Expression Of c-Maf Research Articles

The Transcription Factor c-Maf Promotes the Differentiation of Follicular Helper T Cells.

Follicular helper T cells (Tfh) have been identified as the primary cell subpopulation regulating B cell responses in germinal centers, thus supporting high-affinity antibody production. Among the transcription factors orchestrating Tfh cell differentiation and function, the role played by the proto-oncogene c-Maf remains poorly characterized. We report herein that selective loss of c-Maf expression in the T cell compartment results in defective development of Tfh cells in response to both antigen/adjuvant vaccinations and commensal intestinal bacteria. Accordingly, c-Maf expression in T cells was essential for the development and high-affinity antibody secretion in vaccinated animals. c-Maf was expressed early, concomitantly to BCL6, in Tfh cell precursors and found to regulate Tfh fate in a cell-autonomous fashion. Altogether, our findings reveal a novel, non-redundant, function for c-Maf in the differentiation of Tfh cells and the regulation of humoral immune responses to T-cell-dependent antigens.

Characterization of c-Maf+Foxp3\u2212 Regulatory T Cells Induced by Repeated Stimulation of Antigen-Presenting B Cells

The role of B cells in the development of CD4+ regulatory T cells has been emphasized recently. Our previous studies have demonstrated that the antigen-presenting splenic B cells converted naïve CD4+CD25− T cells into CD4+CD25+Foxp3− T cells without additional cytokines or chemicals with regulatory activity and that referred to as Treg-of-B cells. The present study further showed that Treg-of-B cells increased the IL-10-producing population, and the expression of c-Maf, inducible T-cell co-stimulator (ICOS) as well as cytotoxic T-lymphocyte-associated protein 4 (CTLA4) after repeated stimulation of B cells in a cell-cell contact-dependent manner. Long-term cultured Treg-of-B cells exerted IL-10 and CTLA4-mediated antigen-specific suppressive activity; moreover, the single antigen-specific Treg-of-B cells inhibited in a non-antigen-specific fashion. In conclusion, these results suggest that repeated stimulation of B cells induced IL-10-producing CD4+Foxp3− regulatory T cells in a contact-dependent manner and these Treg-of-B cells possess IL-10 and CTLA4-dependent suppressive function.

Protein kinase C theta is required for efficient induction of IL-10-secreting T cells.

Secretion of interleukin-10 (IL-10) by CD4+ T cells is an essential immunoregulatory mechanism. The work presented here assesses the role of the signaling molecule protein kinase C theta (PKCθ) in the induction of IL-10 expression in CD4+ T cells. Using wildtype and PKCθ-deficient Tg4 T cell receptor transgenic mice, we implemented a well-described protocol of repeated doses of myelin basic protein (MBP)Ac1-9[4Y] antigen to induce Tr1-like IL-10+ T cells. We find that PKCθ is required for the efficient induction of IL-10 following antigen administration. Both serum concentrations of IL-10 and the proportion of IL-10+ T cells were reduced in PKCθ-deficient mice relative to wildtype mice following [4Y] treatment. We further characterized the T cells of [4Y] treated PKCθ-deficient Tg4 mice and found reduced expression of the transcription factors cMaf, Nfil3 and FoxP3 and the surface receptors PD-1 and Tim3, all of which have been associated with the differentiation or function of IL-10+ T cells. Finally, we demonstrated that, unlike [4Y] treated wildtype Tg4 T cells, cells from PKCθ-deficient mice were unable to suppress the priming of naïve T cells in vitro and in vivo. In summary, we present data demonstrating a role for PKCθ in the induction of suppressive, IL-10-secreting T cells induced in TCR-transgenic mice following chronic antigen administration. This should be considered when contemplating PKCθ as a suitable drug target for inducing immune suppression and graft tolerance.

Prohibitin 1 suppresses liver cancer tumorigenesis in mice and human hepatocellular and cholangiocarcinoma cells.

We have identified that PHB1, down-regulated in most human HCC and CCA, heterodimerizes with MAX to repress the E-box and positively regulates MAT1A while suppressing c-MYC, MAFG, and c-MAF expression; in mice, reduced PHB1 expression predisposes to the development of cholestasis-induced CCA. (Hepatology 2017;65:1249-1266).

