBL molecule, SA-4-1BBL, we previously demonstrated that costimulation via 4-1BB receptor renders both CD4(+and CD8(+ T effector (Teff cells refractory to inhibition by Treg cells and increased intratumoral Teff/Treg cell ratio that correlated with therapeutic efficacy in various preclinical tumor models. Building on these studies, we herein show for the first time, to our knowledge, that signaling through 4-1BB inhibits antigen- and TGF-β-driven conversion of naïve CD4(+FoxP3(- T cells into iTreg cells via stimulation of IFN-γ production by CD4(+FoxP3(- T cells. Importantly, treatment with SA-4-1BBL blocked the conversion of CD4(+FoxP3(- T cells into Treg cells by EG.7 tumors. Taken together with our previous studies, these results show that 4-1BB signaling negatively modulate Treg cells by two distinct mechanisms: i inhibiting the conversion of CD4(+FoxP3(- T cells into iTreg cells and ii endowing Teff cells refractory to inhibition by Treg cells. Given the dominant role of Treg cells in tumor immune evasion mechanisms, 4-1BB signaling represents an attractive target for favorably tipping the Teff:Treg balance toward Teff cells with important implications for cancer immunotherapy.

Foxp3 regulates ratio of Treg and NKT cells in a mouse model of asthma.

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Lu, Yanming; Guo, Yinshi; Xu, Linyun; Li, Yaqin; Cao, Lanfang

2015-05-01

Chronic inflammatory disorder of the airways causes asthma. Regulatory T cells (Treg cells) and Natural killer T cells (NKT cells) both play critical roles in the pathogenesis of asthma. Activation of Treg cells requires Foxp3, whereas whether Foxp3 may regulate the ratio of Treg and NKT cells to affect asthma is uncertain. In an ovalbumin (OVA)-induced mouse model of asthma, we either increased Treg cells by lentivirus-mediated forced expression of exogenous Foxp3, or increased NKT cells by stimulation with its activator α-GalCer. We found that the CD4+CD25+ Treg cells increased by forced Foxp3 expression, and decreased by α-GalCer, while the CD3+CD161+ NKT cells decreased by forced Foxp3 expression, and increased by α-GalCer. Moreover, forced Foxp3 expression, but not α-GalCer, significantly alleviated the hallmarks of asthma. Furthermore, forced Foxp3 increased levels of IL_10 and TGFβ1, and α-GalCer increased levels of IL_4 and INFγ in the OVA-treated lung. Taken together, our study suggests that Foxp3 may activate Treg cells and suppress NKT cells in asthma. Treg and NKT cells may antagonize the effects of each other in asthma.

FOXP3 is a novel transcriptional repressor for the breast cancer oncogene SKP2.

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Zuo, Tao; Liu, Runhua; Zhang, Huiming; Chang, Xing; Liu, Yan; Wang, Lizhong; Zheng, Pan; Liu, Yang

2007-12-01

S-phase kinase-associated protein 2 (SKP2) is a component of the E3 ubiquitin ligase SKP1-Cul1-Fbox complex. Overexpression of SKP2 results in cell cycle dysregulation and carcinogenesis; however, the genetic lesions that cause this upregulation are poorly understood. We recently demonstrated that forkhead box P3 (FOXP3) is an X-linked breast cancer suppressor and an important repressor of the oncogene ERBB2/HER2. Since FOXP3 suppresses tumor growth regardless of whether the tumors overexpress ERBB2/HER2, additional FOXP3 targets may be involved in its tumor suppressor activity. Here, we show that mammary carcinomas from mice heterozygous for a Foxp3 mutation exhibited increased Skp2 expression. Ectopic expression of FOXP3 in mouse mammary cancer cells repressed SKP2 expression with a corresponding increase in p27 and polyploidy. Conversely, siRNA silencing of the FOXP3 gene in human mammary epithelial cells increased SKP2 expression. We also show that Foxp3 directly interacted with and repressed the Skp2 promoter. Moreover, the analysis of over 200 primary breast cancer samples revealed an inverse correlation between FOXP3 and SKP2 levels. Finally, we demonstrated that downregulation of SKP2 was critical for FOXP3-mediated growth inhibition in breast cancer cells that do not overexpress ERBB2/HER2. Our data provide genetic, biochemical, and functional evidence that FOXP3 is a novel transcriptional repressor for the oncogene SKP2.

Methionine enkephalin (MENK) inhibits tumor growth through regulating CD4+Foxp3+ regulatory T cells (Tregs) in mice.

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Li, Xuan; Meng, Yiming; Plotnikoff, Nicolas P; Youkilis, Gene; Griffin, Noreen; Wang, Enhua; Lu, Changlong; Shan, Fengping

2015-01-01

Methionine enkephalin (MENK), an endogenous neuropeptide, plays an crucial role in both neuroendocrine and immune systems. CD4+Foxp3+ regulatory T cells (Tregs) are identified as a major subpopulation of T lymphocytes in suppressing immune system to keep balanced immunity. The aim of this research work was to elucidate the mechanisms via which MENK interacts with Tregs in cancer situation. The influence of MENK on transforming growth factor-β (TGF-β) mediated conversion from naïve CD4+CD25- T cells to CD4+CD25+ Tregs was determined and the data from flow cytometry (FCM) analysis indicated that MENK effectively inhibited the expression of Foxp3 during the process of TGF-βinduction. Furthermore, this inhibiting process was accompanied by diminishing phosphorylation and nuclear translocation of Smad2/3, confirmed by western blot (WB) analysis and immunofluorescence (IF) at molecular level. We established sarcoma mice model with S180 to investigate whether MENK could modulate Tregs in tumor circumstance. Our findings showed that MENK delayed the development of tumor in S180 tumor bearing mice and down-regulated level of Tregs. Together, these novel findings reached a conclusion that MENK could inhibit Tregs activity directly and retard tumor development through down-regulating Tregs in mice. This work advances the deepening understanding of the influence of MENK on Tregs in cancer situation, and relation of MENK with immune system, supporting the implication of MENK as a new strategy for cancer immunotherapy.

FOXP3-specific immunity

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Andersen, Mads Hald

2013-01-01

Forkhead box P3 (FOXP3)-specific cytotoxic CD8(+) T cells are present among human peripheral blood mononuclear cells (PBMCs), especially in cancer patients. Such T lymphocytes are able not only to specifically recognize dendritic cells (DCs) that have been exposed to recombinant FOXP3 and regulat...... and regulatory T cells, but also to kill FOXP3(+) malignant T cells. The natural occurrence of FOXP3-specific cytotoxic T lymphocytes among human PBMCs suggests a general role for these cells in the complex network of immune regulation....

Foxp3 Expression is Required for the Induction of Therapeutic Tissue Tolerance1

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Regateiro, Frederico S.; Chen, Ye; Kendal, Adrian R.; Hilbrands, Robert; Adams, Elizabeth; Cobbold, Stephen P.; Ma, Jianbo; Andersen, Kristian G.; Betz, Alexander G.; Zhang, Mindy; Madhiwalla, Shruti; Roberts, Bruce; Waldmann, Herman; Nolan, Kathleen F.; Howie, Duncan

2012-01-01

CD4+Foxp3+ Treg are essential for immune homeostasis and maintenance of self-tolerance. They are produced in the thymus and also generated de novo in the periphery in a TGFβ dependent manner. Foxp3+ Treg are also required to achieve tolerance to transplanted tissues when induced by co receptor or co stimulation blockade. Using TCR transgenic mice to avoid issues of autoimmune pathology, we show that Foxp3 expression is both necessary and sufficient for tissue tolerance by coreceptor blockade. Moreover, the known need in tolerance induction for TGFβ signalling to T cells can wholly be explained by its role in induction of Foxp3, as such signalling proved dispensable for the suppressive process. We analysed the relative contribution of TGFβ and Foxp3 to the transcriptome of TGFβ-induced Treg and showed that TGFβ elicited a large set of down-regulated signature genes. The number of genes uniquely modulated due to the influence of Foxp3 alone was surprisingly limited. Thus, despite the large genetic influence of TGFβ exposure on iTreg, the crucial Foxp3-influenced signature independent of TGFβ is small. Retroviral mediated conditional nuclear expression of Foxp3 proved sufficient to confer transplant-suppressive potency on CD4+ T cells, and was lost once nuclear Foxp3 expression was extinguished. These data support a dual role for TGFβ and Foxp3 in induced tolerance, where TGFβ stimulates Foxp3 expression, whose sustained expression is then associated with acquisition of tolerance. PMID:22988034

Changes in natural Foxp3(+Treg but not mucosally-imprinted CD62L(negCD38(+Foxp3(+Treg in the circulation of celiac disease patients.

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Marieke A van Leeuwen

Full Text Available BACKGROUND: Celiac disease (CD is an intestinal inflammation driven by gluten-reactive CD4(+ T cells. Due to lack of selective markers it has not been determined whether defects in inducible regulatory T cell (Treg differentiation are associated with CD. This is of importance as changes in numbers of induced Treg could be indicative of defects in mucosal tolerance development in CD. Recently, we have shown that, after encounter of retinoic acid during differentiation, circulating gut-imprinted T cells express CD62L(negCD38(+. Using this new phenotype, we now determined whether alterations occur in the frequency of natural CD62L(+Foxp3(+ Treg or mucosally-imprinted CD62L(negCD38(+Foxp3(+ Treg in peripheral blood of CD patients. In particular, we compared pediatric CD, aiming to select for disease at onset, with adult CD. METHODS: Cell surface markers, intracellular Foxp3 and Helios were determined by flow cytometry. Foxp3 expression was also detected by immunohistochemistry in duodenal tissue of CD patients. RESULTS: In children, the percentages of peripheral blood CD4(+Foxp3(+ Treg were comparable between CD patients and healthy age-matched controls. Differentiation between natural and mucosally-imprinted Treg on the basis of CD62L and CD38 did not uncover differences in Foxp3. In adult patients on gluten-free diet and in refractory CD increased percentages of circulating natural CD62L(+Foxp3(+ Treg, but normal mucosally-imprinted CD62L(negCD38(+Foxp3(+ Treg frequencies were observed. CONCLUSIONS: Our data exclude that significant numeric deficiency of mucosally-imprinted or natural Foxp3(+ Treg explains exuberant effector responses in CD. Changes in natural Foxp3(+ Treg occur in a subset of adult patients on a gluten-free diet and in refractory CD patients.

Protein Arginine Methyltransferase 5 Inhibition Upregulates Foxp3+ Regulatory T Cells Frequency and Function during the Ulcerative Colitis

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Yingxia Zheng

2017-05-01

Full Text Available Ulcerative colitis (UC pathogenesis is related to imbalance of immune responses, and the equilibrium between inflammatory T cells and Foxp3+ regulatory T cells (Tregs plays an important role in the intestinal homeostasis. Protein arginine methyltransferases (PRMTs regulate chromatin remodeling and gene expression. Here, we investigated whether inhibition of PRMTs affects colitis pathogenesis in mice and inflammatory bowel disease patients and further explored the underlying mechanisms. In this study, we found that protein arginine N-methyltransferase inhibitor 1 (AMI-1 treatments increased Tregs frequency, function, and reduced colitis incidence. Adoptive transfer of AMI-1-treated Tregs could reduce the colitis incidence. Colitis was associated with increased local PRMT5 expression, which was inhibited by AMI-1 treatment. Additionally, PRMT5 knockdown T cells produced a better response to TGFβ and promoted Tregs differentiation through decreased DNA methyltransferase 1 (DNMT1 expression. PRMT5 also enhanced H3K27me3 and DNMT1 binding to Foxp3 promoter, which restricted Tregs differentiation. Furthermore, PRMT5 knockdown led to decreased Foxp3 promoter methylation during Tregs induction. PRMT5 expression had a negative relationship with Tregs in UC patients, knockdown of PRMT5 expression increased Tregs frequency and decreased TNFα, IL-6, and IL-13 levels. Our study outlines a novel regulation of PRMT5 on Tregs development and function. Strategies to decrease PRMT5 expression might have therapeutic potential to control UC.

Allogeneic effector/memory Th-1 cells impair FoxP3+ regulatory T lymphocytes and synergize with chaperone-rich cell lysate vaccine to treat leukemia.

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Janikashvili, Nona; LaCasse, Collin J; Larmonier, Claire; Trad, Malika; Herrell, Amanda; Bustamante, Sara; Bonnotte, Bernard; Har-Noy, Michael; Larmonier, Nicolas; Katsanis, Emmanuel

2011-02-03

Therapeutic strategies combining the induction of effective antitumor immunity with the inhibition of the mechanisms of tumor-induced immunosuppression represent a key objective in cancer immunotherapy. Herein we demonstrate that effector/memory CD4(+) T helper-1 (Th-1) lymphocytes, in addition to polarizing type-1 antitumor immune responses, impair tumor-induced CD4(+)CD25(+)FoxP3(+) regulatory T lymphocyte (Treg) immunosuppressive function in vitro and in vivo. Th-1 cells also inhibit the generation of FoxP3(+) Tregs from naive CD4(+)CD25(-)FoxP3(-) T cells by an interferon-γ-dependent mechanism. In addition, in an aggressive mouse leukemia model (12B1), Th-1 lymphocytes act synergistically with a chaperone-rich cell lysate (CRCL) vaccine, leading to improved survival and long-lasting protection against leukemia. The combination of CRCL as a source of tumor-specific antigens and Th-1 lymphocytes as an adjuvant has the potential to stimulate efficient specific antitumor immunity while restraining Treg-induced suppression.

TGF-beta and 'adaptive' Foxp3(+) regulatory T cells.

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Chen, Wanjun; Konkel, Joanne E

2010-02-01

In naïve T cells transforming growth factor-beta (TGF-beta) induces Foxp3, a transcription factor essential for programming and developing T regulatory cells (Treg cells). This finding reveals a physiological factor which can turn on the Foxp3 gene and establishes an experimental approach to induce antigen-specific Treg cells as a potential therapy for human diseases. While this role for TGF-beta is well confirmed, several critical questions remain largely unanswered and await further investigation. In this regard, it is imperative to understand the molecular pathways by which TGF-beta signaling initiates and regulates Foxp3 expression. It is also important to elucidate which factors and/or cytokines influence the TGF-beta-mediated conversion of naïve T cells and how to create an immunologically regulatory milieu to facilitate Treg cell generation in vivo. In this short article, we will highlight the key findings and recent progress in the field, discuss the molecular mechanisms underlying the TGF-beta-mediated induction of Foxp3, and attempt to outline the challenges ahead.

Calycosin Inhibits the Migration and Invasion of Human Breast Cancer Cells by Down-Regulation of Foxp3 Expression

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Shuangxi Li

2017-12-01

Full Text Available Background/Aims: Calycosin, a phytoestrogenic compound, has recently emerged as a promising antitumor drug. It has been shown that calycosin suppresses growth and induces apoptosis of breast cancer cells. However, the effect of calycosin on migration and invasion of breast cancer cells and the underlying molecular mechanisms have not been elucidated. Methods: Human breast cancer cells MCF-7 and T47D were treated with, or without, different doses (0, 6.25, 12.5, 25, 50, 100 or 150 μM of calycosin, and the viability of different groups was determined by MTT assay. Next, the inhibitory effect of higher doses (50, 100 or 150 μM of calycosin on migration and invasion of the two cell lines was determined by wound healing and transwell assay. The relative expression levels of forkhead box P3 (Foxp3, vascular endothelial growth factor (VEGF and matrix metalloproteinase-9 (MMP-9 in MCF-7 and T47D cells were determined by quantitative RT-PCR and Western blot. Results: Treatment with lower doses (6.25 or 12.5 μM promoted proliferation of breast cancer cells, but with higher doses significantly reduced the viability of MCF-7 and T47D cells. Furthermore, higher doses of calycosin were found to inhibit migration and invasion of the two cell lines in a dose-dependent manner. Additionally, treatment with a higher dose of calycosin significantly reduced the expression levels of Foxp3, followed by down-regulation of VEGF and MMP-9 in both MCF-7 and T47D breast cancer cells. Conclusion: Treatment with a higher dose of calycosin tends to reduce migration and invasion capacity of human breast cancer cells, by targeting Foxp3-mediated VEGF and MMP-9 expression.

FOXP3 Expression in GARP-Transduced Helper T Cells Is Not Associated with FOXP3 TSDR Demethylation.

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Kehrmann, Jan; Zeschnigk, Michael; Buer, Jan; Probst-Kepper, Michael

2011-10-01

AIM: Glycoprotein A repetitions predominant (GARP or LRRC32) represents a human regulatory CD4+ CD25(hi) FOXP3+ T (T(reg)) cell-specific receptor that controls FOXP3. Ectopic expression of GARP in helper T (T(h)) cells has been shown to be sufficient for the induction of FOXP3 and generation of a stable regulatory phenotype. Since expression of FOXP3 in Treg cells is epigenetically controlled by a conserved motif, the so-called T(reg)-specific demethylated region (TSDR), we asked whether GARP-mediated upregulation of FOXP3 in Th cells is similarly accompanied by demethylation of the TSDR. METHODS: DNA methylation of the FOXP3 TSDR was analyzed by direct sequencing of polymerase chain reaction (PCR) products from bisulfite-treated genomic DNA. RESULTS: Although GARP-transduced T(h) cells exhibit constitutive FOXP3 expression and a regulatory phenotype, the FOXP3 TSDR is completely methylated as in naive T(h) cells. GARP-mediated FOXP3 upregulation in T(h) cells is not associated with T(reg)-specific demethylation of the FOXP3 TSDR. CONCLUSION: Although GARP-engineered T(h) cells exhibit stable FOXP3 expression and a phenotypic reprogramming towards T(reg) cells in vitro, these cells do not completely mimic the epigenotype of natural T(reg) cells. Thus, concepts based on the genetic modification of T(h) cells as cellular therapies to treat autoimmune diseases or to control transplantation tolerance should be critically tested before any clinical application.

Kaempferol enhances the suppressive function of Treg cells by inhibiting FOXP3 phosphorylation.

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Lin, Fang; Luo, Xuerui; Tsun, Andy; Li, Zhiyuan; Li, Dan; Li, Bin

2015-10-01

Kaempferol is a natural flavonoid found in many vegetables and fruits. Epidemiologic studies have described that Kaempferol intake could reduce risk of cancer, especially lung, gastric, pancreatic and ovarian cancers. Recent studies have shown that Kaempferol could also be beneficial to the body to defend against inflammation, and infection by bacteria and viruses; however, the molecular mechanism of its immunoregulatory function remains largely unknown. Through screening a small molecule library of traditional Chinese medicine (TCM), we identified that Kaempferol could enhance the suppressive function of regulatory T cells (Tregs). Kaempferol was found to increase FOXP3 expression level in Treg cells and prevent pathological symptoms of collagen-induced arthritis in a rat animal model. Kaempferol could also reduce PIM1-mediated FOXP3 phosphorylation at S422. Our study reveals a molecular mechanism that underlies the anti-inflammatory action of Kaempferol for the prevention and treatment of inflammatory diseases such as rheumatoid arthritis, systemic lupus erythematosus, and ankylosing spondylitis. Copyright © 2015 Elsevier B.V. All rights reserved.

Transgene IL-6 Enhances DC-Stimulated CTL Responses by Counteracting CD4+25+Foxp3+ Regulatory T Cell Suppression via IL-6-Induced Foxp3 Downregulation

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Kalpana Kalyanasundaram Bhanumathy

2014-03-01

Full Text Available Dendritic cells (DCs, the most potent antigen-presenting cells have been extensively applied in clinical trials for evaluation of antitumor immunity. However, the efficacy of DC-mediated cancer vaccines is still limited as they are unable to sufficiently break the immune tolerance. In this study, we constructed a recombinant adenoviral vector (AdVIL-6 expressing IL-6, and generated IL-6 transgene-engineered DC vaccine (DCOVA/IL-6 by transfection of murine bone marrow-derived ovalbumin (OVA-pulsed DCs (DCOVA with AdVIL-6. We then assessed DCOVA/IL-6-stimulated cytotoxic T-lymphocyte (CTL responses and antitumor immunity in OVA-specific animal tumor model. We demonstrate that DCOVA/IL-6 vaccine up-regulates expression of DC maturation markers, secretes transgene-encoded IL-6, and more efficiently stimulates OVA-specific CTL responses and therapeutic immunity against OVA-expressing B16 melanoma BL6-10OVA in vivo than the control DCOVA/Null vaccine. Moreover, DCOVA/IL-6-stimulated CTL responses were relatively maintained in mice with transfer of CD4+25+Foxp3+ Tr-cells, but significantly reduced when treated with anti-IL-6 antibody. In addition, we demonstrate that IL-6 down-regulates Foxp3-expression of CD4+25+Foxp3+ Tr-cells in vitro. Taken together, our results demonstrate that AdV-mediated IL-6 transgene-engineered DC vaccine stimulates potent CTL responses and antitumor immunity by counteracting CD4+25+ Tr immunosuppression via IL-6-induced Foxp3 down-regulation. Thus, IL-6 may be a good candidate for engineering DCs for cancer immunotherapy.

Zinc supplementation induces CD4+CD25+Foxp3+ antigen-specific regulatory T cells and suppresses IFN-γ production by upregulation of Foxp3 and KLF-10 and downregulation of IRF-1.

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Maywald, Martina; Rink, Lothar

2017-08-01

The essential trace element zinc plays a fundamental role in immune function and regulation since its deficiency is associated with autoimmunity, allergies, and transplant rejection. Thus, we investigated the influence of zinc supplementation on the Th1-driven alloreaction in mixed lymphocyte cultures (MLC), on generation of antigen-specific T cells, and analyzed underlying molecular mechanisms. Cell proliferation and pro-inflammatory cytokine production were monitored by [ 3 H]-thymidine proliferation assay and ELISA, respectively. Analysis of surface and intracellular T cell marker was performed by flow cytometry. Western blotting and mRNA analysis were used for Foxp3, KLF-10, and IRF-1 expression. Zinc supplementation on antigen-specific T cells in physiological doses (50 µM) provokes a significant amelioration of cell proliferation and pro-inflammatory cytokine production after reactivation compared to untreated controls. Zinc administration on MLC results in an increased induction and stabilization of CD4 + CD25 + Foxp3 + and CD4 + CD25 + CTLA-4 + T cells (p zinc-induced upregulation of Foxp3 and KLF-10 and downregulation of IRF-1. However, in resting lymphocytes zinc increases IRF-1. In summary, zinc is capable of ameliorating the allogeneic immune reaction by enhancement of antigen-specific iTreg cells due to modulation of essential molecular targets: Foxp3, KLF-10, and IRF-1. Thus, zinc can be seen as an auspicious tool for inducing tolerance in adverse immune reactions.

Plasmodium falciparum-mediated induction of human CD25Foxp3 CD4 T cells is independent of direct TCR stimulation and requires IL-2, IL-10 and TGFbeta.

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Anja Scholzen

2009-08-01

Full Text Available CD4(+CD25(+Foxp3(+ regulatory T cells (Tregs regulate disease-associated immunity and excessive inflammatory responses, and numbers of CD4(+CD25(+Foxp3(+ Tregs are increased during malaria infection. The mechanisms governing their generation, however, remain to be elucidated. In this study we investigated the role of commonly accepted factors for Foxp3 induction, TCR stimulation and cytokines such as IL-2, TGFbeta and IL-10, in the generation of human CD4(+CD25(+Foxp3(+ T cells by the malaria parasite Plasmodium falciparum. Using a co-culture system of malaria-infected red blood cells (iRBCs and peripheral blood mononuclear cells from healthy individuals, we found that two populations of Foxp3(hi and Foxp3(int CD4(+CD25(hi T cells with a typical Treg phenotype (CTLA-4(+, CD127(low, CD39(+, ICOS(+, TNFRII(+ were induced. Pro-inflammatory cytokine production was confined to the Foxp3(int subset (IFNgamma, IL-4 and IL-17 and inversely correlated with high relative levels of Foxp3(hi cells, consistent with Foxp3(hi CD4 T cell-mediated inhibition of parasite-induced effector cytokine T cell responses. Both Foxp3(hi and Foxp3(int cells were derived primarily from proliferating CD4(+CD25(- T cells with a further significant contribution from CD25(+Foxp3(+ natural Treg cells to the generation of the Foxp3(hi subset. Generation of Foxp3(hi, but not Foxp3(int, cells specifically required TGFbeta1 and IL-10. Add-back experiments showed that monocytes expressing increased levels of co-stimulatory molecules were sufficient for iRBC-mediated induction of Foxp3 in CD4 T cells. Foxp3 induction was driven by IL-2 from CD4 T cells stimulated in an MHC class II-dependent manner. However, transwell separation experiments showed that direct contact of monocytes with the cells that acquire Foxp3 expression was not required. This novel TCR-independent and therefore antigen-non specific mechanism for by-stander CD4(+CD25(hiFoxp3(+ cell induction is likely to reflect a

THE RELATIONSHIP OF FoxP3+ T REGULATORY CELLS TO DISEASE ACTIVITY AND ANTIBODY LEVELS IN EARLY RHEUMATOID ARTHRITIS

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A. S. Avdeeva

2017-01-01

Full Text Available Objective: to analyze the relationship of the count of FoxP3+ T regulatory cells (Tregs to the clinical and laboratory parameters of disease activity and the levels of antibodies in a group of patients with early rheumatoid arthritis (RA.Subjects and methods. The investigation enrolled 45 patients with early RA (2010 ACR/EULAR criteria who had not previously received treatment with methotrexate, including 39 women; median age was 52.0 [32.5; 57.5] years; disease duration, 5 [4; 6] months, DAS28 5.01 [4.18; 5.8]; 71.1% of the patients were rheumatoid factor (RF positive and 88.9% were anti-cyclic citrullinated peptide positive. The relative and absolute counts of Treg (FoxP3+CD25+; CD152+surface; CD152+intracellular; FoxP3+CD127-; CD25+CD127-; FoxP3+ICOS+; FoxP3+CD154+; FoxP3+CD274+ were measured by immunofluorescence staining and multicolor flow cytometry. A control group consisted of 20 healthy donors who were matched for sex and age with the examined patients.Results and discussion. DАS28 was high, moderate, and low in 22 (48.9%, 20 (44.4%, and 3 (6.7% patients, respectively. As compared with the healthy donors, the patients with early RA were observed to have lower values in the percentage of FoxP3+CD25+ cells, in the percentage and absolute count of FoxP3+ICOS+ cells, in the percentage and absolute count of FoxP3+CD154+ and FoxP3+ CD274+ T cells; p<0.05 in all cases. Negative correlation was recorded between the percentage of FoxP3+CD25+ and C-reactive protein (CRP (r=-0.4; that of CD152+intracellular and DAS28 (r=-0.35, ESR (r=-0.46, CRP (r=-0.54; that of FoxP3+CD127 and CRP (r=-0.42; that of CD25+CD127 and DAS28 (r=-0.38, SDAI (r=-0.41, CDAI (r=-0.36, ESR (r=-0.39, CRP (r=-0.47; p<0.05 in all cases.The patients who were seronegative for RF were found to have higher values in the percentage of CD25+CD127, in the percentage and absolute count of Foxp3+CD154+ and Foxp3+CD274+ T lymphocytes.Conclusion. The given data may indicate that the

CD4+CD62L+ Central Memory T Cells Can Be Converted to Foxp3+ T Cells

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Zhang, Xiaolong; Chang Li, Xian; Xiao, Xiang; Sun, Rui; Tian, Zhigang; Wei, Haiming

2013-01-01

The peripheral Foxp3+ Treg pool consists of naturally arising Treg (nTreg) and adaptive Treg cells (iTreg). It is well known that naive CD4+ T cells can be readily converted to Foxp3+ iTreg in vitro, and memory CD4+ T cells are resistant to conversion. In this study, we investigated the induction of Foxp3+ T cells from various CD4+ T-cell subsets in human peripheral blood. Though naive CD4+ T cells were readily converted to Foxp3+ T cells with TGF-β and IL-2 treatment in vitro, such Foxp3+ T cells did not express the memory marker CD45RO as do Foxp3+ T cells induced in the peripheral blood of Hepatitis B Virus (HBV) patients. Interestingly, a subset of human memory CD4+ T cells, defined as CD62L+ central memory T cells, could be induced by TGF-β to differentiate into Foxp3+ T cells. It is well known that Foxp3+ T cells derived from human CD4+CD25- T cells in vitro are lack suppressive functions. Our data about the suppressive functions of CD4+CD62L+ central memory T cell-derived Foxp3+ T cells support this conception, and an epigenetic analysis of these cells showed a similar methylation pattern in the FOXP3 Treg-specific demethylated region as the naive CD4+ T cell-derived Foxp3+ T cells. But further research showed that mouse CD4+ central memory T cells also could be induced to differentiate into Foxp3+ T cells, such Foxp3+ T cells could suppress the proliferation of effector T cells. Thus, our study identified CD4+CD62L+ central memory T cells as a novel potential source of iTreg. PMID:24155942

Immunologic targeting of FOXP3 in inflammatory breast cancer cells.

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Smita Nair

Full Text Available The forkhead transcription factor FOXP3 is necessary for induction of regulatory T lymphocytes (Tregs and their immunosuppressive function. We have previously demonstrated that targeting Tregs by vaccination of mice with murine FOXP3 mRNA-transfected dendritic cells (DCs elicits FOXP3-specific T cell responses and enhances tumor immunity. It is clear that FOXP3 expression is not restricted to T-cell lineage and herein, using RT-PCR, flow cytometry, and western immunoblot we demonstrate for the first time that FOXP3 is expressed in inflammatory breast cancer (IBC cells, SUM149 (triple negative, ErbB1-activated and SUM190 (ErbB2-overexpressing. Importantly, FOXP3-specific T cells generated in vitro using human FOXP3 RNA-transfected DCs as stimulators efficiently lyse SUM149 cells. Interestingly, an isogenic model (rSUM149 derived from SUM149 with an enhanced anti-apoptotic phenotype was resistant to FOXP3-specific T cell mediated lysis. The MHC class I cellular processing mechanism was intact in both cell lines at the protein and transcription levels suggesting that the resistance to cytolysis by rSUM149 cells was not related to MHC class I expression or to the MHC class I antigen processing machinery in these cells. Our data suggest that FOXP3 may be an effective tumor target in IBC cells however increased anti-apoptotic signaling can lead to immune evasion.

CD147 (Basigin/Emmprin) identifies FoxP3+CD45RO+CTLA4+-activated human regulatory T cells.

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Solstad, Therese; Bains, Simer Jit; Landskron, Johannes; Aandahl, Einar Martin; Thiede, Bernd; Taskén, Kjetil; Torgersen, Knut Martin

2011-11-10

Human CD4(+)FoxP3(+) T cells are functionally and phenotypically heterogeneous providing plasticity to immune activation and regulation. To better understand the functional dynamics within this subset, we first used a combined strategy of subcellular fractionation and proteomics to describe differences at the protein level between highly purified human CD4(+)CD25(+) and CD4(+)CD25(-) T-cell populations. This identified a set of membrane proteins highly expressed on the cell surface of human regulatory T cells (Tregs), including CD71, CD95, CD147, and CD148. CD147 (Basigin or Emmprin) divided CD4(+)CD25(+) cells into distinct subsets. Furthermore, CD147, CD25, FoxP3, and in particular CTLA-4 expression correlated. Phenotypical and functional analyses suggested that CD147 marks the switch between resting (CD45RA(+)) and activated (CD45RO(+)) subsets within the FoxP3(+) T-cell population. Sorting of regulatory T cells into CD147(-) and CD147(+) populations demonstrated that CD147 identifies an activated and highly suppressive CD45RO(+) Treg subset. When analyzing CD4(+) T cells for their cytokine producing potential, CD147 levels grouped the FoxP3(+) subset into 3 categories with different ability to produce IL-2, TNF-α, IFN-γ, and IL-17. Together, this suggests that CD147 is a direct marker for activated Tregs within the CD4(+)FoxP3(+) subset and may provide means to manipulate cells important for immune homeostasis.

Foxp3 expression in human cancer cells

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Gourgoulianis Konstantinos I

2008-04-01

Full Text Available Abstract Objective Transcription factor forkhead box protein 3 (Foxp3 specifically characterizes the thymically derived naturally occurring regulatory T cells (Tregs. Limited evidence indicates that it is also expressed, albeit to a lesser extent, in tissues other than thymus and spleen, while, very recently, it was shown that Foxp3 is expressed by pancreatic carcinoma. This study was scheduled to investigate whether expression of Foxp3 transcripts and mature protein occurs constitutively in various tumor types. Materials and methods Twenty five tumor cell lines of different tissue origins (lung cancer, colon cancer, breast cancer, melanoma, erythroid leukemia, acute T-cell leukemia were studied. Detection of Foxp3 mRNA was performed using both conventional RT-PCR and quantitative real-time PCR while protein expression was assessed by immunocytochemistry and flow cytometry, using different antibody clones. Results Foxp3 mRNA as well as Foxp3 protein was detected in all tumor cell lines, albeit in variable levels, not related to the tissue of origin. This expression correlated with the expression levels of IL-10 and TGFb1. Conclusion We offer evidence that Foxp3 expression, characterizes tumor cells of various tissue origins. The biological significance of these findings warrants further investigation in the context of tumor immune escape, and especially under the light of current anti-cancer efforts interfering with Foxp3 expression.

The importance of Foxp3 antibody and fixation/permeabilization buffer combinations in identifying CD4+CD25+Foxp3+ regulatory T cells.

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Law, Jacqueline P; Hirschkorn, Dale F; Owen, Rachel E; Biswas, Hope H; Norris, Philip J; Lanteri, Marion C

2009-12-01

Foxp3 is a key marker for CD4(+) regulatory T cells (T(regs)) and was used in developing a multiparameter flow cytometric panel to identify T(regs). Achieving reproducible staining and analysis first required optimization of Foxp3 staining. We present a comparative study of PCH101, 236A/E7, 3G3, 206D, 150D, and 259D/C7 clones of anti-human-Foxp3 antibodies used in combination with five different fixation/permeabilization buffers. Staining for CD25, CD152, and CD127 was also compared between fixation/permeabilization treatments. Promising antibody/buffer combinations were tested in a panel of peripheral blood mononuclear cells from 10 individuals, and then on fresh versus frozen cells from four individuals. Finally, different fluorochromes coupled to two representative antibodies were compared to optimize separation of Foxp3(+) from Foxp3(-) events. Foxp3 gates were set using two gating strategies based on CD127(+)CD25(-) "non-T(regs)" or based on isotype controls. For Foxp3 staining, the best conditions for fixation/permeabilization were obtained using the eBioscience Foxp3, Imgenex, BioLegend, and BD Foxp3 buffers. Comparing results from 10 subjects, 259D/C7, PCH101, 236A/E7, and 206D antibodies yielded statistically higher levels of Foxp3 cells than those by 150D and 3G3 antibodies (mean = 6.9, 5.1, 4.7, and 3.7% compared with 1.7, and 0.3% of CD25(+)Foxp3(+) events within CD4(+) cells, respectively). Importantly, the "nonspecificity" of some antibodies observed with a Foxp3 gate based on isotype controls could be eliminated by setting the Foxp3 gate on "non-T(regs)". Better separation of Foxp3(+) and Foxp3(-) populations was observed using the PCH101 clone coupled to Alexa647 compared with FITC or the 259D/C7 clone coupled to PE compared with Alexa488 fluorochrome. Foxp3 staining can be highly variable and depends on the choice of antibody/buffer pair and the fluorochrome used. Selecting the correct population for setting the Foxp3 gate is critical to avoid

Dasatinib inhibits both osteoclast activation and prostate cancer PC-3-cell-induced osteoclast formation.

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Araujo, John C; Poblenz, Ann; Corn, Paul; Parikh, Nila U; Starbuck, Michael W; Thompson, Jerry T; Lee, Francis; Logothetis, Christopher J; Darnay, Bryant G

2009-11-01

Therapies to target prostate cancer bone metastases have only limited effects. New treatments are focused on the interaction between cancer cells, bone marrow cells and the bone matrix. Osteoclasts play an important role in the development of bone tumors caused by prostate cancer. Since Src kinase has been shown to be necessary for osteoclast function, we hypothesized that dasatinib, a Src family kinase inhibitor, would reduce osteoclast activity and prostate cancer (PC-3) cell-induced osteoclast formation. Dasatinib inhibited RANKL-induced osteoclast differentiation of bone marrow-derived monocytes with an EC(50) of 7.5 nM. PC-3 cells, a human prostate cancer cell line, were able to differentiate RAW 264.7 cells, a murine monocytic cell line, into osteoclasts, and dasatinib inhibited this differentiation. In addition, conditioned medium from PC-3 cell cultures was able to differentiate RAW 264.7 cells into osteoclasts and this too, was inhibited by dasatinib. Even the lowest concentration of dasatinib, 1.25 nmol, inhibited osteoclast differentiation by 29%. Moreover, dasatinib inhibited osteoclast activity by 58% as measured by collagen 1 release. We performed in vitro experiments utilizing the Src family kinase inhibitor dasatinib to target osteoclast activation as a means of inhibiting prostate cancer bone metastases. Dasatinib inhibits osteoclast differentiation of mouse primary bone marrow-derived monocytes and PC-3 cell-induced osteoclast differentiation. Dasatinib also inhibits osteoclast degradation activity. Inhibiting osteoclast differentiation and activity may be an effective targeted therapy in patients with prostate cancer bone metastases.

circ-SHKBP1 Regulates the Angiogenesis of U87 Glioma-Exposed Endothelial Cells through miR-544a/FOXP1 and miR-379/FOXP2 Pathways

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Qianru He

2018-03-01

Full Text Available Circular RNAs (circRNAs are a type of endogenous non-coding RNAs, which have been considered to mediate diverse tumorigenesis including angiogenesis. The present study aims to elucidate the potential role and molecular mechanism of circ-SHKBP1 in regulating the angiogenesis of U87 glioma-exposed endothelial cells (GECs. The expression of circ-SHKBP1, but not linear SHKBP1, was significantly upregulated in GECs compared with astrocyte-exposed endothelial cells (AECs. circ-SHKBP1 knockdown inhibited the viability, migration, and tube formation of GECs dramatically. The expressions of miR-379/miR-544a were downregulated in GECs, and circ-SHKBP1 functionally targeted miR-544a/miR-379 in an RNA-induced silencing complex (RISC manner. Dual-luciferase reporter assay demonstrated that forkhead box P1/P2 (FOXP1/FOXP2 were targets of miR-544a/miR-379. The expressions of FOXP1/FOXP2 were upregulated in GECs, and silencing of FOXP1/FOXP2 inhibited the viability, migration, and tube formation of GECs. Meanwhile, FOXP1/FOXP2 promoted angiogenic factor with G patch and FHA domains 1 (AGGF1 expression at the transcriptional level. Furthermore, knockdown of AGGF1 suppressed the viability, migration, and tube formation of GECs via phosphatidylinositol 3-kinase (PI3K/AKT and extracellular signal-regulated kinase (ERK1/2 pathways. Taken together, the present study demonstrated that circ-SHKBP1 regulated the angiogenesis of GECs through miR-544a/FOXP1 and miR-379/FOXP2 pathways, and these findings might provide a potential target and effective strategy for combined therapy of gliomas.

Dasatinib inhibits both osteoclast activation and prostate cancer PC-3 cell-induced osteoclast formation

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Araujo, John C.; Poblenz, Ann; Corn, Paul G.; Parikh, Nila U.; Starbuck, Michael W.; Thompson, Jerry T.; Lee, Francis; Logothetis, Christopher J.; Darnay, Bryant G.

2013-01-01

Purpose Therapies to target prostate cancer bone metastases have only limited effects. New treatments are focused on the interaction between cancer cells, bone marrow cells and the bone matrix. Osteoclasts play an important role in the development of bone tumors caused by prostate cancer. Since Src kinase has been shown to be necessary for osteoclast function, we hypothesized that dasatinib, a Src family kinase inhibitor, would reduce osteoclast activity and prostate cancer (PC-3) cell-induced osteoclast formation. Results Dasatinib inhibited RANKL-induced osteoclast differentiation of bone marrow-derived monocytes with an EC50 of 7.5 nM. PC-3 cells, a human prostate cancer cell line, were able to differentiate RAW 264.7 cells, a murine monocytic cell line, into osteoclasts and dasatinib inhibited this differentiation. In addition, conditioned medium from PC-3 cell cultures was able to differentiate RAW 264.7 cells into osteoclasts and this too, was inhibited by dasatinib. Even the lowest concentration of dasatinib, 1.25 nmol, inhibited osteoclast differentiation by 29%. Moreover, dasatinib inhibited osteoclast activity by 58% as measured by collagen 1 release. Experimental design We performed in vitro experiments utilizing the Src family kinase inhibitor dasatinib to target osteoclast activation as a means of inhibiting prostate cancer bone metastases. Conclusion Dasatinib inhibits osteoclast differentiation of mouse primary bone marrow-derived monocytes and PC-3 cell-induced osteoclast differentiation. Dasatinib also inhibits osteoclast degradation activity. Inhibiting osteoclast differentiation and activity may be an effective targeted therapy in patients with prostate cancer bone metastases. PMID:19855158

Micro-RNA 10a Is Increased in Feline T Regulatory Cells and Increases Foxp3 Protein Expression Following In Vitro Transfection

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Yan Wang

2017-02-01

Full Text Available CD4+CD25+Foxp3+ T regulatory (Treg cells are activated during the course of lentiviral infection and exhibit heightened suppressor function when compared to Treg cells from uninfected controls. Foxp3 is essential to Treg cell function and multiple studies have documented that lentivirus-activated Treg cells exhibit heightened Foxp3 expression when compared to Treg cells from uninfected controls. Our hypothesis was that lentivirus-induced micro-RNAs (miRNAs contribute to heightened Treg cell suppressor function by stabilizing Foxp3 expression. We demonstrated that CD4+CD25+ T cells from both feline immunodeficiency virus infected (FIV+ cats and uninfected control cats exhibit increased miRNA 10a and 21 levels compared to autologous CD4+CD25− T cells but there was no difference in the levels of these miRNAs when Treg cells from FIV+ cats were compared to Treg cells from uninfected controls. Further, there was no increase in Foxp3 mRNA following transfection of miRNA 10a or 21 into a feline cell line. However, transfection with miRNA 10a resulted in increased Foxp3 protein expression.

Contribution of Mesenteric Lymph Nodes and GALT to the Intestinal Foxp3+ Regulatory T-Cell CompartmentSummary

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Duke Geem

2016-05-01

Full Text Available Background & Aims: Foxp3+ regulatory T cells (Tregs in the intestine promote immune tolerance to enteric antigens. Previous studies have shown that C-C chemokine receptor 7 (CCR7-dependent migration of intestinal dendritic cells to the mesenteric lymph nodes (mLN is involved in peripheral Foxp3+ Treg accumulation in the intestine and the establishment of oral tolerance. However, the relative contribution of this CCR7+ dendritic cell–mLN–Treg axis to the total intestinal Foxp3+ Treg pool during the steady-state remains unclear. In this study, the contribution of CCR7, as well as the mLN and gut-associated lymphoid tissue (GALT, to the intestinal Foxp3+ Treg compartment in the small intestine (SI and large intestine (LI was assessed. Methods: Intestinal Foxp3+ Tregs were quantitated in Ccr7-/- mice and in mice devoid of secondary lymphoid organs—including mLN and GALT—owing to a deficiency in lymphotoxin (LT signaling. Specific analyses of Foxp3+Helios+ thymically derived (tTregs and Foxp3+Helios- peripherally derived (pTregs in the SI and LI, as well as the role for the mLN in supporting Foxp3+ pTreg development using the B6.Cg-Tg(TcraTcrb425Cbn/J/ovalbumin (OVA feeding system, were performed. Results: Foxp3+ Tregs were enriched in the intestine relative to the mLN, independent of CCR7. In the absence of the mLN and GALT, normal frequency and numbers of Foxp3+ Tregs were observed in LTα-deficient (Lta-/- mice. However, Foxp3+Helios- pTregs were decreased in the SI of Lta-/- mice, corresponding with defective Foxp3+ pTreg expansion to OVA. In the LI, however, the proportion of Foxp3+Helios- pTregs and Foxp3+ pTreg induction to OVA was comparable between Lta-/- and Lta+/+ mice, which coincided with preferential expression of Treg-inducing/immunoregulatory cytokines. Conclusions: The overall size of the intestinal Foxp3+Treg pool is not impacted significantly by CCR7, mLN, or GALT during the steady-state. However, m

Membrane-bound Dickkopf-1 in Foxp3+ regulatory T cells suppresses T-cell-mediated autoimmune colitis.

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Chae, Wook-Jin; Park, Jong-Hyun; Henegariu, Octavian; Yilmaz, Saliha; Hao, Liming; Bothwell, Alfred L M

2017-10-01

Induction of tolerance is a key mechanism to maintain or to restore immunological homeostasis. Here we show that Foxp3 + regulatory T (Treg) cells use Dickkopf-1 (DKK-1) to regulate T-cell-mediated tolerance in the T-cell-mediated autoimmune colitis model. Treg cells from DKK-1 hypomorphic doubleridge mice failed to control CD4 + T-cell proliferation, resulting in CD4 T-cell-mediated autoimmune colitis. Thymus-derived Treg cells showed a robust expression of DKK-1 but not in naive or effector CD4 T cells. DKK-1 expression in Foxp3 + Treg cells was further increased upon T-cell receptor stimulation in vitro and in vivo. Interestingly, Foxp3 + Treg cells expressed DKK-1 in the cell membrane and the functional inhibition of DKK-1 using DKK-1 monoclonal antibody abrogated the suppressor function of Foxp3 + Treg cells. DKK-1 expression was dependent on de novo protein synthesis and regulated by the mitogen-activated protein kinase pathway but not by the canonical Wnt pathway. Taken together, our results highlight membrane-bound DKK-1 as a novel Treg-derived mediator to maintain immunological tolerance in T-cell-mediated autoimmune colitis. © 2017 The Authors. Immunology Published by John Wiley & Sons Ltd.

The Forkhead Transcription Factor FOXP2 Is Required for Regulation of p21WAF1/CIP1 in 143B Osteosarcoma Cell Growth Arrest.

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Gascoyne, Duncan M; Spearman, Hayley; Lyne, Linden; Puliyadi, Rathi; Perez-Alcantara, Marta; Coulton, Les; Fisher, Simon E; Croucher, Peter I; Banham, Alison H

2015-01-01

Mutations of the forkhead transcription factor FOXP2 gene have been implicated in inherited speech-and-language disorders, and specific Foxp2 expression patterns in neuronal populations and neuronal phenotypes arising from Foxp2 disruption have been described. However, molecular functions of FOXP2 are not completely understood. Here we report a requirement for FOXP2 in growth arrest of the osteosarcoma cell line 143B. We observed endogenous expression of this transcription factor both transiently in normally developing murine osteoblasts and constitutively in human SAOS-2 osteosarcoma cells blocked in early osteoblast development. Critically, we demonstrate that in 143B osteosarcoma cells with minimal endogenous expression, FOXP2 induced by growth arrest is required for up-regulation of p21WAF1/CIP1. Upon growth factor withdrawal, FOXP2 induction occurs rapidly and precedes p21WAF1/CIP1 activation. Additionally, FOXP2 expression could be induced by MAPK pathway inhibition in growth-arrested 143B cells, but not in traditional cell line models of osteoblast differentiation (MG-63, C2C12, MC3T3-E1). Our data are consistent with a model in which transient upregulation of Foxp2 in pre-osteoblast mesenchymal cells regulates a p21-dependent growth arrest checkpoint, which may have implications for normal mesenchymal and osteosarcoma biology.

Foxo1 and Foxp1 play opposing roles in regulating the differentiation and antitumor activity of TH9 cells programmed by IL-7

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Bi, Enguang; Ma, Xingzhe; Lu, Yong; Yang, Maojie; Wang, Qiang; Xue, Gang; Qian, Jianfei; Wang, Siqing; Yi, Qing

2018-01-01

Tumor-specific CD4+ T helper 9 (TH9) cells, so-called because of their production of the cytokine interleukin-9 (IL-9), are a powerful effector T cell subset for cancer immunotherapy. We found that pretreatment of naïve CD4+ T cells with IL-7 further enhanced their differentiation into TH9 cells and augmented their antitumor activity. IL-7 markedly increased the abundance of the histone acetyltransferase p300 by activating the STAT5 and PI3K-AKT-mTOR signaling pathways and promoting the acetylation of histones at the Il9 promoter. As a result, the transcriptional regulator Foxo1 was dephosphorylated and translocated to the nucleus, bound to the Il9 promoter, and induced the production of IL-9 protein. In contrast, Foxp1, which bound to the Il9 promoter in naïve CD4+ T cells and inhibited Il9 expression, was outcompeted for binding to the Il9 promoter by Foxo1 and translocated to the cytoplasm. Furthermore, forced expression of Foxo1 or a deficiency in Foxp1 in CD4+ T cells markedly increased the production of IL-9, whereas a deficiency in Foxo1 inhibited the ability of IL-7 to enhance the differentiation and antitumor activity of TH9 cells. Thus, we identified the roles of Foxo1 as a positive regulator and Foxp1 as a negative regulator of TH9 cell differentiation and antitumor activity, which may provide potential targets for cancer immunotherapy. PMID:29018172

Malignant Tregs express low molecular splice forms of FOXP3 in Sézary syndrome

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Krejsgaard, T; Gjerdrum, L M; Ralfkiaer, E

2008-01-01

Sézary syndrome (SS) is an aggressive variant of cutaneous T-cell lymphoma. During disease progression, immunodeficiency develops; however, the underlying molecular and cellular mechanisms are not fully understood. Here, we study the regulatory T cell (Treg) function and the expression of FOXP3...... in SS. We demonstrate that malignant T cells in 8 of 15 patients stain positive with an anti-FOXP3 antibody. Western blotting analysis shows expression of two low molecular splice forms of FOXP3, but not of wild-type (wt) FOXP3. The malignant T cells produce interleukin-10 and TGF-beta and suppress...... the growth of non-malignant T cells. The Treg phenotype and the production of suppressive cytokines are driven by aberrant activation of Jak3 independent of the FOXP3 splice forms. In contrast to wt FOXP3, the low molecular splice forms of FOXP3 have no inhibitory effect on nuclear factor-kappaB (NF...

Inhibition of STAT3 activity delays obesity-induced thyroid carcinogenesis in a mouse model

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Park, Jeong Won; Han, Cho Rong; Zhao, Li; Willingham, Mark C.; Cheng, Sheue-yann

2015-01-01

Compelling epidemiologic studies indicate that obesity is a risk factor for many human cancers, including thyroid cancer. In recent decades, the incidence of thyroid cancer has dramatically increased along with a marked rise in obesity prevalence. We previously demonstrated that a high fat diet (HFD) effectively induced the obese phenotype in a mouse model of thyroid cancer (ThrbPV/PVPten+/− mice). Moreover, HFD activates the STAT3 signal pathway to promote more aggressive tumor phenotypes. The aim of the present study was to evaluate the effect of S3I-201, a specific inhibitor of STAT3 activity, on HFD-induced aggressive cancer progression in the mouse model of thyroid cancer. Wild type and ThrbPV/PVPten+/− mice were treated with HFD together with S3I-201 or vehicle-only as controls. We assessed the effects of S3I-201 on HFD-induced thyroid cancer progression, the leptin-JAK2-STAT3 signaling pathway, and key regulators of epithelial-mesenchymal transition. S3I-201 effectively inhibited HFD-induced aberrant activation of STAT3 and its downstream targets to markedly inhibit thyroid tumor growth and to prolong survival. Decreased protein levels of cyclins D1 and B1, cyclin dependent kinase (CDK) 4, CDK 6, and phosphorylated retinoblastoma protein led to the inhibition of tumor cell proliferation in S3I-201-treated ThrbPV/PVPten+/− mice. Reduced occurrence of vascular invasion and blocking of anaplasia and lung metastasis in thyroid tumors of S3I-201-treated ThrbPV/PVPten+/− mice were mediated via decreased expression of vimentin and matrix metalloproteinases, two key effectors of epithelial-mesenchymal transition. The present findings suggest that inhibition of the STAT3 activity would be a novel treatment strategy for obesity-induced thyroid cancer. PMID:26552408

The role of Foxp3+ regulatory T cells in liver transplant tolerance.

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Li, W; Carper, K; Zheng, X X; Kuhr, C S; Reyes, J D; Liang, Y; Perkins, D L; Thomson, A W; Perkins, J D

2006-12-01

The liver has long been considered a tolerogenic organ that favors the induction of peripheral tolerance. The mechanisms underlying liver tolerogenicity remain largely undefined. In this study, we characterized Foxp3-expressing CD4+ CD25+ regulatory T cells (Treg) in liver allograft recipients and examined the role of Treg in inherent liver tolerogenicity by employing the mouse spontaneous liver transplant tolerance model. Orthotopic liver transplantation was performed from C57BL/10 (H2b) to C3H/HeJ (H2k) mice. The percentage of CD4+ CD25+ Treg was expanded in the liver grafts and recipient spleens from day 5 up to day 100 posttransplantation, associated with high intracellular Foxp3 and CTLA4 expression. Immunohistochemistry further demonstrated significant numbers of Foxp3+ cells in the liver grafts and recipient spleens and increased transforming growth factor beta expression in the recipient spleens throughout the time courses. Adoptive transfer of spleen cells from the long-term liver allograft survivors significantly prolonged donor heart graft survival. Depletion of recipient CD4+ CD25+ Treg using anti-CD25 monoclonal antibody (250 microg/d) induced acute liver allograft rejection, associated with elevated anti-donor T-cell proliferative responses, CTL and natural killer activities, enhanced interleukin (IL)-2, interferon-gamma, IL-10, and decreased IL-4 production, and decreased T-cell apoptotic activity in anti-CD25-treated recipients. Moreover, CTLA4 blockade by anti-CTLA4 monoclonal antibody administration exacerbated liver graft rejection when combined with anti-CD25 monoclonal antibody. Thus, Foxp3+ CD4+ CD25+ Treg appear to underpin spontaneous acceptance of major histocompatability complex- mismatched liver allografts in mice. CTLA4, IL-4, and apoptosis of alloreactive T cells appear to contribute to the function of Treg and regulation of graft outcome.

Deletion of Foxp3+ regulatory T cells in genetically targeted mice supports development of intestinal inflammation

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Boehm Franziska

2012-07-01

Full Text Available Abstract Background Mice lacking Foxp3+ regulatory T (Treg cells develop severe tissue inflammation in lung, skin, and liver with premature death, whereas the intestine remains uninflamed. This study aims to demonstrate the importance of Foxp3+ Treg for the activation of T cells and the development of intestinal inflammation. Methods Foxp3-GFP-DTR (human diphtheria toxin receptor C57BL/6 mice allow elimination of Foxp3+ Treg by treatment with Dx (diphtheria toxin. The influence of Foxp3+ Treg on intestinal inflammation was tested using the CD4+ T-cell transfer colitis model in Rag−/− C57BL/6 mice and the acute DSS-colitis model. Results Continuous depletion of Foxp3+ Treg in Foxp3-GFP-DTR mice led to dramatic weight loss and death of mice by day 28. After 10 days of depletion of Foxp3+ Treg, isolated CD4+ T-cells were activated and produced extensive amounts of IFN-γ, IL-13, and IL-17A. Transfer of total CD4+ T-cells isolated from Foxp3-GFP-DTR mice did not result in any changes of intestinal homeostasis in Rag−/− C57BL/6 mice. However, administration of DTx between days 14 and 18 after T-cell reconstitution, lead to elimination of Foxp3+ Treg and to immediate weight loss due to intestinal inflammation. This pro-inflammatory effect of Foxp3+ Treg depletion consecutively increased inflammatory cytokine production. Further, the depletion of Foxp3+ Treg from Foxp3-GFP-DTR mice increased the severity of acute dSS-colitis accompanied by 80% lethality of Treg-depleted mice. CD4+ effector T-cells from Foxp3+ Treg-depleted mice produced significantly more pro-inflammatory cytokines. Conclusion Intermittent depletion of Foxp3+ Treg aggravates intestinal inflammatory responses demonstrating the importance of Foxp3+ Treg for the balance at the mucosal surface of the intestine.

Phenotypically non-suppressive cells predominate among FoxP3-positive cells in oral lichen planus.

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Schreurs, Olav; Karatsaidis, Andreas; Schenck, Karl

2016-11-01

Oral lichen planus (OLP) is a common T-cell-dominated oral chronic inflammatory disease occurring in periods of remission, quiescence, activity with pronounced inflammation, and acute ulceration. Cell infiltrates in OLP contain varying numbers of CD4 + T cells expressing the transcription factor FoxP3. FoxP3 + CD4 + T cells are, however, a heterogeneous cell population containing suppressive and non-suppressive cells, and their distribution in infiltrates from OLP is unknown. Biopsies were taken from normal oral mucosa (n = 8) and OLP lesions (n = 19), and a set of in situ methods for the determination of the functional phenotype of FoxP3 + CD4 + T cells was applied. Numbers of FoxP3 + CD4 + T cells were highest in the atrophic form of the disease, yet low in the ulcerative form. The main FoxP3 + CD4 + T-cell population observed was FoxP3 + CD45RA - CD25 + CD45RO + and CD15s - , a phenotype delineating a non-suppressive subset. Numbers of cells with an actively suppressing phenotype (FoxP3 + CD45RA - CD25 + CD45RO + and CD15s + ) were, however, about twice as high in reticular lesions as compared with the atrophic form. Many FoxP3 + CD4 + T cells expressed T-bet, the hallmark transcription factor for IFN-γ-producing T cells, indicating that they may enhance immune and inflammatory responses rather than suppress them. The absence of actively suppressing FoxP3 + CD4 + T cells may in part explain why OLP is a remarkably persisting condition, in spite of the presence of substantially high numbers of FoxP3 + CD4 + T cells. The findings emphasize that it is crucial to examine not only numbers but also functional phenotype of FoxP3 + CD4 + T cells in human tissues. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Kaempferol Promotes Transplant Tolerance by Sustaining CD4+FoxP3+ Regulatory T Cells in the Presence of Calcineurin Inhibitor.

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Zeng, Y Q; Liu, X S; Wu, S; Zou, C; Xie, Q; Xu, S M; Jin, X W; Li, W; Zhou, A; Dai, Z

2015-07-01

Calcineurin inhibitor cyclosporine is widely used as an immunosuppressant in clinic. However, mounting evidence has shown that cyclosporine hinders tolerance induction by dampening Tregs. Therefore, it is of paramount importance to overcome this pitfall. Kaempferol was reported to inhibit DC function. Here, we found that kaempferol delayed islet allograft rejection. Combination of kaempferol and low-dose, but not high-dose, of cyclosporine induced allograft tolerance in majority of recipient mice. Although kaempferol plus either dose of cyclosporine largely abrogated proliferation of graft-infiltrating T cells and their CTL activity, both proliferation and CTL activity in mice treated with kaempferol plus low-dose, but not high-dose, cyclosporine reemerged rapidly upon treatment withdrawal. Kaempferol increased CD4+FoxP3+ Tregs both in transplanted mice and in vitro, likely by suppressing DC maturation and their IL-6 expression. Reduction in Tregs by low dose of cyclosporine was reversed by kaempferol. Kaempferol-induced Tregs exhibited both allospecific and non-allospecific suppression. Administering IL-6 abrogated allograft tolerance induced by kaempferol and cyclosporine via diminishing CD4+FoxP3+ Tregs. Thus, for the first time, we demonstrated that kaempferol promotes transplant tolerance in the presence of low dose of cyclosporine, which allows for sufficient Treg generation while minimizing side effects, resulting in much-needed synergy between kaempferol and cyclosporine. © Copyright 2015 The American Society of Transplantation and the American Society of Transplant Surgeons.

FOXP3 and GARP (LRRC32): the master and its minion

OpenAIRE

Buer Jan; Probst-Kepper Michael

2010-01-01

Abstract The transcription factor FOXP3 is essential for the development and function of CD4+CD25hiFOXP3+ regulatory T (Treg) cells, but also expressed in activated human helper T cells without acquisition of a regulatory phenotype. This comment focuses on glycoprotein-A repetitions predominant (GARP or LRRC32) recently identified as specific marker of activated human Treg cells, which may provide the missing link toward a better molecular definition of the regulatory phenotype. Reviewers: Dr...

FOXP3 and GARP (LRRC32): the master and its minion.

Science.gov (United States)

Probst-Kepper, Michael; Buer, Jan

2010-02-05

The transcription factor FOXP3 is essential for the development and function of CD4+CD25hiFOXP3+ regulatory T (T(reg)) cells, but also expressed in activated human helper T cells without acquisition of a regulatory phenotype. This comment focuses on glycoprotein-A repetitions predominant (GARP or LRRC32) recently identified as specific marker of activated human T(reg) cells, which may provide the missing link toward a better molecular definition of the regulatory phenotype.

Berberine suppresses tumorigenicity and growth of nasopharyngeal carcinoma cells by inhibiting STAT3 activation induced by tumor associated fibroblasts

International Nuclear Information System (INIS)

Tsang, Chi Man; Cheung, Yuk Chun; Lui, Vivian Wai-Yan; Yip, Yim Ling; Zhang, Guitao; Lin, Victor Weitao; Cheung, Kenneth Chat-Pan; Feng, Yibin; Tsao, Sai Wah

2013-01-01

Cortidis rhizoma (Huanglian) and its major therapeutic component, berberine, have drawn extensive attention in recent years for their anti-cancer properties. Growth inhibitory effects of berberine on multiple types of human cancer cells have been reported. Berberine inhibits invasion, induces cell cycle arrest and apoptosis in human cancer cells. The anti-inflammatory property of berberine, involving inhibition of Signal Transducer and Activator of Transcription 3 (STAT3) activation, has also been documented. In this study, we have examined the effects of berberine on tumorigenicity and growth of nasopharyngeal carcinoma (NPC) cells and their relationship to STAT3 signaling using both in vivo and in vitro models. Berberine effectively inhibited the tumorigenicity and growth of an EBV-positive NPC cell line (C666-1) in athymic nude mice. Inhibition of tumorigenic growth of NPC cells in vivo was correlated with effective inhibition of STAT3 activation in NPC cells inside the tumor xenografts grown in nude mice. In vitro, berberine inhibited both constitutive and IL-6-induced STAT3 activation in NPC cells. Inhibition of STAT3 activation by berberine induced growth inhibition and apoptotic response in NPC cells. Tumor-associated fibroblasts were found to secret IL-6 and the conditioned medium harvested from the fibroblasts also induced STAT3 activation in NPC cells. Furthermore, STAT3 activation by conditioned medium of tumor-associated fibroblasts could be blocked by berberine or antibodies against IL-6 and IL-6R. Our observation that berberine effectively inhibited activation of STAT3 induced by tumor-associated fibroblasts suggests a role of berberine in modulating the effects of tumor stroma on the growth of NPC cells. The effective inhibition of STAT3 activation in NPC cells by berberine supports its potential use in the treatment of NPC

Activation of aryl hydrocarbon receptor (AhR leads to reciprocal epigenetic regulation of FoxP3 and IL-17 expression and amelioration of experimental colitis.

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Narendra P Singh

Full Text Available Aryl hydrocarbon receptor (AhR, a transcription factor of the bHLH/PAS family, is well characterized to regulate the biochemical and toxic effects of environmental chemicals. More recently, AhR activation has been shown to regulate the differentiation of Foxp3(+ Tregs as well as Th17 cells. However, the precise mechanisms are unclear. In the current study, we investigated the effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, a potent AhR ligand, on epigenetic regulation leading to altered Treg/Th17 differentiation, and consequent suppression of colitis.Dextran sodium sulphate (DSS administration induced acute colitis in C57BL/6 mice, as shown by significant weight loss, shortening of colon, mucosal ulceration, and increased presence of CXCR3(+ T cells as well as inflammatory cytokines. Interestingly, a single dose of TCDD (25 µg/kg body weight was able to attenuate all of the clinical and inflammatory markers of colitis. Analysis of T cells in the lamina propria (LP and mesenteric lymph nodes (MLN, during colitis, revealed decreased presence of Tregs and increased induction of Th17 cells, which was reversed following TCDD treatment. Activation of T cells from AhR(+/+ but not AhR (-/- mice, in the presence of TCDD, promoted increased differentiation of Tregs while inhibiting Th17 cells. Analysis of MLN or LP cells during colitis revealed increased methylation of CpG islands of Foxp3 and demethylation of IL-17 promoters, which was reversed following TCDD treatment.These studies demonstrate for the first time that AhR activation promotes epigenetic regulation thereby influencing reciprocal differentiation of Tregs and Th17 cells, and amelioration of inflammation.

Three distinct domains contribute to nuclear transport of murine Foxp3.

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Wayne W Hancock

2009-11-01

Full Text Available Foxp3, a 47-kDa transcription factor, is necessary for the function of CD4+CD25+ regulatory T cells (Tregs, with an essential role in the control of self-reactive T cells and in preventing autoimmunity. Activation of Tregs by TCR engagement results in upregulation of Foxp3 expression, followed by its rapid nuclear transport and binding to chromatin. Here, we identify three distinct Foxp3 domains that contribute to nuclear transport. The first domain (Domain 1 comprises the C-terminal 12 amino acids. The second domain (Domain 2 is located immediately N-terminal to the forkhead domain (FHD, recently reported to be a binding site for the runt-related transcription factor 1/acute myeloid leukemia 1 (Runx1/AML1. The third domain (Domain 3 is located within the N-terminal first 51 amino acids. Unlike the known nuclear localization signals (NLSs, none of these three regions are rich in basic residues and do not bear any similarity to known monopartite or bipartite NLSs that have one or more clusters of basic amino acids. The basic arginine-lysine-lysine-arginine (RKKR sequence, located 12-aa from the C-terminal end of Foxp3 was previously reported to be a nuclear localization signal (NLS for several proteins, including for a GFP-Foxp3 hybrid. Evidence is provided here that in the full-length native Foxp3 RKKR does not function as an NLS. The data reported in this study indicates that Foxp3 achieves nuclear transport by binding to other nuclear factors and co-transporting with them to the nucleus.

Dominant role of antigen dose in CD4+Foxp3+ regulatory T cell induction and expansion1

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Turner, Michael S.; Kane, Lawrence P.; Morel, Penelope A.

2009-01-01

The definitions of tolerogenic vs. immunogenic dendritic cells (DC) remain controversial. Immature DC have been shown to induce T regulatory cells (Treg) specific for foreign and allo-antigens. However, we have previously reported that mature DC (G4DC) prevented the onset of autoimmune diabetes whereas immature DC (GMDC) were therapeutically ineffective. In this study, islet-specific CD4+ T cells from BDC2.5 TCR Tg mice were stimulated, in the absence of exogenous cytokine, with GMDC or G4DC pulsed with high- or low-affinity antigenic peptides and examined for Treg induction. Both GMDC and G4DC presenting low peptide doses induced weak TCR signaling via the Akt/mTOR pathway, resulting in significant expansion of Foxp3+ Treg. Furthermore, unpulsed G4DC, but not GMDC, also induced Treg. High peptide doses induced strong Akt/mTOR signaling and favored the expansion of Foxp3neg Th cells. The inverse correlation of Foxp3 and Akt/mTOR signaling was also observed in DO11.10 and OT-II TCR-Tg T cells and was recapitulated with anti-CD3/CD28 stimulation in the absence of DC. IL-6 production in these cultures correlated positively with antigen dose and inversely with Treg expansion. Studies with T cells or DC from IL-6−/− mice revealed that IL-6 production by T cells was more important in the inhibition of Treg induction at low antigen doses. These studies indicate that strength of Akt/mTOR signaling, a critical T cell intrinsic determinant for Treg vs Th induction, can be controlled by adjusting the dose of antigenic peptide. Furthermore, this operates in a dominant fashion over DC phenotype and cytokine production. PMID:19801514

Foxp3 (-/ATT polymorphism contributes to the susceptibility of preeclampsia.

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Ximing Chen

Full Text Available OBJECTIVE: To evaluate the potential influence of Foxp3 polymorphism on preeclampsia (PE susceptibility, we conducted a case-control study in Han Chinese women. METHODS: Foxp3 genotyping was determined by polymerase chain reaction with sequence-specific primers (PCR-SSP in 156 PE patients and 252 age-frequency matched controls. Immunohistochemical staining was used to detect the expression of Foxp3 specific transcription factor in 30 PE and 30 normal pregnant women. RESULTS: The positive rate of Foxp3 expression in PE (26.67% was significant difference from that in normal control (63.33%, P<0.05. The frequency of Foxp3-6054 TT genotype was significantly lower in PE patient than that in control. No significant difference was found in Foxp3-3279 genotypes between PE and control, as well as for the variant allele. The frequency of Foxp3-6054A/-3279C haplotype in PE was significantly higher than that in control (P<0.01, while the frequency of Foxp3 6054T/-3279C haplotype was significantly lower in PE patient than that in control (P<0.01. CONCLUSION: Our findings suggest that the immune suppression function in PE patients is weakened, which may result in the occurrence of PE. Foxp3 polymorphism (rs5902434 may be a potential contributor for the development of PE in Han Chinese women.

Fenofibrate Enhances the In Vitro Differentiation of Foxp3+ Regulatory T Cells in Mice

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Zhou Zhou

2012-01-01

Full Text Available Foxp3+ regulatory T cells (Tregs play a critical role in maintaining immune self-tolerance. Reduced number and activity of Tregs are usually found in autoimmune and inflammatory diseases, and enhancing the differentiation of Tregs may be a promising therapeutic strategy. Some reports suggested an anti-inflammatory and anti-autoimmune potential for fenofibrate, a hypolipidemic drug used worldwide, whose lipid effects are mediated by the activation of peroxisome proliferator-activated receptor (PPAR. In the present paper, we found that fenofibrate dose-dependently increased transforming growth factor- and interleukin-2-induced Treg differentiation in vitro, by 1.96-fold from 0 to 20 M (12.59±1.34% to 24.69±3.03%, <0.05. Other PPAR activators, WY14643 (100 M, gemfibrozil (50 M, and bezafibrate (30 M, could not enhance Treg differentiation. In addition, PPAR could not upregulate the promoter activity of the Treg-specific transcription factor Foxp3. Fenofibrate might exert its function by enhancing Smad3 phosphorylation, a critical signal in Treg differentiation, via Akt suppression. Our work reveals a new PPAR independent anti-inflammatory mechanism of fenofibrate in up-regulating mouse Treg differentiation.

FOXP3 and GARP (LRRC32: the master and its minion

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Buer Jan

2010-02-01

Full Text Available Abstract The transcription factor FOXP3 is essential for the development and function of CD4+CD25hiFOXP3+ regulatory T (Treg cells, but also expressed in activated human helper T cells without acquisition of a regulatory phenotype. This comment focuses on glycoprotein-A repetitions predominant (GARP or LRRC32 recently identified as specific marker of activated human Treg cells, which may provide the missing link toward a better molecular definition of the regulatory phenotype. Reviewers: Dr Jim Di Danto, Dr Benedita Rocha and Dr Werner Solbach.

Irreversible inhibition of RANK expression as a possible mechanism for IL-3 inhibition of RANKL-induced osteoclastogenesis

Energy Technology Data Exchange (ETDEWEB)

Khapli, Shruti M.; Tomar, Geetanjali B.; Barhanpurkar, Amruta P.; Gupta, Navita; Yogesha, S.D.; Pote, Satish T. [National Center for Cell Science, University of Pune Campus, Pune 411 007 (India); Wani, Mohan R., E-mail: mohanwani@nccs.res.in [National Center for Cell Science, University of Pune Campus, Pune 411 007 (India)

2010-09-03

Research highlights: {yields} IL-3 inhibits receptor activator of NF-{kappa}B ligand (RANKL)-induced osteoclastogenesis. {yields} IL-3 inhibits RANKL-induced JNK activation. {yields} IL-3 down-regulates expression of c-Fos and NFATc1 transcription factors. {yields} IL-3 down-regulates RANK expression posttranscriptionally and irreversibly. {yields} IL-3 inhibits in vivo RANK expression. -- Abstract: IL-3, a cytokine secreted by activated T lymphocytes, stimulates the proliferation, differentiation and survival of pluripotent hematopoietic stem cells. In this study, we investigated the mechanism of inhibitory action of IL-3 on osteoclast differentiation. We show here that IL-3 significantly inhibits receptor activator of NF-{kappa}B (RANK) ligand (RANKL)-induced activation of c-Jun N-terminal kinase (JNK). IL-3 down-regulates expression of c-Fos and nuclear factor of activated T cells (NFATc1) transcription factors. In addition, IL-3 down-regulates RANK expression posttranscriptionally in both purified osteoclast precursors and whole bone marrow cells. Furthermore, the inhibitory effect of IL-3 on RANK expression was irreversible. Interestingly, IL-3 inhibits in vivo RANK expression in mice. Thus, we provide the first evidence that IL-3 irreversibly inhibits RANK expression that results in inhibition of important signaling molecules induced by RANKL.

Irreversible inhibition of RANK expression as a possible mechanism for IL-3 inhibition of RANKL-induced osteoclastogenesis

International Nuclear Information System (INIS)

Khapli, Shruti M.; Tomar, Geetanjali B.; Barhanpurkar, Amruta P.; Gupta, Navita; Yogesha, S.D.; Pote, Satish T.; Wani, Mohan R.

2010-01-01

Research highlights: → IL-3 inhibits receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis. → IL-3 inhibits RANKL-induced JNK activation. → IL-3 down-regulates expression of c-Fos and NFATc1 transcription factors. → IL-3 down-regulates RANK expression posttranscriptionally and irreversibly. → IL-3 inhibits in vivo RANK expression. -- Abstract: IL-3, a cytokine secreted by activated T lymphocytes, stimulates the proliferation, differentiation and survival of pluripotent hematopoietic stem cells. In this study, we investigated the mechanism of inhibitory action of IL-3 on osteoclast differentiation. We show here that IL-3 significantly inhibits receptor activator of NF-κB (RANK) ligand (RANKL)-induced activation of c-Jun N-terminal kinase (JNK). IL-3 down-regulates expression of c-Fos and nuclear factor of activated T cells (NFATc1) transcription factors. In addition, IL-3 down-regulates RANK expression posttranscriptionally in both purified osteoclast precursors and whole bone marrow cells. Furthermore, the inhibitory effect of IL-3 on RANK expression was irreversible. Interestingly, IL-3 inhibits in vivo RANK expression in mice. Thus, we provide the first evidence that IL-3 irreversibly inhibits RANK expression that results in inhibition of important signaling molecules induced by RANKL.

Active site-targeted covalent irreversible inhibitors of USP7 impair the functions of Foxp3+ T-regulatory cells by promoting ubiquitination of Tip60.

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Feng Wang

Full Text Available Accumulation of Foxp3+ T-regulatory (Treg cells in the tumor microenvironment is associated with tumor immune evasion and poor patient outcome in the case of many solid tumors. Current therapeutic strategies for blocking Treg functions are not Treg-specific, and display only modest and transient efficacy. Recent studies revealed that ubiquitin-specific protease 7 (USP7 is essential for Treg functions by stabilizing expression of Tip60 and Foxp3, which together are central to the development and maintenance of the Treg cell lineage. Pharmacological inhibition of USP7 is therefore a promising strategy for suppressing Treg functions and promoting anti-tumor immunity. Previously, we reported the P5091 series of small molecule USP7 inhibitors and demonstrated their direct anti-tumor activity in vivo using xenograft models. However, the precise mechanism of action of these compounds was not well defined. In this study, we report the development and characterization of P217564, a second-generation USP7 inhibitor with improved potency and selectivity. P217564 selectively targets the catalytic cleft of USP7 and modifies its active site cysteine (C223 by forming a covalent adduct. Irreversible inhibition of USP7 results in durable downstream biological responses in cells, including down-regulation of Tip60 and consequent impairment of Treg suppressive function. In addition, we demonstrate that both USP7 and various USP7 substrates are subjected to Lys48-mediated ubiquitin modification, consistent with increased proteasomal degradation of these proteins because of USP7 inhibition.

Activation of dopamine D3 receptors inhibits reward-related learning induced by cocaine.

Science.gov (United States)

Kong, H; Kuang, W; Li, S; Xu, M

2011-03-10

Memories of learned associations between the rewarding properties of drugs and environmental cues contribute to craving and relapse in humans. The mesocorticolimbic dopamine (DA) system is involved in reward-related learning induced by drugs of abuse. DA D3 receptors are preferentially expressed in mesocorticolimbic DA projection areas. Genetic and pharmacological studies have shown that DA D3 receptors suppress locomotor-stimulant effects of cocaine and reinstatement of cocaine-seeking behaviors. Activation of the extracellular signal-regulated kinase (ERK) induced by acute cocaine administration is also inhibited by D3 receptors. How D3 receptors modulate cocaine-induced reward-related learning and associated changes in cell signaling in reward circuits in the brain, however, have not been fully investigated. In the present study, we show that D3 receptor mutant mice exhibit potentiated acquisition of conditioned place preference (CPP) at low doses of cocaine compared to wild-type mice. Activation of ERK and CaMKIIα, but not the c-Jun N-terminal kinase and p38, in the nucleus accumbens, amygdala and prefrontal cortex is also potentiated in D3 receptor mutant mice compared to that in wild-type mice following CPP expression. These results support a model in which D3 receptors modulate reward-related learning induced by low doses of cocaine by inhibiting activation of ERK and CaMKIIα in reward circuits in the brain. Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.

Functional characterization of Foxp3-specific spontaneous immune responses

DEFF Research Database (Denmark)

Larsen, Susanne Købke; Munir, S; Andersen, Anders Woetmann

2013-01-01

Tumor-infiltrating CD4+CD25+ regulatory T cells (Tregs) are associated with an impaired prognosis in several cancers. The transcription factor forkhead box P3 (Foxp3) is generally expressed in Tregs. Here, we identify and characterize spontaneous cytotoxic immune responses to Foxp3-expressing cel....... Consequently, induction of Foxp3-specific cytotoxic T-cell responses appears as an attractive tool to boost spontaneous or therapeutically provoked immune responses, for example, for the therapy of cancer....

Ubiquitin-specific Protease-7 Inhibition Impairs Tip60-dependent Foxp3+ T-regulatory Cell Function and Promotes Antitumor Immunity

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Liqing Wang

2016-11-01

Full Text Available Foxp3+ T-regulatory (Treg cells are known to suppress protective host immune responses to a wide variety of solid tumors, but their therapeutic targeting is largely restricted to their transient depletion or “secondary” modulation, e.g. using anti-CTLA-4 monoclonal antibody. Our ongoing studies of the post-translational modifications that regulate Foxp3 demonstrated that the histone/protein acetyltransferase, Tip60, plays a dominant role in promoting acetylation, dimerization and function in Treg cells. We now show that the ubiquitin-specific protease, Usp7, controls Treg function largely by stabilizing the expression and promoting the multimerization of Tip60 and Foxp3. Genetic or pharmacologic targeting of Usp7 impairs Foxp3+ Treg suppressive functions, while conventional T cell responses remain intact. As a result, pharmacologic inhibitors of Usp7 can limit tumor growth in immunocompetent mice, and promote the efficacy of antitumor vaccines and immune checkpoint therapy with anti-PD1 monoclonal antibody in murine models. Hence, pharmacologic therapy with Usp7 inhibitors may have an important role in future cancer immunotherapy.

Effects of natalizumab treatment on Foxp3+ T regulatory cells.

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Stenner, Max-Philipp; Waschbisch, Anne; Buck, Dorothea; Doerck, Sebastian; Einsele, Hermann; Toyka, Klaus V; Wiendl, Heinz

2008-10-06

Natalizumab, a monoclonal humanized antibody targeting the alpha-4 chain of very late activation antigen 4 (VLA-4) exerts impressive therapeutic effects in patients with relapsing-remitting multiple sclerosis. Our objective was to study impacts of Natalizumab therapy on Foxp3+ T regulatory cells (Tregs) in multiple sclerosis (MS) patients. A combined approach of in vitro and ex vivo experiments using T cells isolated from the peripheral blood of healthy donors and Natalizumab treated MS patients was chosen. We determined binding of Natalizumab and its effects on the frequency, transmigratory behaviour and suppressive function of Tregs. Binding of Natalizumab and expression of CD49d (alpha-4 chain of VLA-4) differed between non-regulatory and regulatory cells. Albeit Foxp3+ Tregs had lower levels of CD49d, Natalizumab blocked the transmigration of Foxp3+ Tregs similar to non-regulatory T cells. The frequency of peripheral blood Tregs was unaffected by Natalizumab treatment. Natalizumab does not alter the suppressive capacity of CD4+CD25(high)CD127(low)Foxp3+ Tregs under in vitro conditions. Furthermore, the impaired function of Tregs in MS patients is not restored by Natalizumab treatment. We provide a first detailed analysis of Natalizumab effects on the regulatory T cell population. Our prospective study shows that Foxp3+ Tregs express lower levels of VLA-4 and bind less Natalizumab. We further the understanding of the mechanisms of action of Natalizumab by demonstrating that unlike other immunomodulatory drugs the beneficial therapeutic effects of the monoclonal antibody are largely independent of alterations in Treg frequency or function.

Peripherally Generated Foxp3+ Regulatory T Cells Mediate the Immunomodulatory Effects of IVIg in Allergic Airways Disease.

Science.gov (United States)

Massoud, Amir H; Kaufman, Gabriel N; Xue, Di; Béland, Marianne; Dembele, Marieme; Piccirillo, Ciriaco A; Mourad, Walid; Mazer, Bruce D

2017-04-01

IVIg is widely used as an immunomodulatory therapy. We have recently demonstrated that IVIg protects against airway hyperresponsiveness (AHR) and inflammation in mouse models of allergic airways disease (AAD), associated with induction of Foxp3 + regulatory T cells (Treg). Using mice carrying a DTR/EGFP transgene under the control of the Foxp3 promoter (DEREG mice), we demonstrate in this study that IVIg generates a de novo population of peripheral Treg (pTreg) in the absence of endogenous Treg. IVIg-generated pTreg were sufficient for inhibition of OVA-induced AHR in an Ag-driven murine model of AAD. In the absence of endogenous Treg, IVIg failed to confer protection against AHR and airway inflammation. Adoptive transfer of purified IVIg-generated pTreg prior to Ag challenge effectively prevented airway inflammation and AHR in an Ag-specific manner. Microarray gene expression profiling of IVIg-generated pTreg revealed upregulation of genes associated with cell cycle, chromatin, cytoskeleton/motility, immunity, and apoptosis. These data demonstrate the importance of Treg in regulating AAD and show that IVIg-generated pTreg are necessary and sufficient for inhibition of allergen-induced AAD. The ability of IVIg to generate pure populations of highly Ag-specific pTreg represents a new avenue to study pTreg, the cross-talk between humoral and cellular immunity, and regulation of the inflammatory response to Ags. Copyright © 2017 by The American Association of Immunologists, Inc.

The small FOXP1 isoform predominantly expressed in activated B cell-like diffuse large B-cell lymphoma and full-length FOXP1 exert similar oncogenic and transcriptional activity in human B cells.

Science.gov (United States)

van Keimpema, Martine; Grüneberg, Leonie J; Schilder-Tol, Esther J M; Oud, Monique E C M; Beuling, Esther A; Hensbergen, Paul J; de Jong, Johann; Pals, Steven T; Spaargaren, Marcel

2017-03-01

The forkhead transcription factor FOXP1 is generally regarded as an oncogene in activated B cell-like diffuse large B-cell lymphoma. Previous studies have suggested that a small isoform of FOXP1 rather than full-length FOXP1, may possess this oncogenic activity. Corroborating those studies, we herein show that activated B cell-like diffuse large B-cell lymphoma cell lines and primary activated B cell-like diffuse large B-cell lymphoma cells predominantly express a small FOXP1 isoform, and that the 5'-end of the Foxp1 gene is a common insertion site in murine lymphomas in leukemia virus- and transposon-mediated insertional mutagenesis screens. By combined mass spectrometry, (quantative) reverse transcription polymerase chain reaction/sequencing, and small interfering ribonucleic acid-mediated gene silencing, we determined that the small FOXP1 isoform predominantly expressed in activated B cell-like diffuse large B-cell lymphoma lacks the N-terminal 100 amino acids of full-length FOXP1. Aberrant overexpression of this FOXP1 isoform (ΔN100) in primary human B cells revealed its oncogenic capacity; it repressed apoptosis and plasma cell differentiation. However, no difference in potency was found between this small FOXP1 isoform and full-length FOXP1. Furthermore, overexpression of full-length FOXP1 or this small FOXP1 isoform in primary B cells and diffuse large B-cell lymphoma cell lines resulted in similar gene regulation. Taken together, our data indicate that this small FOXP1 isoform and full-length FOXP1 have comparable oncogenic and transcriptional activity in human B cells, suggesting that aberrant expression or overexpression of FOXP1, irrespective of the specific isoform, contributes to lymphomagenesis. These novel insights further enhance the value of FOXP1 for the diagnostics, prognostics, and treatment of diffuse large B-cell lymphoma patients. Copyright© Ferrata Storti Foundation.

COL-3, a chemically modified tetracycline, inhibits lipopolysaccharide-induced microglia activation and cytokine expression in the brain.

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Rawan Abdulhameed Edan

Full Text Available Microglia activation results in release of proinflammatory molecules including cytokines, which contribute to neuronal damage in the central nervous system (CNS if not controlled. Tetracycline antibiotics such as minocycline inhibit microglial activation and cytokine expression during CNS inflammation. In the present study we found that administration of chemically modified tetracycline-3 (COL-3, inhibits lipopolysaccharide (LPS-induced microglial and p38 MAPK activation, as well as the increase in TNF-α, but not IL-1β expression, in the brains of BALB/c mice. COL-3 has been described to have no antibacterial activity. We observed that COL-3 had no activity against a Gram-negative bacteria, Escherichia coli; however surprisingly, COL-3 had antibacterial activity against a Gram-positive bacteria Staphylococcus aureus, with a minimum inhibitory concentration of 1 mg/ml. Our data show that COL-3 has some antibacterial activity against S. aureus, inhibits LPS-induced neuroinflammation, and displays potential as a therapeutic agent for treatment of conditions involving CNS inflammation.

Regulation of CD8+ T cell responses to retinal antigen by local FoxP3+ regulatory T cells

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Scott W McPherson

2012-06-01

Full Text Available While pathogenic CD4 T cells are well known mediators of autoimmune uveoretinitis, CD8 T cells can also be uveitogenic. Since preliminary studies indicated that C57BL/6 mice were minimally susceptible to autoimmune uveoretinitis induction by CD8 T cells, the basis of the retinal disease resistance was sought. Mice that express β-galactosidase (βgal on a retina-specific promoter (arrβgal mice were backcrossed to mice expressing green fluorescent protein and diphtheria toxin receptor under control of the Foxp3 promoter (Foxp3-DTR/GFP mice, and to T cell receptor transgenic mice that produce βgal specific CD8 T cells (BG1 mice. These mice were used to explore the role of regulatory T cells in the resistance to retinal autoimmune disease. Experiments with T cells from double transgenic BG1 x Foxp3-DTR/GFP mice transferred into Foxp3-DTR/GFP x arrβgal mice confirmed that the retina was well protected from attempts to induce disease by adoptive transfer of activated BG1 T cells. The successful induction of retinal disease following unilateral intraocular administration of diphtheria toxin to deplete regulatory T cells showed that the protective activity was dependent on local, toxin-sensitive regulatory T cells; the opposite, untreated eye remained disease-free. Although there were very few Foxp3+ regulatory T cells in the parenchyma of quiescent retina, and they did not accumulate in retina, their depletion by local toxin administration led to disease susceptibility. We propose that these regulatory T cells modulate the pathogenic activity of βgal-specific CD8 T cells in the retinas of arrβgal mice on a local basis, allowing immunoregulation to be responsive to local conditions.

Induced Foxp3+ T cells colonising tolerated allografts exhibit the hypomethylation pattern typical of mature regulatory T cells.

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Robert eHilbrands

2016-04-01

Full Text Available Regulatory T cells expressing the transcription factor Foxp3 require acquisition of a specific hypomethylation pattern to ensure optimal functional commitment, limited lineage plasticity and long-term maintenance of tolerance. A better understanding of the molecular mechanisms involved in the generation of these epigenetic changes in vivo will contribute to the clinical exploitation of Foxp3+Treg. Here we show that both in vitro and in vivo generated antigen specific Foxp3+Treg can acquire Treg-specific epigenetic characteristics and prevent skin graft rejection in an animal model.

Effects of natalizumab treatment on Foxp3+ T regulatory cells.

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Max-Philipp Stenner

Full Text Available BACKGROUND: Natalizumab, a monoclonal humanized antibody targeting the alpha-4 chain of very late activation antigen 4 (VLA-4 exerts impressive therapeutic effects in patients with relapsing-remitting multiple sclerosis. Our objective was to study impacts of Natalizumab therapy on Foxp3+ T regulatory cells (Tregs in multiple sclerosis (MS patients. METHODOLOGY: A combined approach of in vitro and ex vivo experiments using T cells isolated from the peripheral blood of healthy donors and Natalizumab treated MS patients was chosen. We determined binding of Natalizumab and its effects on the frequency, transmigratory behaviour and suppressive function of Tregs. PRINCIPAL FINDINGS: Binding of Natalizumab and expression of CD49d (alpha-4 chain of VLA-4 differed between non-regulatory and regulatory cells. Albeit Foxp3+ Tregs had lower levels of CD49d, Natalizumab blocked the transmigration of Foxp3+ Tregs similar to non-regulatory T cells. The frequency of peripheral blood Tregs was unaffected by Natalizumab treatment. Natalizumab does not alter the suppressive capacity of CD4+CD25(highCD127(lowFoxp3+ Tregs under in vitro conditions. Furthermore, the impaired function of Tregs in MS patients is not restored by Natalizumab treatment. CONCLUSIONS: We provide a first detailed analysis of Natalizumab effects on the regulatory T cell population. Our prospective study shows that Foxp3+ Tregs express lower levels of VLA-4 and bind less Natalizumab. We further the understanding of the mechanisms of action of Natalizumab by demonstrating that unlike other immunomodulatory drugs the beneficial therapeutic effects of the monoclonal antibody are largely independent of alterations in Treg frequency or function.

Neonatal immune responses to TLR2 stimulation: Influence of maternal atopy on Foxp3 and IL-10 expression

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Gold Diane R

2006-03-01

Full Text Available Abstract Background Maternal atopic background and stimulation of the adaptive immune system with allergen interact in the development of allergic disease. Stimulation of the innate immune system through microbial exposure, such as activation of the innate Toll-like-receptor 2 (TLR2, may reduce the development of allergy in childhood. However, little is known about the immunological effects of microbial stimulation on early immune responses and in association with maternal atopy. Methods We analyzed immune responses of cord blood mononuclear cells (CBMC from 50 healthy neonates (31 non-atopic and 19 atopic mothers. Cells were stimulated with the TLR2 agonist peptidoglycan (Ppg or the allergen house dust mite Dermatophagoides farinae (Derf1, and results compared to unstimulated cells. We analyzed lymphocyte proliferation and cytokine secretion of CBMC. In addition, we assessed gene expression associated with T regulatory cells including the transcription factor Foxp3, the glucocorticoid-induced TNF receptor (GITR, and the cytotoxic lymphocyte antigen 4 (CTLA4. Lymphocyte proliferation was measured by 3H-Thymidine uptake, cytokine concentrations determined by ELISA, mRNA expression of T cell markers by real-time RT-PCR. Results Ppg stimulation induced primarily IL-10 cytokine production, in addition to IFN-γ, IL-13 and TNF-α secretion. GITR was increased following Ppg stimulation (p = 0.07. Ppg-induced IL-10 production and induction of Foxp3 were higher in CBMC without, than with maternal atopy (p = 0.04, p = 0.049. IL-10 production was highly correlated with increased expression of Foxp3 (r = 0.53, p = 0.001, GITR (r = 0.47, p = 0.004 and CTLA4 (r = 0.49, p = 0.003, independent of maternal atopy. Conclusion TLR2 stimulation with Ppg induces IL-10 and genes associated with T regulatory cells, influenced by maternal atopy. Increased IL-10 and Foxp3 induction in CBMC of non-atopic compared to atopic mothers, may indicate an increased capacity to

CD4+ Foxp3+ T-cells contribute to myocardial ischemia-reperfusion injury.

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Mathes, Denise; Weirather, Johannes; Nordbeck, Peter; Arias-Loza, Anahi-Paula; Burkard, Matthias; Pachel, Christina; Kerkau, Thomas; Beyersdorf, Niklas; Frantz, Stefan; Hofmann, Ulrich

2016-12-01

The present study analyzed the effect of CD4 + Forkhead box protein 3 negative (Foxp3 - ) T-cells and Foxp3 + CD4 + T-cells on infarct size in a mouse myocardial ischemia-reperfusion model. We examined the infarct size as a fraction of the area-at-risk as primary study endpoint in mice after 30minutes of coronary ligation followed by 24hours of reperfusion. CD4 + T-cell deficient MHC-II KO mice showed smaller histologically determined infarct size (34.5±4.7% in MHCII KO versus 59.4±4.9% in wildtype (WT)) and better preserved ejection fraction determined by magnetic resonance tomography (56.9±2.8% in MHC II KO versus 39.0±4.2% in WT). MHC-II KO mice also displayed better microvascular perfusion than WT mice after 24hours of reperfusion. Also CD4 + T-cell sufficient OT-II mice, which express an in this context irrelevant T-cell receptor, revealed smaller infarct sizes compared to WT mice. However, MHC-II blocking anti-I-A/I-E antibody treatment was not able to reduce infarct size indicating that autoantigen recognition is not required for the activation of CD4 + T-cells during reperfusion. Flow-cytometric analysis also did not detect CD4 + T-cell activation in heart draining lymph nodes in response to 24hours of ischemia-reperfusion. Adoptive transfer of CD4 + T-cells in CD4 KO mice increased the infarct size only when including the Foxp3 + CD25 + subset. Depletion of CD4 + Foxp3 + T-cells in DEREG mice enabling specific conditional ablation of this subset by treatment with diphtheria toxin attenuated infarct size as compared to diphtheria toxin treated WT mice. CD4 + Foxp3 + T-cells enhance myocardial ischemia-reperfusion injury. CD4 + T-cells exert injurious effects without the need for prior activation by MHC-II restricted autoantigen recognition. Copyright © 2016 Elsevier Ltd. All rights reserved.

Induction of Foxp3-expressing regulatory T-cells by donor blood transfusion is required for tolerance to rat liver allografts.

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Yuta Abe

Full Text Available BACKGROUND: Donor-specific blood transfusion (DST prior to solid organ transplantation has been shown to induce long-term allograft survival in the absence of immunosuppressive therapy. Although the mechanisms underlying DST-induced allograft tolerance are not well defined, there is evidence to suggest DST induces one or more populations of antigen-specific regulatory cells that suppress allograft rejection. However, neither the identity nor the regulatory properties of these tolerogenic lymphocytes have been reported. Therefore, the objective of this study was to define the kinetics, phenotype and suppressive function of the regulatory cells induced by DST alone or in combination with liver allograft transplantation (LTx. METHODOLOGY/PRINCIPAL FINDINGS: Tolerance to Dark Agouti (DA; RT1(a rat liver allografts was induced by injection (iv of 1 ml of heparinized DA blood to naïve Lewis (LEW; RT1(l rats once per week for 4 weeks prior to LTx. We found that preoperative DST alone generates CD4(+ T-cells that when transferred into naïve LEW recipients are capable of suppressing DA liver allograft rejection and promoting long-term survival of the graft and recipient. However, these DST-generated T-cells did not express the regulatory T-cell (Treg transcription factor Foxp3 nor did they suppress alloantigen (DA-induced activation of LEW T-cells in vitro suggesting that these lymphocytes are not fully functional regulatory Tregs. We did observe that DST+LTx (but not DST alone induced the time-dependent formation of CD4(+Foxp3(+ Tregs that potently suppressed alloantigen-induced activation of naïve LEW T-cells in vitro and liver allograft rejection in vivo. Finally, we present data demonstrating that virtually all of the Foxp3-expressing Tregs reside within the CD4(+CD45RC(- population whereas in which approximately 50% of these Tregs express CD25. CONCLUSIONS/SIGNIFICANCE: We conclude that preoperative DST, in the absence of liver allograft

FOXP3: required but not sufficient. the role of GARP (LRRC32) as a safeguard of the regulatory phenotype.

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Probst-Kepper, M; Balling, R; Buer, J

2010-08-01

FOXP3 is essential for the development and function of regulatory CD4(+)CD25(hi) T (T(reg)) cells. However, recent evidence suggests that FOXP3 alone is not sufficient to completely explain the regulatory phenotype of these key players in autoimmunity and inflammation: after being activated, conventional human CD4(+) T cells transiently up-regulate FOXP3 without acquiring a regulatory function. Researchers have recently found that glycoprotein A repetitions predominant (GARP, or LRRC32) is a T(reg)-specific receptor that binds latent TGF-beta and dominantly controls FOXP3 and the regulatory phenotype via a positive feedback loop. This finding provides a missing link in our molecular understanding of FOXP3 in T(reg) cells. This viewpoint focuses on GARP as safeguard of FOXP3 and the regulatory phenotype.

Foxp3 overexpression in tumor cells predicts poor survival in oral squamous cell carcinoma

International Nuclear Information System (INIS)

Song, Jing-Jing; Zhao, Si-Jia; Fang, Juan; Ma, Da; Liu, Xiang-Qi; Chen, Xiao-Bing; Wang, Yun; Cheng, Bin; Wang, Zhi

2016-01-01

Forkhead Box P3 (Foxp3) is a regulatory T cells marker, and its expression correlates with prognosis in a number of malignancies. The aim of this study is to determine the relationship of Foxp3 expression with clinicopathological parameters and prognosis in oral squamous cell carcinoma (OSCC). Foxp3 expression was examined using immunohistochemistry (IHC) in paraffin-embedded tissue samples from 273 OSCC patients. Statistical analysis was performed to evaluate the associations between Foxp3 expression, the clinicopathologic characteristics and prognostic factors in OSCC. Foxp3 protein expression was significantly associated with lymph node metastasis (P <0.01). Both univariate and multivariate analyses revealed that Foxp3 was an independent factor for both 5 years overall survival (OS) and relapse-free survival (RFS) (both P <0.01). Patients with Foxp3 overexpression had shorter OS and RFS. Our results determined that elevated Foxp3 protein expression was a predictive factor of outcome in OSCC and could act as a promising therapeutic target

Histone methylation mediates plasticity of human FOXP3(+) regulatory T cells by modulating signature gene expressions.

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He, Haiqi; Ni, Bing; Tian, Yi; Tian, Zhiqiang; Chen, Yanke; Liu, Zhengwen; Yang, Xiaomei; Lv, Yi; Zhang, Yong

2014-03-01

CD4(+) FOXP3(+) regulatory T (Treg) cells constitute a heterogeneous and plastic T-cell lineage that plays a pivotal role in maintaining immune homeostasis and immune tolerance. However, the fate of human Treg cells after loss of FOXP3 expression and the epigenetic mechanisms contributing to such a phenotype switch remain to be fully elucidated. In the current study, we demonstrate that human CD4(+) CD25(high) CD127(low/-) Treg cells convert to two subpopulations with distinctive FOXP3(+) and FOXP3(-) phenotypes following in vitro culture with anti-CD3/CD28 and interleukin-2. Digital gene expression analysis showed that upon in vitro expansion, human Treg cells down-regulated Treg cell signature genes, such as FOXP3, CTLA4, ICOS, IKZF2 and LRRC32, but up-regulated a set of T helper lineage-associated genes, especially T helper type 2 (Th2)-associated, such as GATA3, GFI1 and IL13. Subsequent chromatin immunoprecipitation-sequencing of these subpopulations yielded genome-wide maps of their H3K4me3 and H3K27me3 profiles. Surprisingly, reprogramming of Treg cells was associated with differential histone modifications, as evidenced by decreased abundance of permissive H3K4me3 within the down-regulated Treg cell signature genes, such as FOXP3, CTLA4 and LRRC32 loci, and increased abundance of H3K4me3 within the Th2-associated genes, such as IL4 and IL5; however, the H3K27me3 modification profile was not significantly different between the two subpopulations. In conclusion, this study revealed that loss of FOXP3 expression from human Treg cells during in vitro expansion can induce reprogramming to a T helper cell phenotype with a gene expression signature dominated by Th2 lineage-associated genes, and that this cell type conversion may be mediated by histone methylation events. © 2013 John Wiley & Sons Ltd.

Histone methylation mediates plasticity of human FOXP3+ regulatory T cells by modulating signature gene expressions

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He, Haiqi; Ni, Bing; Tian, Yi; Tian, Zhiqiang; Chen, Yanke; Liu, Zhengwen; Yang, Xiaomei; Lv, Yi; Zhang, Yong

2014-01-01

CD4+ FOXP3+ regulatory T (Treg) cells constitute a heterogeneous and plastic T-cell lineage that plays a pivotal role in maintaining immune homeostasis and immune tolerance. However, the fate of human Treg cells after loss of FOXP3 expression and the epigenetic mechanisms contributing to such a phenotype switch remain to be fully elucidated. In the current study, we demonstrate that human CD4+ CD25high CD127low/− Treg cells convert to two subpopulations with distinctive FOXP3+ and FOXP3− phenotypes following in vitro culture with anti-CD3/CD28 and interleukin-2. Digital gene expression analysis showed that upon in vitro expansion, human Treg cells down-regulated Treg cell signature genes, such as FOXP3, CTLA4, ICOS, IKZF2 and LRRC32, but up-regulated a set of T helper lineage-associated genes, especially T helper type 2 (Th2)-associated, such as GATA3, GFI1 and IL13. Subsequent chromatin immunoprecipitation-sequencing of these subpopulations yielded genome-wide maps of their H3K4me3 and H3K27me3 profiles. Surprisingly, reprogramming of Treg cells was associated with differential histone modifications, as evidenced by decreased abundance of permissive H3K4me3 within the down-regulated Treg cell signature genes, such as FOXP3, CTLA4 and LRRC32 loci, and increased abundance of H3K4me3 within the Th2-associated genes, such as IL4 and IL5; however, the H3K27me3 modification profile was not significantly different between the two subpopulations. In conclusion, this study revealed that loss of FOXP3 expression from human Treg cells during in vitro expansion can induce reprogramming to a T helper cell phenotype with a gene expression signature dominated by Th2 lineage-associated genes, and that this cell type conversion may be mediated by histone methylation events. PMID:24152290

A New Immunosuppressive Molecule Emodin Induces both CD4+FoxP3+ and CD8+CD122+ Regulatory T Cells and Suppresses Murine Allograft Rejection

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Feifei Qiu

2017-11-01

Full Text Available Due to vigorous alloimmunity, an allograft is usually rejected without any conventional immunosuppressive treatment. However, continuous global immunosuppression may cause severe side effects, including tumors and infections. Mounting evidence has shown that cyclosporine (CsA, a common immunosuppressant used in clinic, impedes allograft tolerance by dampening regulatory T cells (Tregs, although it inhibits allograft rejection at the same time. Therefore, it is necessary to seek an alternative immunosuppressive drug that spares Tregs with high efficiency in suppression but low toxicity. In this study, we investigated the capacity of emodin, an anthraquinone molecule originally extracted from certain natural plants, to prolong transplant survival in a mouse model and explored the cellular and molecular mechanisms underlying its action. We found that emodin significantly extended skin allograft survival and hindered CD3+ T cell infiltration in the allograft, accompanied by an increase in CD4+Foxp3+ and CD8+CD122+ Treg frequencies and numbers but a reduction in effector CD8+CD44highCD62Llow T cells in recipient mice. Emodin also inhibited effector CD8+ T cells proliferation in vivo. However, CD4+CD25+, but not CD8+CD122+, Tregs derived from emodin-treated recipients were more potent in suppression of allograft rejection than those isolated from control recipients, suggesting that emodin also enhances the suppressive function of CD4+CD25+ Tregs. Interestingly, depleting CD25+ Tregs largely reversed skin allograft survival prolonged by emodin while depleting CD122+ Tregs only partially abrogated the same allograft survival. Furthermore, we found that emodin hindered dendritic cell (DC maturation and reduced alloantibody production posttransplantation. Finally, we demonstrated that emodin inhibited in vitro proliferation of T cells and blocked their mTOR signaling as well. Therefore, emodin may be a novel mTOR inhibitor that suppresses alloimmunity by

Missed, Not Missing: Phylogenomic Evidence for the Existence of Avian FoxP3.

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Michael P Denyer

Full Text Available The Forkhead box transcription factor FoxP3 is pivotal to the development and function of regulatory T cells (Tregs, which make a major contribution to peripheral tolerance. FoxP3 is believed to perform a regulatory role in all the vertebrate species in which it has been detected. The prevailing view is that FoxP3 is absent in birds and that avian Tregs rely on alternative developmental and suppressive pathways. Prompted by the automated annotation of foxp3 in the ground tit (Parus humilis genome, we have questioned this assumption. Our analysis of all available avian genomes has revealed that the foxp3 locus is missing, incomplete or of poor quality in the relevant genomic assemblies for nearly all avian species. Nevertheless, in two species, the peregrine falcon (Falco peregrinus and the saker falcon (F. cherrug, there is compelling evidence for the existence of exons showing synteny with foxp3 in the ground tit. A broader phylogenomic analysis has shown that FoxP3 sequences from these three species are similar to crocodilian sequences, the closest living relatives of birds. In both birds and crocodilians, we have also identified a highly proline-enriched region at the N terminus of FoxP3, a region previously identified only in mammals.

Altholactone Inhibits NF-κB and STAT3 Activation and Induces Reactive Oxygen Species-Mediated Apoptosis in Prostate Cancer DU145 Cells

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Chunwa Jiang

2017-02-01

Full Text Available Altholactone, a natural compound isolated from Goniothalamus spp., has demonstrated anti-inflammatory and anticancer activities, but its molecular mechanisms are still not fully defined. Nuclear factor kappa B (NF-κB and signal transducer and activator of transcription 3 (STAT3 play pivotal roles in the cell survival of many human tumors. The objective of this study was to elucidate the mechanism of action of altholactone against prostate cancer DU145 cells and to evaluate whether its effects are mediated by inhibition of NF-κB and STAT3 activity. Altholactone inhibited proliferation of DU145 cells and induced cell cycle arrest in S phase and triggered apoptosis. Reporter assays revealed that altholactone repressed p65- and TNF-α-enhanced NF-κB transcriptional activity and also inhibited both constitutive and IL-6-induced transcriptional activity of STAT3. Consistent with this, altholactone down-regulated phosphorylation of STAT3 and moreover, decreased constitutively active mutant of STAT3 (STAT3C-induced transcriptional activity. Altholactone treatment also results in down-regulation of STAT3 target genes such as survivin, and Bcl-2 followed by up regulation of pro-apoptotic Bax protein. However, pre-treatment with the antioxidant N-acetylcysteine (NAC significantly inhibited the activation of Bax and prevented down-regulation of STAT3 target genes. Collectively, our findings suggest that altholactone induces DU145 cells death through inhibition of NF-κB and STAT3 activity.

HTLV-1 modulates the frequency and phenotype of FoxP3+CD4+ T cells in virus-infected individuals

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Satou Yorifumi

2012-05-01

but also wide variation in FoxP3 expression level among individual cases. Conclusions HTLV-1 infection induces an abnormal frequency and phenotype of FoxP3+CD4+ T cells.

FOXP3 expression in papillary thyroid carcinoma with and without Hashimoto's thyroiditis

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Murilo Pedreira Neves Junior

2013-08-01

Full Text Available INTRODUCTION: The forkhead box P3 (FOXP3 plays a role in cell development and control. In the presence of abnormal FOXP3 expression, tumor cells may evade the immunosurveillance of lymphoid cells, the first step for the maintenance of cancer cells in the thyroid tissue. OBJECTIVE: To identify the presence of FOXP3 in papillary thyroid carcinoma (PTC with and without Hashimoto's Thyroiditis (HT. METHODS: We conducted a series study of cases collected from 2000 to 2008, when 1,438 thyroidectomies were performed. We selected those diagnosed with PTC, comprising 466 cases. 30 patients were randomly selected for purposes of immunohistochemistry with antibodies against FOXP3. RESULT: FOXP3 revealed high positivity for PTC and positive immunostaining was present in 21 (72.4% from all analyzed cases. There was no difference regarding coexistent HT or not. DISCUSSION AND CONCLUSION: In the present study, it was evidenced that the focal or diffuse FOXP3 expression was commonly observed in neoplastic cells from PTC, hence indicating that the assessment of this molecule expression in suspected cases of thyroid cancer may contribute to its diagnosis.

Genetic polymorphism in FOXP3 gene: imbalance in regulatory T ...

Indian Academy of Sciences (India)

2013-04-02

Apr 2, 2013 ... In contrast, high Treg levels have been reported in peripheral blood, lymph ... patients with different types of cancer. ... Therefore, in the present study, the relationship between genetic ... (amino acids 240–261) and a carboxy-terminal forkhead ... FOXP3 in tumour cells indicates that FOXP3-targeted drugs.

IL-33 Effect on Quantitative Changes of CD4+CD25highFOXP3+ Regulatory T Cells in Children with Type 1 Diabetes

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Monika Ryba-Stanisławowska

2016-01-01

Full Text Available IL-33 is an IL-1 cytokine family member, with ability to induce both Th1 and Th2 immune responses. It binds to ST2 receptor, whose deficiency is associated with enhanced inflammatory response. The most recent studies have shown the immunoregulatory effect of IL-33 on Tregs in animal models. As type 1 diabetes is an autoimmune, inflammatory disease, where Treg defects have been described, we aimed to analyze the in vitro influence of recombinant IL-33 on quantitative properties of regulatory CD4+CD25highFOXP3+ T cells. CD4+CD25highFOXP3+ as well as CD4+CD25highFOXP3+ST2+ Tregs were analyzed by flow cytometry. In a group of patients with type 1 diabetes in vitro IL-33 treatment induced regulatory CD4+CD25highFOXP3+ cell frequencies as well as upregulating the surface expression of ST2 molecule. In addition, the number of CD4+CD25highFOXP3+ cells carrying ST2 receptor increased significantly. Similar effect was observed in case of the FOXP3 expression. We did not observe any significant changes in IL-33 treated cells of healthy controls. The level of ST2 was higher in serum of patients with type 1 diabetes in comparison to their healthy counterparts. We propose that IL-33 becomes an additional immunostimulatory factor used to induce Treg expansion in future clinical trials of adoptive therapy in type 1 diabetes.

Two FOXP3(+)CD4(+) T cell subpopulations distinctly control the prognosis of colorectal cancers.

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Saito, Takuro; Nishikawa, Hiroyoshi; Wada, Hisashi; Nagano, Yuji; Sugiyama, Daisuke; Atarashi, Koji; Maeda, Yuka; Hamaguchi, Masahide; Ohkura, Naganari; Sato, Eiichi; Nagase, Hirotsugu; Nishimura, Junichi; Yamamoto, Hirofumi; Takiguchi, Shuji; Tanoue, Takeshi; Suda, Wataru; Morita, Hidetoshi; Hattori, Masahira; Honda, Kenya; Mori, Masaki; Doki, Yuichiro; Sakaguchi, Shimon

2016-06-01

CD4(+) T cells that express the forkhead box P3 (FOXP3) transcription factor function as regulatory T (Treg) cells and hinder effective immune responses against cancer cells. Abundant Treg cell infiltration into tumors is associated with poor clinical outcomes in various types of cancers. However, the role of Treg cells is controversial in colorectal cancers (CRCs), in which FOXP3(+) T cell infiltration indicated better prognosis in some studies. Here we show that CRCs, which are commonly infiltrated by suppression-competent FOXP3(hi) Treg cells, can be classified into two types by the degree of additional infiltration of FOXP3(lo) nonsuppressive T cells. The latter, which are distinguished from FOXP3(+) Treg cells by non-expression of the naive T cell marker CD45RA and instability of FOXP3, secreted inflammatory cytokines. Indeed, CRCs with abundant infiltration of FOXP3(lo) T cells showed significantly better prognosis than those with predominantly FOXP3(hi) Treg cell infiltration. Development of such inflammatory FOXP3(lo) non-Treg cells may depend on secretion of interleukin (IL)-12 and transforming growth factor (TGF)-β by tissues and their presence was correlated with tumor invasion by intestinal bacteria, especially Fusobacterium nucleatum. Thus, functionally distinct subpopulations of tumor-infiltrating FOXP3(+) T cells contribute in opposing ways to determining CRC prognosis. Depletion of FOXP3(hi) Treg cells from tumor tissues, which would augment antitumor immunity, could thus be used as an effective treatment strategy for CRCs and other cancers, whereas strategies that locally increase the population of FOXP3(lo) non-Treg cells could be used to suppress or prevent tumor formation.

Autophagy Inhibition Contributes to ROS-Producing NLRP3-Dependent Inflammasome Activation and Cytokine Secretion in High Glucose-Induced Macrophages.

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Dai, Jiezhi; Zhang, Xiaotian; Li, Li; Chen, Hua; Chai, Yimin

2017-01-01

Type 2 diabetes is a persistent inflammatory response that impairs the healing process. We hypothesized that stimulation with high glucose following a pro-inflammatory signal would lead to autophagy inhibition, reactive oxygen species (ROS) production and eventually to the activation of the Nod-like receptor protein (NLRP) -3. Macrophages were isolated from human diabetic wound. We measured the expression of NLRP3, caspase1 and interleukin-1 beta (IL-1β) by western blot and real-time PCR, and the surface markers on cells by flow cytometry. THP-1-derived macrophages exposed to high glucose were applied to study the link between autophagy, ROS and NLRP3 activation. LC3-II, P62, NLRP3 inflammation and IL-1β expression were measured by western blot and real-time PCR. ROS production was measured with a Cellular Reactive Oxygen Species Detection Assay Kit. Macrophages isolated from diabetic wounds exhibited a pro-inflammatory phenotype, including sustained NLRP3 inflammasome activity associated with IL-1β secretion. Our data showed that high glucose inhibited autophagy, induced ROS production, and activated NLRP3 inflammasome and cytokine secretion in THP-1-derived macrophages. To study high glucose-induced NLRP3 inflammasome signalling, we performed studies using an autophagy inducer, a ROS inhibitor and a NLRP3 inhibitor and found that all reduced the NLRP3 inflammasome activation and cytokine secretion. Sustained NLRP3 inflammasome activity in wound-derived macrophages contributes to the hyper-inflammation in human diabetic wounds. Autophagy inhibition and ROS generation play an essential role in high glucose-induced NLRP3 inflammasome activation and cytokine secretion in macrophages. © 2017 The Author(s). Published by S. Karger AG, Basel.

Expansion of CD4+CD25+FOXP3+ regulatory T cells in infants of mothers with type 1 diabetes.

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Luopajärvi, Kristiina; Nieminen, Janne K; Ilonen, Jorma; Akerblom, Hans K; Knip, Mikael; Vaarala, Outi

2012-08-01

Reduced risk for type 1 diabetes (T1D) has been reported in the offspring of mothers with T1D when compared with children of affected fathers. To evaluate the hypothesis that exposure of the offspring to maternal insulin therapy induces regulatory mechanisms in utero, we compared the FOXP3 expressing regulatory T cells in cord blood (CB) of infants born to mothers with or without T1D. Cord blood mononuclear cells (CBMCs) from 20 infants with maternal T1D and from 20 infants with an unaffected mother were analyzed for the numbers of CD4+CD25+FOXP3+ cells ex vivo and after in vitro stimulation with human insulin by flow cytometry. The mRNA expression of FOXP3, NFATc2, STIM1, interleukin (IL)-10, and transforming growth factor (TGF)-β was measured by real-time reverse transcription polymerase chain reaction. The percentage of FOXP3+ cells in CD4+CD25(high) cells was higher in the CB of the infants with maternal T1D when compared with the infants of unaffected mothers (p = 0.023). After in vitro insulin stimulation an increase in the percentage of FOXP3+ cells in CD4+CD25(high) cells (p = 0.0002) as well as upregulation of FOXP3, NFATc2, STIM1, IL-10, and TGF-β transcripts in CBMCs (p mothers with T1D, in whom the disease-related PTPN22 allele was associated with reduced STIM1 and NFATc2 response in insulin-stimulated CBMCs (p = 0.007 and p = 0.014). We suggest that maternal insulin treatment induces expansion of regulatory T cells in the fetus, which might contribute to the lower risk of diabetes in children with maternal vs. paternal diabetes. © 2012 John Wiley & Sons A/S.

Dynamic changes of Foxp3(+) regulatory T cells in spleen and brain of canine distemper virus-infected dogs.

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Qeska, V; Barthel, Y; Iseringhausen, M; Tipold, A; Stein, V M; Khan, M A; Baumgärtner, W; Beineke, A

2013-12-15

Canine distemper virus (CDV) infection causes immunosuppression and demyelinating leukoencephalitis in dogs. In viral diseases, an ambiguous function of regulatory T cells (Treg), with both beneficial effects by reducing immunopathology and detrimental effects by inhibiting antiviral immunity, has been described. However, the role of Treg in the pathogenesis of canine distemper remains unknown. In order to determine the effect of CDV upon immune homeostasis, the amount of Foxp3(+) Treg in spleen and brain of naturally infected dogs has been determined by immunohistochemistry. In addition, splenic cytokine expression has been quantified by reverse transcriptase polymerase chain reaction. Splenic depletion of Foxp3(+) Treg was associated with an increased mRNA-expression of tumor necrosis factor and decreased transcription of interleukin-2 in the acute disease phase, indicative of disturbed immunological counter regulation in peripheral lymphoid organs. In the brain, a lack of Foxp3(+) Treg in predemyelinating and early demyelinating lesions and significantly increased infiltrations of Foxp3(+) Treg in chronic demyelinating lesions were observed. In conclusion, disturbed peripheral and CNS immune regulation associated with a reduction of Treg represents a potential prerequisite for excessive neuroinflammation and early lesion development in canine distemper leukoencephalitis. © 2013 Elsevier B.V. All rights reserved.

Time-resolved transcriptome and proteome landscape of human regulatory T cell (Treg) differentiation reveals novel regulators of FOXP3

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Schmidt, Angelika

2018-04-27

BackgroundRegulatory T cells (Tregs) expressing the transcription factor FOXP3 are crucial mediators of self-tolerance, preventing autoimmune diseases but possibly hampering tumor rejection. Clinical manipulation of Tregs is of great interest, and first-in-man trials of Treg transfer have achieved promising outcomes. Yet, the mechanisms governing induced Treg (iTreg) differentiation and the regulation of FOXP3 are incompletely understood.ResultsTo gain a comprehensive and unbiased molecular understanding of FOXP3 induction, we performed time-series RNA sequencing (RNA-Seq) and proteomics profiling on the same samples during human iTreg differentiation. To enable the broad analysis of universal FOXP3-inducing pathways, we used five differentiation protocols in parallel. Integrative analysis of the transcriptome and proteome confirmed involvement of specific molecular processes, as well as overlap of a novel iTreg subnetwork with known Treg regulators and autoimmunity-associated genes. Importantly, we propose 37 novel molecules putatively involved in iTreg differentiation. Their relevance was validated by a targeted shRNA screen confirming a functional role in FOXP3 induction, discriminant analyses classifying iTregs accordingly, and comparable expression in an independent novel iTreg RNA-Seq dataset.ConclusionThe data generated by this novel approach facilitates understanding of the molecular mechanisms underlying iTreg generation as well as of the concomitant changes in the transcriptome and proteome. Our results provide a reference map exploitable for future discovery of markers and drug candidates governing control of Tregs, which has important implications for the treatment of cancer, autoimmune, and inflammatory diseases.

Single-wall carbon nanohorns inhibited activation of microglia induced by lipopolysaccharide through blocking of Sirt3

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Li, Lihong; Zhang, Jinqian; Yang, Yang; Wang, Qiang; Gao, Li; Yang, Yanlong; Chang, Tao; Zhang, Xingye; Xiang, Guoan; Cao, Yongmei; Shi, Zujin; Zhao, Ming; Gao, Guodong

2013-02-01

Single-wall carbon nanohorns (SWNHs) have been demonstrated to accumulate in cytotoxic levels within organs of various animal models and cell types, which emerge as a wide range of promising biomedical imaging. Septic encephalopathy (SE) is an early sign of sepsis and associated with an increased rate of morbidity and mortality. Microglia activation plays an important role in neuroinflammation, which contributes to neuronal damage. Inhibition of microglia activation may have therapeutic benefits, which can alleviate the progression of neurodegeneration. Therefore, we investigated the functional changes of mice microglia cell lines pre-treated with or without lipopolysaccharide (LPS) induced by SWNHs. To address this question, the research about direct role of SWNHs on the growth, proliferation, and apoptosis of microglia cell lines in mice (N9 and BV2) pre-treated with or without LPS had been performed. Our results indicate that the particle diameter of SWNHs in water is between 342 to 712 nm. The images in scanning electron microscope showed that SWNHs on polystyrene surface are individual particles. LPS induced activation of mice microglia, promoted its growth and proliferation, and inhibited its apoptosis. SWNHs inhibited proliferation, delayed mitotic entry, and promoted apoptosis of mice microglia cells. The effects followed gradually increasing cultured time and concentrations of SWNHs, especially in cells pre-treated with LPS. SWNHs induced a significantly increase in G1 phase and inhibition of S phase of mice microglia cells in a dose-manner dependent of SWNHs, especially in cells pre-treated with LPS. The transmission electron microscope images showed that individual spherical SWNH particles smaller than 100 nm in diameters were localized inside lysosomes of mice microglia cells. SWNHs inhibited mitotic entry, growth and proliferation of mice microglia cells, and promoted its apoptosis, especially in cells pre-treated with LPS. SWNHs inhibited expression

Regulatory function of a novel population of mouse autoantigen-specific Foxp3 regulatory T cells depends on IFN-gamma, NO, and contact with target cells.

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Cyndi Chen

Full Text Available BACKGROUND: Both naturally arising Foxp3(+ and antigen-induced Foxp3(- regulatory T cells (Treg play a critical role in regulating immune responses, as well as in preventing autoimmune diseases and graft rejection. It is known that antigen-specific Treg are more potent than polyclonal Treg in suppressing pathogenic immune responses that cause autoimmunity and inflammation. However, difficulty in identifying and isolating a sufficient number of antigen-specific Treg has limited their use in research to elucidate the mechanisms underlying their regulatory function and their potential role in therapy. METHODOLOGY/PRINCIPAL FINDINGS: Using a novel class II MHC tetramer, we have isolated a population of CD4(+ Foxp3(- T cells specific for the autoantigen glutamic acid decarboxylase p286-300 peptide (NR286 T cells from diabetes-resistant non-obese resistant (NOR mice. These Foxp3(- NR286 T cells functioned as Treg that were able to suppress target T cell proliferation in vitro and inhibit type 1 diabetes in animals. Unexpected results from mechanistic studies in vitro showed that their regulatory function was dependent on not only IFN-gamma and nitric oxide, but also on cell contact with target cells. In addition, separating NR286 Treg from target T cells in transwell assays abolished both production of NO and suppression of target T cells, regardless of whether IFN-gamma was produced in cell cultures. Therefore, production of NO, not IFN-gamma, was cell contact dependent, suggesting that NO may function downstream of IFN-gamma in mediating regulatory function of NR286 Treg. CONCLUSIONS/SIGNIFICANCE: These studies identified a unique population of autoantigen-specific Foxp3(- Treg that can exert their regulatory function dependent on not only IFN-gamma and NO but also cell contact with target cells.

Interleukin 4 promotes the development of ex-Foxp3 Th2 cells during immunity to intestinal helminths

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Coomes, Stephanie M.; Kannan, Yashaswini; Entwistle, Lewis J.; Perez-Lloret, Jimena; Czieso, Stephanie

2017-01-01

Immunity to intestinal helminth infections requires the rapid activation of T helper 2 cells (Th2 cells). However, simultaneous expansion of CD4+Foxp3+ regulatory T cells (T reg cells) impedes protective responses, resulting in chronic infections. The ratio between T reg and effector T cells can therefore determine the outcome of infection. The redifferentiation of T reg cells into Th cells has been identified in hyperinflammatory diseases. In this study, we asked whether ex–T reg Th2 cells develop and contribute to type-2 immunity. Using multigene reporter and fate-reporter systems, we demonstrate that a significant proportion of Th2 cells derive from Foxp3+ cells after Heligmosomoides polygyrus infection and airway allergy. Ex-Foxp3 Th2 cells exhibit characteristic Th2 effector functions and provide immunity to H. polygyrus. Through selective deletion of Il4ra on Foxp3+ cells, we further demonstrate IL-4 is required for the development of ex-Foxp3 Th2 cells. Collectively, our findings indicate that converting T reg cells into Th2 cells could concomitantly enhance Th2 cells and limit T reg cell–mediated suppression. PMID:28507062

Reactive oxygen species-dependent HSP90 protein cleavage participates in arsenical As+3- and MMA+3-induced apoptosis through inhibition of telomerase activity via JNK activation

International Nuclear Information System (INIS)

Shen, S.-C.; Yang, L.-Y.; Lin, H.-Y.; Wu, C.-Y.; Su, T.-H.; Chen, Y.-C.

2008-01-01

The effects of six arsenic compounds including As +3 , MMA +3 , DMA +3 , As +5 , MMA +5 , and DMA +5 on the viability of NIH3T3 cells were examined. As +3 and MMA +3 , but not the others, exhibited significant cytotoxic effects in NIH3T3 cells through apoptosis induction. The apoptotic events such as DNA fragmentation and chromosome condensation induced by As +3 and MMA +3 were prevented by the addition of NAC and CAT, and induction of HO-1 gene expression in accordance with cleavage of the HSP90 protein, and suppression of telomerase activity were observed in NIH3T3 cells under As +3 and MMA +3 treatments. An increase in the intracellular peroxide level was examined in As +3 - and MMA +3 -treated NIH3T3 cells, and As +3 - and MMA +3 -induced apoptotic events were blocked by NAC, CAT, and DPI addition. HSP90 inhibitors, GA and RD, significantly attenuated the telomerase activity in NIH3T3 cells with an enhancement of As +3 - and MMA +3 -induced cytotoxicity. Suppression of JNKs significantly inhibited As +3 - and MMA +3 -induced apoptosis by blocking HSP90 protein cleavage and telomerase reduction in NIH3T3 cells. Furthermore, Hb, SnPP, and dexferosamine showed no effect against As +3 - and MMA +3 -induced apoptosis, and overexpression of HO-1 protein or inhibition of HO-1 protein expression did not affect the apoptosis induced by As +3 or MMA +3 . These data provide the first evidence to indicate that apoptosis induced by As +3 and MMA +3 is mediated by an ROS-dependent degradation of HSP90 protein and reduction of telomerase via JNK activation, and HO-1 induction might not be involved

Platelet-derived-growth-factor-induced signalling in human platelets: phosphoinositide-3-kinase-dependent inhibition of platelet activation.

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Selheim, F; Fukami, M H; Holmsen, H; Vassbotn, F S

2000-09-01

Human platelets release platelet-derived growth factor (PDGF) from alpha-granules during platelet activation. We have previously shown that platelets have PDGF alpha-receptors, a transmembrane tyrosine kinase that takes part in negative feedback regulation during platelet activation. Here we have described a study of PDGF-induced tyrosine phosphorylation of platelet substrates and phosphoinositide 3-kinase (PI-3K) activity in collagen-stimulated platelets. By immunoblotting with phosphotyrosine antibodies of collagen-activated platelets we found that PDGF increased the phosphorylation of several platelet substrates, e.g. pp140, pp120 and pp85. PDGF inhibited collagen-induced platelet activation in the presence of inhibitors of autocrine stimulation, thus blocking the pure collagen-induced signal transduction. PDGF enhanced the collagen-induced formation of PtdIns(3,4)P(2) and PtdIns(3,4,5)P(3) as measured by HPLC. Wortmannin and LY294002, two unrelated inhibitors of PI-3K, were used to investigate the role of PI-3K in PDGF-induced platelet signalling. Incubation of platelets with wortmannin and LY294002 blocked the formation of three phosphorylated inositides as well as the inhibitory effect of PDGF on collagen-induced platelet activation. We conclude that the inhibitory effect of PDGF on platelet activation is PI-3K dependent. This is the first demonstration of a negative regulatory function of 3-phosphorylated inositides in platelets.

GSK621 activates AMPK signaling to inhibit LPS-induced TNFα production

International Nuclear Information System (INIS)

Wu, Yong-hong; Li, Quan; Li, Ping; Liu, Bei

2016-01-01

LPS stimulation in macrophages/monocytes induces TNFα production. We here tested the potential effect of GSK621, a novel AMP-activated protein kinase (AMPK) activator, against the process. In RAW264.7 macrophages, murine bone marrow-derived macrophages (BMDMs), and chronic obstructive pulmonary disease (COPD) patients' monocytes, GSK621 significantly inhibited LPS-induced TNFα protein secretion and mRNA synthesis. Inhibition of AMPK, through AMPKα shRNA knockdown or dominant negative mutation (T172A), almost abolished GSK621's suppression on TNFα in RAW264.7 cells. Reversely, forced-expression of a constitutively-active AMPKα (T172D) mimicked GSK621 actions and reduced LPS-induced TNFα production. Molecularly, GSK621 suppressed LPS-induced reactive oxygen species (ROS) production and nuclear factor kappa B (NFκB) activation. In vivo, GSK621 oral administration inhibited LPS-induced TNFα production and endotoxin shock in mice. In summary, GSK621 activates AMPK signaling to inhibit LPS-induced TNFα production in macrophages/monocytes. - Highlights: • GSK621 inhibits LPS-induced TNFα production/expression in RAW264.7 cells and BMDMs. • GSK621 inhibits LPS-induced TNFα production/expression in COPD patients' PBMCs. • GSK621's inhibition on TNFα production by LPS requires AMPK activation. • GSK621 inhibits LPS-induced ROS production and NFκB activation, dependent on AMPK. • GSK621 oral administration inhibits LPS-induced TNFα production and endotoxin shock in mice.

14-3-3ε boosts bleomycin-induced DNA damage response by inhibiting the drug-resistant activity of MVP.

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Tang, Siwei; Bai, Chen; Yang, Pengyuan; Chen, Xian

2013-06-07

Major vault protein (MVP) is the predominant constituent of the vault particle, the largest known ribonuclear protein complex. Although emerging evidence have been establishing the links between MVP (vault) and multidrug resistance (MDR), little is known regarding exactly how the MDR activity of MVP is modulated during cellular response to drug-induced DNA damage (DDR). Bleomycin (BLM), an anticancer drug, induces DNA double-stranded breaks (DSBs) and consequently triggers the cellular DDR. Due to its physiological implications in hepatocellular carcinoma (HCC) and cell fate decision, 14-3-3ε was chosen as the pathway-specific bait protein to identify the critical target(s) responsible for HCC MDR. By using an LC-MS/MS-based proteomic approach, MVP was first identified in the BLM-induced 14-3-3ε interactome formed in HCC cells. Biological characterization revealed that MVP possesses specific activity to promote the resistance to the BLM-induced DDR. On the other hand, 14-3-3ε enhances BLM-induced DDR by interacting with MVP. Mechanistic investigation further revealed that 14-3-3ε, in a phosphorylation-dependent manner, binds to the phosphorylated sites at both Thr52 and Ser864 of the monomer of MVP. Consequently, the phosphorylation-dependent binding between 14-3-3ε and MVP inhibits the drug-resistant activity of MVP for an enhanced DDR to BLM treatment. Our findings provide an insight into the mechanism underlying how the BLM-induced interaction between 14-3-3ε and MVP modulates MDR, implicating novel strategy to overcome the chemotherapeutic resistance through interfering specific protein-protein interactions.

The Forkhead Transcription Factor, FOXP3, Is Required for Normal Pituitary Gonadotropin Expression in Mice1

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Jung, Deborah O.; Jasurda, Jake S.; Egashira, Noboru; Ellsworth, Buffy S.

2012-01-01

ABSTRACT The hypothalamic-pituitary-gonadal axis is central to normal reproductive function. This pathway begins with the release of gonadotropin-releasing hormone in systematic pulses by the hypothalamus. Gonadotropin-releasing hormone is bound by receptors on gonadotroph cells in the anterior pituitary gland and stimulates the synthesis and secretion of luteinizing hormone and, to some extent, follicle-stimulating hormone. Once stimulated by these glycoprotein hormones, the gonads begin gametogenesis and the synthesis of sex hormones. In humans, mutations of the forkhead transcription factor, FOXP3, lead to an autoimmune disorder known as immunodysregulation, polyendocrinopathy, and enteropathy, X-linked syndrome. Mice with a mutation in the Foxp3 gene have a similar autoimmune syndrome and are infertile. To understand why FOXP3 is required for reproductive function, we are investigating the reproductive phenotype of Foxp3 mutant mice (Foxp3sf/Y). Although the gonadotroph cells appear to be intact in Foxp3sf/Y mice, luteinizing hormone beta (Lhb) and follicle-stimulating hormone beta (Fshb) expression are significantly decreased, demonstrating that these mice exhibit a hypogonadotropic hypogonadism. Hypothalamic expression of gonadotropin-releasing hormone is not significantly decreased in Foxp3sf/Y males. Treatment of Foxp3sf/Y males with a gonadotropin-releasing hormone receptor agonist does not rescue expression of Lhb or Fshb. Interestingly, we do not detect Foxp3 expression in the pituitary or hypothalamus, suggesting that the infertility seen in Foxp3sf/Y males is a secondary effect, possibly due to loss of FOXP3 in immune cells. Pituitary expression of glycoprotein hormone alpha (Cga) and prolactin (Prl) are significantly reduced in Foxp3sf/Y males, whereas the precursor for adrenocorticotropic hormone, pro-opiomelanocortin (Pomc), is increased. Human patients diagnosed with IPEX often exhibit thyroiditis due to destruction of the thyroid gland by

Nonoverlapping roles of PD-1 and FoxP3 in maintaining immune tolerance in a novel autoimmune pancreatitis mouse model.

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Zhang, Baihao; Chikuma, Shunsuke; Hori, Shohei; Fagarasan, Sidonia; Honjo, Tasuku

2016-07-26

PD-1 (programmed-death 1), an immune-inhibitory receptor required for immune self-tolerance whose deficiency causes autoimmunity with variable severity and tissue specificity depending on other genetic factors, is expressed on activated T cells, including the transcription factor FoxP3(+) Treg cells known to play critical roles in maintaining immune tolerance. However, whether PD-1 expression by the Treg cells is required for their immune regulatory function, especially in autoimmune settings, is still unclear. We found that mice with partial FoxP3 insufficiency developed early-onset lympho-proliferation and lethal autoimmune pancreatitis only when PD-1 is absent. The autoimmune phenotype was rescued by the transfer of FoxP3-sufficient T cells, regardless of whether they were derived from WT or PD-1-deficient mice, indicating that Treg cells dominantly protect against development of spontaneous autoimmunity without intrinsic expression of PD-1. The absence of PD-1 combined with partial FoxP3 insufficiency, however, led to generation of ex-FoxP3 T cells with proinflammatory properties and expansion of effector/memory T cells that contributed to the autoimmune destruction of target tissues. Altogether, the results suggest that PD-1 and FoxP3 work collaboratively in maintaining immune tolerance mostly through nonoverlapping pathways. Thus, PD-1 is modulating the activation threshold and maintaining the balance between regulatory and effector T cells, whereas FoxP3 is sufficient for dominant regulation through maintaining the integrity of the Treg function. We suggest that genetic or environmental factors that even moderately affect the expression of both PD-1 and FoxP3 can cause life-threatening autoimmune diseases by disrupting the T-cell homeostasis.

Silencing of Foxp3 delays the growth of murine melanomas and modifies the tumor immunosuppressive environment

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Franco-Molina MA

2016-01-01

and decreased in tumors derived from B16F10.1 cells. Similar data were obtained from spleen cells. These results suggest that, in melanomas, Foxp3 partly induces tumor growth by modifying the immune system at the local and peripheral level, shifting the environment toward an immunosuppressive profile. Therapies incorporating this transcription factor could be strategies for cancer treatment. Keywords: melanoma, Foxp3, cancer, T-regulatory cells

FoxP3+ regulatory T cells are distinct from leukemia cells in HTLV-1-associated adult T-cell leukemia.

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Toulza, Frederic; Nosaka, Kisato; Takiguchi, Masafumi; Pagliuca, Tony; Mitsuya, Hiroaki; Tanaka, Yuetsu; Taylor, Graham P; Bangham, Charles R M

2009-11-15

Human T-lymphotropic virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia/lymphoma (ATLL). It has been postulated that ATLL cells might act as regulatory T cells (T(regs)) which, in common with ATLL cells, express both CD25 and FoxP3, and so contribute to the severe immune suppression typical of ATLL. We report here that the frequency of CD25(+) cells varied independently of the frequency of FoxP3(+) cells in both a cross-sectional study and in a longitudinal study of 2 patients with chronic ATLL. Furthermore, the capacity of ATLL cells to suppress proliferation of heterologous CD4(+)CD25(-) cells correlated with the frequency of CD4(+) FoxP3(+) cells but was independent of CD25 expression. Finally, the frequency of CD4(+)FoxP3(+) cells was inversely correlated with the lytic activity of HTLV-1-specific CTLs in patients with ATLL. We conclude that ATLL is not a tumor of FoxP3(+) regulatory T cells, and that a population of FoxP3(+) cells distinct from ATLL cells has regulatory functions and may impair the cell-mediated immune response to HTLV-1 in patients with ATLL.

Midkine inhibits inducible regulatory T cell differentiation by suppressing the development of tolerogenic dendritic cells.

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Sonobe, Yoshifumi; Li, Hua; Jin, Shijie; Kishida, Satoshi; Kadomatsu, Kenji; Takeuchi, Hideyuki; Mizuno, Tetsuya; Suzumura, Akio

2012-03-15

Midkine (MK), a heparin-binding growth factor, reportedly contributes to inflammatory diseases, including Crohn's disease and rheumatoid arthritis. We previously showed that MK aggravates experimental autoimmune encephalomyelitis (EAE) by decreasing regulatory CD4(+)CD25(+)Foxp3(+) T cells (Tregs), a population that regulates the development of autoimmune responses, although the precise mechanism remains uncertain. In this article, we show that MK produced in inflammatory conditions suppresses the development of tolerogenic dendritic cells (DCregs), which drive the development of inducible Treg. MK suppressed DCreg-mediated expansion of the CD4(+)CD25(+)Foxp3(+) Treg population. DCregs expressed significantly higher levels of CD45RB and produced significantly less IL-12 compared with conventional dendritic cells. However, MK downregulated CD45RB expression and induced IL-12 production by reducing phosphorylated STAT3 levels via src homology region 2 domain-containing phosphatase-2 in DCreg. Inhibiting MK activity with anti-MK RNA aptamers, which bind to the targeted protein to suppress the function of the protein, increased the numbers of CD11c(low)CD45RB(+) dendritic cells and Tregs in the draining lymph nodes and suppressed the severity of EAE, an animal model of multiple sclerosis. Our results also demonstrated that MK was produced by inflammatory cells, in particular, CD4(+) T cells under inflammatory conditions. Taken together, these results suggest that MK aggravates EAE by suppressing DCreg development, thereby impairing the Treg population. Thus, MK is a promising therapeutic target for various autoimmune diseases.

δ-Tocopherol inhibits receptor tyrosine kinase-induced AKT activation in prostate cancer cells.

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Wang, Hong; Hong, Jungil; Yang, Chung S

2016-11-01

The cancer preventive activity of vitamin E is suggested by epidemiological studies and supported by animal studies with vitamin E forms, γ-tocopherol and δ-tocopherol (δ-T). Several recent large-scale cancer prevention trials with high dose of α-tocopherol, however, yielded disappointing results. Whether vitamin E prevents or promotes cancer is a serious concern. A better understanding of the molecular mechanisms of action of the different forms of tocopherols would enhance our understanding of this topic. In this study, we demonstrated that δ-T was the most effective tocopherol form in inhibiting prostate cancer cell growth, by inducing cell cycle arrest and apoptosis. By profiling the effects of δ-T on the cell signaling using the phospho-kinase array, we found that the most inhibited target was the phosphorylation of AKT on T308. Further study on the activation of AKT by EGFR and IGFR revealed that δ-T attenuated the EGF/IGF-induced activation of AKT (via the phosphorylation of AKT on T308 induced by the activation of PIK3). Expression of dominant active PIK3 and AKT in prostate cancer cell line DU145 in which PIK3, AKT, and PTEN are wild type caused the cells to be reflectory to the inhibition of δ-T, supporting that δ-T inhibits the PIK3-mediated activation of AKT. Our data also suggest that δ-T interferes with the EGF-induced EGFR internalization, which leads to the inhibition of the receptor tyrosine kinase-dependent activation of AKT. In summary, our results revealed a novel mechanism of δ-T in inhibiting prostate cancer cell growth, supporting the cancer preventive activity δ-T. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

FOXP3-stained image analysis for follicular lymphoma: optimal adaptive thresholding with maximal nucleus coverage

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Senaras, C.; Pennell, M.; Chen, W.; Sahiner, B.; Shana'ah, A.; Louissaint, A.; Hasserjian, R. P.; Lozanski, G.; Gurcan, M. N.

2017-03-01

Immunohistochemical detection of FOXP3 antigen is a usable marker for detection of regulatory T lymphocytes (TR) in formalin fixed and paraffin embedded sections of different types of tumor tissue. TR plays a major role in homeostasis of normal immune systems where they prevent auto reactivity of the immune system towards the host. This beneficial effect of TR is frequently "hijacked" by malignant cells where tumor-infiltrating regulatory T cells are recruited by the malignant nuclei to inhibit the beneficial immune response of the host against the tumor cells. In the majority of human solid tumors, an increased number of tumor-infiltrating FOXP3 positive TR is associated with worse outcome. However, in follicular lymphoma (FL) the impact of the number and distribution of TR on the outcome still remains controversial. In this study, we present a novel method to detect and enumerate nuclei from FOXP3 stained images of FL biopsies. The proposed method defines a new adaptive thresholding procedure, namely the optimal adaptive thresholding (OAT) method, which aims to minimize under-segmented and over-segmented nuclei for coarse segmentation. Next, we integrate a parameter free elliptical arc and line segment detector (ELSD) as additional information to refine segmentation results and to split most of the merged nuclei. Finally, we utilize a state-of-the-art super-pixel method, Simple Linear Iterative Clustering (SLIC) to split the rest of the merged nuclei. Our dataset consists of 13 region-ofinterest images containing 769 negative and 88 positive nuclei. Three expert pathologists evaluated the method and reported sensitivity values in detecting negative and positive nuclei ranging from 83-100% and 90-95%, and precision values of 98-100% and 99-100%, respectively. The proposed solution can be used to investigate the impact of FOXP3 positive nuclei on the outcome and prognosis in FL.

GSK-3beta inhibition enhances sorafenib-induced apoptosis in melanoma cell lines.

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Panka, David J; Cho, Daniel C; Atkins, Michael B; Mier, James W

2008-01-11

Glycogen synthase kinase-3beta (GSK-3beta) can participate in the induction of apoptosis or, alternatively, provide a survival signal that minimizes cellular injury. We previously demonstrated that the multikinase inhibitor sorafenib induces apoptosis in melanoma cell lines. In this report, we show that sorafenib activates GSK-3beta in multiple subcellular compartments and that this activation undermines the lethality of the drug. Pharmacologic inhibition and/or down-modulation of the kinase enhances sorafenib-induced apoptosis as determined by propidium iodide staining and by assessing the mitochondrial release of apoptosis-inducing factor and Smac/DIABLO. Conversely, the forced expression of a constitutively active form of the enzyme (GSK-3beta(S9A)) protects the cells from the apoptotic effects of the drug. This protective effect is associated with a marked increase in basal levels of Bcl-2, Bcl-x(L), and survivin and a diminution in the degree to which these anti-apoptotic proteins are down-modulated by sorafenib exposure. Sorafenib down-modulates the pro-apoptotic Bcl-2 family member Noxa in cells with high constitutive GSK-3beta activity. Pharmacologic inhibition of GSK-3beta prevents the disappearance of Noxa induced by sorafenib and enhances the down-modulation of Mcl-1. Down-modulation of Noxa largely eliminates the enhancing effect of GSK-3 inhibition on sorafenib-induced apoptosis. These data provide a strong rationale for the use of GSK-3beta inhibitors as adjuncts to sorafenib treatment and suggest that preservation of Noxa may contribute to their efficacy.

Expression of full-length and splice forms of FoxP3 in rheumatoid arthritis

DEFF Research Database (Denmark)

Ryder, L R; Woetmann, A; Madsen, H O

2010-01-01

OBJECTIVE: The aim of our study was to compare the presence of full-length and alternative splice forms of FoxP3 mRNA in CD4 cells from rheumatoid arthritis (RA) patients and healthy controls. METHODS: A quantitative real-time polymerase chain reaction (QRT-PCR) method was used to measure...... the amount of FoxP3 mRNA full-length and splice forms. CD4-positive T cells were isolated from peripheral blood from 50 RA patients by immunomagnetic separation, and the FoxP3 mRNA expression was compared with the results from 10 healthy controls. RESULTS: We observed an increased expression of full......-length FoxP3 mRNA in RA patients when compared to healthy controls, as well as an increase in CD25 mRNA expression, but no corresponding increase in CTLA-4 mRNA expression. The presence of an alternative splice form of FoxP3 lacking exon 2 was confirmed in both RA patients and healthy controls...

Combination of PKCε Activation and PTP1B Inhibition Effectively Suppresses Aβ-Induced GSK-3β Activation and Tau Phosphorylation.

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Kanno, Takeshi; Tsuchiya, Ayako; Tanaka, Akito; Nishizaki, Tomoyuki

2016-09-01

Glycogen synthase kinase-3β (GSK-3β) is a key element to phosphorylate tau and form neurofibrillary tangles (NFTs) found in tauopathies including Alzheimer's disease (AD). A current topic for AD therapy is focused upon how to prevent tau phosphorylation. In the present study, PKCε activated Akt and inactivated GSK-3β by directly interacting with each protein. Inhibition of protein tyrosine phosphatase 1B (PTP1B), alternatively, caused an enhancement in the tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1), allowing activation of Akt through a pathway along an IRS-1/phosphatidylinositol 3 kinase (PI3K)/3-phosphoinositide-dependent protein kinase-1 (PDK1)/Akt axis, to phosphorylate and inactivate GSK-3β. Combination of PKCε activation and PTP1B inhibition more sufficiently activated Akt and inactivated GSK-3β than each independent treatment, to suppress amyloid β (Aβ)-induced tau phosphorylation and ameliorate spatial learning and memory impairment in 5xFAD transgenic mice, an animal model of AD. This may represent an innovative strategy for AD therapy.

Immunohistochemical analysis of FoxP3+ cells in periapical granulomas and radicular cysts.

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Peixoto, Raniel Fernandes; Pereira, Joabe dos Santos; Nonaka, Cassiano Francisco Weege; Silveira, Ericka Janine Dantas da; Miguel, Márcia Cristina da Costa

2012-09-01

To compare the number of FoxP3(+) cells between periapical granulomas (PGs) and radicular cysts (RCs), and to correlate this number with the intensity of the inflammatory infiltrate in these lesions and with epithelial thickness of RCs. Thirty PGs and 30 RCs were submitted to immunohistochemical analysis using an anti-FoxP3 polyclonal antibody. FoxP3(+) cells were counted under a light microscope (×400 magnification) in five fields and the mean value was calculated for each specimen. Statistical tests were used to evaluate differences in the number of FoxP3(+) cells according to type of lesion (PG vs. RC), intensity of the inflammatory infiltrate (grade I/II vs. grade III), and epithelial thickness of RCs (atrophic vs. hyperplastic). FoxP3(+) cells were detected in most PGs (93.3%) and RCs (93.3%). The median number of FoxP3(+) cells was 2.40 in PGs and 1.00 in RCs, with this difference being statistically significant (P=0.005). No significant differences in the number of FoxP3(+) cells were observed in terms of the intensity of the inflammatory infiltrate (P=0.465) or epithelial thickness of RCs (P=0.737). The present results suggest a greater participation of regulatory T cells in the modulation of the inflammatory response in PGs. In addition, the presence of a less effective regulatory environment in RCs, together with the high levels of inflammatory mediators as reported in the literature, may contribute to the greater growth potential of these lesions. Copyright © 2012 Elsevier Ltd. All rights reserved.

PI3K inhibition enhances doxorubicin-induced apoptosis in sarcoma cells.

Directory of Open Access Journals (Sweden)

Diana Marklein

Full Text Available We searched for a drug capable of sensitization of sarcoma cells to doxorubicin (DOX. We report that the dual PI3K/mTOR inhibitor PI103 enhances the efficacy of DOX in several sarcoma cell lines and interacts with DOX in the induction of apoptosis. PI103 decreased the expression of MDR1 and MRP1, which resulted in DOX accumulation. However, the enhancement of DOX-induced apoptosis was unrelated to DOX accumulation. Neither did it involve inhibition of mTOR. Instead, the combination treatment of DOX plus PI103 activated Bax, the mitochondrial apoptosis pathway, and caspase 3. Caspase 3 activation was also observed in xenografts of sarcoma cells in nude mice upon combination of DOX with the specific PI3K inhibitor GDC-0941. Although the increase in apoptosis did not further impact on tumor growth when compared to the efficient growth inhibition by GDC-0941 alone, these findings suggest that inhibition of PI3K may improve DOX-induced proapoptotic effects in sarcoma. Taken together with similar recent studies of neuroblastoma- and glioblastoma-derived cells, PI3K inhibition seems to be a more general option to sensitize tumor cells to anthracyclines.

CD4+CD25+ T cells expressing FoxP3 in Icelandic horses affected with insect bite hypersensitivity.

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Hamza, Eman; Steinbach, Falko; Marti, Eliane

2012-07-15

Insect bite hypersensitivity (IBH) is an IgE-mediated dermatitis caused by bites of midges from the genus Culicoides. We have shown previously that peripheral blood mononuclear cells (PBMC) from IBH-affected horses produce higher levels of IL-4 and lower levels of IL-10 and TGF-β1 than those from healthy horses, suggesting that IBH is associated with a reduced regulatory immune response. FoxP3 is a crucial marker of regulatory T cells (Tregs). Here we have determined the proportion of CD4(+)CD25(+)FoxP3(+) T cells by flow cytometry in PBMC directly after isolation or after stimulation with Culicoides extract or a control antigen (Tetanus Toxoid). There were no differences between healthy and IBH horses either in the proportion of FoxP3(+)CD4(+)CD25(+) cells in freshly isolated PBMC or in the following stimulation with Tetanus Toxoid. However, upon stimulation of PBMC with the allergen, expression of FoxP3 by CD4(+)CD25(+high) and CD4(+)CD25(+dim) cells was significantly higher in healthy than in IBH horses. Addition of recombinant IL-4 to PBMC from healthy horses stimulated with the allergen significantly decreased the proportion of FoxP3 expressing cells within CD4(+)CD25(+high). These results suggest that IBH is associated with a decreased number of allergen-induced Tregs. This could be a consequence of the increased IL-4 production by PBMC of IBH-affected horses. Copyright © 2011 Elsevier B.V. All rights reserved.

Association of aplastic anemia and FoxP3 gene polymorphisms in Koreans.

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In, Ji Won; Lee, Nuri; Roh, Eun Youn; Shin, Sue; Park, Kyoung Un; Song, Eun Young

2017-04-01

Aplastic anemia (AA) is characterized by pancytopenia and bone marrow failure, and most acquired AA is an immune-mediated disorder. Regulatory T cells (T regs ) suppressing autoreactive T cells were decreased in AA patients. FoxP3 is a major regulator for the development and function of T regs . Polymorphism in FoxP3 was shown to be associated with various autoimmune diseases, however, has not yet been studied in AA. In this study, we examined the association between FoxP3 polymorphisms and AA in Korean patients. The study population consisted of 94 patients diagnosed by bone marrow examination in Seoul National University Hospital (SNUH) during 1997-2012 and 195 healthy controls. FoxP3 polymorphisms (rs5902434 del/ATT, rs3761548 C/A, rs3761549 C/T, rs2232365 A/G) were analyzed by PCR-sequencing method. We analyzed differences of genotype and allele frequencies between patients and controls. We also compared differences of genotype and allele frequencies between responder and non-responder in patients treated with immunosuppressive therapy (IST). For the statistical analysis, the chi-square test and Fisher's exact test were used and P < 0.05 was regarded as statistically significant. There was no significant difference in the genotype frequencies of FoxP3 polymorphisms between patients and controls. With regards to the allele frequencies, rs3761548 C allele was significantly higher in AA patients than in controls (87.4% vs. 79.7%, P = 0.047). In patients treated with IST, rs3761549 C allele was significantly higher in non-responder patients than in responders (89.6% vs. 66.7%, P = 0.036) and female rs3761549 C/C genotype carriers were associated with greater risk for non-response to IST (84.2% vs. 16.7%, P = 0.006). Polymorphisms in rs3761548 and rs3761549 of FoxP3 in our population were associated with disease susceptibility and response for IST, respectively. This study suggests an association between FoxP3 polymorphisms and AA in Korean patients

ChIP-on-chip analysis identifies IL-22 as direct target gene of ectopically expressed FOXP3 transcription factor in human T cells

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Jeron Andreas

2012-12-01

Full Text Available Abstract Background The transcription factor (TF forkhead box P3 (FOXP3 is constitutively expressed at high levels in naturally occurring CD4+CD25+ regulatory T cells (nTregs. It is not only the most accepted marker for that cell population but is also considered lineage determinative. Chromatin immunoprecipitation (ChIP of TFs in combination with genomic tiling microarray analysis (ChIP-on-chip has been shown to be an appropriate tool for identifying FOXP3 transcription factor binding sites (TFBSs on a genome-wide scale. In combination with microarray expression analysis, the ChIP-on-chip technique allows identification of direct FOXP3 target genes. Results ChIP-on-chip analysis of the human FOXP3 expressed in resting and PMA/ionomycin–stimulated Jurkat T cells revealed several thousand putative FOXP3 binding sites and demonstrated the importance of intronic regions for FOXP3 binding. The analysis of expression data showed that the stimulation-dependent down-regulation of IL-22 was correlated with direct FOXP3 binding in the IL-22 promoter region. This association was confirmed by real-time PCR analysis of ChIP-DNA. The corresponding ChIP-region also contained a matching FOXP3 consensus sequence. Conclusions Knowledge of the general distribution patterns of FOXP3 TFBSs in the human genome under resting and activated conditions will contribute to a better understanding of this TF and its influence on direct target genes, as well as its importance for the phenotype and function of Tregs. Moreover, FOXP3-dependent repression of Th17-related IL-22 may be relevant to an understanding of the phenomenon of Treg/Th17 cell plasticity.

Neuropilin 1 deficiency on CD4+Foxp3+ regulatory T cells impairs mouse melanoma growth

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Hutzler, Marina; Abel, Simone; Alter, Christina; Stockmann, Christian; Kliche, Stefanie; Albert, Juliane; Sparwasser, Tim; Sakaguchi, Shimon; Westendorf, Astrid M.; Schadendorf, Dirk; Buer, Jan; Helfrich, Iris

2012-01-01

Infiltration of Foxp3+ regulatory T (T reg) cells is considered to be a critical step during tumor development and progression. T reg cells supposedly suppress locally an effective anti-tumor immune response within tumor tissues, although the precise mechanism by which T reg cells infiltrate the tumor is still unclear. We provide evidence that Neuropilin 1 (Nrp-1), highly expressed by Foxp3+ T reg cells, regulates the immunological anti-tumor control by guiding T reg cells into the tumor in response to tumor-derived vascular endothelial growth factor (VEGF). We demonstrate for the first time that T cell–specific ablation of Nrp-1 expression results in a significant breakdown in tumor immune escape in various transplantation models and in a spontaneous, endogenously driven melanoma model associated with strongly reduced tumor growth and prolonged tumor-free survival. Strikingly, numbers of tumor-infiltrating Foxp3+ T reg cells were significantly reduced accompanied by enhanced activation of CD8+ T cells within tumors of T cell–specific Nrp-1–deficient mice. This phenotype can be reversed by adoptive transfer of Nrp-1+ T reg cells from wild-type mice. Thus, our data strongly suggest that Nrp-1 acts as a key mediator of Foxp3+ T reg cell infiltration into the tumor site resulting in a dampened anti-tumor immune response and enhanced tumor progression. PMID:23045606

Caffeic acid phenethyl ester inhibits 3-MC-induced CYP1A1 expression through induction of hypoxia-inducible factor-1α

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Kim, Hyung Gyun [Department of Toxicology, College of Pharmacy, Chungnam National University, Daejeon (Korea, Republic of); Han, Eun Hee [Division of Life Science, Korea Basic Science Institute, Daejeon (Korea, Republic of); Im, Ji Hye; Lee, Eun Ji; Jin, Sun Woo [Department of Toxicology, College of Pharmacy, Chungnam National University, Daejeon (Korea, Republic of); Jeong, Hye Gwang, E-mail: hgjeong@cnu.ac.kr [Department of Toxicology, College of Pharmacy, Chungnam National University, Daejeon (Korea, Republic of)

2015-09-25

Caffeic acid phenethyl ester (CAPE), a natural component of propolis, is reported to have anticarcinogenic properties, although its precise chemopreventive mechanism remains unclear. In this study, we examined the effects of CAPE on 3-methylcholanthrene (3-MC)-induced CYP1A1 expression and activities. CAPE reduced the formation of the benzo[a]pyrene-DNA adduct. Moreover, CAPE inhibited 3-MC-induced CYP1A1 activity, mRNA expression, protein level, and promoter activity. CAPE treatment also decreased 3-MC-inducible xenobiotic-response element (XRE)-linked luciferase, aryl hydrocarbons receptor (AhR) transactivation and nuclear localization. CAPE induced hypoxia inducible factor-1α (HIF-1α) protein level and HIF-1α responsible element (HRE) transcriptional activity. CAPE-mediated HIF-1α reduced 3-MC-inducible CYP1A1 protein expression. Taken together, CAPE decreases 3-MC-mediated CYP1A1 expression, and this inhibitory response is associated with inhibition of AhR and HIF-1α induction. - Highlights: • CAPE reduced the formation of the benzo[a]pyrene-DNA adduct. • CAPE inhibited 3-MC-induced CYP1A1 expression. • CAPE induced HIF-1α induction. • CAPE-mediated HIF-1α reduced 3-MC-inducible CYP1A1 expression.

Lactobacillus reuteri DSM 17938 changes the frequency of Foxp3+ regulatory T cells in the intestine and mesenteric lymph node in experimental necrotizing enterocolitis.

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Yuying Liu

Full Text Available Necrotizing enterocolitis (NEC is an inflammatory disease of the intestine in premature infants. Lactobacillus reuteri DSM 17938 improves survival and reduces the incidence and severity of NEC in a rodent model. Foxp3(+ regulatory T cells (Tregs maintain intestinal homeostasis by controlling inflammation and inducing tolerance. To determine whether there are insufficient numbers of Tregs to control inflammation in NEC and to determine if LR17938 increases the frequency of Tregs, we studied selected groups of newborn Sprague-Dawley rats according to feeding plan: dam±LR17938, formula±LR17938, and NEC±LR17938. NEC was induced by gavage feeding with special formula and exposure to hypoxic conditions. Lymphocytes isolated from ileum, mesenteric lymph nodes (MLN, spleen and thymus were labeled for T cell surface markers (CD3, CD4, CD8 and intracellular Foxp3; and labeled cells were analyzed by flow cytometry. The percentage of CD3(+ T cells and Foxp3(+ Tregs in the ileum significantly decreased in pups with NEC, compared to normal controls. Feeding LR17938 to neonatal rats with NEC increased the % of Foxp3(+ T cells in the ileum while decreasing the percentage of cells in the MLN. Administration of LR17938 to dam-fed rats significantly increased Foxp3(+Tregs in the ileum as early as day of life (DOL1 but did not produce an increase in Tregs in formula-fed rats on DOL1. These results suggest that factors in breast milk may enhance the early immunomodulatory effects of LR17938. An anti-inflammatory effect of LR17938 in NEC was associated with the modulation of immune responses and induction and what appears to be migration of Foxp3(+ Tregs to the diseased gut. Probiotic-facilitated development of Tregs might play an important role in the prevention of NEC.

Increased FOXP3 expression in tumour-associated tissues of horses affected with equine sarcoid disease.

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Mählmann, K; Hamza, E; Marti, E; Dolf, G; Klukowska, J; Gerber, V; Koch, C

2014-12-01

Recent studies suggest that regulatory T cells (Tregs) are associated with disease severity and progression in papilloma virus induced neoplasia. Bovine papilloma virus (BPV) is recognised as the most important aetiological factor in equine sarcoid (ES) disease. The aim of this study was to compare expression levels of Treg markers and associated cytokines in tissue samples of ES-affected equids with skin samples of healthy control horses. Eleven ES-affected, and 12 healthy horses were included in the study. Expression levels of forkhead box protein 3 (FOXP3), interleukin 10 (IL10), interleukin 4 (IL4) and interferon gamma (IFNG) mRNA in lesional and tumour-distant samples from ES-affected horses, as well as in dermal samples of healthy control horses were measured using quantitative reverse transcription polymerase chain reaction (PCR). Expression levels were compared between lesional and tumour-distant as well as between tumour-distant and control samples. Furthermore, BPV-1 E5 DNA in samples of ES-affected horses was quantified using quantitative PCR, and possible associations of viral load, disease severity and gene expression levels were evaluated. Expression levels of FOXP3, IL10 and IFNG mRNA and BPV-1 E5 copy numbers were significantly increased in lesional compared to tumour-distant samples. There was no difference in FOXP3 and cytokine expression in tumour-distant samples from ES- compared with control horses. In tumour-distant samples viral load was positively correlated with IL10 expression and severity score. The increased expression of Treg markers in tumour-associated tissues of ES-affected equids indicates a local, Treg-induced immune suppression. Copyright © 2014 Elsevier Ltd. All rights reserved.

(99) Tc-methylene diphosphonate improves rheumatoid arthritis disease activity by increasing the frequency of peripheral γδ T cells and CD4(+) CD25(+) Foxp3(+) Tregs.

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Su, Dinglei; Shen, Minning; Gu, Bingjie; Wang, Xiaoqin; Wang, Dandan; Li, Xia; Sun, Lingyun

2016-06-01

γδ T cells exhibit important functions in the pathogenesis of rheumatoid arthritis (RA). In recent years, numerous studies harnessed the γδ T cell-activating capacity of aminobiphosphonates for the treatment of malignant tumors. As (99) Tc-methylene diphosphonate ((99) Tc-MDP) has long been widely used for the treatment of RA in China with good efficacy, we are interested in whether this drug exerts its therapeutic effect on RA by modulating peripheral γδ T cells of RA patients. To investigate the effect of (99) Tc-MDP on the frequency of γδ T cells and CD4(+) CD25(+) Foxp3(+) Tregs in the peripheral blood of patients with active RA. Nineteen patients with active RA were treated with (99) Tc-MDP intravenously at a dose of 20 μg/day consecutively for 10-14 days. Before and after treatment, the main clinical and laboratory parameters for each patient were evaluated. The frequency of CD3(+) γδ(+) T cells and CD4(+) CD25(+) Foxp3(+) Tregs was detected by flow cytometry. Serum levels of interferon (IFN)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-10 and transforming growth factor (TGF)-β were measured with enzyme-linked immunosorbent assay. After intravenous (99) Tc-MDP therapy, the frequency of peripheral CD3(+) γδ(+) T cells and CD4(+) CD25(+) Foxp3(+) Tregs were significantly elevated, paralleled with decreased serum levels of TNF-α and IL-6 and increased level of serum TGF-β. The elevation of peripheral CD3(+) γδ(+) T cells was positively correlated with increased serum TGF-β and decreased disease activity. (99) Tc-MDP may improve the activity of RA through upregulating the frequency of peripheral γδ T cells and CD4(+) CD25(+) Foxp3(+) Tregs as well as affecting the serum cytokine environment by increasing TGF-β and decreasing TNF-α and IL-6. © 2014 Asia Pacific League of Associations for Rheumatology and Wiley Publishing Asia Pty Ltd.

TRPC3 Overexpression Promotes the Progression of Inflammation-Induced Preterm Labor and Inhibits T Cell Activation.

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Jing, Chen; Dongming, Zheng; Hong, Cui; Quan, Na; Sishi, Liu; Caixia, Liu

2018-01-01

To detect the expression of the TRPC3 channel protein in the tissues of women experiencing preterm labor and investigate its interaction with T lymphocytes, providing a theoretical basis for the clinical prevention of threatened preterm labor and the development of drug-targeted therapy. Forty-seven women experiencing preterm labor and 47 women experiencing normal full-term labor were included in this study. All included women underwent delivery via cesarean section; uterine samples were obtained at delivery. The expression of TRPC3 in uterine tissue was detected by immunohistochemistry, real-time quantitative reverse transcription-PCR, and western blot assay. Activation of T lymphocytes in peripheral blood and uterine tissue were detected by flow cytometry. A TRPC3-/- mouse model of inflammation-induced preterm labor was established; expression of TRPC3, Cav3.1, and Cav3.2 were analyzed in mouse uterine tissue. Activation of T lymphocytes in female mouse and human peripheral blood samples was determined using flow cytometry. In women experiencing preterm labor, expression of TRPC3 and the Cav3.1 and Cav3.2 proteins was significantly increased; in addition, the percentage of CD3+, CD4+, and CD8+ T cells in peripheral blood was significantly decreased. TRPC3 knockout significantly delayed the occurrence of preterm labor in mice. The muscle tension of ex vivo uterine strips was lower, Cav3.1 and Cav3.2 protein expression was lower, and the percentage of CD8+ T lymphocytes was significantly increased in wild-type mice subjected to an inflammation-induced preterm labor than in wild-type mice experiencing normal full-term labor. TRPC3 is closely related to the initiation of labor. TRPC3 relies on Cav3.1 and Cav3.2 proteins to inhibit inflammation-induced preterm labor by inhibiting the activation of T cells, in particular CD8+ T lymphocytes. © 2018 The Author(s). Published by S. Karger AG, Basel.

GSK-3β Inhibition Attenuates CLP-Induced Liver Injury by Reducing Inflammation and Hepatic Cell Apoptosis

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Hui Zhang

2014-01-01

Full Text Available Liver dysfunction has been known to occur frequently in cases of sepsis. Excessive inflammation and apoptosis are pathological features of acute liver failure. Recent studies suggest that activation of glycogen synthase kinase- (GSK- 3β is involved in inflammation and apoptosis. We aimed to investigate the protective effects of GSK-3β inhibition on polymicrobial sepsis-induced liver injury and to explore the possible mechanisms. Polymicrobial sepsis was induced by cecal ligation and puncture (CLP, and SB216763 was used to inhibit GSK-3β in C57BL/6 mice. GSK-3β was activated following CLP. Administration of SB216763 decreased mortality, ameliorated liver injury, and reduced hepatic apoptosis. The inhibition of GSK-3β also reduced leukocyte infiltration and hepatic inflammatory cytokine expression and release. Moreover, GSK-3β inhibition suppressed the transcriptional activity of nuclear factor-kappa B (NF-κB but enhanced the transcriptional activity of cAMP response element binding protein (CREB in the liver. In in vitro studies, GSK-3β inhibition reduced inflammatory cytokine production via modulation of NF-κB and CREB signaling pathways in lipopolysaccharide-stimulated macrophages. In conclusion, these findings suggest that GSK-3β blockade protects against CLP-induced liver via inhibition of inflammation by modulating NF-κB and CREB activity and suppression of hepatic apoptosis.

Hydroxysafflor Yellow A Inhibits LPS-Induced NLRP3 Inflammasome Activation via Binding to Xanthine Oxidase in Mouse RAW264.7 Macrophages

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Xiaolong Xu

2016-01-01

Full Text Available Hydroxysafflor yellow A (HSYA is an effective therapeutic agent for inflammatory diseases and autoimmune disorders; however, its regulatory effect on NLRP3 inflammasome activation in macrophages has not been investigated. In this study, we predicted the potential interaction between HSYA and xanthine oxidase (XO via PharmMapper inverse docking and confirmed the binding inhibition via inhibitory test (IC50 = 40.04 μM. Computation docking illustrated that, in this HSYA-XO complex, HSYA was surrounded by Leu 648, Leu 712, His 875, Leu 873, Ser 876, Glu 879, Phe 649, and Asn 650 with a binding energy of −5.77 kcal/M and formed hydrogen bonds with the hydroxyl groups of HSYA at Glu 879, Asn 650, and His 875. We then found that HSYA significantly decreased the activity of XO in RAW264.7 macrophages and suppressed LPS-induced ROS generation. Moreover, we proved that HSYA markedly inhibited LPS-induced cleaved caspase-1 activation via suppressing the sensitization of NLRP3 inflammasome and prevented the mature IL-1β formation from pro-IL-1β form. These findings suggest that XO may be a potential target of HSYA via direct binding inhibition and the combination of HSYA-XO suppresses LPS-induced ROS generation, contributing to the depression of NLRP3 inflammasome and inhibition of IL-1β secretion in macrophages.

Eosinophils from eosinophilic oesophagitis patients have T cell suppressive capacity and express FOXP3.

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Lingblom, C; Wallander, J; Ingelsten, M; Bergquist, H; Bove, M; Saalman, R; Welin, A; Wennerås, C

2017-03-01

Eosinophilic esophagitis (EoE) is an antigen-driven T cell-mediated chronic inflammatory disease where food and environmental antigens are thought to have a role. Human eosinophils express the immunoregulatory protein galectin-10 and have T cell suppressive capacity similar to regulatory T cells (T regs ). We hypothesized that one function of eosinophils in EoE might be to regulate the T cell-driven inflammation in the oesophagus. This was tested by evaluating the suppressive capacity of eosinophils isolated from the blood of adult EoE patients in a mixed lymphocyte reaction. In addition, eosinophilic expression of forkhead box protein 3 (FOXP3), the canonical transcription factor of T regs , was determined by conventional and imaging flow cytometry, quantitative polymerase chain reaction (qPCR), confocal microscopy and immunoblotting. It was found that blood eosinophils from EoE patients had T cell suppressive capacity, and that a fraction of the eosinophils expressed FOXP3. A comparison of EoE eosinophils with healthy control eosinophils indicated that the patients' eosinophils had inferior suppressive capacity. Furthermore, a higher percentage of the EoE eosinophils expressed FOXP3 protein compared with the healthy eosinophils, and they also had higher FOXP3 protein and mRNA levels. FOXP3 was found in the cytosol and nucleus of the eosinophils from both the patients and healthy individuals, contrasting with the strict nuclear localization of FOXP3 in T regs . To conclude, these findings suggest that the immunoregulatory function of eosinophils may be impaired in EoE. © 2016 British Society for Immunology.

Helios expression and Foxp3 TSDR methylation of IFNy+ and IFNy- Treg from kidney transplant recipients with good long-term graft function.

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Karina Trojan

Full Text Available There is circumstantial evidence that IFNy+ Treg might have clinical relevance in transplantation. IFNy+ Treg express IFNy receptors and are induced by IFNy. In the present study we investigated in kidney transplant recipients with good long-term stable graft function the absolute cell counts of IFNy+ Treg subsets and whether their expression of Foxp3 is stable or transient.Helios expression determined by eight-color-fluorescence flow cytometry and methylation status of the Foxp3 Treg specific demethylation region (TSDR served as indicators for stability of Foxp3 expression. Methylation status was investigated in enriched IFNy+ and IFNy- Treg preparations originating from peripheral blood using high resolution melt analysis. A total of 136 transplant recipients and 52 healthy controls were studied.Proportions of IFNy+ Treg were similar in patients and healthy controls (0.05% and 0.04% of all CD4+ lymphocytes; p = n.s.. Patients also had similar absolute counts of IFNy producing Helios+ and Helios- Treg (p = n.s.. Most of the IFNy+ and IFNy- Treg in transplant recipients had a methylated Foxp3 TSDR, however, there was a sizeable proportion of IFNy+ and IFNy- Treg with demethylated Foxp3 TSDR. Male and female patients showed more frequently methylated IFNy+ and IFNy- Treg than male and female controls (all p<0.05.Kidney transplant recipients with good long-term stable graft function have similar levels of IFNy+ Treg as healthy controls. IFNy+ and IFNy- Treg subsets in patients consist of cells with stable and cells with transient Foxp3 expression; however, patients showed more frequently methylated IFNy+ and IFNy- Treg than controls. The data show increased levels of Treg subsets with stable as well as transient Foxp3 expression in patients with stable allograft acceptance compared to healthy controls.

Propofol and magnesium attenuate isoflurane-induced caspase-3 activation via inhibiting mitochondrial permeability transition pore

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Zhang Yiying

2012-08-01

Full Text Available Abstract Background The inhalation anesthetic isoflurane has been shown to open the mitochondrial permeability transition pore (mPTP and induce caspase activation and apoptosis, which may lead to learning and memory impairment. Cyclosporine A, a blocker of mPTP opening might attenuate the isoflurane-induced mPTP opening, lessening its ripple effects. Magnesium and anesthetic propofol are also mPTP blockers. We therefore set out to determine whether propofol and magnesium can attenuate the isoflurane-induced caspase activation and mPTP opening. Methods We investigated the effects of magnesium sulfate (Mg2+, propofol, and isoflurane on the opening of mPTP and caspase activation in H4 human neuroglioma cells stably transfected to express full-length human amyloid precursor protein (APP (H4 APP cells and in six day-old wild-type mice, employing Western blot analysis and flowcytometry. Results Here we show that Mg2+ and propofol attenuated the isoflurane-induced caspase-3 activation in H4-APP cells and mouse brain tissue. Moreover, Mg2+ and propofol, the blockers of mPTP opening, mitigated the isoflurane-induced mPTP opening in the H4-APP cells. Conclusion These data illustrate that Mg2+ and propofol may ameliorate the isoflurane-induced neurotoxicity by inhibiting its mitochondrial dysfunction. Pending further studies, these findings may suggest the use of Mg2+ and propofol in preventing and treating anesthesia neurotoxicity.

FOXP3 positive regulatory T-cells in cutaneous and systemic CD30 positive T-cell lymphoproliferations

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Gjerdrum, Lise Mette; Woetmann, Anders; Ødum, Niels

2008-01-01

for FOXP3 expression in tumour cells and tumour infiltrating Tregs. Labelling of a majority of the neoplastic cells was seen in one case of C-ALCL. Another three cases (one LyP and two C-ALCL) displayed weak labelling of very occasional atypical T-cells. In the remaining 38 cases the atypical lymphoid...... infiltrate was FOXP3 negative. By contrast, all biopsies contained tumour infiltrating FOXP3-positive Tregs. Significant higher numbers were recorded in ALK negative S-ALCL and LyP than in C-ALCL and S-ALCL positive for ALK. In conclusion, it is shown that FOXP3 expression in cutaneous and systemic CD30...

Chikusetsu saponin IVa ameliorates high fat diet-induced inflammation in adipose tissue of mice through inhibition of NLRP3 inflammasome activation and NF-κB signaling.

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Yuan, Chengfu; Liu, Chaoqi; Wang, Ting; He, Yumin; Zhou, Zhiyong; Dun, Yaoyan; Zhao, Haixia; Ren, Dongming; Wang, Junjie; Zhang, Changcheng; Yuan, Ding

2017-05-09

Chronic metabolic inflammation in adipose tissue plays an important role in the development of obesity-associated diseases. Our previous study indicated that total saponins of Panax japonicus (SPJ) rhizoma and Chikusetsu saponin V, one main component of SPJ, could exert the anti-oxidative and anti-inflammatory effects. The present study aimed to investigate the in vivo and Ex vivo anti-inflammatory activities of another main component of SPJ, namely Chikusetsu saponin IVa (CS). CS could significantly inhibited HFD-induced lipid homeostasis, and inhibited inflammation in adipose tissue, as reflected by the decreased mRNA expression levels of inflammation-related genes and secretion of the chemokines/cytokines, inhibited the accumulation of adipose tissue macrophages (ATMs) and shifted their polarization from M1 to M2, suppressed HFD-induced expression of NLRP3 inflammasome component genes and decreased IL-1β and Caspase-1 production in mice. Moreover, CS treatment also inhibited the activation of NLRP3 inflammasome in bone marrow-derived macrophages (BMDMs). Meanwhile, CS treatment inhibited an NLRP3-induced ASC pyroptosome formation and lipopolysaccharide (LPS)-induced pyroptosis. Furthermore, CS treatment suppressed HFD-induced NF-κB signaling in vivo and LPS-induced NF-κB activation as reflected by the fact that their phosphorylated forms and the ratios of pNF-κB/NF-κB, pIKK/IKK, and pIκB/IκB were all decreased in EAT from HFD-fed mice treated with CS as compared with those of HFD mice. Taking together, this study has revealed that CS effectively inhibits HFD-induced inflammation in adipose tissue of mice through inhibiting both NLRP3 inflammasome activation and NF-κB signaling. Thus, CS can serve as a potential therapeutic drug in the prevention and treatment of inflammation-associated diseases.

β3-Adrenoceptor activation relieves oxidative inhibition of the cardiac Na+-K+ pump in hyperglycemia induced by insulin receptor blockade.

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Karimi Galougahi, Keyvan; Liu, Chia-Chi; Garcia, Alvaro; Fry, Natasha A; Hamilton, Elisha J; Figtree, Gemma A; Rasmussen, Helge H

2015-09-01

Dysregulated nitric oxide (NO)- and superoxide (O2 (·-))-dependent signaling contributes to the pathobiology of diabetes-induced cardiovascular complications. We examined if stimulation of β3-adrenergic receptors (β3-ARs), coupled to endothelial NO synthase (eNOS) activation, relieves oxidative inhibition of eNOS and the Na(+)-K(+) pump induced by hyperglycemia. Hyperglycemia was established in male New Zealand White rabbits by infusion of the insulin receptor antagonist S961 for 7 days. Hyperglycemia increased tissue and blood indexes of oxidative stress. It induced glutathionylation of the Na(+)-K(+) pump β1-subunit in cardiac myocytes, an oxidative modification causing pump inhibition, and reduced the electrogenic pump current in voltage-clamped myocytes. Hyperglycemia also increased glutathionylation of eNOS, which causes its uncoupling, and increased coimmunoprecipitation of cytosolic p47(phox) and membranous p22(phox) NADPH oxidase subunits, consistent with NADPH oxidase activation. Blocking translocation of p47(phox) to p22(phox) with the gp91ds-tat peptide in cardiac myocytes ex vivo abolished the hyperglycemia-induced increase in glutathionylation of the Na(+)-K(+) pump β1-subunit and decrease in pump current. In vivo treatment with the β3-AR agonist CL316243 for 3 days eliminated the increase in indexes of oxidative stress, decreased coimmunoprecipitation of p22(phox) with p47(phox), abolished the hyperglycemia-induced increase in glutathionylation of eNOS and the Na(+)-K(+) pump β1-subunit, and abolished the decrease in pump current. CL316243 also increased coimmunoprecipitation of glutaredoxin-1 with the Na(+)-K(+) pump β1-subunit, which may reflect facilitation of deglutathionylation. In vivo β3-AR activation relieves oxidative inhibition of key cardiac myocyte proteins in hyperglycemia and may be effective in targeting the deleterious cardiac effects of diabetes. Copyright © 2015 the American Physiological Society.

Yogurt Feeding Induced the Prolongation of Fully Major Histocompatibility Complex-Mismatched Murine Cardiac Graft Survival by Induction of CD4+Foxp3+ Cells.

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Uchiyama, M; Yin, E; Yanagisawa, T; Jin, X; Hara, M; Matsuyama, S; Imazuru, T; Uchida, K; Kawamura, M; Niimi, M

Yogurt is a nutrient-rich food and the beneficial effects of yogurt on both health and immunomodulatory effects are well documented. In this pilot study, we investigated the effects of commercially produced yogurt R-1 on alloimmune responses in a murine cardiac transplantation model. The R-1 is produced by Meiji Co., Ltd., and contains live and active lactic acid bacteria (lactobacillus bulgaricus OLL1073R-1) mainly. CBA (H2 k ) mice underwent transplantation of a C57BL/6 (H2 b ; B6) heart and received oral administration of 1 mL, 0.1 mL, and 0.01 mL of R-1 from the day of transplantation until 7 days afterward. Additionally, we prepared one group of CBA recipients given 1 mL of R-1 sterilized by microwave for 7 days. Histological and immunohistochemical studies were performed. Naïve CBA mice rejected B6 cardiac graft acutely (median survival time [MST]: 7 days). CBA recipients given of 1 mL of R-1 had significantly prolonged B6 allograft survival (MST, 27 days). However, other doses of 0.1 mL and 0.01 mL of R-1 did not prolonged allograft survival (MSTs, 9 days and 8.5 days, respectively). Also, CBA recipients administered microwaved R-1 had no prolongation of B6 allograft (MST, 9 days). Histological and immunohistochemical studies showed the cardiac allograft from R-1-exposed CBA recipients had preserved graft and vessel structure and the number of infiltrated CD4 + , CD8 + , and Foxp3 + cells in R-1-exposed CBA recipients increased, respectively. In conclusion, our findings imply that yogurt containing active lactic acid bacteria could change alloimmune responses partially and induce the prolongation of cardiac allograft survival via CD4 + Foxp3 + regulatory cells. Copyright © 2017 Elsevier Inc. All rights reserved.

Lithium potentiates GSK-3β activity by inhibiting phosphoinositide 3-kinase-mediated Akt phosphorylation

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Tian, Nie; Kanno, Takeshi; Jin, Yu; Nishizaki, Tomoyuki

2014-01-01

Highlights: • Lithium suppresses Akt activity by reducing PI3K-mediated Akt phosphorylation. • Lithium enhances GSK-3β activity by reducing Akt-mediated GSK-3β phosphorylation. • Lithium suppresses GSK-3β activity through its direct inhibition. - Abstract: Accumulating evidence has pointed to the direct inhibitory action of lithium, an anti-depressant, on GSK-3β. The present study investigated further insight into lithium signaling pathways. In the cell-free assay Li 2 CO 3 significantly inhibited phosphoinositide 3-kinase (PI3K)-mediated phosphorylation of Akt1 at Ser473, but Li 2 CO 3 did not affect PI3K-mediated PI(3,4,5)P 3 production and 3-phosphoinositide-dependent protein kinase 1 (PDK1)-mediated phosphorylation of Akt1 at Thr308. This indicates that lithium could enhance GSK-3β activity by suppressing Akt-mediated Ser9 phosphorylation of GSK-3β in association with inhibition of PI3K-mediated Akt activation. There was no direct effect of Li 2 CO 3 on Akt1-induced phosphorylation of GSK-3β at Ser9, but otherwise Li 2 CO 3 significantly reduced GSK-3β-mediated phosphorylation of β-catenin at Ser33/37 and Thr41. This indicates that lithium directly inhibits GSK-3β in an Akt-independent manner. In rat hippocampal slices Li 2 CO 3 significantly inhibited phosphorylation of Akt1/2 at Ser473/474, GSK-3β at Ser9, and β-catenin at Ser33/37 and Thr41. Taken together, these results indicate that lithium exerts its potentiating and inhibiting bidirectional actions on GSK-3β activity

A dynamic dual role of IL-2 signaling in the two-step differentiation process of adaptive regulatory T cells.

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Guo, Zhiyong; Khattar, Mithun; Schroder, Paul M; Miyahara, Yoshihiro; Wang, Guohua; He, Xiaoshung; Chen, Wenhao; Stepkowski, Stanislaw M

2013-04-01

The molecular mechanism of the extrathymic generation of adaptive, or inducible, CD4(+)Foxp3(+) regulatory T cells (iTregs) remains incompletely defined. We show that exposure of splenic CD4(+)CD25(+)Foxp3(-) cells to IL-2, but not other common γ-chain cytokines, resulted in Stat5 phosphorylation and induced Foxp3 expression in ∼10% of the cells. Thus, IL-2/Stat5 signaling may be critical for Foxp3 induction in peripheral CD4(+)CD25(+)Foxp3(-) iTreg precursors. In this study, to further define the role of IL-2 in the formation of iTreg precursors as well as their subsequent Foxp3 expression, we designed a two-step iTreg differentiation model. During the initial "conditioning" step, CD4(+)CD25(-)Foxp3(-) naive T cells were activated by TCR stimulation. Inhibition of IL-2 signaling via Jak3-Stat5 was required during this step to generate CD4(+)CD25(+)Foxp3(-) cells containing iTreg precursors. During the subsequent Foxp3-induction step driven by cytokines, IL-2 was the most potent cytokine to induce Foxp3 expression in these iTreg precursors. This two-step method generated a large number of iTregs with relatively stable expression of Foxp3, which were able to prevent CD4(+)CD45RB(high) cell-mediated colitis in Rag1(-/-) mice. In consideration of this information, whereas initial inhibition of IL-2 signaling upon T cell priming generates iTreg precursors, subsequent activation of IL-2 signaling in these precursors induces the expression of Foxp3. These findings advance the understanding of iTreg differentiation and may facilitate the therapeutic use of iTregs in immune disorders.

FOXP3 Transcription Factor: A Candidate Marker for Susceptibility and Prognosis in Triple Negative Breast Cancer

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Leandra Fiori Lopes

2014-01-01

Full Text Available Triple negative breast cancer (TNBC is a relevant subgroup of neoplasia which presents negative phenotype of estrogen and progesterone receptors and has no overexpression of the human epidermal growth factor 2 (HER2. FOXP3 (forkhead transcription factor 3 is a marker of regulatory T cells (Tregs, whose expression may be increased in tumor cells. This study aimed to investigate a polymorphism (rs3761548 and the protein expression of FOXP3 for a possible involvement in TNBC susceptibility and prognosis. Genetic polymorphism was evaluated in 50 patients and in 115 controls by allele-specific PCR (polymerase chain reaction. Protein expression was evaluated in 38 patients by immunohistochemistry. It was observed a positive association for homozygous AA (OR = 3.78; 95% CI = 1.02–14.06 in relation to TNBC susceptibility. Most of the patients (83% showed a strong staining for FOXP3 protein in the tumor cells. In relation to FOXP3-positive infiltrate, 47% and 58% of patients had a moderate or intense intratumoral and peritumoral mononuclear infiltrate cells, respectively. Tumor size was positively correlated to intratumoral FOXP3-positive infiltrate (P=0.026. In conclusion, since FOXP3 was positively associated with TNBC susceptibility and prognosis, it seems to be a promising candidate for further investigation in larger TNBC samples.

FoxP3 mRNA splice forms in synovial CD4+ T cells in rheumatoid arthritis and psoriatic arthritis

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Ryder, L Rebekka; Bartels, Else Marie; Woetmann, Anders

2012-01-01

Our aim was to elucidate the relative amount of the different splice forms of FoxP3 mRNA in CD4+ T cells in peripheral blood (PB) compared to synovial fluid (SF) in RA and PsA patients. FoxP3 mRNA was measured using a quantitative real-time PCR method. CD4+ T cells were isolated from 17 paired...... samples of PB and SF from RA and PsA patients, and PB from 10 controls. FoxP3fl and FoxP3Δ2 mRNA was significantly increased (6.7 and 2.1-fold, respectively) in PB CD4+ T cells from RA patients compared to controls. FoxP3fl and Δ2 mRNA in SF CD4+ T cells was increased compared to controls in sero......-negative RA and PsA, but not in sero-positive RA patients, who had a high FoxP3 expression in both PB and SF. The FoxP3Δ2Δ7 mRNA was barely detectable in patient samples, and not at all in healthy individuals. We provide evidence of an increased expression of FoxP3 splice forms in synovial CD4+ T cells from...

Salmon cartilage proteoglycan suppresses mouse experimental colitis through induction of Foxp3+ regulatory T cells

International Nuclear Information System (INIS)

Mitsui, Toshihito; Sashinami, Hiroshi; Sato, Fuyuki; Kijima, Hiroshi; Ishiguro, Yoh; Fukuda, Shinsaku; Yoshihara, Shuichi; Hakamada, Ken-Ichi; Nakane, Akio

2010-01-01

Research highlights: → Salmon proteoglycan suppresses IL-10 -/- cell transfer-induced colitis progression. → Salmon proteoglycan suppresses Th1- and Th17-related factors in colitis mice. → Salmon proteoglycan enhances Foxp3 expression. -- Abstract: Proteoglycans (PGs) are complex glycohydrates which are widely distributed in extracellular matrix (ECM). PGs are involved in the construction of ECM, cell proliferation and differentiation. ECM components are involved in transduction of proinflammatory responses, but it is still unknown whether PGs are involved in inflammatory response. In this study, we investigated the effect of PG extracted from salmon cartilage on the progression of experimental colitis-induced in severe combined immunodeficiency mice by cell transfer from interleukin-10 (IL-10) -/- mice. IL-10 -/- cell-transferred mice showed weight loss, colon shortening and histological appearance of mild colitis. Daily oral administration of PG attenuated the clinical progression of colitis in a dose-dependent manner. Colitis-induced mice showed the elevated expression of IFN-γ, IL-12, TNF-α, IL-21, IL-23p19, IL-6, IL-17A and retinoic acid-related orphan receptor γt (RORγt) in lamina propria mononuclear cells (LPMCs) and oral administration of PG suppressed the expression of these factors. Conversely, expression of Foxp3 that induces CD4 + CD25 + regulatory T cells in LPMCs was enhanced by PG administration. These findings suggested that salmon PG attenuated the progression of colitis due to suppression of inflammatory response by enhancement of regulatory T cell induction.

LPS inhibits caspase 3-dependent apoptosis in RAW264.7 macrophages induced by the AMPK activator AICAR

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Russe, Otto Quintus; Möser, Christine V.; Kynast, Katharina L.; King, Tanya S.; Olbrich, Katrin; Grösch, Sabine; Geisslinger, Gerd; Niederberger, Ellen

2014-01-01

Highlights: • AMPK-activation induces caspase 3-dependent apoptosis in macrophages. • Apoptosis is associated with decreased mTOR and increased p21 levels. • All effects can be significantly inhibited by the TLR4 agonist lipopolysaccharide. - Abstract: AMP-activated kinase is a cellular energy sensor which is activated in stages of increased ATP consumption. Its activation has been associated with a number of beneficial effects such as decreasing inflammatory processes and the disease progress of diabetes and obesity, respectively. Furthermore, AMPK activation has been linked with induction of cell cycle arrest and apoptosis in cancer and vascular cells, indicating that it might have a therapeutic impact for the treatment of cancer and atherosclerosis. However, the impact of AMPK on the proliferation of macrophages, which also play a key role in the formation of atherosclerotic plaques and in inflammatory processes, has not been focused so far. We have assessed the influence of AICAR- and metformin-induced AMPK activation on cell viability of macrophages with and without inflammatory stimulation, respectively. In cells without inflammatory stimulation, we found a strong induction of caspase 3-dependent apoptosis associated with decreased mTOR levels and increased expression of p21. Interestingly, these effects could be inhibited by co-stimulation with bacterial lipopolysaccharide (LPS) but not by other proinflammatory cytokines suggesting that AICAR induces apoptosis via AMPK in a TLR4-pathway dependent manner. In conclusion, our results revealed that AMPK activation is not only associated with positive effects but might also contribute to risk factors by disturbing important features of macrophages. The fact that LPS is able to restore AMPK-associated apoptosis might indicate an important role of TLR4 agonists in preventing unfavorable cell death of immune cells

LPS inhibits caspase 3-dependent apoptosis in RAW264.7 macrophages induced by the AMPK activator AICAR

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Russe, Otto Quintus, E-mail: quintus@russe.eu; Möser, Christine V., E-mail: chmoeser@hotmail.com; Kynast, Katharina L., E-mail: katharina.kynast@googlemail.com; King, Tanya S., E-mail: tanya.sarah.king@googlemail.com; Olbrich, Katrin, E-mail: Katrin.olbrich@gmx.net; Grösch, Sabine, E-mail: groesch@em.uni-frankfurt.de; Geisslinger, Gerd, E-mail: geisslinger@em.uni-frankfurt.de; Niederberger, Ellen, E-mail: e.niederberger@em.uni-frankfurt.de

2014-05-09

Highlights: • AMPK-activation induces caspase 3-dependent apoptosis in macrophages. • Apoptosis is associated with decreased mTOR and increased p21 levels. • All effects can be significantly inhibited by the TLR4 agonist lipopolysaccharide. - Abstract: AMP-activated kinase is a cellular energy sensor which is activated in stages of increased ATP consumption. Its activation has been associated with a number of beneficial effects such as decreasing inflammatory processes and the disease progress of diabetes and obesity, respectively. Furthermore, AMPK activation has been linked with induction of cell cycle arrest and apoptosis in cancer and vascular cells, indicating that it might have a therapeutic impact for the treatment of cancer and atherosclerosis. However, the impact of AMPK on the proliferation of macrophages, which also play a key role in the formation of atherosclerotic plaques and in inflammatory processes, has not been focused so far. We have assessed the influence of AICAR- and metformin-induced AMPK activation on cell viability of macrophages with and without inflammatory stimulation, respectively. In cells without inflammatory stimulation, we found a strong induction of caspase 3-dependent apoptosis associated with decreased mTOR levels and increased expression of p21. Interestingly, these effects could be inhibited by co-stimulation with bacterial lipopolysaccharide (LPS) but not by other proinflammatory cytokines suggesting that AICAR induces apoptosis via AMPK in a TLR4-pathway dependent manner. In conclusion, our results revealed that AMPK activation is not only associated with positive effects but might also contribute to risk factors by disturbing important features of macrophages. The fact that LPS is able to restore AMPK-associated apoptosis might indicate an important role of TLR4 agonists in preventing unfavorable cell death of immune cells.

Nicotinamide Inhibits Ethanol-Induced Caspase-3 and PARP-1 Over-activation and Subsequent Neurodegeneration in the Developing Mouse Cerebellum.

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Ieraci, Alessandro; Herrera, Daniel G

2018-06-01

Fetal alcohol spectrum disorder (FASD) is the principal preventable cause of mental retardation in the western countries resulting from alcohol exposure during pregnancy. Ethanol-induced massive neuronal cell death occurs mainly in immature neurons during the brain growth spurt period. The cerebellum is one of the brain areas that are most sensitive to ethanol neurotoxicity. Currently, there is no effective treatment that targets the causes of these disorders and efficient treatments to counteract or reverse FASD are desirable. In this study, we investigated the effects of nicotinamide on ethanol-induced neuronal cell death in the developing cerebellum. Subcutaneous administration of ethanol in postnatal 4-day-old mice induced an over-activation of caspase-3 and PARP-1 followed by a massive neurodegeneration in the developing cerebellum. Interestingly, treatment with nicotinamide, immediately or 2 h after ethanol exposure, diminished caspase-3 and PARP-1 over-activation and reduced ethanol-induced neurodegeneration. Conversely, treatment with 3-aminobenzadine, a specific PARP-1 inhibitor, was able to completely block PARP-1 activation, but not caspase-3 activation or ethanol-induced neurodegeneration in the developing cerebellum. Our results showed that nicotinamide reduces ethanol-induced neuronal cell death and inhibits both caspase-3 and PARP-1 alcohol-induced activation in the developing cerebellum, suggesting that nicotinamide might be a promising and safe neuroprotective agent for treating FASD and other neurodegenerative disorders in the developing brain that shares similar cell death pathways.

LY294002 inhibits glucocorticoid-induced COX-2 gene expression in cardiomyocytes through a phosphatidylinositol 3 kinase-independent mechanism

International Nuclear Information System (INIS)

Sun Haipeng; Xu Beibei; Sheveleva, Elena; Chen, Qin M.

2008-01-01

Glucocorticoids induce COX-2 expression in rat cardiomyocytes. While investigating whether phosphatidylinositol 3 kinase (PI3K) plays a role in corticosterone (CT)-induced COX-2, we found that LY294002 (LY29) but not wortmannin (WM) attenuates CT from inducing COX-2 gene expression. Expression of a dominant-negative mutant of p85 subunit of PI3K failed to inhibit CT from inducing COX-2 expression. CT did not activate PI3K/AKT signaling pathway whereas LY29 and WM decreased the activity of PI3K. LY303511 (LY30), a structural analogue and a negative control for PI3K inhibitory activity of LY29, also suppressed COX-2 induction. These data suggest PI3K-independent mechanisms in regulating CT-induced COX-2 expression. LY29 and LY30 do not inhibit glucocorticoid receptor transactivity. Both compounds have been reported to inhibit Casein Kinase 2 activity and modulate potassium and calcium levels independent of PI3K, while LY29 has been reported to inhibit mammalian Target of Rapamycin (mTOR), and DNA-dependent Protein Kinase (DNA-PK). Inhibitor of Casein Kinase 2 (CK2), mTOR or DNA-PK failed to prevent CT from inducing COX-2 expression. Tetraethylammonium (TEA), a potassium channel blocker, and nimodipine, a calcium channel blocker, both attenuated CT from inducing COX-2 gene expression. CT was found to increase intracellular Ca 2+ concentration, which can be inhibited by LY29, TEA or nimodipine. These data suggest a possible role of calcium instead of PI3K in CT-induced COX-2 expression in cardiomyocytes

Thorax irradiation triggers a local and systemic accumulation of immunosuppressive CD4+ FoxP3+ regulatory T cells

International Nuclear Information System (INIS)

Wirsdörfer, Florian; Cappuccini, Federica; Niazman, Muska; Leve, Simone de; Westendorf, Astrid M; Lüdemann, Lutz; Stuschke, Martin; Jendrossek, Verena

2014-01-01

Lymphocyte infiltration is a common feature of radiation-induced pneumonitis and fibrosis, but their contribution to the pathogenic processes is still unclear. Here, we addressed the impact of thorax irradiation on the T cell compartment with a focus on immunosuppressive regulatory T cells (Treg). C57BL/6 wild type mice (WT) received anesthesia only (sham controls, 0 Gy) or were exposed to a single dose of whole thorax irradiation (15 Gy). Immune cells from lung tissue, spleen, and cervical lymph nodes were collected 10 to 84 days post-irradiation and phenotypically characterized by flow cytometry. Whole thorax irradiation provoked an increased influx of CD3+ T cells at 42 and 84 days post-irradiation. In contrast, local irradiation caused a sustained reduction in CD3+ T cells in peripheral lymphoid tissues. Interestingly, we observed a significant local and systemic increase in the fraction of CD4+ T cells expressing the transcription factor forkhead box P3 (FoxP3), the phenotypic marker for murine Treg, at day 21 post-irradiation. The accumulation of Treg was associated with increased levels of T cells expressing surface proteins characteristic for recruitment and immunosuppressive activity, e.g. CD103, CTLA-4 and CD73. Importantly, Treg isolated at this time point were able to suppress CD4+ effector T cells to a similar extent as Treg isolated from control mice. The response of the adaptive immune system to whole thorax irradiation is characterized by local immunoactivation and systemic immunosuppression. The transient accumulation of immunosuppressive CD4+ FoxP3+ Treg may be required to protect the lung against excessive inflammation-induced tissue damage. Further investigations shall define the mechanisms underlying the accumulation of Treg and their role for the pathogenesis of radiation-induced lung disease

Insulin Induces an Increase in Cytosolic Glucose Levels in 3T3-L1 Cells with Inhibited Glycogen Synthase Activation

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Helena H. Chowdhury

2014-10-01

Full Text Available Glucose is an important source of energy for mammalian cells and enters the cytosol via glucose transporters. It has been thought for a long time that glucose entering the cytosol is swiftly phosphorylated in most cell types; hence the levels of free glucose are very low, beyond the detection level. However, the introduction of new fluorescence resonance energy transfer-based glucose nanosensors has made it possible to measure intracellular glucose more accurately. Here, we used the fluorescent indicator protein (FLIPglu-600µ to monitor cytosolic glucose dynamics in mouse 3T3-L1 cells in which glucose utilization for glycogen synthesis was inhibited. The results show that cells exhibit a low resting cytosolic glucose concentration. However, in cells with inhibited glycogen synthase activation, insulin induced a robust increase in cytosolic free glucose. The insulin-induced increase in cytosolic glucose in these cells is due to an imbalance between the glucose transported into the cytosol and the use of glucose in the cytosol. In untreated cells with sensitive glycogen synthase activation, insulin stimulation did not result in a change in the cytosolic glucose level. This is the first report of dynamic measurements of cytosolic glucose levels in cells devoid of the glycogen synthesis pathway.

TNFR2 expression on CD25hiFOXP3+ T cells induced upon TCR stimulation of CD4 T cells identifies maximal cytokine-producing effectors.

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Chindu eGovindaraj

2013-08-01

Full Text Available In this study, we show that CD25hiTNFR2+ cells can be rapidly generated in vitro from circulating CD4 lymphocytes by polyclonal stimuli anti-CD3 in the presence of anti-CD28. The in vitro induced CD25hiTNFR2+ T cells express a conventional Treg phenotype FOXP3+CTLA4+CD127lo/-, but produce effector and immunoregulatory cytokines including IL-2, IL-10 and IFN-g. These induced CD25hiTNFR2+ T cells do not suppress target cell proliferation, but enhance it instead. Thus the CD25hiTNFR2+ phenotype induced rapidly following CD3/28 cross linking of CD4 T cells identifies cells with maximal proliferative and effector cytokine producing capability. The in vivo counterpart of this cell population may play an important role in immune response initiation.

Expression of Foxp3 in colorectal cancer but not in Treg cells correlates with disease progression in patients with colorectal cancer.

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Mia Kim

Full Text Available BACKGROUND: Regulatory T cells (Treg expressing the transcription factor forkhead-box protein P3 (Foxp3 have been identified to counteract anti-tumor immune responses during tumor progression. Besides, Foxp3 presentation by cancer cells itself may also allow them to evade from effector T-cell responses, resulting in a survival benefit of the tumor. For colorectal cancer (CRC the clinical relevance of Foxp3 has not been evaluated in detail. Therefore the aim of this study was to study its impact in colorectal cancer (CRC. METHODS AND FINDINGS: Gene and protein analysis of tumor tissues from patients with CRC was performed to quantify the expression of Foxp3 in tumor infiltrating Treg and colon cancer cells. The results were correlated with clinicopathological parameters and patients overall survival. Serial morphological analysis demonstrated Foxp3 to be expressed in cancer cells. High Foxp3 expression of the cancer cells was associated with poor prognosis compared to patients with low Foxp3 expression. In contrast, low and high Foxp3 level in tumor infiltrating Treg cells demonstrated no significant differences in overall patient survival. CONCLUSIONS: Our findings strongly suggest that Foxp3 expression mediated by cancer cells rather than by Treg cells contribute to disease progression.

Tiron Inhibits UVB-Induced AP-1 Binding Sites Transcriptional Activation on MMP-1 and MMP-3 Promoters by MAPK Signaling Pathway in Human Dermal Fibroblasts.

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Jing Lu

Full Text Available Recent research found that Tiron was an effective antioxidant that could act as the intracellular reactive oxygen species (ROS scavenger or alleviate the acute toxic metal overload in vivo. In this study, we investigated the inhibitory effect of Tiron on matrix metalloproteinase (MMP-1 and MMP-3 expression in human dermal fibroblast cells. Western blot and ELISA analysis revealed that Tiron inhibited ultraviolet B (UVB-induced protein expression of MMP-1 and MMP-3. Real-time quantitative PCR confirmed that Tiron could inhibit UVB-induced mRNA expression of MMP-1 and MMP-3. Furthermore, Tiron significantly blocked UVB-induced activation of the MAPK signaling pathway and activator protein (AP-1 in the downstream of this transduction pathway in fibroblasts. Through the AP-1 binding site mutation, it was found that Tiron could inhibit AP-1-induced upregulation of MMP-1 and MMP-3 expression through blocking AP-1 binding to the AP-1 binding sites in the MMP-1 and MMP-3 promoter region. In conclusion, Tiron may be a novel antioxidant for preventing and treating skin photoaging UV-induced.

A Bivalent Securinine Compound SN3-L6 Induces Neuronal Differentiation via Translational Upregulation of Neurogenic Transcription Factors

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Yumei Liao

2018-04-01

Full Text Available Developing therapeutic approaches that target neuronal differentiation will be greatly beneficial for the regeneration of neurons and synaptic networks in neurological diseases. Protein synthesis (mRNA translation has recently been shown to regulate neurogenesis of neural stem/progenitor cells (NSPCs. However, it has remained unknown whether engineering translational machinery is a valid approach for manipulating neuronal differentiation. The present study identifies that a bivalent securinine compound SN3-L6, previously designed and synthesized by our group, induces potent neuronal differentiation through a novel translation-dependent mechanism. An isobaric tag for relative and absolute quantitation (iTRAQ-based proteomic analysis in Neuro-2a progenitor cells revealed that SN3-L6 upregulated a group of neurogenic transcription regulators, and also upregulated proteins involved in RNA processing, translation, and protein metabolism. Notably, puromycylation and metabolic labeling of newly synthesized proteins demonstrated that SN3-L6 induced rapid and robust activation of general mRNA translation. Importantly, mRNAs of the proneural transcription factors Foxp1, Foxp4, Hsf1, and Erf were among the targets that were translationally upregulated by SN3-L6. Either inhibition of translation or knockdown of these transcription factors blocked SN3-L6 activity. We finally confirmed that protein synthesis of a same set of transcription factors was upregulated in primary cortical NPCs. These findings together identify a new compound for translational activation and neuronal differentiation, and provide compelling evidence that reprogramming transcriptional regulation network at translational levels is a promising strategy for engineering NSPCs.

Silymarin attenuated paraquat-induced cytotoxicity in macrophage by regulating Trx/TXNIP complex, inhibiting NLRP3 inflammasome activation and apoptosis.

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Liu, Zhenning; Sun, Mingli; Wang, Yu; Zhang, Lichun; Zhao, Hang; Zhao, Min

2018-02-01

Oxidative stress and inflammation are involved in paraquat-induced cytotoxicity. Silymarin can exert a potent antioxidative and anti-inflammatory effect in various pathophysiological processes. The aim of this current study is to explore the protective effect and potential mechanism of silymarin in paraquat-induced macrophage injury. Cells were pretreated with different doses of silymarin for 3h before exposure to paraquat. At 24h after exposure to paraquat, the paraquat-induced cytotoxicity to macrophage was measured via the MTT assay and LDH release. The levels of intracellular reactive oxygen species, GSH-Px, SOD, and lipid peroxidation product malondialdehyde were measured to evaluate the oxidative effect of paraquat. NLRP3 inflammasome and cytokines secretion in macrophage exposed to paraquat at 24h were measured via immunofluorescence microscopy, western blot or Elisa. Our results revealed that paraquat could dramatically cause cytotoxicity and reactive oxygen species generation, enhance TXNIP expression, and induce NLRP3 inflammasome activation and cytokines secretion. The pretreatment with silymarin could remarkably reduce the cytotoxicity, promote the expression of Trx and antioxidant enzymes, and suppress the TXNIP and NLRP3 inflammasome activation. In conclusion, silymarin attenuated paraquat-induced cytotoxicity in macrophage by inhibiting oxidative stress, NLRP3 inflammasome activation, cytokines secretion and apoptosis. Copyright © 2017 Elsevier Ltd. All rights reserved.

Reishi Protein LZ-8 Induces FOXP3+ Treg Expansion via a CD45-Dependent Signaling Pathway and Alleviates Acute Intestinal Inflammation in Mice

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Hsien-Yeh Hsu

2013-01-01

Full Text Available LZ-8, an immunomodulatory protein isolated from Ganoderma lucidum (also known as Ling-Zhi or Reishi, has been shown to promote cell proliferation and IL-2 production in T cells. In this study, we show that LZ-8 induces the expansion of both murine and human CD4+ T cells into FOXP3+ regulatory T (Treg cells. LZ-8 treatment was found to stimulate a 4-fold and a 10-fold expansion in the Treg populations of murine and human primary CD4+ T cells, respectively. In addition, the expression of CTLA-4 and IL-10 was induced in LZ-8-treated CD4+ T cells. Using neutralizing antibodies and gene-deficient T-cell lines, we also found that LZ-8 promotes Treg expansion through a CD45-mediated signaling pathway and that the CD18-dependent induction of IL-2 was involved in Treg formation and IL-10 production. The suppressive activity of LZ-8 was confirmed using a murine model of DSS-induced colitis; the disease was alleviated by the adoptive transfer of LZ-8-treated CD4+ T cells. In conclusion, a new regulatory function for LZ-8 was identified, and the molecular mechanisms underlying this function were elucidated.

Maternal variant in the upstream of FOXP3 gene on the X chromosome is associated with recurrent infertility in Japanese Black cattle.

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Arishima, Taichi; Sasaki, Shinji; Isobe, Tomohiro; Ikebata, Yoshihisa; Shimbara, Shinichi; Ikeda, Shogo; Kawashima, Keisuke; Suzuki, Yutaka; Watanabe, Manabu; Sugano, Sumio; Mizoshita, Kazunori; Sugimoto, Yoshikazu

2017-12-06

Repeat breeding, which is defined as cattle failure to conceive after three or more inseminations in the absence of clinical abnormalities, is a substantial problem in cattle breeding. To identify maternal genetic variants of repeat breeding in Japanese Black cattle, we selected 29 repeat-breeding heifers that failed to conceive following embryo transfer (ET) and conducted a genome-wide association study (GWAS) using the traits. We found that a single-nucleotide polymorphism (SNP; g.92,377,635A > G) in the upstream region of the FOXP3 gene on the X chromosome was highly associated with repeat breeding and failure to conceive following ET (P = 1.51 × 10 -14 ). FOXP3 is a master gene for differentiation of regulatory T (T reg ) cells that function in pregnancy maintenance. Reporter assay results revealed that the activity of the FOXP3 promoter was lower in reporter constructs with the risk-allele than in those with the non-risk-allele by approximately 0.68 fold. These findings suggest that the variant in the upstream region of FOXP3 with the risk-allele decreased FOXP3 transcription, which in turn, could reduce the number of maternal T reg cells and lead to infertility. The frequency of the risk-allele in repeat-breeding heifers is more than that in cows, suggesting that the risk-allele could be associated with infertility in repeat-breeding heifers. This GWAS identified a maternal variant in the upstream region of FOXP3 that was associated with infertility in repeat-breeding Japanese Black cattle that failed to conceive using ET. The variant affected the level of FOXP3 mRNA expression. Thus, the results suggest that the risk-allele could serve as a useful marker to reduce and eliminate animals with inferior fertility in Japanese Black cattle.

FOXP3+ regulatory T cells in cutaneous T-cell lymphomas: association with disease stage and survival

DEFF Research Database (Denmark)

Gjerdrum, L M; Woetmann, A; Odum, Niels

2007-01-01

FOXP3 is a unique marker for CD4+CD25+ regulatory T cells (Tregs). In solid tumours, high numbers of Tregs are associated with a poor prognosis. Knowledge about the implications of Tregs for the behaviour of haematological malignancies is limited. In this study, skin biopsies from 86 patients...... with mycosis fungoides (MF) and cutaneous T-cell lymphoma (CTCL) unspecified were analysed for the expression of FOXP3 on tumour cells and tumour-infiltrating Tregs. Labelling of above 10% of the neoplastic cells was seen in one case classified as an aggressive epidermotropic CD8+ cytotoxic CTCL....... In the remaining 85 cases, the atypical neoplastic infiltrate was either FOXP3 negative (n=80) or contained only very occasional weakly positive cells (n=5). By contrast, all biopsies showed varying numbers of strongly FOXP3+ tumour-infiltrating Tregs. MF with early or infiltrated plaques had significantly higher...

Sulforaphane inhibits CYP1A1 activity and promotes genotoxicity induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin in vitro

Energy Technology Data Exchange (ETDEWEB)

Yang, Fangxing, E-mail: fxyang@zju.edu.cn [MOE Key Laboratory of Environmental Remediation and Ecosystem Health, College of Environmental and Resource Sciences, Zhejiang University, Hangzhou, 310058 (China); Zhuang, Shulin [MOE Key Laboratory of Environmental Remediation and Ecosystem Health, College of Environmental and Resource Sciences, Zhejiang University, Hangzhou, 310058 (China); Zhang, Chao; Dai, Heping [State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, 430072 (China); Liu, Weiping, E-mail: wliu@zju.edu.cn [MOE Key Laboratory of Environmental Remediation and Ecosystem Health, College of Environmental and Resource Sciences, Zhejiang University, Hangzhou, 310058 (China)

2013-06-15

Increasing environmental pollution by carcinogens such as some of persistent organic pollutants (POPs) has prompted growing interest in searching for chemopreventive compounds which are readily obtainable. Sulforaphane (SFN) is isolated from cruciferous vegetables and has the potentials to reduce carcinogenesis through various pathways. In this study, we studied the effects of SFN on CYP1A1 activity and genotoxicity induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The results showed that SFN inhibited TCDD-induced CYP1A1 activity in H4IIE cells by directly inhibiting CYP1A1 activity, probably through binding to aryl hydrocarbon receptor and/or CYP1A1 revealed by molecular docking. However, SFN promoted TCDD-induced DNA damage in yeast cells and reduced the viability of initiated yeast cells. Besides, it is surprising that SFN also failed to reduce genotoxicity induced by other genotoxic reagents which possess different mechanisms to lead to DNA damage. Currently, it is difficult to predict whether SFN has the potentials to reduce the risk of TCDD based on the conflicting observations in the study. Therefore, further studies should be urgent to reveal the function and mechanism of SFN in the stress of such POPs on human health. - Highlights: • Sulforaphane inhibited TCDD-induced CYP1A1 activity in H4IIE cells. • Sulforaphane may bind to aryl hydrocarbon receptor and/or CYP1A1. • Sulforaphane promoted TCDD-induced DNA damage in yeast cells. • Sulforaphane may promote DNA damage by DNA strand breaks or DNA alkylation.

Synergistic Interactions with PI3K Inhibition that Induce Apoptosis. | Office of Cancer Genomics

Science.gov (United States)

Activating mutations involving the PI3K pathway occur frequently in human cancers. However, PI3K inhibitors primarily induce cell cycle arrest, leaving a significant reservoir of tumor cells that may acquire or exhibit resistance. We searched for genes that are required for the survival of PI3K mutant cancer cells in the presence of PI3K inhibition by conducting a genome scale shRNA-based apoptosis screen in a PIK3CA mutant human breast cancer cell. We identified 5 genes (PIM2, ZAK, TACC1, ZFR, ZNF565) whose suppression induced cell death upon PI3K inhibition.

Mercuric ions inhibit mitogen-activated protein kinase dephosphorylation by inducing reactive oxygen species

International Nuclear Information System (INIS)

Haase, Hajo; Engelhardt, Gabriela; Hebel, Silke; Rink, Lothar

2011-01-01

Mercury intoxication profoundly affects the immune system, in particular, signal transduction of immune cells. However, the mechanism of the interaction of mercury with cellular signaling pathways, such as mitogen activated protein kinases (MAPK), remains elusive. Therefore, the objective of this study is to investigate three potential ways in which Hg 2+ ions could inhibit MAPK dephosphorylation in the human T-cell line Jurkat: (1) by direct binding to phosphatases; (2) by releasing cellular zinc (Zn 2+ ); and (3) by inducing reactive oxygen species (ROS). Hg 2+ causes production of ROS, measured by dihydrorhodamine 123, and triggers ROS-mediated Zn 2+ release, detected with FluoZin-3. Yet, phosphatase-inhibition is not mediated by binding of Zn 2+ or Hg 2+ . Rather, phosphatases are inactivated by at least two forms of thiol oxidation; initial inhibition is reversible with reducing agents such as Tris(2-carboxyethyl)phosphine. Prolonged inhibition leads to non-reversible phosphatase oxidation, presumably oxidizing the cysteine thiol to sulfinic- or sulfonic acid. Notably, phosphatases are a particularly sensitive target for Hg 2+ -induced oxidation, because phosphatase activity is inhibited at concentrations of Hg 2+ that have only minor impact on over all thiol oxidation. This phosphatase inhibition results in augmented, ROS-dependent MAPK phosphorylation. MAPK are important regulators of T-cell function, and MAPK-activation by inhibition of phosphatases seems to be one of the molecular mechanisms by which mercury affects the immune system.

Circulating gluten-specific FOXP3+CD39+ regulatory T cells have impaired suppressive function in patients with celiac disease.

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Cook, Laura; Munier, C Mee Ling; Seddiki, Nabila; van Bockel, David; Ontiveros, Noé; Hardy, Melinda Y; Gillies, Jana K; Levings, Megan K; Reid, Hugh H; Petersen, Jan; Rossjohn, Jamie; Anderson, Robert P; Zaunders, John J; Tye-Din, Jason A; Kelleher, Anthony D

2017-12-01

Celiac disease is a chronic immune-mediated inflammatory disorder of the gut triggered by dietary gluten. Although the effector T-cell response in patients with celiac disease has been well characterized, the role of regulatory T (Treg) cells in the loss of tolerance to gluten remains poorly understood. We sought to define whether patients with celiac disease have a dysfunction or lack of gluten-specific forkhead box protein 3 (FOXP3) + Treg cells. Treated patients with celiac disease underwent oral wheat challenge to stimulate recirculation of gluten-specific T cells. Peripheral blood was collected before and after challenge. To comprehensively measure the gluten-specific CD4 + T-cell response, we paired traditional IFN-γ ELISpot with an assay to detect antigen-specific CD4 + T cells that does not rely on tetramers, antigen-stimulated cytokine production, or proliferation but rather on antigen-induced coexpression of CD25 and OX40 (CD134). Numbers of circulating gluten-specific Treg cells and effector T cells both increased significantly after oral wheat challenge, peaking at day 6. Surprisingly, we found that approximately 80% of the ex vivo circulating gluten-specific CD4 + T cells were FOXP3 + CD39 + Treg cells, which reside within the pool of memory CD4 + CD25 + CD127 low CD45RO + Treg cells. Although we observed normal suppressive function in peripheral polyclonal Treg cells from patients with celiac disease, after a short in vitro expansion, the gluten-specific FOXP3 + CD39 + Treg cells exhibited significantly reduced suppressive function compared with polyclonal Treg cells. This study provides the first estimation of FOXP3 + CD39 + Treg cell frequency within circulating gluten-specific CD4 + T cells after oral gluten challenge of patients with celiac disease. FOXP3 + CD39 + Treg cells comprised a major proportion of all circulating gluten-specific CD4 + T cells but had impaired suppressive function, indicating that Treg cell dysfunction might be a key

A short caspase-3 isoform inhibits chemotherapy-induced apoptosis by blocking apoptosome assembly.

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Frédérique Végran

Full Text Available Alternative splicing of caspase-3 produces a short isoform caspase-3s that antagonizes caspase-3 apoptotic activity. However, the mechanism of apoptosis inhibition by caspase-3s remains unknown. Here we show that exogenous caspase-3 sensitizes MCF-7 and HBL100 breast cancers cells to chemotherapeutic treatments such as etoposide and methotrexate whereas co-transfection with caspase-3s strongly inhibits etoposide and methotrexate-induced apoptosis underlying thus the anti-apoptotic role of caspase-3s. In caspase-3 transfected cells, lamin-A and α-fodrin were cleaved when caspase-3 was activated by etoposide or methotrexate. When caspase-3s was co-transfected, this cleavage was strongly reduced. Depletion of caspase-3 by RNA interference in HBL100 containing endogenous caspase-3s caused reduction in etoposide and methotrexate-induced apoptosis, whereas the depletion of caspase-3s sensitized cells to chemotherapy. In the presence of caspase-3s, a lack of interaction between caspase-3 and caspase-9 was observed. Immunoprecipitation assays showed that caspase-3s binds the pro-forms of caspase-3. This result suggested that the absence of interaction with caspase-9 when both variants of caspase-3 are present contribute to block the apoptosome assembly and inhibit apoptosis. These data support that caspases-3s negatively interferes with caspase-3 activation and apoptosis in breast cancer, and that it can play key roles in the modulation of response to chemotherapeutic treatments.

EFFECTS OF HISTONE DEACETYLASE INHIBITOR, SAHA, ON EFFECTOR AND FOXP3+ REGULATORY T CELLS IN RHESUS MACAQUES

Science.gov (United States)

Johnson, Jennifer; Pahuja, Anil; Graham, Melanie; Hering, Bernhard; Hancock, Wayne W.; Pratima, Bansal-Pakala

2008-01-01

SAHA, a histone deacetylase inhibitor (HDACi), is clinically approved for treatment of cutaneous T-cell lymphoma. Although the exact underlying mechanisms are unknown, HDACi arrest the cell cycle in rapidly proliferating tumor cells and promote their apoptosis. HDACi were also recently shown to enhance the production and suppressive functions of Foxp3+ regulatory T (Treg) cells in rodents, leading us to begin to investigate the actions of HDACi on rhesus monkey T cells for the sake of potential preclinical applications. In this study, we show that SAHA inhibits polyclonal activation and proliferation of rhesus T cells and that the anti-proliferative effects are due to inhibition of T effector (Teff) cells and enhancement of Treg cells. Cryopreserved rhesus macaque splenocytes were CFSE labeled, stimulated with anti-CD3/anti-CD28 and cultured for 5 days in the presence of varying concentrations of SAHA. Samples were then co-stained to evaluate CD4 and CD8 expression. 10 and 5μM concentrations of SAHA were toxic to splenocytes. Proliferation was inhibited by 57% in CD4 cells and 47% in CD8 cells when unseparated splenocytes were cultured with 3 μM SAHA. Effector cells alone showed a decreased inhibition to proliferation when cultured with 3 μM and 1 μM SAHA when compared to Teff plus Treg cells. Our data suggest that SAHA can be used as part of an immunosuppressive protocol to enhance graft survival by limiting Teff cell proliferation as well as increasing Treg cells, thereby promoting tolerance. PMID:18374101

Smad3 induces atrogin-1, inhibits mTOR and protein synthesis, and promotes muscle atrophy in vivo.

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Goodman, Craig A; McNally, Rachel M; Hoffmann, F Michael; Hornberger, Troy A

2013-11-01

Myostatin, a member of the TGF superfamily, is sufficient to induce skeletal muscle atrophy. Myostatin-induced atrophy is associated with increases in E3-ligase atrogin-1 expression and protein degradation and decreases in Akt/mechanistic target of rapamycin (mTOR) signaling and protein synthesis. Myostatin signaling activates the transcription factor Smad3 (Small Mothers Against Decapentaplegic), which has been shown to be necessary for myostatin-induced atrogin-1 expression and atrophy; however, it is not known whether Smad3 is sufficient to induce these events or whether Smad3 simply plays a permissive role. Thus, the aim of this study was to address these questions with an in vivo model. To accomplish this goal, in vivo transfection of plasmid DNA was used to create transient transgenic mouse skeletal muscles, and our results show for the first time that Smad3 expression is sufficient to stimulate atrogin-1 promoter activity, inhibit Akt/mTOR signaling and protein synthesis, and induce muscle fiber atrophy. Moreover, we propose that Akt/mTOR signaling is inhibited by a Smad3-induced decrease in microRNA-29 (miR-29) expression and a subsequent increase in the translation of phosphatase and tensin homolog (PTEN) mRNA. Smad3 is also sufficient to inhibit peroxisome proliferator-activated receptor-γ coactivator-1α (PGC1α) promoter activity and to increase FoxO (Forkhead Box Protein, Subclass O)-mediated signaling and the promoter activity of plasminogen activator inhibitor 1 (PAI-1). Combined, this study provides the first evidence that Smad3 is sufficient to regulate many of the events associated with myostatin-induced atrophy and therefore suggests that Smad3 signaling may be a viable target for therapies aimed at preventing myostatin-induced muscle atrophy.

Analysis of FOXP3+ regulatory T cells that display apparent viral antigen specificity during chronic hepatitis C virus infection.

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Shuo Li

2009-12-01

Full Text Available We reported previously that a proportion of natural CD25(+ cells isolated from the PBMC of HCV patients can further upregulate CD25 expression in response to HCV peptide stimulation in vitro, and proposed that virus-specific regulatory T cells (Treg were primed and expanded during the disease. Here we describe epigenetic analysis of the FOXP3 locus in HCV-responsive natural CD25(+ cells and show that these cells are not activated conventional T cells expressing FOXP3, but hard-wired Treg with a stable FOXP3 phenotype and function. Of approximately 46,000 genes analyzed in genome wide transcription profiling, about 1% were differentially expressed between HCV-responsive Treg, HCV-non-responsive natural CD25(+ cells and conventional T cells. Expression profiles, including cell death, activation, proliferation and transcriptional regulation, suggest a survival advantage of HCV-responsive Treg over the other cell populations. Since no Treg-specific activation marker is known, we tested 97 NS3-derived peptides for their ability to elicit CD25 response (assuming it is a surrogate marker, accompanied by high resolution HLA typing of the patients. Some reactive peptides overlapped with previously described effector T cell epitopes. Our data offers new insights into HCV immune evasion and tolerance, and highlights the non-self specific nature of Treg during infection.

FOXP2 promotes the nuclear translocation of POT1, but FOXP2(R553H), mutation related to speech-language disorder, partially prevents it

Energy Technology Data Exchange (ETDEWEB)

Tanabe, Yuko [Division of Development and Differentiation, National Institute of Neuroscience, NCNP, 4-1-1 Ogawahigasi, Kodaira 187-8511 (Japan); Fujita, Eriko [Division of Development and Differentiation, National Institute of Neuroscience, NCNP, 4-1-1 Ogawahigasi, Kodaira 187-8511 (Japan); Department of Pediatrics, Jichi Medical University, 3311-1 Yakushiji, Shimotsuke, Tochigi 329-0498 (Japan); Momoi, Takashi, E-mail: momoi@iuhw.ac.jp [Division of Development and Differentiation, National Institute of Neuroscience, NCNP, 4-1-1 Ogawahigasi, Kodaira 187-8511 (Japan); Center for Medical Science, International University of Health and Welfare, 2600-1 Kitakanamaru, Otawara, Tochigi 324-8501 (Japan)

2011-07-08

Highlights: {yields} We isolated protection of telomeres 1 (POT1) as a FOXP2-associated protein by a yeast two-hybrid. {yields} FOXP2 associated and co-localized with POT1 in the nuclei. {yields} FOXP2(R553H) also co-localized with POT1 in both the cytoplasm and nuclei. {yields} FOXP2(R553H) partially prevented the nuclear translocation of POT1. {yields} FOXP2(R553H) mutation may be associated with the pathogenesis of speech-language disorder. -- Abstract: FOXP2 is a forkhead box-containing transcription factor with several recognizable sequence motifs. However, little is known about the FOXP2-associated proteins except for C-terminal binding protein (CtBP). In the present study, we attempted to isolate the FOXP2-associated protein with a yeast two-hybrid system using the C-terminal region, including the forkhead domain, as a bait probe, and identified protection of telomeres 1 (POT1) as a FOXP2-associated protein. Immunoprecipitation assay confirmed the association with FOXP2 and POT1. POT1 alone localized in the cytoplasm but co-localized with FOXP2 and the forkhead domain of FOXP2 in nuclei. However, both FOXP2 with mutated nuclear localization signals and (R553H) mutated forkhead, which is associated with speech-language disorder, prevented the nuclear translocation of POT1. These results suggest that FOXP2 is a binding partner for the nuclear translocation of POT1. As loss of POT1 function induces the cell arrest, the impaired nuclear translocation of POT1 in the developing neuronal cells may be associated with the pathogenesis of speech-language disorder with FOXP2(R553H) mutation.

FOXP2 promotes the nuclear translocation of POT1, but FOXP2(R553H), mutation related to speech-language disorder, partially prevents it

International Nuclear Information System (INIS)

Tanabe, Yuko; Fujita, Eriko; Momoi, Takashi

2011-01-01

Highlights: → We isolated protection of telomeres 1 (POT1) as a FOXP2-associated protein by a yeast two-hybrid. → FOXP2 associated and co-localized with POT1 in the nuclei. → FOXP2(R553H) also co-localized with POT1 in both the cytoplasm and nuclei. → FOXP2(R553H) partially prevented the nuclear translocation of POT1. → FOXP2(R553H) mutation may be associated with the pathogenesis of speech-language disorder. -- Abstract: FOXP2 is a forkhead box-containing transcription factor with several recognizable sequence motifs. However, little is known about the FOXP2-associated proteins except for C-terminal binding protein (CtBP). In the present study, we attempted to isolate the FOXP2-associated protein with a yeast two-hybrid system using the C-terminal region, including the forkhead domain, as a bait probe, and identified protection of telomeres 1 (POT1) as a FOXP2-associated protein. Immunoprecipitation assay confirmed the association with FOXP2 and POT1. POT1 alone localized in the cytoplasm but co-localized with FOXP2 and the forkhead domain of FOXP2 in nuclei. However, both FOXP2 with mutated nuclear localization signals and (R553H) mutated forkhead, which is associated with speech-language disorder, prevented the nuclear translocation of POT1. These results suggest that FOXP2 is a binding partner for the nuclear translocation of POT1. As loss of POT1 function induces the cell arrest, the impaired nuclear translocation of POT1 in the developing neuronal cells may be associated with the pathogenesis of speech-language disorder with FOXP2(R553H) mutation.

Autotaxin is induced by TSA through HDAC3 and HDAC7 inhibition and antagonizes the TSA-induced cell apoptosis.

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Li, Song; Wang, Baolu; Xu, Yan; Zhang, Junjie

2011-02-12

Autotaxin (ATX) is a secreted glycoprotein with the lysophospholipase D (lysoPLD) activity to convert lysophosphatidylcholine (LPC) into lysophosphatidic acid (LPA), a bioactive lysophospholipid involved in diverse biological actions. ATX is highly expressed in some cancer cells and contributes to their tumorigenesis, invasion, and metastases, while in other cancer cells ATX is silenced or expressed at low level. The mechanism of ATX expression regulation in cancer cells remains largely unknown. In the present study, we demonstrated that trichostatin A (TSA), a well-known HDAC inhibitor (HDACi), significantly induced ATX expression in SW480 and several other cancer cells with low or undetectable endogenous ATX expression. ATX induction could be observed when HDAC3 and HDAC7 were down-regulated by their siRNAs. It was found that HDAC7 expression levels were low in the cancer cells with high endogenous ATX expression. Exogenous over-expression of HDAC7 inhibited ATX expression in these cells in a HDAC3-dependent manner. These data indicate that HDAC3 and HDAC7 collaboratively suppress ATX expression in cancer cells, and suggest that TSA induce ATX expression by inhibiting HDAC3 and HDAC7. The biological significance of this regulation mechanism was revealed by demonstrating that TSA-induced ATX protected cancer cells against TSA-induced apoptosis by producing LPA through its lysoPLD activity, which could be reversed by BrP-LPA and S32826, the inhibitors of the ATX-LPA axis. We have demonstrated that ATX expression is repressed by HDAC3 and HDAC7 in cancer cells. During TSA treatment, ATX is induced due to the HDAC3 and HDAC7 inhibition and functionally antagonizes the TSA-induced apoptosis. These results reveal an internal HDACi-resistant mechanism in cancer cells, and suggest that the inhibition of ATX-LPA axis would be helpful to improve the efficacy of HDACi-based therapeutics against cancer.

Curcumin blocks interleukin (IL)-2 signaling in T-lymphocytes by inhibiting IL-2 synthesis, CD25 expression, and IL-2 receptor signaling

International Nuclear Information System (INIS)

Forward, Nicholas A.; Conrad, David M.; Power Coombs, Melanie R.; Doucette, Carolyn D.; Furlong, Suzanne J.; Lin, Tong-Jun; Hoskin, David W.

2011-01-01

Highlights: → Curcumin inhibits CD4 + T-lymphocyte proliferation. → Curcumin inhibits interleukin-2 (IL-2) synthesis and CD25 expression by CD4 + T-lymphocytes. → Curcumin interferes with IL-2 receptor signaling by inhibiting JAK3 and STAT5 phosphorylation. → IL-2-dependent regulatory T-lymphocyte function and Foxp3 expression is downregulated by curcumin. -- Abstract: Curcumin (diferulomethane) is the principal curcuminoid in the spice tumeric and a potent inhibitor of activation-induced T-lymphocyte proliferation; however, the molecular basis of this immunosuppressive effect has not been well studied. Here we show that micromolar concentrations of curcumin inhibited DNA synthesis by mouse CD4 + T-lymphocytes, as well as interleukin-2 (IL-2) and CD25 (α chain of the high affinity IL-2 receptor) expression in response to antibody-mediated cross-linking of CD3 and CD28. Curcumin acted downstream of protein kinase C activation and intracellular Ca 2+ release to inhibit IκB phosphorylation, which is required for nuclear translocation of the transcription factor NFκB. In addition, IL-2-dependent DNA synthesis by mouse CTLL-2 cells, but not constitutive CD25 expression, was impaired in the presence of curcumin, which demonstrated an inhibitory effect on IL-2 receptor (IL-2R) signaling. IL-2-induced phosphorylation of STAT5A and JAK3, but not JAK1, was diminished in the presence of curcumin, indicating inhibition of critical proximal events in IL-2R signaling. In line with the inhibitory action of curcumin on IL-2R signaling, pretreatment of CD4 + CD25 + regulatory T-cells with curcumin downregulated suppressor function, as well as forkhead box p3 (Foxp3) expression. We conclude that curcumin inhibits IL-2 signaling by reducing available IL-2 and high affinity IL-2R, as well as interfering with IL-2R signaling.

FOXP3 is a novel transcriptional repressor for the breast cancer oncogene SKP2

OpenAIRE

Zuo, Tao; Liu, Runhua; Zhang, Huiming; Chang, Xing; Liu, Yan; Wang, Lizhong; Zheng, Pan; Liu, Yang

2007-01-01

S-phase kinase-associated protein 2 (SKP2) is a component of the E3 ubiquitin ligase SKP1-Cul1-Fbox complex. Overexpression of SKP2 results in cell cycle dysregulation and carcinogenesis; however, the genetic lesions that cause this upregulation are poorly understood. We recently demonstrated that forkhead box P3 (FOXP3) is an X-linked breast cancer suppressor and an important repressor of the oncogene ERBB2/HER2. Since FOXP3 suppresses tumor growth regardless of whether the tumors overexpres...

FOXP3+ T Regulatory Cell Modifications in Inflammatory Bowel Disease Patients Treated with Anti-TNFα Agents

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Luisa Guidi

2013-01-01

Full Text Available Treg modulation has been hypothesized as one of the mechanisms by which antitumor necrosis factor α (TNFα agents exert their action in rheumatoid arthritis (RA and inflammatory bowel disease (IBD. However, data in IBD are still conflicting. We evaluated CD4+CD25+FOXP3+ (Tregs by flow cytometry in peripheral blood from 32 adult IBD patient before (T0 and after the induction of anti-TNFα therapy (T1. Eight healthy controls (HCs were included. We also evaluated the number of FOXP3+ cells in the lamina propria (LP in biopsies taken in a subset of patients and controls. Treg frequencies were significantly increased in peripheral blood from our patients after anti-TNFα therapy compared to T0. T1 but not T0 levels were higher than HC. The increase was detectable only in clinical responders to the treatment. A negative correlation was found among delta Treg levels and the age of patients or disease duration and with the activity score of Crohn’s disease (CD. No significant differences were found in LP FOXP3+ cells. Our data suggest the possibility that in IBD patients the treatment with anti-TNFα may affect Treg percentages and that Treg modifications may correlate with clinical response, but differently in early versus late disease.

CD4~+Foxp3~+ regulatory T cells converted by rapamycin from peripheral CD4~+ CD25~-naive T cells display more potent regulatory ability in vitro

Institute of Scientific and Technical Information of China (English)

CHEN Jian-fei; GAO Jie; ZHANG Dong; WANG Zi-han; ZHU Ji-ye

2010-01-01

Background Rapamycin (RAPA) is a relatively new immunosuppressant drug that functions as a serine/threonine kinase inhibitor to prevent rejection in organ transplantation. RAPA blocks activation of T-effector (Teff) cells by inhibiting the response to interleukin-2. Recently, RAPA was also shown to selectively expand the T-regulator (Treg) cell population. To date, no studies have examined the mechanism by which RAPA converts Teff cells to Treg cells. Methods Peripheral CD4~+CD25~- naive T cells were cultivated with RAPA and B cells as antigen-presenting cells (APCs) in vitro. CD4~+CD25~- T cells were harvested after 6 days and analyzed for expression of forkhead box protein 3 (Foxp3) using flow cytometry. CD4~+CD25~+CD127~- subsets as the converted Tregs were isolated from the mixed lymphocyte reactions (MLR) with CD127 negative selection, followed by CD4 and CD25 positive selection using microbeads and magnetic separation column (MSC). Moreover, mRNA was extracted from converted Tregs and C57BL/6 naive CD4~+CD25~+ T cells and Foxp3 levels were examined by quantitative real-time polymerase chain reaction (rt-PCR). A total of 1×10~5 carboxyfluorescein succinimidyl ester (CFSE)-labeled naive CD4~+CD25~- T cells/well from C57BL/6 mice were cocultured with DBA/2 or C3H maturation of dendritic cells (mDCs) (0.25×10~5/well) in 96-well round-bottom plates for 6 days. Then 1×10~5 or 0.25×10~5 converted Treg cells were added to every well as regulatory cells. Cells were harvested after 6 days of culture and analyzed for proliferation of CFSE-labeled naive CD4~+CD25~- T cells using flow cytometry. Data were analyzed using CellQuest software.Results We found that RAPA can convert peripheral CD4~+CD25~- naive T Cells to CD4~+Foxp3~+ Treg cells using B cells as APCs, and this subtype of Treg can potently suppress Teff proliferation and maintain antigenic specificity. Conclusion Our findings provide evidence that RAPA induces Treg cell conversion from Teff cells and

Salmon cartilage proteoglycan suppresses mouse experimental colitis through induction of Foxp3{sup +} regulatory T cells

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Mitsui, Toshihito [Department of Microbiology and Immunology, Hirosaki University Graduate School of Medicine, Zaifu-cho 5, Hirosaki, Aomori 036-8562 (Japan); Department of Digestive Surgery, Hirosaki University Graduate School of Medicine, Hirosaki, Aomori 036-8562 (Japan); Sashinami, Hiroshi [Department of Microbiology and Immunology, Hirosaki University Graduate School of Medicine, Zaifu-cho 5, Hirosaki, Aomori 036-8562 (Japan); Sato, Fuyuki; Kijima, Hiroshi [Department of Pathology and Bioscience, Hirosaki University Graduate School of Medicine, Hirosaki, Aomori 036-8562 (Japan); Ishiguro, Yoh; Fukuda, Shinsaku [Department of Digestive Internal Medicine, Hirosaki University Graduate School of Medicine, Hirosaki, Aomori 036-8562 (Japan); Yoshihara, Shuichi [Department of Glycomedicine, Hirosaki University Graduate School of Medicine, Hirosaki, Aomori 036-8562 (Japan); Hakamada, Ken-Ichi [Department of Digestive Surgery, Hirosaki University Graduate School of Medicine, Hirosaki, Aomori 036-8562 (Japan); Nakane, Akio, E-mail: a27k03n0@cc.hirosaki-u.ac.jp [Department of Microbiology and Immunology, Hirosaki University Graduate School of Medicine, Zaifu-cho 5, Hirosaki, Aomori 036-8562 (Japan)

2010-11-12

Research highlights: {yields} Salmon proteoglycan suppresses IL-10{sup -/-} cell transfer-induced colitis progression. {yields} Salmon proteoglycan suppresses Th1- and Th17-related factors in colitis mice. {yields} Salmon proteoglycan enhances Foxp3 expression. -- Abstract: Proteoglycans (PGs) are complex glycohydrates which are widely distributed in extracellular matrix (ECM). PGs are involved in the construction of ECM, cell proliferation and differentiation. ECM components are involved in transduction of proinflammatory responses, but it is still unknown whether PGs are involved in inflammatory response. In this study, we investigated the effect of PG extracted from salmon cartilage on the progression of experimental colitis-induced in severe combined immunodeficiency mice by cell transfer from interleukin-10 (IL-10){sup -/-} mice. IL-10{sup -/-} cell-transferred mice showed weight loss, colon shortening and histological appearance of mild colitis. Daily oral administration of PG attenuated the clinical progression of colitis in a dose-dependent manner. Colitis-induced mice showed the elevated expression of IFN-{gamma}, IL-12, TNF-{alpha}, IL-21, IL-23p19, IL-6, IL-17A and retinoic acid-related orphan receptor {gamma}t (ROR{gamma}t) in lamina propria mononuclear cells (LPMCs) and oral administration of PG suppressed the expression of these factors. Conversely, expression of Foxp3 that induces CD4{sup +}CD25{sup +} regulatory T cells in LPMCs was enhanced by PG administration. These findings suggested that salmon PG attenuated the progression of colitis due to suppression of inflammatory response by enhancement of regulatory T cell induction.

β-cell-specific IL-2 therapy increases islet Foxp3+Treg and suppresses type 1 diabetes in NOD mice.

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Johnson, Mark C; Garland, Alaina L; Nicolson, Sarah C; Li, Chengwen; Samulski, R Jude; Wang, Bo; Tisch, Roland

2013-11-01

Interleukin-2 (IL-2) is a critical cytokine for the homeostasis and function of forkhead box p3-expressing regulatory T cells (Foxp3(+)Tregs). Dysregulation of the IL-2-IL-2 receptor axis is associated with aberrant Foxp3(+)Tregs and T cell-mediated autoimmune diseases such as type 1 diabetes. Treatment with recombinant IL-2 has been reported to enhance Foxp3(+)Tregs and suppress different models of autoimmunity. However, efficacy of IL-2 therapy is dependent on achieving sufficient levels of IL-2 to boost tissue-resident Foxp3(+)Tregs while avoiding the potential toxic effects of systemic IL-2. With this in mind, adeno-associated virus (AAV) vector gene delivery was used to localize IL-2 expression to the islets of NOD mice. Injection of a double-stranded AAV vector encoding IL-2 driven by a mouse insulin promoter (dsAAVmIP-IL2) increased Foxp3(+)Tregs in the islets but not the draining pancreatic lymph nodes. Islet Foxp3(+)Tregs in dsAAVmIP-IL2-treated NOD mice exhibited enhanced fitness marked by increased expression of Bcl-2, proliferation, and suppressor function. In contrast, ectopic IL-2 had no significant effect on conventional islet-infiltrating effector T cells. Notably, β-cell-specific IL-2 expression suppressed late preclinical type 1 diabetes in NOD mice. Collectively, these findings demonstrate that β-cell-specific IL-2 expands an islet-resident Foxp3(+)Tregs pool that effectively suppresses ongoing type 1 diabetes long term.

EFFECT OF LIPOSOMAL CLODRONATE-DEPENDENT DEPLETION OF PROFESSIONAL ANTIGEN PRESENTING CELLS ON NUMBERS AND PHENOTYPE OF CANINE CD4+CD25+FOXP3+ REGULATORY T CELLS

Science.gov (United States)

Weaver, Kriston F.; Stokes, John V.; Gunnoe, Sagen A.; Follows, Joyce S.; Shafer, Lydia; Ammari, Mais G.; Archer, Todd M.; Thomason, John M.; Mackin, Andrew J.; Pinchuk, Lesya M.

2015-01-01

Regulatory T cells (Tregs) are known to control autoreactivity during and subsequent to the development of the peripheral immune system. Professional antigen presenting cells (APCs), dendritic cells (DCs) and monocytes, have an important role in inducing Tregs. For the first time, this study evaluated proportions and phenotypes of Tregs in canine peripheral blood depleted of professional APCs, utilizing liposomal clodronate (LC) and multicolor flow cytometry analysis. Our results demonstrate that LC exposure promoted short term decreases followed by significant increases in the proportions or absolute numbers of CD4+CD25+FOXP3+ Tregs in dogs. In general, the LC-dependent Treg fluctuations were similar to the changes in the levels of CD14+ monocytes in Walker hounds. However, the proportions of monocytes showed more dramatic changes compared to the proportions of Tregs that were visually unchanged after LC treatment over the study period. At the same time, absolute Treg numbers showed, similarly to the levels of CD14+ monocytes, significant compensatory gains as well as the recovery during the normalization period. We confirm the previous data that CD4+ T cells with the highest CD25 expression were highly enriched for FOXP3. Furthermore, for the first time, we report that CD4+CD25lowFOXP3+ is the major regulatory T cell subset affected by LC exposure. The increases within the lowest CD25 expressers of CD4+FOXP3+ cells together with compensatory gains in the proportion of CD14+ monocytes during compensatory and normalization periods suggest the possible direct or indirect roles of monocytes in active recruitment and generation of Tregs from naïve CD4+ T cells. PMID:25950023

The Living Eye “Disarms” Uncommitted Autoreactive T Cells by Converting Them to FoxP3+ Regulatory Cells Following Local Antigen Recognition

Science.gov (United States)

Zhou, Ru; Horai, Reiko; Silver, Phyllis B; Mattapallil, Mary J; Zárate-Bladés, Carlos R; Chong, Wai Po; Chen, Jun; Rigden, Rachael C; Villasmil, Rafael; Caspi, Rachel R

2011-01-01

Immune privilege is used by the eye, brain, reproductive organs and gut to preserve structural and functional integrity in the face of inflammation. The eye is arguably the most vulnerable, and therefore also the most “privileged” of tissues, but paradoxically, remains subject to destructive autoimmunity. It has been proposed, although never proven in vivo, that the eye can induce T regulatory cells (Tregs) locally. Using FoxP3-GFP reporter mice expressing a retina-specific T cell receptor, we now show that uncommitted T cells rapidly convert in the living eye to FoxP3+ Tregs in a process involving retinal antigen recognition, de novo FoxP3 induction and proliferation. This takes place within the ocular tissue and is supported by retinoic acid, which is normally present in the eye due to its function in the chemistry of vision. Non-converted T cells showed evidence of priming, but appeared restricted from expressing effector function in the eye. Preexisting ocular inflammation impeded conversion of uncommitted T cells into Tregs. Importantly, retina-specific T cells primed in vivo before introduction into the eye were resistant to Treg conversion in the ocular environment, and instead caused severe uveitis. Thus, uncommitted T cells can be disarmed, but immune privilege is unable to protect from uveitogenic T cells that have acquired effector function prior to entering the eye. These findings shed new light on the phenomenon of immune privilege and on its role, as well as its limitations, in actively controlling immune responses in the tissue. PMID:22238462

IP-10, p53, and Foxp3 Expression in Hepatocytes of Chronic Hepatitis B Patients with Cirrhosis and Hepatocellular Carcinoma.

Science.gov (United States)

Shahera, Umme; Munshi, Saifullah; Jahan, Munira; Nessa, Afzalun; Alam, Shahinul; Tabassum, Shahina

2016-01-01

Elucidating differences in gene expression may be useful in understanding the molecular pathogenesis and for developing specific markers for the outcome of hepatitis B virus (HBV) infection. In the present study, expressions of host gene interferon gamma-inducible protein (IP-10), p53, and Foxp3 were studied in hepatocytes of patients with chronic HBV infection to determine a possible link between selected host gene expression and the outcome of HBV infection. The study was conducted in 60 patients with chronic HBV infection and they were divided into four groups: HBV-positive cirrhosis (n = 15), HBV-negative cirrhosis (n = 15), HBV-positive hepatocellular carcinoma (HCC) (n = 15) and HBV-negative HCC (n = 15). Total messenger ribonucleic acid (mRNA) extraction was done followed by complementary deoxyribonucleic acid (cDNA) synthesis, and finally gene expression was performed using real-time polymerase chain reaction (PCR) technique. IP-10 and p53 gene expressions were lower in HBV-positive cirrhosis, and Foxp3 gene expression was upregulated in HBV-positive cirrhosis in comparison to HBV-negative cirrhosis. The expressions of all the three genes were upregulated among HBV-positive HCC in comparison to HBV-negative HCC. The expression of IP-10, p53, and Foxp3 genes was upregulated in HBV-positive HCC in comparison to HBV-positive cirrhosis. This study indicates that there are variations in the expression of the selected genes among cirrhosis and HCC patients with or without HBV. All the three selected genes were more or less upregulated in HBV-positive HCC patients, but only Foxp3 expression was upregulated in HBV-positive cirrhosis. These three particular genes may have a role in the molecular pathogenesis and clinical outcome of HBV-positive cirrhosis and HCC patients. These aspects need further evaluation by studies with larger numbers of cirrhosis and HCC patients. Shahera U, Munshi S, Jahan M, Nessa A, Alam S, Tabassum S. IP-10, p53, and Foxp3 Expression in

Glycogen synthase kinase-3 inhibition sensitizes human induced pluripotent stem cells to thiol-containing antioxidants induced apoptosis.

Science.gov (United States)

Tu, Chengyi; Xu, Robert; Koleti, Meghana; Zoldan, Janet

2017-08-01

Inhibition of glycogen synthase kinase 3 (GSK3) is an extensively used strategy to activate Wnt pathway for pluripotent stem cell (PSC) differentiation. However, the effects of such inhibition on PSCs, besides upregulating the Wnt pathway, have rarely been investigated despite that GSK3 is broadly involved in other cellular activities such as insulin signaling and cell growth/survival regulation. Here we describe a previously unknown synergistic effect between GSK3 inhibition (e.g., Chir99021 and LY2090314) and various normally non-toxic thiol-containing antioxidants (e.g., N-acetylcysteine, NAC) on the induction of apoptosis in human induced pluripotent stem cells (iPSCs). Neither Chir99021 nor the antioxidants individually induced significant apoptosis, whereas their combined treatment resulted in rapid and extensive apoptosis, with substantial caspase 3 activity observed within 3h and over 90% decrease in cell viability after 24h. We confirmed the generality of this phenomenon with multiple independent iPSCs lines, various thiol-based antioxidants and distinct GSK3 inhibitors. Mechanistically, we demonstrated that rapamycin treatment could substantially reduce cell death, suggesting the critical role of mammalian target of rapamycin (mTOR). Akt dysregulation was also found to partially contribute to cell apoptosis but was not the primary cause. Further, this coordinated proapoptotic effect was not detected in mouse ESCs but was present in another human cells line: a breast cancer cell line (MDA-MB-231). Given the wide use of GSK3 inhibition in biomedical research: from iPSC differentiation to cancer intervention and the treatment of neuronal diseases, researchers can potentially take advantage of or avoid this synergistic effect for improved experimental or clinical outcome. Copyright © 2017. Published by Elsevier B.V.

Eriocalyxin B Inhibits STAT3 Signaling by Covalently Targeting STAT3 and Blocking Phosphorylation and Activation of STAT3.

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Xiaokui Yu

Full Text Available Activated STAT3 plays an important role in oncogenesis by stimulating cell proliferation and resisting apoptosis. STAT3 therefore is an attractive target for cancer therapy. We have screened a traditional Chinese herb medicine compound library and found Eriocalyxin B (EB, a diterpenoid from Isodon eriocalyx, as a specific inhibitor of STAT3. EB selectively inhibited constitutive as well as IL-6-induced phosphorylation of STAT3 and induced apoptosis of STAT3-dependent tumor cells. EB did not affect the upstream protein tyrosine kinases or the phosphatase (PTPase of STAT3, but rather interacted directly with STAT3. The effects of EB could be abolished by DTT or GSH, suggesting a thiol-mediated covalent linkage between EB and STAT3. Site mutagenesis of cysteine in and near the SH2 domain of STAT3 identified Cys712 to be the critical amino acid for the EB-induced inactivation of STAT3. Furthermore, LC/MS/MS analyses demonstrated that an α, β-unsaturated carbonyl of EB covalently interacted with the Cys712 of STAT3. Computational modeling analyses also supported a direct interaction between EB and the Cys712 of STAT3. These data strongly suggest that EB directly targets STAT3 through a covalent linkage to inhibit the phosphorylation and activation of STAT3 and induces apoptosis of STAT3-dependent tumor cells.

Natural indoles, indole-3-carbinol and 3,3′-diindolymethane, inhibit T cell activation by staphylococcal enterotoxin B through epigenetic regulation involving HDAC expression

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Busbee, Philip B.; Nagarkatti, Mitzi; Nagarkatti, Prakash S.

2014-01-01

Staphylococcal enterotoxin B (SEB) is a potent exotoxin produced by the Staphylococcus aureus. This toxin is classified as a superantigen because of its ability to directly bind with MHC-II class molecules followed by activation of a large proportion of T cells bearing specific Vβ-T cell receptors. Commonly associated with classic food poisoning, SEB has also been shown to induce toxic shock syndrome, and is also considered to be a potential biological warfare agent because it is easily aerosolized. In the present study, we assessed the ability of indole-3-carbinol (I3C) and one of its byproducts, 3,3′-diindolylmethane (DIM), found in cruciferous vegetables, to counteract the effects of SEB-induced activation of T cells in mice. Both I3C and DIM were found to decrease the activation, proliferation, and cytokine production by SEB-activated Vβ8 + T cells in vitro and in vivo. Interestingly, inhibitors of histone deacetylase class I (HDAC-I), but not class II (HDAC-II), showed significant decrease in SEB-induced T cell activation and cytokine production, thereby suggesting that epigenetic modulation plays a critical role in the regulation of SEB-induced inflammation. In addition, I3C and DIM caused a decrease in HDAC-I but not HDAC-II in SEB-activated T cells, thereby suggesting that I3C and DIM may inhibit SEB-mediated T cell activation by acting as HDAC-I inhibitors. These studies not only suggest for the first time that plant-derived indoles are potent suppressors of SEB-induced T cell activation and cytokine storm but also that they may mediate these effects by acting as HDAC inhibitors. - Highlights: • I3C and DIM reduce SEB-induced T cell activation and inflammatory cytokines. • Inhibiting class I HDACs reduces T cell activation and inflammatory cytokines. • Inhibiting class II HDACs increases T cell activation and inflammatory cytokines. • I3C and DIM selectively reduce mRNA expression of class I HDACs. • Novel use and mechanism to counteract SEB

Nuclear protein IκB-ζ inhibits the activity of STAT3

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Wu, Zhihao; Zhang, Xiaoai; Yang, Juntao; Wu, Guangzhou; Zhang, Ying; Yuan, Yanzhi; Jin, Chaozhi; Chang, Zhijie; Wang, Jian; Yang, Xiaoming; He, Fuchu

2009-01-01

STAT3 (Signal transducer and activator of transcription 3) is a key transcription factor of the JAK-STAT (Janus kinase/signal transducer and activator of transcription) pathway that regulates cell proliferation and apoptosis. Activation of STAT3 is under tight regulation, and yet the different signaling pathways and the mechanisms that regulate its activity remain to be elucidated. Using a yeast two-hybrid screening, we have identified a nuclear protein IκB-ζ that interacts in a novel way with STAT3. This physical interaction was further confirmed by co-immunoprecipitation assays. The interaction regions were mapped to the coiled-coil domain of STAT3 and the C-terminal of IκB-ζ. Overexpression of IκB-ζ inhibited the transcriptional activity of STAT3. It also suppressed cell growth and induced cell apoptosis in SRC-simulated cells, which is partially mediated by down-regulation of expression of a known STAT3 target gene, MCL1. Our results suggest that IκB-ζ is a negative regulator of STAT3, and demonstrate a novel mechanism in which a component of the NF-κB signaling pathway inhibits the activation of STAT3.

Epigallocatechin gallate protects dopaminergic neurons against 1-methyl-4- phenyl-1,2,3,6-tetrahydropyridine-induced neurotoxicity by inhibiting microglial cell activation.

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Li, Rui; Peng, Ning; Du, Fang; Li, Xu-ping; Le, Wei-dong

2006-04-01

To observe whether the dopaminergic neuroprotective effect of (-)-epigallocatechin gallate (EGCG) is associated with its inhibition of microglial cell activation in vivo. The effects of EGCG at different doses on dopaminergic neuronal survival were tested in a methyl-4-phenyl-pyridinium (MPP+)-induced dopaminergic neuronal injury model in the primary mesencephalic cell cultures. With unbiased stereological method, tyrosine hydroxylase-immunoreactive (TH-ir) cells were counted in the A8, A9 and A10 regions of the substantia nigra (SN) in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated C57BL/6 mice. The effect of EGCG on microglial activation in the SN was also investigated. Pretreatment with EGCG (1 to 100 micromol/L) significantly attenuated MPP+-induced TH-ir cell loss by 22.2% to 80.5% in the mesencephalic cell cultures. In MPTP-treated C57BL/6 mice, EGCG at a low concentration (1 mg/kg) provided significant protection against MPTP-induced TH-ir cell loss by 50.9% in the whole nigral area and by 71.7% in the A9 region. EGCG at 5 mg/kg showed more prominent protective effect than at 1 or 10 mg/kg. EGCG pretreatment significantly inhibited microglial activation and CD11b expression induced by MPTP. EGCG exerts potent dopaminergic neuroprotective activity by means of microglial inhibition, which shed light on the potential use of EGCG in treatment of Parkinson's disease.

Effects of Forsythia suspensa on expression of NF-κB and Foxp3 during liver injury in rats with severe acute pancreatitis

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FAN Xiaobin

2013-07-01

Full Text Available ObjectiveTo investigate the roles of nuclear factor-kappa B (NF-κB and forkhead box P3 (Foxp3 during liver injury in rats with severe acute pancreatitis (SAP and the effects of Forsythia suspensa on their expression. MethodsEighty male Wistar rats were randomly allocated into sham operation (SO group, SAP group, and intervention group. The intervention group was further divided into high-dose, middle-dose, and low-dose Forsythia suspensa subgroups and positive control (PDTC subgroup. A rat model was induced by injecting sodium taurocholate into the bile-pancreatic duct. The rats in SO and SAP groups were sacrificed at 3, 6, and 12 h after operation, and those in intervention group were sacrificed at 12 h after operation. The serum levels of amylase (Amy, alanine aminotransferase (ALT, and tumor necrosis factor (TNFα were measured. The endotoxin content in plasma was determined using a limulus reagent. The percentage of Treg cells among peripheral blood lymphocytes was measured by flow cytometry. The pathological examination and scoring of the liver and pancreas were performed. The mRNA expression levels of NF-κB and Foxp3 in liver tissue were measured by RT-PCR. One-way analysis of variance was used for comparison among groups; multiple comparison was performed by LSD test; the indices were subjected to linear correlation analysis. ResultsCompared with those in the SO group, all the indices in the SAP group increased over time and reached the peak levels at 12 h after operation. Compared with the rats in SAP group that were sacrificed at 12 h after operation, the intervention group (mortality = 0 had significantly decreased mRNA expression of NF-κB and Foxp3 in liver tissue (P＜0.01. The NF-κB mRNA level was positively correlated with the percentage of Treg cells (r=0.738, P＜0.01. As the dose of Forsythia suspensa rose, Amy, ALT, and TNFα levels decreased significantly, and the inflammation of liver and pancreas was significantly

Resveratrol protects vascular endothelial cells from high glucose-induced apoptosis through inhibition of NADPH oxidase activation-driven oxidative stress.

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Chen, Feng; Qian, Li-Hua; Deng, Bo; Liu, Zhi-Min; Zhao, Ying; Le, Ying-Ying

2013-09-01

Hyperglycemia-induced oxidative stress has been implicated in diabetic vascular complications in which NADPH oxidase is a major source of reactive oxygen species (ROS) generation. Resveratrol is a naturally occurring polyphenol, which has vasoprotective effects in diabetic animal models and inhibits high glucose (HG)-induced oxidative stress in endothelial cells. We aimed to examine whether HG-induced NADPH oxidase activation and ROS production contribute to glucotoxicity to endothelial cells and the effect of resveratrol on glucotoxicity. Using a murine brain microvascular endothelial cell line bEnd3, we found that NADPH oxidase inhibitor (apocynin) and resveratrol both inhibited HG-induced endothelial cell apoptosis. HG-induced elevation of NADPH oxidase activity and production of ROS were inhibited by apocynin, suggesting that HG induces endothelial cell apoptosis through NADPH oxidase-mediated ROS production. Mechanistic studies revealed that HG upregulated NADPH oxidase subunit Nox1 but not Nox2, Nox4, and p22(phox) expression through NF-κB activation, which resulted in elevation of NADPH oxidase activity and consequent ROS production. Resveratrol prevented HG-induced endothelial cell apoptosis through inhibiting HG-induced NF-κB activation, NADPH oxidase activity elevation, and ROS production. HG induces endothelial cell apoptosis through NF-κB/NADPH oxidase/ROS pathway, which was inhibited by resveratrol. Our findings provide new potential therapeutic targets against brain vascular complications of diabetes. © 2013 John Wiley & Sons Ltd.

Aloin Suppresses Lipopolysaccharide-Induced Inflammatory Response and Apoptosis by Inhibiting the Activation of NF-κB

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Xuan Luo

2018-02-01

Full Text Available Numerous herbal-derived natural products are excellent anti-inflammatory agents. Several studies have reported that aloin, the major anthraquinone glycoside obtained from the Aloe species, exhibits anti-inflammatory activity. However, the molecular mechanism of this activity is not well understood. In this report, we found that aloin suppresses lipopolysaccharide-induced pro-inflammatory cytokine secretion and nitric oxide production, and downregulates the expression of tumor necrosis factor alpha (TNF-α, interleukin 6 (IL-6, inducible nitric oxide synthase (iNOS, and cyclooxygenase-2 (COX-2. Aloin inhibits the phosphorylation and acetylation of the NF-κB p65 subunit by suppressing the upstream kinases p38 and Msk1, preventing LPS-induced p65 translocation to the nucleus. We have also shown that aloin inhibits LPS-induced caspase-3 activation and apoptotic cell death. Collectively, these findings suggest that aloin effectively suppresses the inflammatory response, primarily through the inhibition of NF-κB signaling.

Fucoxanthin prevents H2O2-induced neuronal apoptosis via concurrently activating the PI3-K/Akt cascade and inhibiting the ERK pathway.

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Yu, Jie; Lin, Jia-Jia; Yu, Rui; He, Shan; Wang, Qin-Wen; Cui, Wei; Zhang, Jin-Rong

2017-01-01

Background : As a natural carotenoid abundant in chloroplasts of edible brown algae, fucoxanthin possesses various health benefits, including anti-oxidative activity in particular. Objective : In the present study, we studied whether fucoxanthin protected against hydrogen peroxide (H 2 O 2 )-induced neuronal apoptosis. Design : The neuroprotective effects of fucoxanthin on H 2 O 2 -induced toxicity were studied in both SH-SY5Y cells and primary cerebellar granule neurons. Results : Fucoxanthin significantly protected against H 2 O 2 -induced neuronal apoptosis and intracellular reactive oxygen species. H 2 O 2 treatment led to the reduced activity of phosphoinositide 3-kinase (PI3-K)/Akt cascade and the increased activity of extracellular signal-regulated kinase (ERK) pathway in SH-SY5Y cells. Moreover, fucoxanthin significantly restored the altered activities of PI3-K/Akt and ERK pathways induced by H 2 O 2 . Both specific inhibitors of glycogen synthase kinase 3β (GSK3β) and mitogen-activated protein kinase kinase (MEK) significantly protected against H 2 O 2 -induced neuronal death. Furthermore, the neuroprotective effects of fucoxanthin against H 2 O 2 -induced neuronal death were abolished by specific PI3-K inhibitors. Conclusions : Our data strongly revealed that fucoxanthin protected against H 2 O 2 -induced neurotoxicity via concurrently activating the PI3-K/Akt cascade and inhibiting the ERK pathway, providing support for the use of fucoxanthin to treat neurodegenerative disorders induced by oxidative stress.

[Increased expressions of peripheral PD-1+ lymphocytes and CD4+CD25+FOXP3+ T cells in gastric adenocarcinoma patients].

Science.gov (United States)

Li, Hao; Li, Songyan; Hu, Shidong; Zou, Guijun; Hu, Zilong; Wei, Huahua; Wang, Yufeng; Du, Xiaohui

2017-01-01

Objective To detect the frequencies of peripheral programmed death-1 + (PD-1 + ) lymphocytes and CD4 + CD25 + FOXP3 + regulatory T cells in patients with gastric adenocarcinoma. Methods The study enrolled 29 patients with gastric adenocarcinoma and 29 age- and sex-matched healthy controls. Frequencies of PD-1 + lymphocytes and CD4 + CD25 + FOXP3 + regulatory T cells were detected using flow cytometry. Results The number of PD-1 + lymphocytes and CD4 + CD25 + FOXP3 + regulatory T cells in peripheral blood was higher in patients with gastric adenocarcinoma than that in the control group. Moreover, linear correlation analysis indicated a positive correlation between PD-1 expression and frequency of CD4 + CD25 + FOXP3 + regulatory T cells in peripheral blood of the patients. Conclusion Gastric adenocarcinoma patients present with increased PD-1 + lymphocytes and CD4 + CD25 + FOXP3 + regulatory T cells in the peripheral blood.

Autophagy contributes to gefitinib-induced glioma cell growth inhibition

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Chang, Cheng-Yi [Department of Surgery, Fong-Yuan Hospital, Taichung 420, Taiwan (China); Graduate Institute of Pharmaceutical Science and Technology, Central Taiwan University of Science and Technology, Taichung 406, Taiwan (China); Kuan, Yu-Hsiang [Department of Pharmacology, School of Medicine, Chung Shan Medical University, Taichung 402, Taiwan (China); Department of Pharmacy, Chung Shan Medical University Hospital, Taichung 402, Taiwan (China); Ou, Yen-Chuan; Li, Jian-Ri [Division of Urology, Taichung Veterans General Hospital, Taichung 407, Taiwan (China); Wu, Chih-Cheng [Department of Anesthesiology, Taichung Veterans General Hospital, Taichung 407, Taiwan (China); Department of Financial and Computational Mathematics, Providence University, Taichung 433, Taiwan (China); Pan, Pin-Ho [Department of Pediatrics, Tungs’ Taichung MetroHarbor Hospital, Taichung 435, Taiwan (China); Chen, Wen-Ying [Department of Veterinary Medicine, National Chung Hsing University, Taichung 402, Taiwan (China); Huang, Hsuan-Yi [Department of Surgery, Fong-Yuan Hospital, Taichung 420, Taiwan (China); Chen, Chun-Jung, E-mail: cjchen@vghtc.gov.tw [Department of Medical Research, Taichung Veterans General Hospital, Taichung 407, Taiwan (China); Institute of Biomedical Sciences, National Chung Hsing University, Taichung 402, Taiwan (China); Rong Hsing Research Center for Translational Medicine, National Chung Hsing University, Taichung 402, Taiwan (China); Center for General Education, Tunghai University, Taichung 407, Taiwan (China); Department of Nursing, HungKuang University, Taichung 433, Taiwan (China)

2014-09-10

Epidermal growth factor receptor tyrosine kinase inhibitors, including gefitinib, have been evaluated in patients with malignant gliomas. However, the molecular mechanisms involved in gefitinib-mediated anticancer effects against glioma are incompletely understood. In the present study, the cytostatic potential of gefitinib was demonstrated by the inhibition of glioma cell growth, long-term clonogenic survival, and xenograft tumor growth. The cytostatic consequences were accompanied by autophagy, as evidenced by monodansylcadaverine staining of acidic vesicle formation, conversion of microtubule-associated protein-1 light chain 3-II (LC3-II), degradation of p62, punctate pattern of GFP-LC3, and conversion of GFP-LC3 to cleaved-GFP. Autophagy inhibitor 3-methyladenosine and chloroquine and genetic silencing of LC3 or Beclin 1 attenuated gefitinib-induced growth inhibition. Gefitinib-induced autophagy was not accompanied by the disruption of the Akt/mammalian target of rapamycin signaling. Instead, the activation of liver kinase-B1/AMP-activated protein kinase (AMPK) signaling correlated well with the induction of autophagy and growth inhibition caused by gefitinib. Silencing of AMPK suppressed gefitinib-induced autophagy and growth inhibition. The crucial role of AMPK activation in inducing glioma autophagy and growth inhibition was further supported by the actions of AMP mimetic AICAR. Gefitinib was shown to be capable of reducing the proliferation of glioma cells, presumably by autophagic mechanisms involving AMPK activation. - Highlights: • Gefitinib causes cytotoxic and cytostatic effect on glioma. • Gefitinib induces autophagy. • Gefitinib causes cytostatic effect through autophagy. • Gefitinib induces autophagy involving AMPK.

Autophagy contributes to gefitinib-induced glioma cell growth inhibition

International Nuclear Information System (INIS)

Chang, Cheng-Yi; Kuan, Yu-Hsiang; Ou, Yen-Chuan; Li, Jian-Ri; Wu, Chih-Cheng; Pan, Pin-Ho; Chen, Wen-Ying; Huang, Hsuan-Yi; Chen, Chun-Jung

2014-01-01

Epidermal growth factor receptor tyrosine kinase inhibitors, including gefitinib, have been evaluated in patients with malignant gliomas. However, the molecular mechanisms involved in gefitinib-mediated anticancer effects against glioma are incompletely understood. In the present study, the cytostatic potential of gefitinib was demonstrated by the inhibition of glioma cell growth, long-term clonogenic survival, and xenograft tumor growth. The cytostatic consequences were accompanied by autophagy, as evidenced by monodansylcadaverine staining of acidic vesicle formation, conversion of microtubule-associated protein-1 light chain 3-II (LC3-II), degradation of p62, punctate pattern of GFP-LC3, and conversion of GFP-LC3 to cleaved-GFP. Autophagy inhibitor 3-methyladenosine and chloroquine and genetic silencing of LC3 or Beclin 1 attenuated gefitinib-induced growth inhibition. Gefitinib-induced autophagy was not accompanied by the disruption of the Akt/mammalian target of rapamycin signaling. Instead, the activation of liver kinase-B1/AMP-activated protein kinase (AMPK) signaling correlated well with the induction of autophagy and growth inhibition caused by gefitinib. Silencing of AMPK suppressed gefitinib-induced autophagy and growth inhibition. The crucial role of AMPK activation in inducing glioma autophagy and growth inhibition was further supported by the actions of AMP mimetic AICAR. Gefitinib was shown to be capable of reducing the proliferation of glioma cells, presumably by autophagic mechanisms involving AMPK activation. - Highlights: • Gefitinib causes cytotoxic and cytostatic effect on glioma. • Gefitinib induces autophagy. • Gefitinib causes cytostatic effect through autophagy. • Gefitinib induces autophagy involving AMPK

cGMP-Phosphodiesterase Inhibition Prevents Hypoxia-Induced Cell Death Activation in Porcine Retinal Explants.

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Lorena Olivares-González

Full Text Available Retinal hypoxia and oxidative stress are involved in several retinal degenerations including diabetic retinopathy, glaucoma, central retinal artery occlusion, or retinopathy of prematurity. The second messenger cyclic guanosine monophosphate (cGMP has been reported to be protective for neuronal cells under several pathological conditions including ischemia/hypoxia. The purpose of this study was to evaluate whether the accumulation of cGMP through the pharmacological inhibition of phosphodiesterase (PDE with Zaprinast prevented retinal degeneration induced by mild hypoxia in cultures of porcine retina. Exposure to mild hypoxia (5% O2 for 24h reduced cGMP content and induced retinal degeneration by caspase dependent and independent (PARP activation mechanisms. Hypoxia also produced a redox imbalance reducing antioxidant response (superoxide dismutase and catalase activities and increasing superoxide free radical release. Zaprinast reduced mild hypoxia-induced cell death through inhibition of caspase-3 or PARP activation depending on the cell layer. PDE inhibition also ameliorated the effects of mild hypoxia on antioxidant response and the release of superoxide radical in the photoreceptor layer. The use of a PKG inhibitor, KT5823, suggested that cGMP-PKG pathway is involved in cell survival and antioxidant response. The inhibition of PDE, therefore, could be useful for reducing retinal degeneration under hypoxic/ischemic conditions.

Hyperin protects against LPS-induced acute kidney injury by inhibiting TLR4 and NLRP3 signaling pathways

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Chunzhi, Gong; Zunfeng, Li; Chengwei, Qin; Xiangmei, Bu; Jingui, Yu

2016-01-01

Hyperin is a flavonoid compound derived from Ericaceae, Guttifera, and Celastraceae that has been shown to have various biological effects, such as anti-inflammatory and anti-oxidant effects. However, there is no evidence to show the protective effects of hyperin on lipopolysaccharide (LPS)-induced acute kidney injury (AKI). Therefore, we investigated the protective effects and mechanism of hyperin on LPS-induced AKI in mice. The levels of TNF-α, IL-6, and IL-1β were tested by ELISA. The effects of hyperin on blood urea nitrogen (BUN) and serum creatinine were also detected. In addition, the expression of TLR4, NF-κB, and NLRP3 were detected by western blot analysis. The results showed that hyperin significantly inhibited LPS-induced TNF-α, IL-6, and IL-1β production. The levels of BUN and creatinine were also suppressed by hyperin. Furthermore, LPS-induced TLR4 expression and NF-κB activation were also inhibited by hyperin. In addition, treatment of hyperin dose-dependently inhibited LPS-induced NLRP3 signaling pathway. In conclusion, the results showed that hyperin inhibited LPS-induced inflammatory response by inhibiting TLR4 and NLRP3 signaling pathways. Hyperin has potential application prospects in the treatment of sepsis-induced AKI. PMID:27813491

MCL Plays an Anti-Inflammatory Role in Mycobacterium tuberculosis-Induced Immune Response by Inhibiting NF-κB and NLRP3 Inflammasome Activation

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Qingwen Zhang

2017-01-01

Full Text Available Mycobacterium tuberculosis (Mtb remains a significant menace to global health as it induces granulomatous lung lesions and systemic inflammatory responses during active tuberculosis (TB. Micheliolide (MCL, a sesquiterpene lactone, was recently reported to have a function of relieving LPS-induced inflammatory response, but the regulative role of MCL on the immunopathology of TB still remains unknown. In this experiment, we examined the inhibitory effect of MCL on Mtb-induced inflammatory response in mouse macrophage-like cell line Raw264.7 by downregulating the activation of nuclear factor kappa B (NF-κB and NLRP3 inflammasome. Evidences showed that MCL decreased the secretion of Mtb-induced inflammatory cytokines (IL-1β and TNF-α in a dose-dependent manner. Meanwhile, MCL dramatically suppressed Mtb-induced activation of iNOS and COX2 as well as subsequent production of NO. Furthermore, MCL inhibited Mtb-induced phosphorylation of Akt (Ser 473 in Raw264.7. According to our results, MCL plays an important role in modulating Mtb-induced inflammatory response through PI3K/Akt/NF-κB pathway and subsequently downregulating the activation of NLRP3 inflammasome. Therefore, MCL may represent as a potential drug candidate in the adjuvant treatment of TB by regulating host immune response.

Improved efficacy of allergen-specific immunotherapy by JAK inhibition in a murine model of allergic asthma

DEFF Research Database (Denmark)

Aguilar-Pimentel, Juan Antonio; Graessel, Anke; Alessandrini, Francesca

2017-01-01

)-induced allergic airway inflammation and allergen-specific immunotherapy was combined with the administration of Tofacitinib (TOFA, a FDA-approved JAK inhibitor) from 48 hours prior to 48 hours after therapeutic OVA-injection. The effect of TOFA on human FOXP3+CD4+ T cells was studied in vitro. RESULTS: AIT...... combined with short-term TOFA administration was significantly more effective in suppressing total cell and eosinophil infiltration into the lung, local cytokine production including IL-1β and CXCL1 and showed a trend for the reduction of IL-4, IL-13, TNF-α and IL-6 compared to AIT alone. Furthermore, TOFA...... co-administration significantly reduced systemic IL-6, IL-1β and OVA-specific IgE levels and induced IgG1 to the same extent as AIT alone. Additionally, TOFA enhanced the induction of human FOXP3+CD4+ T cells. CONCLUSIONS: This proof of concept study shows that JAK inhibition did not inhibit...

Arecoline inhibits the 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced cytochrome P450 1A1 activation in human hepatoma cells

International Nuclear Information System (INIS)

Chang, Eddy Essen; Miao Zhifeng; Lee, W.-J.; Chao, H.-R.; Li, Lih-Ann; Wang, Y.-F.; Ko, Y.-C.; Tsai, F.-Y.; Yeh, S.C.; Tsou, T.-C.

2007-01-01

In the present study, we investigated the effect of arecoline, a major areca nut alkaloid, on the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced activation of cytochrome P4501A1 (CYP1A1) in a human hepatoma cell line Huh-7. We treated Huh-7 cells with 10 nM TCDD in the presence of different concentrations of arecoline (50-300 μM). Our results indicated that arecoline attenuated the TCDD-induced CYP1A1 enzyme activation with an inhibitory effect on cell proliferation. By using real-time RT-PCR, we demonstrated that arecoline inhibited the TCDD-induced activations of CYP1A1 and AhR repressor (AhRR) mRNA expression in a similar pattern. Our results revealed that arecoline inhibited AhR mRNA expression with no direct effect on CYP1A1 enzyme activity. Therefore, in our present study, the observed inhibitory effect of arecoline on CYP1A1 activation was not due to the up-regulation of AhRR or direct inhibitory effect on CYP1A1. Taken together, here we have demonstrated that arecoline attenuates the TCDD-induced CYP1A1 activation mainly via down-regulation of AhR expression in human hepatoma cells, suggesting the possible involvement of arecoline in the AhR-mediated metabolism of environmental toxicants in liver

Fluoxetine-induced inhibition of synaptosomal [3H]5-HT release: Possible Ca2+-channel inhibition

International Nuclear Information System (INIS)

Stauderman, K.A.; Gandhi, V.C.; Jones, D.J.

1992-01-01

Fluoxetine, a selective 5-Ht uptake inhibitor, inhibited 15 mM K + -induced [ 3 H]5-HT release from rat spinal cord and cortical synaptosomes at concentrations > 0.5 uM. This effect reflected a property shared by another selective 5-HT uptake inhibitor paroxetine but not by less selective uptake inhibitors such as amitriptyline, desipramine, imipramine or nortriptyline. Inhibition of release by fluoxetine was inversely related to both the concentration of K + used to depolarize the synaptosomes and the concentration of external Ca 2+ . Experiments aimed at determining a mechanism of action revealed that fluoxetine did not inhibit voltage-independent release of [ 3 H]5-HT release induced by the Ca 2+ -ionophore A 23187 or Ca 2+ -independent release induced by fenfluramine. Moreover the 5-HT autoreceptor antagonist methiothepin did not reverse the inhibitory actions of fluoxetine on K + -induced release. Further studies examined the effects of fluoxetine on voltage-dependent Ca 2+ channels and Ca 2+ entry

Foxp3(+) T cells regulate immunoglobulin a selection and facilitate diversification of bacterial species responsible for immune homeostasis.

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Kawamoto, Shimpei; Maruya, Mikako; Kato, Lucia M; Suda, Wataru; Atarashi, Koji; Doi, Yasuko; Tsutsui, Yumi; Qin, Hongyan; Honda, Kenya; Okada, Takaharu; Hattori, Masahira; Fagarasan, Sidonia

2014-07-17

Foxp3(+) T cells play a critical role for the maintenance of immune tolerance. Here we show that in mice, Foxp3(+) T cells contributed to diversification of gut microbiota, particularly of species belonging to Firmicutes. The control of indigenous bacteria by Foxp3(+) T cells involved regulatory functions both outside and inside germinal centers (GCs), consisting of suppression of inflammation and regulation of immunoglobulin A (IgA) selection in Peyer's patches, respectively. Diversified and selected IgAs contributed to maintenance of diversified and balanced microbiota, which in turn facilitated the expansion of Foxp3(+) T cells, induction of GCs, and IgA responses in the gut through a symbiotic regulatory loop. Thus, the adaptive immune system, through cellular and molecular components that are required for immune tolerance and through the diversification as well as selection of antibody repertoire, mediates host-microbial symbiosis by controlling the richness and balance of bacterial communities required for homeostasis. Copyright © 2014 Elsevier Inc. All rights reserved.

GARP (LRRC32) is essential for the surface expression of latent TGF-β on platelets and activated FOXP3+ regulatory T cells

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Tran, Dat Q.; Andersson, John; Wang, Rui; Ramsey, Heather; Unutmaz, Derya; Shevach, Ethan M.

2009-01-01

TGF-β family members are highly pleiotropic cytokines with diverse regulatory functions. TGF-β is normally found in the latent form associated with latency-associated peptide (LAP). This latent complex can associate with latent TGFβ-binding protein (LTBP) to produce a large latent form. Latent TGF-β is also found on the surface of activated FOXP3+ regulatory T cells (Tregs), but it is unclear how it is anchored to the cell membrane. We show that GARP or LRRC32, a leucine-rich repeat molecule ...

Delphinidin inhibits BDNF-induced migration and invasion in SKOV3 ovarian cancer cells.

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Lim, Won-Chul; Kim, Hyunhee; Kim, Young-Joo; Park, Seung-Ho; Song, Ji-Hye; Lee, Ki Heon; Lee, In Ho; Lee, Yoo-Kyung; So, Kyeong A; Choi, Kyung-Chul; Ko, Hyeonseok

2017-12-01

Brain-derived neurotrophic factor (BDNF), the TrkB ligand, is associated with aggressive malignant behavior, including migration and invasion, in tumor cells and a poor prognosis in patients with various types of cancer. Delphinidin is a diphenylpropane-based polyphenolic ring structure-harboring compound, which exhibits a wide range of pharmacological activities, anti-tumor, anti-oxidant, anti-inflammatory, anti-angiogenic and anti-mutagenic activity. However, the possible role of delphinidin in the cancer migration and invasion is unclear. We investigated the suppressive effect of delphinidin on the cancer migration and invasion. Thus, we found that BDNF enhanced cancer migration and invasion in SKOV3 ovarian cancer cell. To exam the inhibitory role of delphinidin in SKOV3 ovarian cancer migration and invasion, we investigated the use of delphinidin as inhibitors of BDNF-induced motility and invasiveness in SKOV3 ovarian cancer cells in vitro. Here, we found that delphinidin prominently inhibited the BDNF-induced increase in cell migration and invasion of SKOV3 ovarian cancer cells. Furthermore, delphinidin remarkably inhibited BDNF-stimulated expression of MMP-2 and MMP-9. Also, delphinidin antagonized the phosphorylation of Akt and nuclear translocation of NF-κB permitted by the BDNF in SKOV3 ovarian cancer cells. Taken together, our findings provide new evidence that delphinidin suppressed the BDNF-induced ovarian cancer migration and invasion through decreasing of Akt activation. Copyright © 2017 Elsevier Ltd. All rights reserved.

Tob1 induces apoptosis and inhibits proliferation, migration and invasion of gastric cancer cells by activating Smad4 and inhibiting β‑catenin signaling.

Science.gov (United States)

Kundu, Juthika; Wahab, S M Riajul; Kundu, Joydeb Kumar; Choi, Yoon-La; Erkin, Ozgur Cem; Lee, Hun Seok; Park, Sang Gyu; Shin, Young Kee

2012-09-01

Transducer of ErbB-2.1 (Tob1), a tumor suppressor protein, is inactivated in a variety of cancers including stomach cancer. However, the role of Tob1 in gastric carcinogenesis remains elusive. The present study aimed to investigate whether Tob1 could inhibit gastric cancer progression in vitro, and to elucidate its underlying molecular mechanisms. We found differential expression of Tob1 in human gastric cancer (MKN28, AGS and MKN1) cells. The overexpression of Tob1 induced apoptosis in MKN28 and AGS cells, which was associated with sub-G1 arrest, activation of caspase-3, induction of Bax, inhibition of Bcl-2 and cleavage of poly (ADP-ribose) polymerase (PARP). In addition, Tob1 inhibited proliferation, migration and invasion, which were reversed in MKN1 and AGS cells transfected with Tob1 siRNA. Overexpression of Tob1 in MKN28 and AGS cells induced the expression of Smad4, leading to the increased expression and the promoter activity of p15, which was diminished by silencing of Tob1 using specific siRNA. Tob1 decreased the phosphorylation of Akt and glycogen synthase kinase-3β (GSK3β) in MKN28 and AGS cells, resulting in the reduced protein expression and the transcriptional activity of β‑catenin, which in turn decreased the expression of cyclin D1, cyclin-dependent kinase-4 (CDK4), urokinase plasminogen activator receptor (uPAR) and peroxisome proliferator and activator receptor-δ (PPARδ). Conversely, silencing of Tob1 induced the phosphorylation of Akt and GSK-3β, and increased the expression of β‑catenin and its target genes. Collectively, our study demonstrates that the overexpression of Tob1 inhibits gastric cancer progression by activating Smad4- and inhibiting β‑catenin-mediated signaling pathways.

Aromatic hydrocarbon receptor inhibits lysophosphatidic acid-induced vascular endothelial growth factor-A expression in PC-3 prostate cancer cells

International Nuclear Information System (INIS)

Wu, Pei-Yi; Lin, Yueh-Chien; Lan, Shun-Yan; Huang, Yuan-Li; Lee, Hsinyu

2013-01-01

Highlights: •LPA-induced VEGF-A expression was regulated by HIF-1α and ARNT. •PI3K mediated LPA-induced VEGF-A expression. •AHR signaling inhibited LPA-induced VEGF-A expression in PC-3 cells. -- Abstract: Lysophosphatidic acid (LPA) is a lipid growth factor with multiple biological functions and has been shown to stimulate cancer cell secretion of vascular endothelial growth factor-A (VEGF-A) and trigger angiogenesis. Hypoxia-inducible factor-1 (HIF-1), a heterodimer consisting of HIF-1α and HIF-1β (also known as aromatic hydrocarbon receptor nuclear translocator (ARNT)) subunits, is an important regulator of angiogenesis in prostate cancer (PC) through the enhancement of VEGF-A expression. In this study, we first confirmed the ability of LPA to induce VEGF-A expression in PC-3 cells and then validated that LPA-induced VEGF-A expression was regulated by HIF-1α and ARNT through phosphatidylinositol 3-kinase activation. Aromatic hydrocarbon receptor (AHR), a receptor for dioxin-like compounds, functions as a transcription factor through dimerization with ARNT and was found to inhibit prostate carcinogenesis and vanadate-induced VEGF-A production. Since ARNT is a common dimerization partner of AHR and HIF-1α, we hypothesized that AHR might suppress LPA-induced VEGF-A expression in PC-3 cells by competing with HIF-1α for ARNT. Here we demonstrated that overexpression and ligand activation of AHR inhibited HIF-1-mediated VEGF-A induction by LPA treatment of PC-3 cells. In conclusion, our results suggested that AHR activation may inhibit LPA-induced VEGF-A expression in PC-3 cells by attenuating HIF-1α signaling, and subsequently, suppressing angiogenesis and metastasis of PC. These results suggested that AHR presents a potential therapeutic target for the prevention of PC metastasis

Natural indoles, indole-3-carbinol and 3,3′-diindolymethane, inhibit T cell activation by staphylococcal enterotoxin B through epigenetic regulation involving HDAC expression

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Busbee, Philip B.; Nagarkatti, Mitzi; Nagarkatti, Prakash S., E-mail: prakash@mailbox.sc.edu

2014-01-01

Staphylococcal enterotoxin B (SEB) is a potent exotoxin produced by the Staphylococcus aureus. This toxin is classified as a superantigen because of its ability to directly bind with MHC-II class molecules followed by activation of a large proportion of T cells bearing specific Vβ-T cell receptors. Commonly associated with classic food poisoning, SEB has also been shown to induce toxic shock syndrome, and is also considered to be a potential biological warfare agent because it is easily aerosolized. In the present study, we assessed the ability of indole-3-carbinol (I3C) and one of its byproducts, 3,3′-diindolylmethane (DIM), found in cruciferous vegetables, to counteract the effects of SEB-induced activation of T cells in mice. Both I3C and DIM were found to decrease the activation, proliferation, and cytokine production by SEB-activated Vβ8{sup +} T cells in vitro and in vivo. Interestingly, inhibitors of histone deacetylase class I (HDAC-I), but not class II (HDAC-II), showed significant decrease in SEB-induced T cell activation and cytokine production, thereby suggesting that epigenetic modulation plays a critical role in the regulation of SEB-induced inflammation. In addition, I3C and DIM caused a decrease in HDAC-I but not HDAC-II in SEB-activated T cells, thereby suggesting that I3C and DIM may inhibit SEB-mediated T cell activation by acting as HDAC-I inhibitors. These studies not only suggest for the first time that plant-derived indoles are potent suppressors of SEB-induced T cell activation and cytokine storm but also that they may mediate these effects by acting as HDAC inhibitors. - Highlights: • I3C and DIM reduce SEB-induced T cell activation and inflammatory cytokines. • Inhibiting class I HDACs reduces T cell activation and inflammatory cytokines. • Inhibiting class II HDACs increases T cell activation and inflammatory cytokines. • I3C and DIM selectively reduce mRNA expression of class I HDACs. • Novel use and mechanism to counteract

Arctigenin Inhibits Adipogenesis by Inducing AMPK Activation and Reduces Weight Gain in High-Fat Diet-Induced Obese Mice.

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Han, Yo-Han; Kee, Ji-Ye; Park, Jinbong; Kim, Hye-Lin; Jeong, Mi-Young; Kim, Dae-Seung; Jeon, Yong-Deok; Jung, Yunu; Youn, Dong-Hyun; Kang, JongWook; So, Hong-Seob; Park, Raekil; Lee, Jong-Hyun; Shin, Soyoung; Kim, Su-Jin; Um, Jae-Young; Hong, Seung-Heon

2016-09-01

Although arctigenin (ARC) has been reported to have some pharmacological effects such as anti-inflammation, anti-cancer, and antioxidant, there have been no reports on the anti-obesity effect of ARC. The aim of this study is to investigate whether ARC has an anti-obesity effect and mediates the AMP-activated protein kinase (AMPK) pathway. We investigated the anti-adipogenic effect of ARC using 3T3-L1 pre-adipocytes and human adipose tissue-derived mesenchymal stem cells (hAMSCs). In high-fat diet (HFD)-induced obese mice, whether ARC can inhibit weight gain was investigated. We found that ARC reduced weight gain, fat pad weight, and triglycerides in HFD-induced obese mice. ARC also inhibited the expression of peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT/enhancer-binding protein alpha (C/EBPα) in in vitro and in vivo. Furthermore, ARC induced the AMPK activation resulting in down-modulation of adipogenesis-related factors including PPARγ, C/EBPα, fatty acid synthase, adipocyte fatty acid-binding protein, and lipoprotein lipase. This study demonstrates that ARC can reduce key adipogenic factors by activating the AMPK in vitro and in vivo and suggests a therapeutic implication of ARC for obesity treatment. J. Cell. Biochem. 117: 2067-2077, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Selective inhibition by harmane of the apurinic apyrimidinic endonuclease activity of phage T4-induced UV endonuclease.

Science.gov (United States)

Warner, H R; Persson, M L; Bensen, R J; Mosbaugh, D W; Linn, S

1981-11-25

1-Methyl-9H-pyrido-[3,4-b]indole (harmane) inhibits the apurinic/apyrimidinic (AP) endonuclease activity of the UV endonuclease induced by phage T4, whereas it stimulates the pyrimidine dimer-DNA glycosylase activity of that enzyme. E. coli endonuclease IV, E. coli endonuclease VI (the AP endonuclease activity associated with E. coli exonuclease III), and E. coli uracil-DNA glycosylase were not inhibited by harmane. Human fibroblast AP endonucleases I and II also were only slightly inhibited. Therefore, harmane is neither a general inhibitor of AP endonucleases, nor a general inhibitor of Class I AP endonucleases which incise DNA on the 3'-side of AP sites. However, E. coli endonuclease III and its associated dihydroxythymine-DNA glycosylase activity were both inhibited by harmane. This observation suggests that harmane may inhibit only AP endonucleases which have associated glycosylase activities.

Inhibition of glycogen synthase kinase 3beta ameliorates triptolide-induced acute cardiac injury by desensitizing mitochondrial permeability transition

International Nuclear Information System (INIS)

Wang, Wenwen; Yang, Yanqin; Xiong, Zhewen; Kong, Jiamin; Fu, Xinlu; Shen, Feihai; Huang, Zhiying

2016-01-01

Triptolide (TP), a diterpene triepoxide, is a major active component of Tripterygium wilfordii extracts, which are prepared as tablets and has been used clinically for the treatment of inflammation and autoimmune disorders. However, TP's therapeutic potential is limited by severe adverse effects. In a previous study, we reported that TP induced mitochondria dependent apoptosis in cardiomyocytes. Glycogen synthase kinase-3β (GSK-3β) is a multifunctional serine/threonine kinase that plays important roles in the necrosis and apoptosis of cardiomyocytes. Our study aimed to investigate the role of GSK-3β in TP-induced cardiotoxicity. Inhibition of GSK-3β activity by SB 216763, a potent and selective GSK-3 inhibitor, prominently ameliorated the detrimental effects in C57BL/6J mice with TP administration, which was associated with a correction of GSK-3β overactivity. Consistently, in TP-treated H9c2 cells, SB 216763 treatment counteracted GSK-3β overactivity, improved cell viability, and prevented apoptosis by modulating the expression of Bcl-2 family proteins. Mechanistically, GSK-3β interacted with and phosphorylated cyclophilin F (Cyp-F), a key regulator of mitochondrial permeability transition pore (mPTP). GSK-3β inhibition prevented the phosphorylation and activation of Cyp-F, and desensitized mPTP. Our findings suggest that pharmacological targeting of GSK-3β could represent a promising therapeutic strategy for protecting against cardiotoxicity induced by TP. - Highlights: • GSK-3β inhibition ameliorates TP-induced cardiotoxicity in vitro and in vivo. • GSK-3β controls Cyp-F activation, and regulates mPTP and apoptosis in H9c2 cells. • The protective effect is attributed to GSK-3β activity rather than to protein level. • GSK-3β may be a promising target against TP-induced cardiotoxicity.

Inhibition of glycogen synthase kinase 3beta ameliorates triptolide-induced acute cardiac injury by desensitizing mitochondrial permeability transition

Energy Technology Data Exchange (ETDEWEB)

Wang, Wenwen; Yang, Yanqin; Xiong, Zhewen; Kong, Jiamin [School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006 (China); Fu, Xinlu [Laboratory Animals Center, Sun Yat-sen University, Guangzhou 510006 (China); Shen, Feihai, E-mail: shenfh3@mail.sysu.edu.cn [School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006 (China); Huang, Zhiying, E-mail: hzhiying@mail.sysu.edu.cn [School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006 (China)

2016-12-15

Triptolide (TP), a diterpene triepoxide, is a major active component of Tripterygium wilfordii extracts, which are prepared as tablets and has been used clinically for the treatment of inflammation and autoimmune disorders. However, TP's therapeutic potential is limited by severe adverse effects. In a previous study, we reported that TP induced mitochondria dependent apoptosis in cardiomyocytes. Glycogen synthase kinase-3β (GSK-3β) is a multifunctional serine/threonine kinase that plays important roles in the necrosis and apoptosis of cardiomyocytes. Our study aimed to investigate the role of GSK-3β in TP-induced cardiotoxicity. Inhibition of GSK-3β activity by SB 216763, a potent and selective GSK-3 inhibitor, prominently ameliorated the detrimental effects in C57BL/6J mice with TP administration, which was associated with a correction of GSK-3β overactivity. Consistently, in TP-treated H9c2 cells, SB 216763 treatment counteracted GSK-3β overactivity, improved cell viability, and prevented apoptosis by modulating the expression of Bcl-2 family proteins. Mechanistically, GSK-3β interacted with and phosphorylated cyclophilin F (Cyp-F), a key regulator of mitochondrial permeability transition pore (mPTP). GSK-3β inhibition prevented the phosphorylation and activation of Cyp-F, and desensitized mPTP. Our findings suggest that pharmacological targeting of GSK-3β could represent a promising therapeutic strategy for protecting against cardiotoxicity induced by TP. - Highlights: • GSK-3β inhibition ameliorates TP-induced cardiotoxicity in vitro and in vivo. • GSK-3β controls Cyp-F activation, and regulates mPTP and apoptosis in H9c2 cells. • The protective effect is attributed to GSK-3β activity rather than to protein level. • GSK-3β may be a promising target against TP-induced cardiotoxicity.

Inhibition of Hypoxia Inducible Factor Alpha and Astrocyte-Elevated Gene-1 Mediates Cryptotanshinone Exerted Antitumor Activity in Hypoxic PC-3 Cells

Directory of Open Access Journals (Sweden)

Hyo-Jeong Lee

2012-01-01

Full Text Available Although cryptotanshinone (CT was known to exert antitumor activity in several cancers, its molecular mechanism under hypoxia still remains unclear. Here, the roles of AEG-1 and HIF-1α in CT-induced antitumor activity were investigated in hypoxic PC-3 cells. CT exerted cytotoxicity against prostate cancer cells and suppressed HIF-1α accumulation and AEG-1 expression in hypoxic PC-3 cells. Also, AEG-1 was overexpressed in prostate cancer cells. Interestingly, HIF-1α siRNA transfection enhanced the cleavages of caspase-9,3, and PAPR and decreased expression of Bcl-2 and AEG1 induced by CT in hypoxic PC-3 cells. Of note, DMOG enhanced the stability of AEG-1 and HIF-1α during hypoxia. Additionally, CT significantly reduced cellular level of VEGF in PC-3 cells and disturbed tube formation of HUVECs. Consistently, ChIP assay revealed that CT inhibited the binding of HIF-1α to VEGF promoter. Furthermore, CT at 10 mg/kg suppressed the growth of PC-3 cells in BALB/c athymic nude mice by 46.4% compared to untreated control. Consistently, immunohistochemistry revealed decreased expression of Ki-67, CD34, VEGF, carbonic anhydrase IX, and AEG-1 indices in CT-treated group compared to untreated control. Overall, our findings suggest that CT exerts antitumor activity via inhibition of HIF-1α, AEG1, and VEGF as a potent chemotherapeutic agent.

Leishmania donovani isolates with antimony-resistant but not -sensitive phenotype inhibit sodium antimony gluconate-induced dendritic cell activation.

Directory of Open Access Journals (Sweden)

Arun Kumar Haldar

2010-05-01

Full Text Available The inability of sodium antimony gluconate (SAG-unresponsive kala-azar patients to clear Leishmania donovani (LD infection despite SAG therapy is partly due to an ill-defined immune-dysfunction. Since dendritic cells (DCs typically initiate anti-leishmanial immunity, a role for DCs in aberrant LD clearance was investigated. Accordingly, regulation of SAG-induced activation of murine DCs following infection with LD isolates exhibiting two distinct phenotypes such as antimony-resistant (Sb(RLD and antimony-sensitive (Sb(SLD was compared in vitro. Unlike Sb(SLD, infection of DCs with Sb(RLD induced more IL-10 production and inhibited SAG-induced secretion of proinflammatory cytokines, up-regulation of co-stimulatory molecules and leishmanicidal effects. Sb(RLD inhibited these effects of SAG by blocking activation of PI3K/AKT and NF-kappaB pathways. In contrast, Sb(SLD failed to block activation of SAG (20 microg/ml-induced PI3K/AKT pathway; which continued to stimulate NF-kappaB signaling, induce leishmanicidal effects and promote DC activation. Notably, prolonged incubation of DCs with Sb(SLD also inhibited SAG (20 microg/ml-induced activation of PI3K/AKT and NF-kappaB pathways and leishmanicidal effects, which was restored by increasing the dose of SAG to 40 microg/ml. In contrast, Sb(RLD inhibited these SAG-induced events regardless of duration of DC exposure to Sb(RLD or dose of SAG. Interestingly, the inhibitory effects of isogenic Sb(SLD expressing ATP-binding cassette (ABC transporter MRPA on SAG-induced leishmanicidal effects mimicked that of Sb(RLD to some extent, although antimony resistance in clinical LD isolates is known to be multifactorial. Furthermore, NF-kappaB was found to transcriptionally regulate expression of murine gammaglutamylcysteine synthetase heavy-chain (mgammaGCS(hc gene, presumably an important regulator of antimony resistance. Importantly, Sb(RLD but not Sb(SLD blocked SAG-induced mgammaGCS expression in DCs by

DNA polymerase gamma inhibition by vitamin K3 induces mitochondria-mediated cytotoxicity in human cancer cells.

Science.gov (United States)

Sasaki, Ryohei; Suzuki, Yoko; Yonezawa, Yuko; Ota, Yosuke; Okamoto, Yoshiaki; Demizu, Yusuke; Huang, Peng; Yoshida, Hiromi; Sugimura, Kazuro; Mizushina, Yoshiyuki

2008-05-01

Among the vitamin K (VK) compounds, VK3 exhibits distinct cytotoxic activity in cancer cells and is thought to affect redox cycling; however, the underlying mechanisms remain unclear. Here we demonstrate that VK3 selectively inhibits DNA polymerase (pol) gamma, the key enzyme responsible for mitochondrial DNA replication and repair. VK3 at 30 microM inhibited pol gamma by more than 80%, caused impairment of mitochondrial DNA replication and repair, and induced a significant increase in reactive oxygen species (ROS), leading to apoptosis. At a lower concentration (3 microM), VK3 did not cause a significant increase in ROS, but was able to effectively inhibit cell proliferation, which could be reversed by supplementing glycolytic substrates. The cytotoxic action of VK3 was independent of p53 tumor suppressor gene status. Interestingly, VK3 only inhibited pol gamma but did not affect other pol including human pol alpha, pol beta, pol delta, and pol epsilon. VK1 and VK2 exhibited no inhibitory effect on any of the pol tested. These data together suggest that the inhibition of pol gamma by VK3 is relatively specific, and that this compound seems to exert its anticancer activity by two possible mechanisms in a concentration-dependent manner: (1) induction of ROS-mediated cell death at high concentrations; and (2) inhibition of cell proliferation at lower concentrations likely through the suppression of mitochondrial respiratory function. These findings may explain various cytotoxic actions induced by VK3, and may pave the way for the further use of VK3.

Curcumin blocks interleukin (IL)-2 signaling in T-lymphocytes by inhibiting IL-2 synthesis, CD25 expression, and IL-2 receptor signaling

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Forward, Nicholas A.; Conrad, David M. [Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia (Canada); Power Coombs, Melanie R.; Doucette, Carolyn D. [Department of Pathology, Dalhousie University, Halifax, Nova Scotia (Canada); Furlong, Suzanne J. [Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia (Canada); Lin, Tong-Jun [Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia (Canada); Department of Pediatrics, Dalhousie University, Halifax, Nova Scotia (Canada); Hoskin, David W., E-mail: d.w.hoskin@dal.ca [Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia (Canada); Department of Pathology, Dalhousie University, Halifax, Nova Scotia (Canada); Department of Surgery, Dalhousie University, Halifax, Nova Scotia (Canada)

2011-04-22

Highlights: {yields} Curcumin inhibits CD4{sup +} T-lymphocyte proliferation. {yields} Curcumin inhibits interleukin-2 (IL-2) synthesis and CD25 expression by CD4{sup +} T-lymphocytes. {yields} Curcumin interferes with IL-2 receptor signaling by inhibiting JAK3 and STAT5 phosphorylation. {yields} IL-2-dependent regulatory T-lymphocyte function and Foxp3 expression is downregulated by curcumin. -- Abstract: Curcumin (diferulomethane) is the principal curcuminoid in the spice tumeric and a potent inhibitor of activation-induced T-lymphocyte proliferation; however, the molecular basis of this immunosuppressive effect has not been well studied. Here we show that micromolar concentrations of curcumin inhibited DNA synthesis by mouse CD4{sup +} T-lymphocytes, as well as interleukin-2 (IL-2) and CD25 ({alpha} chain of the high affinity IL-2 receptor) expression in response to antibody-mediated cross-linking of CD3 and CD28. Curcumin acted downstream of protein kinase C activation and intracellular Ca{sup 2+} release to inhibit I{kappa}B phosphorylation, which is required for nuclear translocation of the transcription factor NF{kappa}B. In addition, IL-2-dependent DNA synthesis by mouse CTLL-2 cells, but not constitutive CD25 expression, was impaired in the presence of curcumin, which demonstrated an inhibitory effect on IL-2 receptor (IL-2R) signaling. IL-2-induced phosphorylation of STAT5A and JAK3, but not JAK1, was diminished in the presence of curcumin, indicating inhibition of critical proximal events in IL-2R signaling. In line with the inhibitory action of curcumin on IL-2R signaling, pretreatment of CD4{sup +}CD25{sup +} regulatory T-cells with curcumin downregulated suppressor function, as well as forkhead box p3 (Foxp3) expression. We conclude that curcumin inhibits IL-2 signaling by reducing available IL-2 and high affinity IL-2R, as well as interfering with IL-2R signaling.

Commensal-induced regulatory T cells mediate protection against pathogen-stimulated NF-kappaB activation.

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Caitlin O'Mahony

Full Text Available Host defence against infection requires a range of innate and adaptive immune responses that may lead to tissue damage. Such immune-mediated pathologies can be controlled with appropriate T regulatory (Treg activity. The aim of the present study was to determine the influence of gut microbiota composition on Treg cellular activity and NF-kappaB activation associated with infection. Mice consumed the commensal microbe Bifidobacterium infantis 35624 followed by infection with Salmonella typhimurium or injection with LPS. In vivo NF-kappaB activation was quantified using biophotonic imaging. CD4+CD25+Foxp3+ T cell phenotypes and cytokine levels were assessed using flow cytometry while CD4+ T cells were isolated using magnetic beads for adoptive transfer to naïve animals. In vivo imaging revealed profound inhibition of infection and LPS induced NF-kappaB activity that preceded a reduction in S. typhimurium numbers and murine sickness behaviour scores in B. infantis-fed mice. In addition, pro-inflammatory cytokine secretion, T cell proliferation, and dendritic cell co-stimulatory molecule expression were significantly reduced. In contrast, CD4+CD25+Foxp3+ T cell numbers were significantly increased in the mucosa and spleen of mice fed B. infantis. Adoptive transfer of CD4+CD25+ T cells transferred the NF-kappaB inhibitory activity. Consumption of a single commensal micro-organism drives the generation and function of Treg cells which control excessive NF-kappaB activation in vivo. These cellular interactions provide the basis for a more complete understanding of the commensal-host-pathogen trilogue that contribute to host homeostatic mechanisms underpinning protection against aberrant activation of the innate immune system in response to a translocating pathogen or systemic LPS.

Dehydration-induced modulation of κ-opioid inhibition of vasopressin neurone activity

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Scott, Victoria; Bishop, Valerie R; Leng, Gareth; Brown, Colin H

2009-01-01

Dehydration increases vasopressin (antidiuretic hormone) secretion from the posterior pituitary gland to reduce water loss in the urine. Vasopressin secretion is determined by action potential firing in vasopressin neurones, which can exhibit continuous, phasic (alternating periods of activity and silence), or irregular activity. Autocrine κ-opioid inhibition contributes to the generation of activity patterning of vasopressin neurones under basal conditions and so we used in vivo extracellular single unit recording to test the hypothesis that changes in autocrine κ-opioid inhibition drive changes in activity patterning of vasopressin neurones during dehydration. Dehydration increased the firing rate of rat vasopressin neurones displaying continuous activity (from 7.1 ± 0.5 to 9.0 ± 0.6 spikes s−1) and phasic activity (from 4.2 ± 0.7 to 7.8 ± 0.9 spikes s−1), but not those displaying irregular activity. The dehydration-induced increase in phasic activity was via an increase in intraburst firing rate. The selective κ-opioid receptor antagonist nor-binaltorphimine increased the firing rate of phasic neurones in non-dehydrated rats (from 3.4 ± 0.8 to 5.3 ± 0.6 spikes s−1) and dehydrated rats (from 6.4 ± 0.5 to 9.1 ± 1.2 spikes s−1), indicating that κ-opioid feedback inhibition of phasic bursts is maintained during dehydration. In a separate series of experiments, prodynorphin mRNA expression was increased in vasopressin neurones of hyperosmotic rats, compared to hypo-osmotic rats. Hence, it appears that dynorphin expression in vasopressin neurones undergoes dynamic changes in proportion to the required secretion of vasopressin so that, even under stimulated conditions, autocrine feedback inhibition of vasopressin neurones prevents over-excitation. PMID:19822541

Phenylarsine Oxide Inhibits the Fusicoccin-Induced Activation of Plasma Membrane H+-ATPase1

Science.gov (United States)

Olivari, Claudio; Albumi, Cristina; Pugliarello, Maria Chiara; De Michelis, Maria Ida

2000-01-01

To investigate the mechanism by which fusicoccin (FC) induces the activation of the plasma membrane (PM) H+-ATPase, we used phenylarsine oxide (PAO), a known inhibitor of protein tyrosine-phosphatases. PAO was supplied in vivo in the absence or presence of FC to radish (Raphanus sativus L.) seedlings and cultured Arabidopsis cells prior to PM extraction. Treatment with PAO alone caused a slight decrease of PM H+-ATPase activity and, in radish, a decrease of PM-associated 14-3-3 proteins. When supplied prior to FC, PAO drastically inhibited FC-induced activation of PM H+-ATPase, FC binding to the PM, and the FC-induced increase of the amount of 14-3-3 associated with the PM. On the contrary, PAO was completely ineffective on all of the above-mentioned parameters when supplied after FC. The H+-ATPase isolated from PAO-treated Arabidopsis cells maintained the ability to respond to FC if supplied with exogenous, nonphosphorylated 14-3-3 proteins. Altogether, these results are consistent with a model in which the dephosphorylated state of tyrosine residues of a protein(s), such as 14-3-3 protein, is required to permit FC-induced association between the 14-3-3 protein and the PM H+-ATPase. PMID:10677439

Protectin D1 reduces concanavalin A-induced liver injury by inhibiting NF-κB-mediated CX3CL1/CX3CR1 axis and NLR family, pyrin domain containing 3 inflammasome activation.

Science.gov (United States)

Ren, Jun; Meng, Shanshan; Yan, Bingdi; Yu, Jinyan; Liu, Jing

2016-04-01

Protectin D1 (PD1) is a bioactive product generated from docosahexaenoic acid, which may exert anti-inflammatory effects in various inflammatory diseases. However, the underlying molecular mechanism of its anti‑inflammatory activity on concanavalin A (Con A)-induced hepatitis remains unknown. The aim of the present study was to investigate the protective effects of PD1 against Con A‑induced liver injury and the underlying mechanisms via intravenous injection of PD1 prior to Con A administration. C57BL/6 mice were randomly divided into four experimental groups as follows: Control group, Con A group (30 mg/kg), 20 µg/kg PD1 + Con A (30 mg/kg) group and 10 µg/kg PD1 + Con A (30 mg/kg) group. PD1 pretreatment was demonstrated to significantly inhibit elevated plasma aminotransferase levels, high mobility group box 1 and liver necrosis, which were observed in Con A‑induced hepatitis. Furthermore, compared with the Con A group, PD1 pretreatment prevented the production of pro‑inflammatory cytokines, including tumor necrosis factor‑α, interferon‑γ and interleukin‑2, ‑1β and ‑6. In addition, pretreatment with PD1 markedly downregulated cluster of differentiation (CD)4+, CD8+ and natural killer T (NKT) cell infiltration in the liver. PD1 pretreatment was observed to suppress the messenger RNA and protein expression levels of NLR family, pyrin domain containing 3 and Toll‑like receptor (TLR) 4 in liver tissue samples. Further data indicated that PD1 pretreatment inhibited the activation of the nuclear factor κ‑light‑chain‑enhancer of activated B cells (NF‑κB) signaling pathway and chemokine (C‑X3‑C motif) ligand 1 (CX3CL1)/chemokine (C-X3-C motif) receptor 1 (CX3CR1) axis by preventing phosphorylation of nuclear factor of κ light polypeptide gene enhancer in B-cells inhibitor, α and NF‑κB in Con A‑induced liver injury. Therefore, these results suggest that PD1 administration protects mice against Con A‑induced liver injury via

Calcineurin inhibitor-induced complement system activation via ERK1/2 signalling is inhibited by SOCS-3 in human renal tubule cells.

Science.gov (United States)

Loeschenberger, Beatrix; Niess, Lea; Würzner, Reinhard; Schwelberger, Hubert; Eder, Iris E; Puhr, Martin; Guenther, Julia; Troppmair, Jakob; Rudnicki, Michael; Neuwirt, Hannes

2018-02-01

One factor that significantly contributes to renal allograft loss is chronic calcineurin inhibitor (CNI) nephrotoxicity (CIN). Among other factors, the complement (C-) system has been proposed to be involved CIN development. Hence, we investigated the impact of CNIs on intracellular signalling and the effects on the C-system in human renal tubule cells. In a qPCR array, CNI treatment upregulated C-factors and downregulated SOCS-3 and the complement inhibitors CD46 and CD55. Additionally, ERK1/-2 was required for these regulations. Following knock-down and overexpression of SOCS-3, we found that SOCS-3 inhibits ERK1/-2 signalling. Finally, we assessed terminal complement complex formation, cell viability and apoptosis. Terminal complement complex formation was induced by CNIs. Cell viability was significantly decreased, whereas apoptosis was increased. Both effects were reversed under complement component-depleted conditions. In vivo, increased ERK1/-2 phosphorylation and SOCS-3 downregulation were observed at the time of transplantation in renal allograft patients who developed a progressive decline of renal function in the follow-up compared to stable patients. The progressive cohort also had lower total C3 levels, suggesting higher complement activity at baseline. In conclusion, our data suggest that SOCS-3 inhibits CNI-induced ERK1/-2 signalling, thereby blunting the negative control of C-system activation. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Scoparone exerts anti-tumor activity against DU145 prostate cancer cells via inhibition of STAT3 activity.

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Jeong-Kook Kim

Full Text Available Scoparone, a natural compound isolated from Artemisia capillaris, has been used in Chinese herbal medicine to treat neonatal jaundice. Signal transducer and activator of transcription 3 (STAT3 contributes to the growth and survival of many human tumors. This study was undertaken to investigate the anti-tumor activity of scoparone against DU145 prostate cancer cells and to determine whether its effects are mediated by inhibition of STAT3 activity. Scoparone inhibited proliferation of DU145 cells via cell cycle arrest in G1 phase. Transient transfection assays showed that scoparone repressed both constitutive and IL-6-induced transcriptional activity of STAT3. Western blot and quantitative real-time PCR analyses demonstrated that scoparone suppressed the transcription of STAT3 target genes such as cyclin D1, c-Myc, survivin, Bcl-2, and Socs3. Consistent with this, scoparone decreased phosphorylation and nuclear accumulation of STAT3, but did not reduce phosphorylation of janus kinase 2 (JAK2 or Src, the major upstream kinases responsible for STAT3 activation. Moreover, transcriptional activity of a constitutively active mutant of STAT3 (STAT3C was inhibited by scoparone, but not by AG490, a JAK2 inhibitor. Furthermore, scoparone treatment suppressed anchorage-independent growth in soft agar and tumor growth of DU145 xenografts in nude mice, concomitant with a reduction in STAT3 phosphorylation. Computational modeling suggested that scoparone might bind the SH2 domain of STAT3. Our findings suggest that scoparone elicits an anti-tumor effect against DU145 prostate cancer cells in part through inhibition of STAT3 activity.

Camptothecin inhibits platelet-derived growth factor-BB-induced proliferation of rat aortic vascular smooth muscle cells through inhibition of PI3K/Akt signaling pathway

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Park, Eun-Seok [Department of Applied Biochemistry, Division of Life Science, College of Health and Biomedical Science, Konkuk University, Chungju, Chungbuk (Korea, Republic of); Kang, Shin-il [College of Pharmacy Medical Research Center, Chungbuk National University, Cheongju (Korea, Republic of); Yoo, Kyu-dong [Hazardous Substances Analysis Division, Gwangju Regional Food and Drug Administration, Gwangju (Korea, Republic of); Lee, Mi-Yea [Department of Nursing Kyungbok University, Pocheon (Korea, Republic of); Yoo, Hwan-Soo; Hong, Jin-Tae [College of Pharmacy Medical Research Center, Chungbuk National University, Cheongju (Korea, Republic of); Shin, Hwa-Sup [Department of Applied Biochemistry, Division of Life Science, College of Health and Biomedical Science, Konkuk University, Chungju, Chungbuk (Korea, Republic of); Kim, Bokyung [Department of Physiology, Konkuk Medical School, Konkuk University, Chungju, Chungbuk (Korea, Republic of); Yun, Yeo-Pyo, E-mail: ypyun@chungbuk.ac.kr [College of Pharmacy Medical Research Center, Chungbuk National University, Cheongju (Korea, Republic of)

2013-04-15

The abnormal proliferation of vascular smooth muscle cells (VSMCs) in arterial wall is a major cause of vascular disorders such as atherosclerosis and restenosis after angioplasty. In this study, we investigated not only the inhibitory effects of camptothecin (CPT) on PDGF-BB-induced VSMC proliferation, but also its molecular mechanism of this inhibition. CPT significantly inhibited proliferation with IC50 value of 0.58 μM and the DNA synthesis of PDGF-BB-stimulated VSMCs in a dose-dependent manner (0.5–2 μM ) without any cytotoxicity. CPT induced the cell cycle arrest at G0/G1 phase. Also, CPT decreased the expressions of G0/G1-specific regulatory proteins including cyclin-dependent kinase (CDK)2, cyclin D1 and PCNA in PDGF-BB-stimulated VSMCs. Pre-incubation of VSMCs with CPT significantly inhibited PDGF-BB-induced Akt activation, whereas CPT did not affect PDGF-receptor beta phosphorylation, extracellular signal-regulated kinase (ERK) 1/2 phosphorylation and phospholipase C (PLC)-γ1 phosphorylation in PDGF-BB signaling pathway. Our data showed that CPT pre-treatment inhibited VSMC proliferation, and that the inhibitory effect of CPT was enhanced by LY294002, a PI3K inhibitor, on PDGF-BB-induced VSMC proliferation. In addition, inhibiting the PI3K/Akt pathway by LY294002 significantly enhanced the suppression of PCNA expression and Akt activation by CPT. These results suggest that the anti-proliferative activity of CPT is mediated in part by downregulating the PI3K/Akt signaling pathway. - Highlights: ► CPT inhibits proliferation of PDGF-BB-induced VSMC without cytotoxicity. ► CPT arrests the cell cycle in G0/G1 phase by downregulation of cyclin D1 and CDK2. ► CPT significantly attenuates Akt phosphorylation in PDGF-BB signaling pathway. ► LY294002 enhanced the inhibitory effect of CPT on VSMC proliferation. ► Thus, CPT is mediated by downregulating the PI3K/Akt signaling pathway.

Camptothecin inhibits platelet-derived growth factor-BB-induced proliferation of rat aortic vascular smooth muscle cells through inhibition of PI3K/Akt signaling pathway

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Park, Eun-Seok; Kang, Shin-il; Yoo, Kyu-dong; Lee, Mi-Yea; Yoo, Hwan-Soo; Hong, Jin-Tae; Shin, Hwa-Sup; Kim, Bokyung; Yun, Yeo-Pyo

2013-01-01

The abnormal proliferation of vascular smooth muscle cells (VSMCs) in arterial wall is a major cause of vascular disorders such as atherosclerosis and restenosis after angioplasty. In this study, we investigated not only the inhibitory effects of camptothecin (CPT) on PDGF-BB-induced VSMC proliferation, but also its molecular mechanism of this inhibition. CPT significantly inhibited proliferation with IC50 value of 0.58 μM and the DNA synthesis of PDGF-BB-stimulated VSMCs in a dose-dependent manner (0.5–2 μM ) without any cytotoxicity. CPT induced the cell cycle arrest at G0/G1 phase. Also, CPT decreased the expressions of G0/G1-specific regulatory proteins including cyclin-dependent kinase (CDK)2, cyclin D1 and PCNA in PDGF-BB-stimulated VSMCs. Pre-incubation of VSMCs with CPT significantly inhibited PDGF-BB-induced Akt activation, whereas CPT did not affect PDGF-receptor beta phosphorylation, extracellular signal-regulated kinase (ERK) 1/2 phosphorylation and phospholipase C (PLC)-γ1 phosphorylation in PDGF-BB signaling pathway. Our data showed that CPT pre-treatment inhibited VSMC proliferation, and that the inhibitory effect of CPT was enhanced by LY294002, a PI3K inhibitor, on PDGF-BB-induced VSMC proliferation. In addition, inhibiting the PI3K/Akt pathway by LY294002 significantly enhanced the suppression of PCNA expression and Akt activation by CPT. These results suggest that the anti-proliferative activity of CPT is mediated in part by downregulating the PI3K/Akt signaling pathway. - Highlights: ► CPT inhibits proliferation of PDGF-BB-induced VSMC without cytotoxicity. ► CPT arrests the cell cycle in G0/G1 phase by downregulation of cyclin D1 and CDK2. ► CPT significantly attenuates Akt phosphorylation in PDGF-BB signaling pathway. ► LY294002 enhanced the inhibitory effect of CPT on VSMC proliferation. ► Thus, CPT is mediated by downregulating the PI3K/Akt signaling pathway

ATF3 activates Stat3 phosphorylation through inhibition of p53 expression in skin cancer cells.

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Hao, Zhen-Feng; Ao, Jun-Hong; Zhang, Jie; Su, You-Ming; Yang, Rong-Ya

2013-01-01

ATF3, a member of the ATF/CREB family of transcription factors, has been found to be selectively induced by calcineurin/NFAT inhibition and to enhance keratinocyte tumor formation, although the precise role of ATF3 in human skin cancer and possible mechanisms remain unknown. In this study, clinical analysis of 30 skin cancer patients and 30 normal donors revealed that ATF3 was accumulated in skin cancer tissues. Functional assays demonstrated that ATF3 significantly promoted skin cancer cell proliferation. Mechanically, ATF3 activated Stat3 phosphorylation in skin cancer cell through regulation of p53 expression. Moreover, the promotion effect of ATF3 on skin cancer cell proliferation was dependent on the p53-Stat3 signaling cascade. Together, the results indicate that ATF3 might promote skin cancer cell proliferation and enhance skin keratinocyte tumor development through inhibiting p53 expression and then activating Stat3 phosphorylation.

Vitamin K3 attenuates cerulein-induced acute pancreatitis through inhibition of the autophagic pathway.

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Chinzei, Ryo; Masuda, Atsuhiro; Nishiumi, Shin; Nishida, Masayuki; Onoyama, Mitsuko; Sanuki, Tsuyoshi; Fujita, Tsuyoshi; Moritoh, Satoshi; Itoh, Tomoo; Kutsumi, Hiromu; Mizuno, Shigeto; Azuma, Takeshi; Yoshida, Masaru

2011-01-01

The discovery of novel and effective treatment methods would be of great help to patients with acute pancreatitis. The aims of this study were to determine the inhibitory effects of vitamin K3 (VK3) against cerulein-induced acute pancreatitis in mice and to examine the mechanisms behind these effects. Acute pancreatitis in mice was induced by intraperitoneal injection of cerulein 6 times at hourly intervals. Vitamin K3 was administered once before the first injection of cerulein or twice before and after the first injection of cerulein. The degrees of inflammation and autophagy in the pancreatic tissue were estimated by histological examination, measurement of enzyme activity, confocal microscopy, and Western blotting. The inhibitory effects of VK3 against rapamycin-induced autophagy were also examined using HeLa cells stably expressing green fluorescent protein LC3. Cerulein-induced acute pancreatitis was markedly attenuated by the administration of VK3. In addition, VK3 led to the inhibition of cerulein-evoked autophagic changes and colocalization of autophagosomes and lysosomes in the pancreatic tissue. Vitamin K3 also reduced rapamycin-induced autophagy in HeLa/green fluorescent protein LC3 cells. Our data suggest that the administration of VK3 reduces pancreatic inflammation in acute pancreatitis through inhibition of the autophagic pathway. Vitamin K3 may be an effective therapeutic strategy against acute pancreatitis.

IGF-1 protects against Aβ25-35-induced neuronal cell death via inhibition of PUMA expression and Bax activation.

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Hou, Xunyao; Jin, Yan; Chen, Jian; Hong, Yan; Luo, Dingzhen; Yin, Qingqing; Liu, Xueping

2017-01-10

Amyloid-β-peptide (Aβ) is considered to be the toxic species in AD and causes cell death in the affected areas of patient's brain. Insulin-like growth factor 1 (IGF-1) has been reported to attenuate Aβ toxicity in neuronal cells. However, the molecular mechanisms involved in the neuroprotective function of IGF-1 remain largely unknown. In the present study, we for the first time demonstrated that IGF-1 protects against Aβ-induced neurotoxicity via inhibition of PUMA expression and Bax activation. We found that IGF-1 could activate Akt, which in turn inhibited Aβ-induced FOXO3a nuclear translocation and thus decreased the binding ability of FOXO3a to PUMA promoter, leading to decreased PUMA expression. In addition, IGF-1 inhibited the translocation of Bax to the mitochondria induced by Aβ. Notably, addition of wortmannin, a specific inhibitor of PI3K, significantly abolished the neuroprotective effect of IGF-1, suggesting that IGF-1 exerts its anti-apoptotic effect depend on PI3K activity. Our findings may provide new insights into molecular mechanisms mediated by IGF-1 in cell survival against Aβ-induced apoptosis. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Differential pattern and prognostic significance of CD4+, FOXP3+ and IL-17+ tumor infiltrating lymphocytes in ductal and lobular breast cancers

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Droeser, Raoul; Zlobec, Inti; Kilic, Ergin; Güth, Uwe; Heberer, Michael; Spagnoli, Giulio; Oertli, Daniel; Tapia, Coya

2012-01-01

Clinical relevance of tumor infiltrating lymphocytes (TILs) in breast cancer is controversial. Here, we used a tumor microarray including a large series of ductal and lobular breast cancers with long term follow up data, to analyze clinical impact of TIL expressing specific phenotypes and distribution of TILs within different tumor compartments and in different histological subtypes. A tissue microarray (TMA) including 894 ductal and 164 lobular breast cancers was stained with antibodies recognizing CD4, FOXP3, and IL-17 by standard immunohistochemical techniques. Lymphocyte counts were correlated with clinico-pathological parameters and survival. CD4 + lymphocytes were more prevalent than FOXP3 + TILs whereas IL-17 + TILs were rare. Increased numbers of total CD4 + and FOXP3 + TIL were observed in ductal, as compared with lobular carcinomas. High grade (G3) and estrogen receptor (ER) negative ductal carcinomas displayed significantly (p < 0.001) higher CD4 + and FOXP3 + lymphocyte infiltration while her2/neu over-expression in ductal carcinomas was significantly (p < 0.001) associated with higher FOXP3 + TIL counts. In contrast, lymphocyte infiltration was not linked to any clinico-pathological parameters in lobular cancers. In univariate but not in multivariate analysis CD4 + infiltration was associated with significantly shorter survival in patients bearing ductal, but not lobular cancers. However, a FOXP3 + /CD4 + ratio > 1 was associated with improved overall survival even in multivariate analysis (p = 0.033). Ductal and lobular breast cancers appear to be infiltrated by different lymphocyte subpopulations. In ductal cancers increased CD4 + and FOXP3 + TIL numbers are associated with more aggressive tumor features. In survival analysis, absolute numbers of TILs do not represent major prognostic indicators in ductal and lobular breast cancer. Remarkably however, a ratio > 1 of total FOXP3 + /CD4 + TILs in ductal carcinoma appears to represent an independent

Novel derivative of aminobenzenesulfonamide (3c) induces apoptosis in colorectal cancer cells through ROS generation and inhibits cell migration.

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Al-Khayal, Khayal; Alafeefy, Ahmed; Vaali-Mohammed, Mansoor-Ali; Mahmood, Amer; Zubaidi, Ahmed; Al-Obeed, Omar; Khan, Zahid; Abdulla, Maha; Ahmad, Rehan

2017-01-03

Colorectal cancer (CRC) is the 3 rd most common type of cancer worldwide. New anti-cancer agents are needed for treating late stage colorectal cancer as most of the deaths occur due to cancer metastasis. A recently developed compound, 3c has shown to have potent antitumor effect; however the mechanism underlying the antitumor effect remains unknown. 3c-induced inhibition of proliferation was measured in the absence and presence NAC using MTT in HT-29 and SW620 cells and xCELLigence RTCA DP instrument. 3c-induced apoptotic studies were performed using flow cytometry. 3c-induced redox alterations were measured by ROS production using fluorescence plate reader and flow cytometry and mitochondrial membrane potential by flow cytometry; NADPH and GSH levels were determined by colorimetric assays. Bcl2 family protein expression and cytochrome c release and PARP activation was done by western blotting. Caspase activation was measured by ELISA. Cell migration assay was done using the real time xCELLigence RTCA DP system in SW620 cells and wound healing assay in HT-29. Many anticancer therapeutics exert their effects by inducing reactive oxygen species (ROS). In this study, we demonstrate that 3c-induced inhibition of cell proliferation is reversed by the antioxidant, N-acetylcysteine, suggesting that 3c acts via increased production of ROS in HT-29 cells. This was confirmed by the direct measurement of ROS in 3c-treated colorectal cancer cells. Additionally, treatment with 3c resulted in decreased NADPH and glutathione levels in HT-29 cells. Further, investigation of the apoptotic pathway showed increased release of cytochrome c resulting in the activation of caspase-9, which in turn activated caspase-3 and -6. 3c also (i) increased p53 and Bax expression, (ii) decreased Bcl2 and BclxL expression and (iii) induced PARP cleavage in human colorectal cancer cells. Confirming our observations, NAC significantly inhibited induction of apoptosis, ROS production, cytochrome c

A synthetic peptide blocking TRPV1 activation inhibits UV-induced skin responses.

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Kang, So Min; Han, Sangbum; Oh, Jang-Hee; Lee, Young Mee; Park, Chi-Hyun; Shin, Chang-Yup; Lee, Dong Hun; Chung, Jin Ho

2017-10-01

Transient receptor potential type 1 (TRPV1) can be activated by ultraviolet (UV) irradiation, and mediates UV-induced matrix metalloproteinase (MMP)-1 and proinflammatory cytokines in keratinocytes. Various chemicals and compounds targeting TRPV1 activation have been developed, but are not in clinical use mostly due to their safety issues. We aimed to develop a novel TRPV1-targeting peptide to inhibit UV-induced responses in human skin. We designed and generated a novel TRPV1 inhibitory peptide (TIP) which mimics the specific site in TRPV1 (aa 701-709: Gln-Arg-Ala-Ile-Thr-Ile-Leu-Asp-Thr, QRAITILDT), Thr 705 , and tested its efficacy of blocking UV-induced responses in HaCaT, mouse, and human skin. TIP effectively inhibited capsaicin-induced calcium influx and TRPV1 activation. Treatment of HaCaT with TIP prevented UV-induced increases of MMP-1 and pro-inflammatory cytokines such as interleukin (IL)-6 and tumor necrosis factor-α. In mouse skin in vivo, TIP inhibited UV-induced skin thickening and prevented UV-induced expression of MMP-13 and MMP-9. Moreover, TIP attenuated UV-induced erythema and the expression of MMP-1, MMP-2, IL-6, and IL-8 in human skin in vivo. The novel synthetic peptide targeting TRPV1 can ameliorate UV-induced skin responses in vitro and in vivo, providing a promising therapeutic approach against UV-induced inflammation and photoaging. Copyright © 2017 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.

RNCR3 knockdown inhibits diabetes mellitus-induced retinal reactive gliosis

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Liu, Chang; Li, Chao-peng; Wang, Jia-Jian; Shan, Kun; Liu, Xin; Yan, Biao

2016-01-01

Retinal reactive gliosis is an important pathological feature of diabetic retinopathy. Identifying the underlying mechanisms causing reactive gliosis will be important for developing new therapeutic strategies for treating diabetic retinopathy. Herein, we show that long noncoding RNA-RNCR3 knockdown significantly inhibits retinal reactive gliosis. RNCR3 knockdown leads to a marked reduction in the release of several cytokines. RNCR3 knockdown alleviates diabetes mellitus-induced retinal neurodegeneration, as shown by less apoptotic retinal cells and ameliorative visual function. RNCR3 knockdown could also decrease Müller glial cell viability and proliferation, and reduce the expression of glial reactivity-related genes including GFAP and vimentin in vitro. Collectively, this study shows that RNCR3 knockdown may be a promising strategy for the prevention of diabetes mellitus-induced retinal neurodegeneration. - Highlights: • RNCR3 knockdown inhibits retinal reactive gliosis. • RNCR3 knockdown causes a significant change in cytokine profile. • RNCR3 knockdown alleviates diabetes mellitus-induced retinal neurodegeneration. • RNCR3 knockdown affects Müller glial cell function in vitro.

Low-dose temozolomide before dendritic-cell vaccination reduces (specifically) CD4+CD25++Foxp3+ regulatory T-cells in advanced melanoma patients.

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Ridolfi, Laura; Petrini, Massimiliano; Granato, Anna Maria; Gentilcore, Giusy; Simeone, Ester; Ascierto, Paolo Antonio; Pancisi, Elena; Ancarani, Valentina; Fiammenghi, Laura; Guidoboni, Massimo; de Rosa, Francesco; Valmorri, Linda; Scarpi, Emanuela; Nicoletti, Stefania Vittoria Luisa; Baravelli, Stefano; Riccobon, Angela; Ridolfi, Ruggero

2013-05-31

In cancer immunotherapy, dendritic cells (DCs) play a fundamental role in the dialog between innate and adaptive immune response, but several immunosuppressive mechanisms remain to be overcome. For example, a high number of CD4+CD25++Foxp3+ regulatory T-cells (Foxp3+Tregs) have been observed in the peripheral blood and tumor microenvironment of cancer patients. On the basis of this, we conducted a study on DC-based vaccination in advanced melanoma, adding low-dose temozolomide to obtain lymphodepletion. Twenty-one patients were entered onto our vaccination protocol using autologous DCs pulsed with autologous tumor lysate and keyhole limpet hemocyanin. Patients received low-dose temozolomide before vaccination and 5 days of low-dose interleukin-2 (IL-2) after vaccination. Circulating Foxp3+Tregs were evaluated before and after temozolomide, and after IL-2. Among the 17 evaluable patients we observed 1 partial response (PR), 6 stable disease (SD) and 10 progressive disease (PD). The disease control rate (PR+SD = DCR) was 41% and median overall survival was 10 months. Temozolomide reduced circulating Foxp3+Treg cells in all patients. A statistically significant reduction of 60% was observed in Foxp3+Tregs after the first cycle, whereas the absolute lymphocyte count decreased by only 14%. Conversely, IL-2 increased Foxp3+Treg cell count by 75.4%. Of note the effect of this cytokine, albeit not statistically significant, on the DCR subgroup led to a further 33.8% reduction in Foxp3+Treg cells. Our results suggest that the combined immunological therapy, at least as far as the DCR subgroup is concerned, effectively reduced the number of Foxp3+Treg cells, which exerted a blunting effect on the growth-stimulating effect of IL-2. However, this regimen, with its current modality, would not seem to be capable of improving clinical outcome.

Forkhead box P3 regulates ARHGAP15 expression and affects migration of glioma cells through the Rac1 signaling pathway.

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Sun, Zhen; Zhang, Biao; Wang, Chen; Fu, Tao; Li, Lianling; Wu, Qiaoli; Cai, Ying; Wang, Jinhuan

2017-01-01

Forkhead box P3 (FOXP3) plays a crucial role in the development and function of regulatory T cells and was recently identified as a tumor suppressor in different cancer types. Forkhead box P3 is expressed in normal brain tissues, but is strongly downregulated or absent in glioblastomas. In order to understand the FOXP3 adjustment mechanisms in glioma cells, we performed a DNA microarray in U87 cells overexpressing FOXP3 and validated the differences using quantitative real-time PCR, Western blot analysis, and immunohistochemistry in vitro and in vivo. We found that FOXP3 can regulate the expression of ARHGAP15. Expression of FOXP3 was also correlated with ARHGAP15 in glioma samples. Overexpression of FOXP3 inhibited glioma cell migration through ARHGAP15 upregulation and Rac1 inactivation. Silencing of FOXP3 promoted migration through ARHGAP15 downregulation and Rac1 activation. ARHGAP15, a GTPase-activating protein for Rac1, inhibits small GTPase signaling in a dual negative manner. We found that there is a correlation between expression of ARHGAP15 and glioma level. The small GTPase Rac1 plays an important role in cell migration. In addition, we found that FOXP3 regulates expression of epithelial-mesenchymal transition markers E-cadherin and N-cadherin, which is important given that epithelial-mesenchymal transition is critically involved in tumor spreading and dissemination. Thus, FOXP3 or ARHGAP15 may serve as a new molecular target for antimetastatic therapies in treating glioma. © 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

DMH1 (4-[6-(4-isopropoxyphenylpyrazolo[1,5-a]pyrimidin-3-yl]quinoline inhibits chemotherapeutic drug-induced autophagy

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Yue Sheng

2015-07-01

Full Text Available Our previous work found that DMH1 (4-[6-(4-isopropoxyphenylpyrazolo [1,5-a]pyrimidin-3-yl]quinoline was a novel autophagy inhibitor. Here, we aimed to investigate the effects of DMH1 on chemotherapeutic drug-induced autophagy as well as the efficacy of chemotherapeutic drugs in different cancer cells. We found that DMH1 inhibited tamoxifen- and cispcis-diaminedichloroplatinum (II (CDDP-induced autophagy responses in MCF-7 and HeLa cells, and potentiated the anti-tumor activity of tamoxifen and CDDP for both cells. DMH1 inhibited 5-fluorouracil (5-FU-induced autophagy responses in MCF-7 and HeLa cells, but did not affect the anti-tumor activity of 5-FU for these two cell lines. DMH1 itself did not induce cell death in MCF-7 and HeLa cells, but inhibited the proliferation of these cells. In conclusion, DMH1 inhibits chemotherapeutic drug-induced autophagy response and the enhancement of efficacy of chemotherapeutic drugs by DMH1 is dependent on the cell sensitivity to drugs.

In vivo fluctuation of Tax, Foxp3, CTLA-4, and GITR mRNA expression in CD4(+)CD25(+) T cells of patients with human T-lymphotropic virus type 1-associated myelopathy.

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Ramirez, E; Cartier, L; Rodriguez, L; Alberti, C; Valenzuela, M A

2010-11-01

HTLV-1 Tax expression exerts an inhibitory effect on the Foxp3 transcription factor in CD4(+)CD25(+) T-regulatory cells (Treg). For a better understanding of the role of Tax mRNA in the gene expression of cellular markers we measured Tax, Foxp3, CTLA-4, GITR, TGF-β, and IL-10 mRNA in Treg cells of 50 patients with human T-lymphotropic virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP; 27 women and 23 men; mean age: 56.7 years). The control group consisted of 23 non-infected subjects (12 women and 11 men) with a mean age of 51.3 years. Real-time PCR was used to measure mRNA of Tax proteins and several cellular markers of Treg function. Determinations revealed a high level of Tax mRNA in HAM/TSP (124.35 copies/100 CD4(+)CD25(+) T cells). Foxp3, GITR, and CTLA-4 mRNA levels were lower in HAM/TSP patients (mean ± SD, 22.07 ± 0.78, 9.63 ± 0.36, and 4.54 ± 0.39, respectively) than in non-infected controls (47.15 ± 12.94, 22.14 ± 1.91, and 21.07 ± 2.31). Both groups had similar levels of TGF-β and IL-10. An inverse relationship was found between Tax levels and Foxp3, CTLA-4, and GITR levels. Conversely, there was a direct correlation between levels of Foxp3, GITR, and CTLA-4. Disease severity and evolution time did not correlate with Tax or Foxp3 levels. The present results suggest that Tax and Foxp3 mRNA vary with the same degree of disease severity in HAM/TSP patients. Tax fluctuations may affect CTLA-4 and GITR expression via the Foxp3 pathway, causing virus-induced dysfunction of CD4(+)CD25(+) T cells in HAM/TSP patients.

In vivo fluctuation of Tax, Foxp3, CTLA-4, and GITR mRNA expression in CD4+CD25+ T cells of patients with human T-lymphotropic virus type 1-associated myelopathy

Directory of Open Access Journals (Sweden)

E. Ramirez

2010-11-01

Full Text Available HTLV-1 Tax expression exerts an inhibitory effect on the Foxp3 transcription factor in CD4+CD25+ T-regulatory cells (Treg. For a better understanding of the role of Tax mRNA in the gene expression of cellular markers we measured Tax, Foxp3, CTLA-4, GITR, TGF-β, and IL-10 mRNA in Treg cells of 50 patients with human T-lymphotropic virus type 1 (HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP; 27 women and 23 men; mean age: 56.7 years. The control group consisted of 23 non-infected subjects (12 women and 11 men with a mean age of 51.3 years. Real-time PCR was used to measure mRNA of Tax proteins and several cellular markers of Treg function. Determinations revealed a high level of Tax mRNA in HAM/TSP (124.35 copies/100 CD4+CD25+ T cells. Foxp3, GITR, and CTLA-4 mRNA levels were lower in HAM/TSP patients (mean ± SD, 22.07 ± 0.78, 9.63 ± 0.36, and 4.54 ± 0.39, respectively than in non-infected controls (47.15 ± 12.94, 22.14 ± 1.91, and 21.07 ± 2.31. Both groups had similar levels of TGF-β and IL-10. An inverse relationship was found between Tax levels and Foxp3, CTLA-4, and GITR levels. Conversely, there was a direct correlation between levels of Foxp3, GITR, and CTLA-4. Disease severity and evolution time did not correlate with Tax or Foxp3 levels. The present results suggest that Tax and Foxp3 mRNA vary with the same degree of disease severity in HAM/TSP patients. Tax fluctuations may affect CTLA-4 and GITR expression via the Foxp3 pathway, causing virus-induced dysfunction of CD4+CD25+ T cells in HAM/TSP patients.

Inhibition of Rac1 activity induces G1/S phase arrest through the GSK3/cyclin D1 pathway in human cancer cells.

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Liu, Linna; Zhang, Hongmei; Shi, Lei; Zhang, Wenjuan; Yuan, Juanli; Chen, Xiang; Liu, Juanjuan; Zhang, Yan; Wang, Zhipeng

2014-10-01

Rac1 has been shown to regulate the cell cycle in cancer cells. Yet, the related mechanism remains unclear. Thus, the present study aimed to investigate the mechanism involved in the regulation of G1/S phase transition by Rac1 in cancer cells. Inhibition of Rac1 by inhibitor NSC23766 induced G1/S phase arrest and inhibited the proliferation of A431, SW480 and U2-OS cells. Suppression of GSK3 by shRNA partially rescued G1/S phase arrest and inhibition of proliferation. Incubation of cells with NSC23766 reduced p-AKT and inactivated p-GSK3α and p-GSK3β, increased p-cyclin D1 expression and decreased the level of cyclin D1 protein. Consequently, cyclin D1 targeting transcriptional factor E2F1 expression, which promotes G1 to S phase transition, was also reduced. In contrast, constitutive active Rac1 resulted in increased p-AKT and inactivated p-GSK3α and p-GSK3β, decreased p-cyclin D1 expression and enhanced levels of cyclin D1 and E2F1 expression. Moreover, suppression of GSK3 did not alter p-AKT or Rac1 activity, but decreased p-cyclin D1 and increased total cyclin D1 protein. However, neither Rac1 nor GSK3 inhibition altered cyclin D1 at the RNA level. Moreover, after inhibition of Rac1 or GSK3 following proteasome inhibitor MG132 treatment, cyclin D1 expression at the protein level remained constant, indicating that Rac1 and GSK3 may regulate cyclin D1 turnover through phosphorylation and degradation. Therefore, our findings suggest that inhibition of Rac1 induces cell cycle G1/S arrest in cancer cells by regulation of the GSK3/cyclin D1 pathway.

An Anti-C1s Monoclonal, TNT003, Inhibits Complement Activation Induced by Antibodies Against HLA.

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Thomas, K A; Valenzuela, N M; Gjertson, D; Mulder, A; Fishbein, M C; Parry, G C; Panicker, S; Reed, E F

2015-08-01

Antibody-mediated rejection (AMR) of solid organ transplants (SOT) is characterized by damage triggered by donor-specific antibodies (DSA) binding donor Class I and II HLA (HLA-I and HLA-II) expressed on endothelial cells. While F(ab')2 portions of DSA cause cellular activation and proliferation, Fc regions activate the classical complement cascade, resulting in complement deposition and leukocyte recruitment, both hallmark features of AMR. We characterized the ability of an anti-C1s monoclonal antibody, TNT003, to inhibit HLA antibody (HLA-Ab)-induced complement activation. Complement deposition induced by HLA-Ab was evaluated using novel cell- and bead-based assays. Human aortic endothelial cells (HAEC) were cultured with HLA-Ab and human complement; production of activated complement proteins was measured by flow cytometry. Additionally, C3d deposition was measured on single antigen beads (SAB) mixed with HLA-Ab and human complement. TNT003 inhibited HLA-Ab mediated complement deposition on HAEC in a concentration-dependent manner; C3a, C4a and C5a anaphylatoxin production was also diminished by TNT003. Finally, TNT003 blocked C3d deposition induced by Class I (HLAI-Ab)- and Class II (HLAII-Ab)-specific antibodies on SAB. These data suggest TNT003 may be useful for modulating the effects of DSA, as TNT003 inhibits complement deposition and split product formation generated by HLA-I/II-Ab in vitro. © 2015 The Authors. American Journal of Transplantation Published by Wiley Periodicals, Inc.

Cyclic AMP-Responsive Element-Binding Protein (CREB is Critical in Autoimmunity by Promoting Th17 but Inhibiting Treg Cell Differentiation

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Xiaohu Wang

2017-11-01

Full Text Available The molecular mechanisms that govern differential T cell development into pro-inflammatory Th17 vs. regulatory T (Treg cells remain unclear. Here, we show that selective deletion of CREB in T cells or Th17 cells impaired Th17 cell differentiation in vitro and in vivo, and led to resistance to autoimmune diseases. Mechanistically, CREB, activated by CD3-PKC-ϴ signaling, plays a key role in regulating Th17 cell differentiation, at least in part through directly binding to the Il17-Il17f gene locus. Unexpectedly, although dispensable for FOXP3 expression and for the homeostasis and suppressive function of thymus-derived Treg cells, CREB negatively regulates the survival of TGF-β-induced Treg cells, and deletion of CREB resulted in increased FOXP3+ Treg cells in the intestine and protection in a colitis model. Thus, CREB is critical in autoimmune diseases by promoting Th17 cell and inhibiting de novo Treg cell generation.

FOXP1 suppresses immune response signatures and MHC class II expression in activated B-cell-like diffuse large B-cell lymphomas

DEFF Research Database (Denmark)

Brown, P J; Wong, K K; Felce, S L

2016-01-01

The FOXP1 (forkhead box P1) transcription factor is a marker of poor prognosis in diffuse large B-cell lymphoma (DLBCL). Here microarray analysis of FOXP1-silenced DLBCL cell lines identified differential regulation of immune response signatures and major histocompatibility complex class II (MHC II......) genes as some of the most significant differences between germinal center B-cell (GCB)-like DLBCL with full-length FOXP1 protein expression versus activated B-cell (ABC)-like DLBCL expressing predominantly short FOXP1 isoforms. In an independent primary DLBCL microarray data set, multiple MHC II genes......, including human leukocyte antigen DR alpha chain (HLA-DRA), were inversely correlated with FOXP1 transcript expression (PABC-DLBCL cells led to increased cell-surface expression of HLA-DRA and CD74. In R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone...

CR108, a novel vitamin K3 derivative induces apoptosis and breast tumor inhibition by reactive oxygen species and mitochondrial dysfunction

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Yang, Chun-Ru [Department of Biological Science and Technology, National Chiao Tung University, Hsinchu 30068, Taiwan (China); Liao, Wei-Siang [Institute of Molecular Medicine and Bioengineering, National Chiao Tung University, Hsinchu 30068, Taiwan (China); Wu, Ya-Hui [Department of Biological Science and Technology, National Chiao Tung University, Hsinchu 30068, Taiwan (China); Murugan, Kaliyappan [Department of Chemistry, National Dong Hwa University, Hualien 974, Taiwan (China); Chen, Chinpiao, E-mail: chinpiao@mail.ndhu.edu.tw [Department of Chemistry, National Dong Hwa University, Hualien 974, Taiwan (China); Chao, Jui-I, E-mail: jichao@faculty.nctu.edu.tw [Department of Biological Science and Technology, National Chiao Tung University, Hsinchu 30068, Taiwan (China); Institute of Molecular Medicine and Bioengineering, National Chiao Tung University, Hsinchu 30068, Taiwan (China)

2013-12-15

Vitamin K3 derivatives have been shown to exert anticancer activities. Here we show a novel vitamin K3 derivative (S)-2-(2-hydroxy-3-methylbutylthio)naphthalene-1,4-dione, which is named as CR108 that induces apoptosis and tumor inhibition through reactive oxygen species (ROS) and mitochondrial dysfunction in human breast cancer. CR108 is more effective on the breast cancer cell death than other vitamin K3 derivatives. Moreover, CR108 induced apoptosis in both the non-HER-2-overexpressed MCF-7 and HER-2-overexpressed BT-474 breast cancer cells. CR108 caused the loss of mitochondrial membrane potential, cytochrome c released from mitochondria to cytosol, and cleaved PARP proteins for apoptosis induction. CR108 markedly increased ROS levels in breast cancer cells. N-acetylcysteine (NAC), a general ROS scavenger, completely blocked the CR108-induced ROS levels, mitochondrial dysfunction and apoptosis. Interestingly, CR108 increased the phosphorylation of p38 MAP kinase but conversely inhibited the survivin protein expression. NAC treatment prevented the activation of p38 MAP kinase and rescued the survivin protein levels. SB202190, a specific p38 MAP kinase inhibitor, recovered the survivin protein levels and attenuated the cytotoxicity of CR108-treated cells. Furthermore, CR108 inhibited the xenografted human breast tumor growth in nude mice. Together, we demonstrate that CR108 is a novel vitamin K3 derivative that induces apoptosis and tumor inhibition by ROS production and mitochondrial dysfunction and associates with the phosphorylation of p38 MAP kinase and the inhibition of survivin in the human breast cancer. - Highlights: • CR108 is more effective on the cell death than other vitamin K3 derivatives. • CR108 induces apoptosis and tumor inhibition by ROS and mitochondrial dysfunction. • CR108 induces apoptosis by p38 kinase activation and survivin inhibition. • CR108 is a potent vitamin K3 analog that can develop for breast cancer therapy.

CR108, a novel vitamin K3 derivative induces apoptosis and breast tumor inhibition by reactive oxygen species and mitochondrial dysfunction

International Nuclear Information System (INIS)

Yang, Chun-Ru; Liao, Wei-Siang; Wu, Ya-Hui; Murugan, Kaliyappan; Chen, Chinpiao; Chao, Jui-I

2013-01-01

Vitamin K3 derivatives have been shown to exert anticancer activities. Here we show a novel vitamin K3 derivative (S)-2-(2-hydroxy-3-methylbutylthio)naphthalene-1,4-dione, which is named as CR108 that induces apoptosis and tumor inhibition through reactive oxygen species (ROS) and mitochondrial dysfunction in human breast cancer. CR108 is more effective on the breast cancer cell death than other vitamin K3 derivatives. Moreover, CR108 induced apoptosis in both the non-HER-2-overexpressed MCF-7 and HER-2-overexpressed BT-474 breast cancer cells. CR108 caused the loss of mitochondrial membrane potential, cytochrome c released from mitochondria to cytosol, and cleaved PARP proteins for apoptosis induction. CR108 markedly increased ROS levels in breast cancer cells. N-acetylcysteine (NAC), a general ROS scavenger, completely blocked the CR108-induced ROS levels, mitochondrial dysfunction and apoptosis. Interestingly, CR108 increased the phosphorylation of p38 MAP kinase but conversely inhibited the survivin protein expression. NAC treatment prevented the activation of p38 MAP kinase and rescued the survivin protein levels. SB202190, a specific p38 MAP kinase inhibitor, recovered the survivin protein levels and attenuated the cytotoxicity of CR108-treated cells. Furthermore, CR108 inhibited the xenografted human breast tumor growth in nude mice. Together, we demonstrate that CR108 is a novel vitamin K3 derivative that induces apoptosis and tumor inhibition by ROS production and mitochondrial dysfunction and associates with the phosphorylation of p38 MAP kinase and the inhibition of survivin in the human breast cancer. - Highlights: • CR108 is more effective on the cell death than other vitamin K3 derivatives. • CR108 induces apoptosis and tumor inhibition by ROS and mitochondrial dysfunction. • CR108 induces apoptosis by p38 kinase activation and survivin inhibition. • CR108 is a potent vitamin K3 analog that can develop for breast cancer therapy

Effects of oral Lactobacillus administration on antioxidant activities and CD4+CD25+forkhead box P3 (FoxP3)+ T cells in NZB/W F1 mice.

Science.gov (United States)

Tzang, Bor-Show; Liu, Chung-Hsien; Hsu, Kuo-Ching; Chen, Yi-Hsing; Huang, Chih-Yang; Hsu, Tsai-Ching

2017-09-01

Systemic lupus erythematosus (SLE) is an autoimmune disease that is characterised by a dysregulation of the immune system, which causes inflammation responses, excessive oxidative stress and a reduction in the number of cluster of differentiation (CD)4+CD25+forkhead box P3 (FoxP3)+ T cells. Supplementation with certain Lactobacillus strains has been suggested to be beneficial in the comprehensive treatment of SLE. However, little is known about the effect and mechanism of certain Lactobacillus strains on SLE. To investigate the effects of Lactobacillus on SLE, NZB/W F1 mice were orally gavaged with Lactobacillus paracasei GMNL-32 (GMNL-32), Lactobacillus reuteri GMNL-89 (GMNL-89) and L. reuteri GMNL-263 (GMNL-263). Supplementation with GMNL-32, GMNL-89 and GMNL-263 significantly increased antioxidant activity, reduced IL-6 and TNF-α levels and significantly decreased the toll-like receptors/myeloid differentiation primary response gene 88 signalling in NZB/W F1 mice. Notably, supplementation with GMNL-263, but not GMNL-32 and GMNL-89, in NZB/W F1 mice significantly increased the differentiation of CD4+CD25+FoxP3+ T cells. These findings reveal beneficial effects of GMNL-32, GMNL-89 and GMNL-263 on NZB/W F1 mice and suggest that these specific Lactobacillus strains can be used as part of a comprehensive treatment of SLE patients.

Comparative Approach to Define Increased Regulatory T Cells in Different Cancer Subtypes by Combined Assessment of CD127 and FOXP3

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Marc Beyer

2011-01-01

Full Text Available In recent years an increase of functional CD4+CD25+ regulatory T cells (Treg cells has been established for patients with solid tumors, acute leukemias, and lymphomas. We have reported an expanded pool of CD4+CD25high Treg cells in patients with chronic lymphatic leukemia (CLL, multiple myeloma (MM as well as its premalignant precursor monoclonal gammopathy of undetermined significance (MGUS. In healthy individuals, low-level expression of CD127 on T cells in addition to the expression of FOXP3 has been associated with Treg cells. Here, we demonstrate that the expanded FOXP3+ T-cell population in patients with colorectal cancer, CLL, MGUS, MM, follicular lymphoma, and Hodgkin's disease are exclusively CD127low Treg cells and were strongly suppressive. A significant portion of CD127lowFOXP3+ Treg cells expressed only low levels of CD25 suggesting that the previously reported expansion of CD25+ Treg cells underestimates the true expansion. The assessment of CCR7 and CD45RA expression on the expanded CD4+CD127lowFOXP3+ Treg cells revealed an increase of both naïve as well as central and effector memory Treg cells in peripheral blood. Our data strongly support superiority of combined CD127 and FOXP3 analysis in comparison to CD25 and FOXP3 assessment for further quantification of Treg cells in malignant diseases.

SPARC, FOXP3, CD8 and CD45 correlation with disease recurrence and long-term disease-free survival in colorectal cancer.

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Angela Chew

Full Text Available BACKGROUND: SPARC is a matricellular protein involved in tissue remodelling, cell migration and angiogenesis, while forkhead box P3 (FOXP3 protein functions as a transcription factor involved in immune cell regulation. Both SPARC and FOXP3 can play an anti-tumorigenic role in cancer progression. The aim was to determine if SPARC, FOXP3, CD8 and CD45RO expression levels are associated with colorectal cancer (CRC stage, disease outcome and long-term cancer-specific survival (CSS in stage II and III CRC. METHODS AND FINDINGS: SPARC expression was initially assessed in 120 paired normal and stage I-IV CRCs. Subsequently, approximately 1000 paired patient samples of stage II or III CRCs in tissue microarrays were stained for SPARC, FOXP3, CD8 or CD45RO. Proportional hazards modelling assessed correlations between these markers and clinicopathological data, including disease outcome and cancer specific survival (CSS. Both SPARC and FOXP3 expression were significantly greater in CRC than normal colon (p<0.0001. High SPARC expression correlated with good disease outcome (≥60 mths without disease recurrence, p = 0.0039 and better long-term CSS in stage II CRC (<0.0001. In stage III CRC, high SPARC expression correlated with better long-term CSS (p<0.0001 and less adjuvant chemotherapy use (p = 0.01. High FOXP3 correlated with a good disease outcome, better long-term CSS and less adjuvant chemotherapy use in stage II (p<0.0037, <0.0001 and p = 0.04 respectively, but not in stage III CRC. High CD8 and CD45RO expression correlated with better disease outcome in stage II CRC, and better CSS, but the differences were not as marked as for SPARC and FOXP3. CONCLUSIONS: These data suggest that high SPARC and FOXP3 are associated with better disease outcome in stage II CRC and may be prognostic indicators of CSS. Further assessment of whether these markers predict patients at high risk of recurrence with stage II CRC and functional studies of these

Andrographolide inhibits adipogenesis of 3T3-L1 cells by suppressing C/EBPβ expression and activation

International Nuclear Information System (INIS)

Chen, Ching-Chu; Chuang, Wei-Ting; Lin, Ai-Hsuan; Tsai, Chia-Wen; Huang, Chin-Shiu; Chen, Yun-Ting; Chen, Haw-Wen; Lii, Chong-Kuei

2016-01-01

�� Andrographolide inhibits adipogenesis of 3 T3-L1 adipocytes. • Andrographolide suppresses differentiation cocktail-induced C/EBPβ expression. • Andrographolide attenuates ERK and GSK3β-dependent C/EBPβ activation. • Andrographolide arrests 3 T3-L1 adipocytes at G0/G1 phase.

Andrographolide inhibits adipogenesis of 3T3-L1 cells by suppressing C/EBPβ expression and activation

Energy Technology Data Exchange (ETDEWEB)

Chen, Ching-Chu [Division of Endocrinology and Metabolism, Department of Medicine, China Medical University Hospital, Taichung, Taiwan (China); Division of Endocrinology and Metabolism, Department of Chinese Medicine, China Medical University, China Medical University, Taichung, Taiwan (China); Chuang, Wei-Ting; Lin, Ai-Hsuan; Tsai, Chia-Wen [Department of Nutrition, China Medical University, Taichung, Taiwan (China); Huang, Chin-Shiu [Department of Health and Nutrition Biotechnology, Asia University, Taichung, Taiwan (China); Department of Medical Research, China Medical University Hospital, Taichung, Taiwan (China); Chen, Yun-Ting [Department of Nutrition, China Medical University, Taichung, Taiwan (China); Chen, Haw-Wen, E-mail: chenhw@mail.cmu.edu.tw [Department of Nutrition, China Medical University, Taichung, Taiwan (China); Lii, Chong-Kuei, E-mail: cklii@mail.cmu.edu.tw [Department of Nutrition, China Medical University, Taichung, Taiwan (China); Department of Health and Nutrition Biotechnology, Asia University, Taichung, Taiwan (China)

2016-09-15

�� Andrographolide inhibits adipogenesis of 3 T3-L1 adipocytes. • Andrographolide suppresses differentiation cocktail-induced C/EBPβ expression. • Andrographolide attenuates ERK and GSK3β-dependent C/EBPβ activation. • Andrographolide arrests 3 T3-L1 adipocytes at G0/G1 phase.

mTOR Inhibition Induces EGFR Feedback Activation in Association with Its Resistance to Human Pancreatic Cancer

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Feng Wei

2015-02-01

Full Text Available The mammalian target of rapamycin (mTOR is dysregulated in diverse cancers and contributes to tumor progression and drug resistance. The first generation of mTOR inhibitors have failed to show clinical efficiency in treating pancreatic cancers due in part to the feedback relief of the insulin-like growth factor-1 receptor (IGF-1R-AKT signaling pathway. The second generation of mTOR inhibitors, such as AZD8055, could inhibit AKT activation upon mTOR complex 2 (mTORC2 inhibition. However, whether this generation of mTOR inhibitors can obtain satisfactory activities in pancreatic cancer therapy remains unclear. In this study, we found AZD8055 did not show great improvement compared with everolimus, AZD8055 induced a temporal inhibition of AKT kinase activities and AKT was then rephosphorylated. Additionally, we found that AZD8055-induced transient AKT inhibition increased the expression and activation of epidermal growth factor receptor (EGFR by releasing its transcriptional factors Fork-head box O 1/3a (FoxO1/3a, which might contribute to cell resistance to AZD8055. The in vitro and in vivo experiments further indicated the combination of AZD8055 and erlotinib synergistically inhibited the mTORC1/C2 signaling pathway, EGFR/AKT feedback activation, and cell growth, as well as suppressed the progression of pancreatic cancer in a xenograft model. This study provides a rationale and strategy for overcoming AZD8055 resistance by a combined treatment with the EGFR inhibitor erlotinib in pancreatic cancer therapy.

Belinostat-induced apoptosis and growth inhibition in pancreatic cancer cells involve activation of TAK1-AMPK signaling axis

International Nuclear Information System (INIS)

Wang, Bing; Wang, Xin-bao; Chen, Li-yu; Huang, Ling; Dong, Rui-zen

2013-01-01

Highlights: •Belinostat activates AMPK in cultured pancreatic cancer cells. •Activation of AMPK is important for belinostat-induced cytotoxic effects. •ROS and TAK1 are involved in belinostat-induced AMPK activation. •AMPK activation mediates mTOR inhibition by belinostat. -- Abstract: Pancreatic cancer accounts for more than 250,000 deaths worldwide each year. Recent studies have shown that belinostat, a novel pan histone deacetylases inhibitor (HDACi) induces apoptosis and growth inhibition in pancreatic cancer cells. However, the underlying mechanisms are not fully understood. In the current study, we found that AMP-activated protein kinase (AMPK) activation was required for belinostat-induced apoptosis and anti-proliferation in PANC-1 pancreatic cancer cells. A significant AMPK activation was induced by belinostat in PANC-1 cells. Inhibition of AMPK by RNAi knockdown or dominant negative (DN) mutation significantly inhibited belinostat-induced apoptosis in PANC-1 cells. Reversely, AMPK activator AICAR and A-769662 exerted strong cytotoxicity in PANC-1 cells. Belinostat promoted reactive oxygen species (ROS) production in PANC-1 cells, increased ROS induced transforming growth factor-β-activating kinase 1 (TAK1)/AMPK association to activate AMPK. Meanwhile, anti-oxidants N-Acetyl-Cysteine (NAC) and MnTBAP as well as TAK1 shRNA knockdown suppressed belinostat-induced AMPK activation and PANC-1 cell apoptosis. In conclusion, we propose that belinostat-induced apoptosis and growth inhibition require the activation of ROS-TAK1-AMPK signaling axis in cultured pancreatic cancer cells

Belinostat-induced apoptosis and growth inhibition in pancreatic cancer cells involve activation of TAK1-AMPK signaling axis

Energy Technology Data Exchange (ETDEWEB)

Wang, Bing, E-mail: wangbin69@yahoo.com; Wang, Xin-bao; Chen, Li-yu; Huang, Ling; Dong, Rui-zen

2013-07-19

Highlights: •Belinostat activates AMPK in cultured pancreatic cancer cells. •Activation of AMPK is important for belinostat-induced cytotoxic effects. •ROS and TAK1 are involved in belinostat-induced AMPK activation. •AMPK activation mediates mTOR inhibition by belinostat. -- Abstract: Pancreatic cancer accounts for more than 250,000 deaths worldwide each year. Recent studies have shown that belinostat, a novel pan histone deacetylases inhibitor (HDACi) induces apoptosis and growth inhibition in pancreatic cancer cells. However, the underlying mechanisms are not fully understood. In the current study, we found that AMP-activated protein kinase (AMPK) activation was required for belinostat-induced apoptosis and anti-proliferation in PANC-1 pancreatic cancer cells. A significant AMPK activation was induced by belinostat in PANC-1 cells. Inhibition of AMPK by RNAi knockdown or dominant negative (DN) mutation significantly inhibited belinostat-induced apoptosis in PANC-1 cells. Reversely, AMPK activator AICAR and A-769662 exerted strong cytotoxicity in PANC-1 cells. Belinostat promoted reactive oxygen species (ROS) production in PANC-1 cells, increased ROS induced transforming growth factor-β-activating kinase 1 (TAK1)/AMPK association to activate AMPK. Meanwhile, anti-oxidants N-Acetyl-Cysteine (NAC) and MnTBAP as well as TAK1 shRNA knockdown suppressed belinostat-induced AMPK activation and PANC-1 cell apoptosis. In conclusion, we propose that belinostat-induced apoptosis and growth inhibition require the activation of ROS-TAK1-AMPK signaling axis in cultured pancreatic cancer cells.

Inhibition of Allograft Inflammatory Factor-1 in Dendritic Cells Restrains CD4+ T Cell Effector Responses and Induces CD25+Foxp3+ T Regulatory Subsets

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Diana M. Elizondo

2017-11-01

Full Text Available Allograft inflammatory factor-1 (AIF1 is a cytoplasmic scaffold protein shown to influence immune responses in macrophages and microglial cells. The protein contains Ca2+ binding EF-hand and PDZ interaction domains important for mediating intracellular signaling complexes. This study now reports that AIF1 is expressed in CD11c+ dendritic cells (DC and silencing of expression restrains induction of antigen-specific CD4+ T cell effector responses. AIF1 knockdown in murine DC resulted in impaired T cell proliferation and skewed polarization away from T helper type 1 and 17 fates. In turn, there was a parallel expansion of IL-10-producing and CD25+Foxp3+ T regulatory subsets. These studies are the first to demonstrate that AIF1 expression in DC serves as a potent governor of cognate T cell responses and presents a novel target for engineering tolerogenic DC-based immunotherapies.

Histamine H3 receptor activation selectively inhibits dopamine D1 receptor-dependent [3H]GABA release from depolarization-stimulated slices of rat substantia nigra pars reticulata

International Nuclear Information System (INIS)

Aceves, J.; Young, J.M.; Arias-Montano, J.A.; Floran, B.; Garcia, M.

1997-01-01

The release of [ 3 H]GABA from slices of rat substantia nigra pars reticulata induced by increasing extracellular K + from 6 to 15 mM in the presence of 10 μM sulpiride was inhibited by 73±3% by 1 μM SCH 23390, consistent with a large component of release dependent upon D 1 receptor activation. The histamine H 3 receptor-selective agonist immepip (1 μM) and the non-selective agonist histamine (100 μM) inhibited [ 3 H]GABA release by 78±2 and 80±2%, respectively. The inhibition by both agonists was reversed by the H 3 receptor antagonist thioperamide (1 μM). However, in the presence of 1 μM SCH 23390 depolarization-induced release of [ 3 H]GABA was not significantly decreased by 1 μM immepip. In rats depleted of dopamine by pretreatment with reserpine, immepip no longer inhibited control release of [ 3 H]GABA, but in the presence of 1 μM SKF 38393, which produced a 7±1-fold stimulation of release, immepip reduced the release to a level not statistically different from that in the presence of immepip alone. Immepip (1 μM) also inhibited the depolarization-induced release of [ 3 H]dopamine from substantia nigra pars reticulata slices, by 38±3%.The evidence is consistent with the proposition that activation of histamine H 3 receptors leads to the selective inhibition of the component of depolarization-induced [ 3 H]GABA release in substantia nigra pars reticulata slices which is dependent upon D 1 receptor activation. This appears to be largely an action at the terminals of the striatonigral GABA projection neurons, which may be enhanced by a partial inhibition of dendritic [ 3 H]dopamine release. (Copyright (c) 1997 Elsevier Science B.V., Amsterdam. All rights reserved.)

Inhibition of glycogen synthase kinase-3β attenuates glucocorticoid-induced suppression of myogenic differentiation in vitro.

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Zhenyu Ma

Full Text Available Glucocorticoids are the only therapy that has been demonstrated to alter the progress of Duchenne muscular dystrophy (DMD, the most common muscular dystrophy in children. However, glucocorticoids disturb skeletal muscle metabolism and hamper myogenesis and muscle regeneration. The mechanisms involved in the glucocorticoid-mediated suppression of myogenic differentiation are not fully understood. Glycogen synthase kinase-3β (GSK-3β is considered to play a central role as a negative regulator in myogenic differentiation. Here, we showed that glucocorticoid treatment during the first 48 h in differentiation medium decreased the level of phosphorylated Ser9-GSK-3β, an inactive form of GSK-3β, suggesting that glucocorticoids affect GSK-3β activity. We then investigated whether GSK-3β inhibition could regulate glucocorticoid-mediated suppression of myogenic differentiation in vitro. Two methods were employed to inhibit GSK-3β: pharmacological inhibition with LiCl and GSK-3β gene knockdown. We found that both methods resulted in enhanced myotube formation and increased levels of muscle regulatory factors and muscle-specific protein expression. Importantly, GSK-3β inhibition attenuated glucocorticoid-induced suppression of myogenic differentiation. Collectively, these data suggest the involvement of GSK-3β in the glucocorticoid-mediated impairment of myogenic differentiation. Therefore, the inhibition of GSK-3β may be a strategy for preventing glucocorticoid-induced muscle degeneration.

Ctla-4 modulates the differentiation of inducible Foxp3+ Treg cells but IL-10 mediates their function in experimental autoimmune encephalomyelitis.

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Johan Verhagen

Full Text Available In vitro induced Foxp3+ T regulatory (iTreg cells form a novel and promising target for therapeutic tolerance induction. However, the potential of these cells as a target for the treatment of various immune diseases, as well as the factors involved in their development and function, remain debated. Here, we demonstrate in a myelin basic protein (MBP-specific murine model of CNS autoimmune disease that adoptive transfer of antigen-specific iTreg cells ameliorates disease progression. Moreover, we show that the co-stimulatory molecule CTLA-4 mediates in vitro differentiation of iTreg cells. Finally, we demonstrate that the secreted, immunosuppressive cytokine IL-10 controls the ability of antigen-specific iTreg cells to suppress autoimmune disease. Overall, we conclude that antigen-specific iTreg cells, which depend on various immune regulatory molecules for their differentiation and function, represent a major target for effective immunotherapy of autoimmune disease.

TGF-β2 inhibits AKT activation and FGF-2-induced corneal endothelial cell proliferation

International Nuclear Information System (INIS)

Lu Jiawei; Lu Zhenyu; Reinach, Peter

2006-01-01

The corneal endothelial cells form a boundary layer between anterior chamber and cornea. This single cell layer is important to maintain cornea transparency by eliciting net fluid transport into the anterior chamber. Injuries of the corneal endothelial layer in humans lead to corneal swelling and translucence. This hindrance is thought to be due to limited proliferative capacity of the endothelial layer. Fibroblast growth factor 2 (FGF-2) and transforming growth factor-beta 2 (TGF-β2) are both found in aqueous humor, and these two cytokines promote and inhibit cell growth, respectively. The intracellular signaling mechanisms by which TGF-β2 suppresses the mitogenic response to FGF-2, however, remain unclear. We have addressed this question by investigating potential crosstalk between FGF-2-induced and TGF-β2-regulated intracellular signaling events in cultured bovine corneal endothelial (BCE) cells. We found that TGF-β2 and FGF-2 oppositely affect BCE cell proliferation and TGF-β2 can override the stimulating effects of FGF-2 by increasing COX-2 expression in these cells. Consistent with these findings, overexpression of COX-2 significantly reduced FGF-2-induced cell proliferation whereas a COX-2 specific inhibitor NS398 reversed the effect of TGF-β2 on FGF-2-induced cell proliferation. The COX-2 product prostaglandin E2 (PGE-2) blocks FGF-2-induced cell proliferation. Whereas FGF-2 stimulates cell proliferation by activating the AKT pathway, TGF-β2 and PGE-2 both inhibit this pathway. In accordance with the effect of PGE-2, cAMP also inhibits FGF-2-induced AKT activation. These findings suggest that the mitogenic response to FGF-2 in vivo in the corneal endothelial layer may be inhibited by TGF-β2-induced suppression of the PI3-kinase/AKT signaling pathway

ERK inhibition sensitizes CZ415-induced anti-osteosarcoma activity in vitro and in vivo.

Science.gov (United States)

Yin, Gang; Fan, Jin; Zhou, Wei; Ding, Qingfeng; Zhang, Jun; Wu, Xuan; Tang, Pengyu; Zhou, Hao; Wan, Bowen; Yin, Guoyong

2017-10-10

mTOR is a valuable oncotarget for osteosarcoma. The anti-osteosarcoma activity by a novel mTOR kinase inhibitor, CZ415, was evaluated. We demonstrated that CZ415 potently inhibited survival and proliferation of known osteosarcoma cell lines (U2OS, MG-63 and SaOs2), and primary human osteosarcoma cells. Further, CZ415 provoked apoptosis and disrupted cell cycle progression in osteosarcoma cells. CZ415 treatment in osteosarcoma cells concurrently blocked mTORC1 and mTORC2 activation. Intriguingly, ERK-MAPK activation could be a major resistance factor of CZ415. ERK inhibition (by MEK162/U0126) or knockdown (by targeted ERK1/2 shRNAs) dramatically sensitized CZ415-induced osteosarcoma cell apoptosis. In vivo , CZ415 oral administration efficiently inhibited U2OS tumor growth in mice. Its activity was further potentiated with co-administration of MEK162. Collectively, we demonstrate that ERK inhibition sensitizes CZ415-induced anti-osteosarcoma activity in vitro and in vivo . CZ415 could be further tested as a promising anti-osteosarcoma agent, alone or in combination of ERK inhibition.

Prognostic and predictive role of FOXP3 positive tumor infiltrating lymphocytes (TILs in curatively resected non small cell lung cancer other than stage IA

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Fatih Kose

2017-12-01

Full Text Available Lung cancer is the leading cause of cancer-related mortality and responsible for 1.6 million deaths per year through world-wide. Surgical resection with negative margin combined with the adjuvant therapy [except for stage IA and IB (<4 cm] is the Standard treatment for early-stage Non-small cell lung cancer (NSCLC. Early-stage NSCLC, however, has relapse rate over 40% mostly at distant sites. Therefore, high relapse rate necessitates urgent novel biomarker for these patients. In this study, we aim to evaluate the predictive and prognostic role of FOXP3+ Treg cells along with well defined Clinicohistopathological factors in early-stage non-small cell lung cancer (NSCLC. FOXP3 expression in tumor infiltrating lymphocytes (TIL was examined by immunohistochemical staining from resected early-stage 48 NSCLC patients. Data of patients and FOXP3 expression status along with common clinicohistopathological prognostic factors were evaluated retrospectively. Median age of patients was 62 years-old (range 43–78. Mean follow-up, median overall survival (OS, and disease-free survival (DFS were 49, 49 and 30 months, respectively. FOXP3 expression was positive in 23 (47.9% patients. Adjuvant chemotherapy (4 cycles of cisplatin-vinorelbine was given to 16 patients (33.3% at physician discretion. Patients with a FOXP3 expression of 25% or higher significantly lower OS and DFS when compared with patients with a FOXP3 staining lower than 25% with p-value of 0.016 and 0.032, respectively. In the patients with high FOXP3 expression, platin-based adjuvant chemotherapy had showed a detrimental effect on DFS and OS. These results suggest that FOXP3 expression may be used as useful prognostic biomarker in resected NSCLC. Our findings also suggest that resected NSCLC patients with FOXP3 expression of 25% or higher staining intensity may not get any benefit even disfavor from adjuvant platin chemotherapy.

H32, a non-quinone sulfone analog of vitamin K3, inhibits human hepatoma cell growth by inhibiting Cdc25 and activating ERK.

Science.gov (United States)

Kar, Siddhartha; Wang, Meifang; Ham, Seung Wook; Carr, Brian I

2006-10-01

We previously synthesized a K-vitamin derivative, Cpd 5, which was a potent growth inhibitor of human tumor cells, including Hep3B hepatoma cells. However, being a quinone compound, Cpd 5 has the potential for generating toxic reactive oxygen species (ROS). We therefore synthesized a nonquinone sulfone derivative, H32, which has a sufone group substituting the quinone. The IC50 of H32 for Hep3B cells was found to be 2.5 microM, which was 2.5 and 3.2 times more potent than Cpd 5 and vitamin K3 respectively. It induced apoptosis in Hep3B cells but did not generate ROS when compared to Cpd 5. Interestingly, under similar culture conditions, normal rat hepatocytes were 14-fold more and 7-fold more resistant to the growth inhibitory effects of H32 than Hep3B and PLC/PRF5 cells respectively. H32 preferentially inhibited the activities of the cell cycle controlling Cdc25A phosphatase likely by binding to its catalytic cysteine. As a consequence, it induced inhibitory tyrosine phosphorylation of the Cdc25 substrate kinases Cdk2 and Cdk4 in Hep3B cells and the cells undergo an arrest in the G1 phase of the cell cycle. H32 also induced persistent phosphorylation of the MAPK protein ERK1/2, but marginal JNK1/2 and p38 phosphorylation. The ERK inhibitor U0126, added at least 30 min prior to H32, antagonized the growth inhibition induced by H32. However, the JNK and p38 inhibitors, JNKI-II and SB203580, were not able to antagonize H32 induced growth inhibition. Thus, H32 differentially inhibited growth of normal and liver tumor cells by preferentially inhibiting the actions of Cdc25 phosphatases and inducing persistent ERK phosphorylation.

Lycopene inhibits PDGF-BB-induced retinal pigment epithelial cell migration by suppression of PI3K/Akt and MAPK pathways

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Chan, Chi-Ming [School of Medicine, Fu Jen Catholic University, Taipei Hsien, Taiwan, ROC (China); Department of Ophthalmology, Cardinal Tien Hospital, Taipei Hsien, Taiwan, ROC (China); Fang, Jia-You [Pharmaceutics Laboratory, Graduate Institute of Natural Products, Chang Gung University, Kweishan, Taoyuan, Taiwan, ROC (China); Lin, Hsin-Huang [School of Medicine, Fu Jen Catholic University, Taipei Hsien, Taiwan, ROC (China); Yang, Chi-Yea [Department of Biotechnology, Vanung University, Taoyuan, Taiwan, ROC (China); Hung, Chi-Feng, E-mail: 054317@mail.fju.edu.tw [School of Medicine, Fu Jen Catholic University, Taipei Hsien, Taiwan, ROC (China)

2009-10-09

Retinal pigment epithelial (RPE) cells play a dominant role in the development of proliferative vitreoretinopathy (PVR), which is the leading cause of failure in retinal reattachment surgery. Several studies have shown that platelet-derived growth factor (PDGF) exhibits chemotaxis and proliferation effects on RPE cells in PVR. In this study, the inhibitory effect of lycopene on PDGF-BB-induced ARPE19 cell migration is examined. In electric cell-substrate impedance sensing (ECIS) and Transwell migration assays, significant suppression of PDGF-BB-induced ARPE19 cell migration by lycopene is observed. Cell viability assays show no cytotoxicity of lycopene on RPE cells. Lycopene shows no effect on ARPE19 cell adhesion and is found to inhibit PDGF-BB-induced tyrosine phosphorylation and the underlying signaling pathways of PI3K, Akt, ERK and p38 activation. However, PDGF-BB and lycopene show no effects on JNK activation. Taken together, our results demonstrate that lycopene inhibits PDGF-BB-induced ARPE19 cell migration through inhibition of PI3K/Akt, ERK and p38 activation.

Lycopene inhibits PDGF-BB-induced retinal pigment epithelial cell migration by suppression of PI3K/Akt and MAPK pathways

International Nuclear Information System (INIS)

Chan, Chi-Ming; Fang, Jia-You; Lin, Hsin-Huang; Yang, Chi-Yea; Hung, Chi-Feng

2009-01-01

Retinal pigment epithelial (RPE) cells play a dominant role in the development of proliferative vitreoretinopathy (PVR), which is the leading cause of failure in retinal reattachment surgery. Several studies have shown that platelet-derived growth factor (PDGF) exhibits chemotaxis and proliferation effects on RPE cells in PVR. In this study, the inhibitory effect of lycopene on PDGF-BB-induced ARPE19 cell migration is examined. In electric cell-substrate impedance sensing (ECIS) and Transwell migration assays, significant suppression of PDGF-BB-induced ARPE19 cell migration by lycopene is observed. Cell viability assays show no cytotoxicity of lycopene on RPE cells. Lycopene shows no effect on ARPE19 cell adhesion and is found to inhibit PDGF-BB-induced tyrosine phosphorylation and the underlying signaling pathways of PI3K, Akt, ERK and p38 activation. However, PDGF-BB and lycopene show no effects on JNK activation. Taken together, our results demonstrate that lycopene inhibits PDGF-BB-induced ARPE19 cell migration through inhibition of PI3K/Akt, ERK and p38 activation.

[Changes of CD(4)(+)Foxp3(+) regulatory T cells and CD(4)(+)IL-17(+)T cells in cigarette smoke-exposed rats].

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Meng, Jing-jing; Zhong, Xiao-ning; Bai, Jing; He, Zhi-yi; Zhang, Jian-quan; Huang, Qiu-pin

2012-01-01

To evaluate the changes of CD(4)(+)IL-17(+) T (Th17) and CD(4)(+)Foxp3(+) regulatory T (Treg) cells in peripheral blood and bronchoalveolar lavage fluid (BALF), and therefore to explore the role of Th17 and Treg in cigarette smoke-induced airway inflammation/COPD in rats. Forty male Wistar rats were randomly divided into 4 groups: a 12 wk smoke-exposure group, a 24 wk smoke-exposure group, a 12 wk control group and a 24 wk control group (n = 10 each). Cells in BALF were collected and analyzed by absolute and differential cell counts. IL-17 and IL-6 levels in serum and BALF were tested by enzyme linked immunosorbent assay (ELISA). The proportion of CD(4)(+)IL-17(+) T and CD(4)(+)Foxp3(+) Treg in peripheral blood and BALF were determined by flow cytometry. The mRNA expressions of IL-17 and Foxp3 were measured by real-time PCR. Comparisons of the data between different groups were performed using one-way ANOVA, and SNK and Games-Howell test were used for comparison between 2 groups. Levels of IL-17 were remarkable increased in the 12 wk smoke-exposure group and the 24 wk smoke-exposure group in serum [(52.6 ± 1.8) ng/L, (75.4 ± 6.0) ng/L] and BALF [(78.1 ± 5.8) ng/L, (95.0 ± 6.8) ng/L] compared with the 12 wk control group [(40.0 ± 3.2)ng/L, (54.5 ± 4.6) ng/L] and the 24 wk control group [(36.7 ± 3.2) ng/L, (53.9 ± 3.7) ng/L], all P cells and macrophages (r = 0.512, 0.543, all P cells and an increase of inflammatory cytokines were evident in airway inflammation of cigarette smoke-exposed rats, suggesting that Treg was involved in the immunological regulation and Th17 was associated with the persistent inflammation in cigarette smoke-induced airway inflammation in rats.

Silencing of Foxp3 enhances the antitumor efficacy of GM-CSF genetically modified tumor cell vaccine against B16 melanoma

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Miguel A

2017-01-01

Full Text Available Antonio Miguel,1 Luis Sendra,1 Verónica Noé,2 Carles J Ciudad,2 Francisco Dasí,3,4 David Hervas,5 María José Herrero,1,6 Salvador F Aliño17 1Department of Pharmacology, Faculty of Medicine, University of Valencia, 2Department of Biochemistry and Molecular Biology, Faculty of Pharmacy, University of Barcelona, 3Research University Hospital of Valencia, INCLIVA Health Research Institute, 4Department of Physiology, Faculty of Medicine, University of Valencia Foundation, 5Biostatistics Unit, 6Pharmacogenetics Unit, Instituto de Investigación Sanitaria La Fe (IIS La Fe, 7Clinical Pharmacology Unit, ACM Hospital Universitario y Politécnico La Fe, Valencia, Spain Abstract: The antitumor response after therapeutic vaccination has a limited effect and seems to be related to the presence of T regulatory cells (Treg, which express the immunoregulatory molecules CTLA4 and Foxp3. The blockage of CTLA4 using antibodies has shown an effective antitumor response conducing to the approval of the human anti-CTLA4 antibody ipilimumab by the US Food and Drug Administration. On the other hand, Foxp3 is crucial for Treg development. For this reason, it is an attractive target for cancer treatment. This study aims to evaluate whether combining therapeutic vaccination with CTLA4 or Foxp3 gene silencing enhances the antitumor response. First, the “in vitro” cell entrance and gene silencing efficacy of two tools, 2'-O-methyl phosphorotioate-modified oligonucleotides (2'-OMe-PS-ASOs and polypurine reverse Hoogsteen hairpins (PPRHs, were evaluated in EL4 cells and cultured primary lymphocytes. Following B16 tumor transplant, C57BL6 mice were vaccinated with irradiated B16 tumor cells engineered to produce granulocyte-macrophage colony-stimulating factor (GM-CSF and were intraperitoneally treated with CTLA4 and Foxp3 2'-OMe-PS-ASO before and after vaccination. Tumor growth, mice survival, and CTLA4 and Foxp3 expression in blood cells were measured. The following

Ripk3 induces mitochondrial apoptosis via inhibition of FUNDC1 mitophagy in cardiac IR injury

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Hao Zhou

2017-10-01

Full Text Available Ripk3-required necroptosis and mitochondria-mediated apoptosis are the predominant types of cell death that largely account for the development of cardiac ischemia reperfusion injury (IRI. Here, we explored the effect of Ripk3 on mitochondrial apoptosis. Compared with wild-type mice, the infarcted area in Ripk3-deficient (Ripk3-/- mice had a relatively low abundance of apoptotic cells. Moreover, the loss of Ripk3 protected the mitochondria against IRI and inhibited caspase9 apoptotic pathways. These protective effects of Ripk3 deficiency were relied on mitophagy activation. However, inhibition of mitophagy under Ripk3 deficiency enhanced cardiomyocyte and endothelia apoptosis, augmented infarcted area and induced microvascular dysfunction. Furthermore, ischemia activated mitophagy by modifying FUNDC1 dephosphorylation, which substantively engulfed mitochondria debris and cytochrome-c, thus blocking apoptosis signal. However, reperfusion injury elevated the expression of Ripk3 which disrupted FUNDC1 activation and abated mitophagy, increasing the likelihood of apoptosis. In summary, this study confirms the promotive effect of Ripk3 on mitochondria-mediated apoptosis via inhibition of FUNDC1-dependent mitophagy in cardiac IRI. These findings provide new insight into the roles of Ripk3-related necroptosis, mitochondria-mediated apoptosis and FUNDC1-required mitophagy in cardiac IRI.

Inhibition of interleukin-3- and interferon- α-induced JAK/STAT signaling by the synthetic α-X-2',3,4,4'-tetramethoxychalcones α-Br-TMC and α-CF3-TMC.

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Jobst, Belinda; Weigl, Julia; Michl, Carina; Vivarelli, Fabio; Pinz, Sophia; Amslinger, Sabine; Rascle, Anne

2016-11-01

The JAK/STAT pathway is an essential mediator of cytokine signaling, often upregulated in human diseases and therefore recognized as a relevant therapeutic target. We previously identified the synthetic chalcone α-bromo-2',3,4,4'-tetramethoxychalcone (α-Br-TMC) as a novel JAK2/STAT5 inhibitor. We also found that treatment with α-Br-TMC resulted in a downward shift of STAT5 proteins in SDS-PAGE, suggesting a post-translational modification that might affect STAT5 function. In the present study, we show that a single cysteine within STAT5 is responsible for the α-Br-TMC-induced protein shift, and that this modification does not alter STAT5 transcriptional activity. We also compared the inhibitory activity of α-Br-TMC to that of another synthetic chalcone, α-trifluoromethyl-2',3,4,4'-tetramethoxychalcone (α-CF3-TMC). We found that, like α-Br-TMC, α-CF3-TMC inhibits JAK2 and STAT5 phosphorylation in response to interleukin-3, however without altering STAT5 mobility in SDS-PAGE. Moreover, we demonstrate that both α-Br-TMC and α-CF3-TMC inhibit interferon-α-induced activation of STAT1 and STAT2, by inhibiting their phosphorylation and the expression of downstream interferon-stimulated genes. Together with the previous finding that α-Br-TMC and α-CF3-TMC inhibit the response to inflammation by inducing Nrf2 and blocking NF-κB activities, our data suggest that synthetic chalcones might be useful as anti-inflammatory, anti-cancer and immunomodulatory agents in the treatment of human diseases.

Regulatory T Cell Induced by Poria cocos Bark Exert Therapeutic Effects in Murine Models of Atopic Dermatitis and Food Allergy

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Min-Jung Bae

2016-01-01

Full Text Available The prevalence of allergic disorders including atopic dermatitis (AD and food allergy (FA has increased dramatically in pediatric populations, but there is no effective drug available for their management. Therefore, trials are required for the development of safe therapeutic agents such as herbal medicines. We determined whether orally administered Poria cocos bark (PCB extract could exert immunosuppressive effects on allergic and inflammatory symptoms of AD and FA. For both AD, which was induced using house dust mite extract, and FA, which was induced by exposure to ovalbumin, model mice were orally treated with PCB extract for 62 days and 18 days, respectively. We also investigated the inductive effect of PCB extract on the generation and maintenance of Foxp3+CD4+ regulatory T cells (Tregs. The symptoms of AD and FA were ameliorated by the administration of PCB extract. Furthermore, PCB extract inhibited the Th2-related cytokines and increased the population of Foxp3+CD4+ Tregs in both AD and FA models. In ex vivo experiments, PCB extract promoted the functional differentiation of Foxp3+CD4+ Tregs, which is dependent on aryl hydrocarbon receptor activation. Thus, PCB extract has potential as an oral immune suppressor for the treatment of AD and FA through the generation of Tregs.

Technetium-99 conjugated with methylene diphosphonate inhibits receptor activator of nuclear factor-κB ligand-induced osteoclastogenesis.

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Gong, Wei; Dou, Huan; Liu, Xianqin; Sun, Lingyun; Hou, Yayi

2012-10-01

1. In the present study, we investigated the effects of technetium-99 conjugated with methylene diphosphonate ((99)Tc-MDP), an agent used in radionuclide therapy, on receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis and explored the underlying mechanisms. 2. The murine macrophage cell line RAW264.7 and bone marrow-derived-macrophages from C57BL/6 mice (BMM) were used as models for osteoclastogenesis in vitro. The expression of some key factors in RANKL (50 ng/mL)-induced osteoclastogenesis in RAW264.7 cells was investigated by flow cytometry and real-time reverse transcription-polymerase chain reaction (RT-PCR). To detect multinucleated osteoclast formation, RAW264.7 cells were induced with RANKL for 4 days, whereas BMM were induced by 50 ng/mL RANKL and 20 ng/mL macrophage colony-stimulating factor for 7 days, before being stained with tartrate-resistant acid phosphatase. 3. Osteoclastogenesis was evaluated using the osteoclast markers CD51, matrix metalloproteinase (MMP)-9 and cathepsin K. At 0.01 μg/mL, (99)Tc-MDP significantly inhibited RANKL-induced osteoclastogenesis without any cytotoxicity. In addition, (99)Tc-MDP abolished the appearance of multinucleated osteoclasts. 4. Real-time RT-PCR analysis of transcription factor expression revealed that (99)Tc-MDP inhibited the expression of c-Fos and nuclear factor of activated T cells. In addition, (99)Tc-MDP inhibited the expression of the inflammatory factors interleukin (IL)-6, tumour necrosis factor-α and IL-1β. Finally, (99)Tc-MDP inhibited the activation of mitogen-activated protein kinases in RAW264.7 cells following RANKL stimulation. 5. In conclusion, (99)Tc-MDP possesses anti-osteoclastogenic activity against RANKL-induced osteoclast formation. © 2012 The Authors Clinical and Experimental Pharmacology and Physiology © 2012 Wiley Publishing Asia Pty Ltd.

Effect of everolimus initiation and early calcineurin inhibitor withdrawal on myocardial FOXP3+ regulatory T cells in heart transplantation

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Mirza, Kiran; Gustafsson, Finn; Gullestad, Lars

2016-01-01

BACKGROUND: Through immunosuppression CD4+FoxP3+ regulatory T-cells (Tregs) play an indispensable role in allograft rejection. Post-HTx treatment with everolimus is associated with slower progression of cardiac allograft vasculopathy (CAV) - chronic rejection - than CNI based therapy. We hypothes......BACKGROUND: Through immunosuppression CD4+FoxP3+ regulatory T-cells (Tregs) play an indispensable role in allograft rejection. Post-HTx treatment with everolimus is associated with slower progression of cardiac allograft vasculopathy (CAV) - chronic rejection - than CNI based therapy. We...... hypothesized treatment with everolimus reduced the risk of CAV by modulating myocardial FoxP3 levels. METHODS: 15 patients from the Schedule trial comparing everolimus, MMF, steroid and early CNI (Everolimus, n=8) withdrawal to conventional CNI based immunosuppression (Controls, n=7) after de novo HTx were...

Galectin-3 silencing inhibits epirubicin-induced ATP binding cassette transporters and activates the mitochondrial apoptosis pathway via β-catenin/GSK-3β modulation in colorectal carcinoma.

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Yung-Kuo Lee

Full Text Available Multidrug resistance (MDR, an unfavorable factor compromising the treatment efficacy of anticancer drugs, involves the upregulation of ATP binding cassette (ABC transporters and induction of galectin-3 signaling. Galectin-3 plays an anti-apoptotic role in many cancer cells and regulates various pathways to activate MDR. Thus, the inhibition of galectin-3 has the potential to enhance the efficacy of the anticancer drug epirubicin. In this study, we examined the effects and mechanisms of silencing galectin-3 via RNA interference (RNAi on the β-catenin/GSK-3β pathway in human colon adenocarcinoma Caco-2 cells. Galectin-3 knockdown increased the intracellular accumulation of epirubicin in Caco-2 cells; suppressed the mRNA expression of galectin-3, β-catenin, cyclin D1, c-myc, P-glycoprotein (P-gp, MDR-associated protein (MRP 1, and MRP2; and downregulated the protein expression of P-gp, cyclin D1, galectin-3, β-catenin, c-Myc, and Bcl-2. Moreover, galectin-3 RNAi treatment significantly increased the mRNA level of GSK-3β, Bax, caspase-3, and caspase-9; remarkably increased the Bax-to-Bcl-2 ratio; and upregulated the GSK-3β and Bax protein expressions. Apoptosis was induced by galectin-3 RNAi and/or epirubicin as demonstrated by chromatin condensation, a higher sub-G1 phase proportion, and increased caspase-3 and caspase-9 activity, indicating an intrinsic/mitochondrial apoptosis pathway. Epirubicin-mediated resistance was effectively inhibited via galectin-3 RNAi treatment. However, these phenomena could be rescued after galectin-3 overexpression. We show for the first time that the silencing of galectin-3 sensitizes MDR cells to epirubicin by inhibiting ABC transporters and activating the mitochondrial pathway of apoptosis through modulation of the β-catenin/GSK-3β pathway in human colon cancer cells.

Docosahexaenoic Acid Induces Cell Death in Human Non-Small Cell Lung Cancer Cells by Repressing mTOR via AMPK Activation and PI3K/Akt Inhibition

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Nayeong Kim

2015-01-01

Full Text Available The anticancer properties and mechanism of action of omega-3 polyunsaturated fatty acids (ω3-PUFAs have been demonstrated in several cancers; however, the mechanism in lung cancer remains unclear. Here, we show that docosahexaenoic acid (DHA, a ω3-PUFA, induced apoptosis and autophagy in non-small cell lung cancer (NSCLC cells. DHA-induced cell death was accompanied by AMP-activated protein kinase (AMPK activation and inactivated phosphatidylinositol 3-kinase (PI3K/Akt/mammalian target of rapamycin (mTOR signaling. Knocking down AMPK and overexpressing Akt increased mTOR activity and attenuated DHA-induced cell death, suggesting that DHA induces cell death via AMPK- and Akt-regulated mTOR inactivation. This was confirmed in Fat-1 transgenic mice, which produce ω3-PUFAs. Lewis lung cancer (LLC tumor cells implanted into Fat-1 mice showed slower growth, lower phospho-Akt levels, and higher levels of apoptosis and autophagy than cells implanted into wild-type mice. Taken together, these data suggest that DHA-induced apoptosis and autophagy in NSCLC cells are associated with AMPK activation and PI3K/Akt inhibition, which in turn lead to suppression of mTOR; thus ω3-PUFAs may be utilized as potential therapeutic agents for NSCLC treatment.

Effector and naturally occurring regulatory T cells display no abnormalities in activation induced cell death in NOD mice.

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Ayelet Kaminitz

Full Text Available BACKGROUND: Disturbed peripheral negative regulation might contribute to evolution of autoimmune insulitis in type 1 diabetes. This study evaluates the sensitivity of naïve/effector (Teff and regulatory T cells (Treg to activation-induced cell death mediated by Fas cross-linking in NOD and wild-type mice. PRINCIPAL FINDINGS: Both effector (CD25(-, FoxP3(- and suppressor (CD25(+, FoxP3(+ CD4(+ T cells are negatively regulated by Fas cross-linking in mixed splenocyte populations of NOD, wild type mice and FoxP3-GFP trangeneess. Proliferation rates and sensitivity to Fas cross-linking are dissociated in Treg cells: fast cycling induced by IL-2 and CD3/CD28 stimulation improve Treg resistance to Fas-ligand (FasL in both strains. The effector and suppressor CD4(+ subsets display balanced sensitivity to negative regulation under baseline conditions, IL-2 and CD3/CD28 stimulation, indicating that stimulation does not perturb immune homeostasis in NOD mice. Effective autocrine apoptosis of diabetogenic cells was evident from delayed onset and reduced incidence of adoptive disease transfer into NOD.SCID by CD4(+CD25(- T cells decorated with FasL protein. Treg resistant to Fas-mediated apoptosis retain suppressive activity in vitro. The only detectable differential response was reduced Teff proliferation and upregulation of CD25 following CD3-activation in NOD mice. CONCLUSION: These data document negative regulation of effector and suppressor cells by Fas cross-linking and dissociation between sensitivity to apoptosis and proliferation in stimulated Treg. There is no evidence that perturbed AICD in NOD mice initiates or promotes autoimmune insulitis.

Foxp3+ Regulatory T Cells Delay Expulsion of Intestinal Nematodes by Suppression of IL-9-Driven Mast Cell Activation in BALB/c but Not in C57BL/6 Mice

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Brenz, Yannick; Eschbach, Marie-Luise; Hartmann, Wiebke; Haben, Irma; Sparwasser, Tim; Huehn, Jochen; Kühl, Anja; Feyerabend, Thorsten B.; Rodewald, Hans-Reimer; Breloer, Minka

2014-01-01

Accumulating evidence suggests that IL-9-mediated immunity plays a fundamental role in control of intestinal nematode infection. Here we report a different impact of Foxp3+ regulatory T cells (Treg) in nematode-induced evasion of IL-9-mediated immunity in BALB/c and C57BL/6 mice. Infection with Strongyloides ratti induced Treg expansion with similar kinetics and phenotype in both strains. Strikingly, Treg depletion reduced parasite burden selectively in BALB/c but not in C57BL/6 mice. Treg function was apparent in both strains as Treg depletion increased nematode-specific humoral and cellular Th2 response in BALB/c and C57BL/6 mice to the same extent. Improved resistance in Treg-depleted BALB/c mice was accompanied by increased production of IL-9 and accelerated degranulation of mast cells. In contrast, IL-9 production was not significantly elevated and kinetics of mast cell degranulation were unaffected by Treg depletion in C57BL/6 mice. By in vivo neutralization, we demonstrate that increased IL-9 production during the first days of infection caused accelerated mast cell degranulation and rapid expulsion of S. ratti adults from the small intestine of Treg-depleted BALB/c mice. In genetically mast cell-deficient (Cpa3-Cre) BALB/c mice, Treg depletion still resulted in increased IL-9 production but resistance to S. ratti infection was lost, suggesting that IL-9-driven mast cell activation mediated accelerated expulsion of S. ratti in Treg-depleted BALB/c mice. This IL-9-driven mast cell degranulation is a central mechanism of S. ratti expulsion in both, BALB/c and C57BL/6 mice, because IL-9 injection reduced and IL-9 neutralization increased parasite burden in the presence of Treg in both strains. Therefore our results suggest that Foxp3+ Treg suppress sufficient IL-9 production for subsequent mast cell degranulation during S. ratti infection in a non-redundant manner in BALB/c mice, whereas additional regulatory pathways are functional in Treg-depleted C57BL/6

Indirubin-3-Oxime Prevents H2O2-Induced Neuronal Apoptosis via Concurrently Inhibiting GSK3β and the ERK Pathway.

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Yu, Jie; Zheng, Jiacheng; Lin, Jiajia; Jin, Linlu; Yu, Rui; Mak, Shinghung; Hu, Shengquan; Sun, Hongya; Wu, Xiang; Zhang, Zaijun; Lee, Mingyuen; Tsim, Wahkeung; Su, Wei; Zhou, Wenhua; Cui, Wei; Han, Yifan; Wang, Qinwen

2017-05-01

Oxidative stress-induced neuronal apoptosis plays an important role in many neurodegenerative disorders. In this study, we have shown that indirubin-3-oxime, a derivative of indirubin originally designed for leukemia therapy, could prevent hydrogen peroxide (H 2 O 2 )-induced apoptosis in both SH-SY5Y cells and primary cerebellar granule neurons. H 2 O 2 exposure led to the increased activities of glycogen synthase kinase 3β (GSK3β) and extracellular signal-regulated kinase (ERK) in SH-SY5Y cells. Indirubin-3-oxime treatment significantly reversed the altered activity of both the PI3-K/Akt/GSK3β cascade and the ERK pathway induced by H 2 O 2 . In addition, both GSK3β and mitogen-activated protein kinase inhibitors significantly prevented H 2 O 2 -induced neuronal apoptosis. Moreover, specific inhibitors of the phosphoinositide 3-kinase (PI3-K) abolished the neuroprotective effects of indirubin-3-oxime against H 2 O 2 -induced neuronal apoptosis. These results strongly suggest that indirubin-3-oxime prevents H 2 O 2 -induced apoptosis via concurrent inhibiting GSK3β and the ERK pathway in SH-SY5Y cells, providing support for the use of indirubin-3-oxime to treat neurodegenerative disorders caused or exacerbated by oxidative stress.

Activation of Plant Innate Immunity by Extracellular High Mobility Group Box 3 and Its Inhibition by Salicylic Acid.

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Hyong Woo Choi

2016-03-01

Full Text Available Damage-associated molecular pattern molecules (DAMPs signal the presence of tissue damage to induce immune responses in plants and animals. Here, we report that High Mobility Group Box 3 (HMGB3 is a novel plant DAMP. Extracellular HMGB3, through receptor-like kinases BAK1 and BKK1, induced hallmark innate immune responses, including i MAPK activation, ii defense-related gene expression, iii callose deposition, and iv enhanced resistance to Botrytis cinerea. Infection by necrotrophic B. cinerea released HMGB3 into the extracellular space (apoplast. Silencing HMGBs enhanced susceptibility to B. cinerea, while HMGB3 injection into apoplast restored resistance. Like its human counterpart, HMGB3 binds salicylic acid (SA, which results in inhibition of its DAMP activity. An SA-binding site mutant of HMGB3 retained its DAMP activity, which was no longer inhibited by SA, consistent with its reduced SA-binding activity. These results provide cross-kingdom evidence that HMGB proteins function as DAMPs and that SA is their conserved inhibitor.

Lactococcus lactis KR-050L inhibit IL-6/STAT3 activation.

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Hwang, J T; Jang, H-J; Kim, J H; Park, C S; Kim, Y; Lim, C-H; Lee, S W; Rho, M-C

2017-05-01

The purpose of this study was to investigate IL-6/STAT3 inhibitory activity using lactic acid bacteria (LABs) isolated from Gajuknamu kimchi. Six LABs were isolated from Gajuknamu kimchi and identified through 16S rRNA sequencing. Among them, the culture broth of Lactococcus lactis KR-050L inhibited IL-6-induced STAT3 luciferase activity. Fifteen compounds were isolated from the EtOAc extract of culture broth though column chromatography and preparative high-performance liquid chromatography, and they were identified as 2,5-diketopipperazine structures by spectroscopic analyses (MS, 1 H- and 13 C-NMR). They also showed inhibitory activities on IL-6-induced STAT3 activation, and showed the different in activity according to the presence of a phenylalanine residue, hydroxyl groups and isometric structure. The six new LABs isolated from Gajuknamu kimchi, and Lc. lactis KR-050L was selected as candidate IL-6/STAT3 inhibitors. The activity levels of 15 2,5-DKPs isolated from Lc. lactis KR-050L were verified. This study constitutes the first attempt to isolate various LABs from Gajuknamu kimchi and to discover IL-6/STAT3 inhibitors in the EtOAc extract of Lc. lactis KR-050L culture broth. Moreover, our data provide useful biochemical information regarding the commercialization of Lc. lactis isolated from Gajuknamu kimchi as an approach to use functional foods for the treatment of various diseases via IL-6/STAT3 activation. © 2017 The Society for Applied Microbiology.

Effect of Lactobacillus salivarius on Th1/Th2 cytokines and the number of spleen CD4⁺ CD25⁺ Foxp3⁺ Treg in asthma Balb/c mouse.

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Yun, Xiang; Shang, Yunxiao; Li, Miao

2015-01-01

Bronchial asthma is a chronic airway inflammatory disease that involves T lymphocytes. In order to explore the effect of Lactobacillus salivarius on Th1/Th2 cytokines and the number of spleen CD4(+) CD25(+) Foxp3(+) Treg in asthma Balb/c mouse, we constructed acute asthma model with ovalbumin to observe the mouse behavior change in Balb/c mice. The expression of GATA-3 mRNA and T-bet mRNA was measured by real-time PCR. The proportion of CD4(+) CD25(+) Foxp3(+) Treg/CD4(+) was determined by flow cytometry. The results demonstrated that oral gavage with Lactobacillus salivarius before sensitization could alleviate the clinical symptoms, airway hyper-reactivity and airway inflammation in asthma mouse to some extent; Lactobacillus salivarius may improve the imbalance of Th1/Th2 in asthma mouse through increasing the expression of T-bet mRNA at the transcriptional level and inhibiting the expression of GATA-3 mRNA simultaneously. CD4(+) CD25(+) Foxp3(+) Treg cells may be involved in the pathogenesis of bronchial asthma, and may be the upstream regulatory mechanism of the improvement of Th1/Th2 imbalance by Lactobacillus salivarius.

Prospective investigation of FOXP1 syndrome.

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Siper, Paige M; De Rubeis, Silvia; Trelles, Maria Del Pilar; Durkin, Allison; Di Marino, Daniele; Muratet, François; Frank, Yitzchak; Lozano, Reymundo; Eichler, Evan E; Kelly, Morgan; Beighley, Jennifer; Gerdts, Jennifer; Wallace, Arianne S; Mefford, Heather C; Bernier, Raphael A; Kolevzon, Alexander; Buxbaum, Joseph D

2017-01-01

Haploinsufficiency of the forkhead-box protein P1 ( FOXP1 ) gene leads to a neurodevelopmental disorder termed FOXP1 syndrome. Previous studies in individuals carrying FOXP1 mutations and deletions have described the presence of autism spectrum disorder (ASD) traits, intellectual disability, language impairment, and psychiatric features. The goal of the present study was to comprehensively characterize the genetic and clinical spectrum of FOXP1 syndrome. This is the first study to prospectively examine the genotype-phenotype relationship in multiple individuals with FOXP1 syndrome, using a battery of standardized clinical assessments. Genetic and clinical data was obtained and analyzed from nine children and adolescents between the ages of 5-17 with mutations in FOXP1 . Phenotypic characterization included gold standard ASD testing and norm-referenced measures of cognition, adaptive behavior, language, motor, and visual-motor integration skills. In addition, psychiatric, medical, neurological, and dysmorphology examinations were completed by a multidisciplinary team of clinicians. A comprehensive review of reported cases was also performed. All missense and in-frame mutations were mapped onto the three-dimensional structure of DNA-bound FOXP1. We have identified nine de novo mutations, including three frameshift, one nonsense, one mutation in an essential splice site resulting in frameshift and insertion of a premature stop codon, three missense, and one in-frame deletion. Reviewing prior literature, we found seven instances of recurrent mutations and another 34 private mutations. The majority of pathogenic missense and in-frame mutations, including all four missense mutations in our cohort, lie in the DNA-binding domain. Through structural analyses, we show that the mutations perturb amino acids necessary for binding to the DNA or interfere with the domain swapping that mediates FOXP1 dimerization. Individuals with FOXP1 syndrome presented with delays in early