Changes of circulating Tfr and Tfh cells in children with myasthenia gravis

Objective To investigate the changes of follicular regulatory T cells (Tfr cells) and follicular T helper cells (Tfh cells) in peripheral blood of children with myasthenia gravis (MG). Methods We recruited 28 MG patients and 20 healthy subjects in this study. The percentages of Tfh and Tfr cells in peripheral blood samples were measured by flow cytometry. Real-time PCR was performed to detect the expression of transcription factors and regulatory factors of Bcl-6, c-MAF, Blimp-1 and PD-1 at mRNA level. ELISA was used to detect the levels of IL-2, IL-6, IL-10 and IL-21 in plasma samples and the titers of Ach-Rab and PsMab. Results Compared with the healthy subjects, the MG patients showed higher percentages of Tfh cells and lower percentages of Tfr cells before receiving treatment. The expression of Bcl-6 and c-MAF on CD4+ T lymphocytes cells at transcriptional level were significantly enhanced, while the expression of Blimp-1 on CD4+ T cells and the expression of PD-1 on Treg cells at transcriptional level were inhibited in the MG patients in comparison with those in healthy subjects. Moreover, decreased levels of IL-2 and increased levels of IL-21 were found in plasma samples collected from the MG patients. Conclusion The decreased percentages of Tfr cells and increased percentages of Tfh cells in patients with MG resulted in abnormal ratios of Tfr/Tfh cells, which might be involved in the immunological pathogenesis of MG. Several changes in the patients with MG might be responsible for the imbalanced ratio of Tfr/Tfh cells, which included changes of IL-2 and IL-21 in microenvironment, enhanced expression of Bcl-6 and c-MAF at mRNA level and inhibited expression of Blimp-1 at mRNA level on CD4+ T cells as well as over-expression of PD-1 at mRNA level on Treg cells. Key words: Myasthenia gravis; Tfr cells; Tfh cells

Differences in frequency and regulation of T follicular helper cells between newly diagnosed and chronic pediatric immune thrombocytopenia

ObjectiveThis study aims to investigate the role of T follicular helper (TFH) cells in the immunopathogenesis of pediatric immune thrombocytopenia (ITP), as well as differences in TFH expansion and its regulation between newly diagnosed ITP (nITP) and chronic pediatric ITP (cITP). MethodsEighty-five children with ITP and 20 age-matched healthy controls were enrolled into this study. TFH cell frequencies and TFH cell-associated regulatory factors before and after treatment were analyzed by flow cytometry, RT-PCR and ELISA. ResultsThe percentages of TFH cells were significantly elevated in both nITP and cITP compared with controls. RT-PCR revealed significant differences in Bcl-6, c-Maf, Blimp-1, ICOSL, TACI and BAFFR mRNA expression in CD4+ T or CD19+ B cells between patients and controls, and further between nITP and cITP, before and after treatment. Moreover, there were significant differences in serum IL-4, IL-21 and BAFF between patients and controls. ConclusionThe overactivation of TFH cells may contribute to the immunopathogenesis of pediatric ITP. IL-21 and IL-4 serum levels may affect the differentiation of TFH cells in ITP patients. The aberrant balance between BAFFR-BAFF/TACI-BAFF may be a factor that caused the persistent high expression of ICOSL in pediatric cITP, which consequently lead to the over activation of TFH cells in pediatric cITP.

T follicular helper cells in patients with acute schistosomiasis.

BackgroundThe role of T follicular helper (Tfh) cells in schistosome infection is not fully defined. In a previous study, a higher frequency of circulating PD-1+CXCR5+CD4+ Tfh cells was observed in patients with chronic schistosomiasis relative to healthy controls (HCs) and it correlated positively with the level of soluble egg antigen (SEA) specific antibodies in serum. However, the function of Tfh cells in patients with acute schistosomiasis remains elusive; this was investigated in the present study.MethodsThe frequency of circulating Tfh cells and the expression of inducible T cell co-stimulator (ICOS), programmed cell death 1 (PD-1) and B cell subsets were analyzed in 12 patients with acute schistosomiasis and 10 HCs by flow cytometry. The expression of Bcl6, c-Maf and IL-21 mRNA were detected by quantitative real-time reverse transcriptase PCR (qRT-PCR). The concentration of serum IL-21 and IgG specific to Schistosoma japonicum antigen were then determined by enzyme linked immunosorbent assay (ELISA). Correlations between PD-1+CXCR5+CD4+ Tfh cells, memory B cells and IgG specific to S. japonicum were analyzed by Spearman’s rank correlation.ResultsThe frequency of PD-1+CXCR5+CD4+ Tfh and memory B cells was increased in acute schistosomiasis patients relative to HCs. Moreover, the levels of IL-21 in serum and the expression of IL-21 mRNA were higher in acute schistosomiasis patients. However, there was no significant correlation between PD-1+CXCR5+CD4+ Tfh cells, memory B cells and IgG specific to S. japonicum antigen in patients with acute schistosomiasis.ConclusionsPD-1+CXCR5+CD4+ Tfh cells in peripheral blood are involved in the immune response of patients with acute schistosomiasis. Understanding the immunological mechanism is helpful for the development of vaccination strategies to control schistosomiasis.

ICOS+CD38+ cTfh in peripheral blood and thoracic duct lymph.

Abstract T follicular helper cells provide essential B cell help within the germinal center (GC), and these cells have been shown to populate the follicular mantel once the GC reaction is complete. The significance and ontology of circulating Tfh (cTfh) is less clear. Studies in humans have suggested subsets of cTfh that correlate with humoral responses in chronic infection (HIV, malaria) and in response to acute challenge (influenza vaccination). We have previously described a subset of CXCR5+PD1+ cTfh that expresses both ICOS and CD38. This population was significantly increased 7 days after influenza vaccination as well as in patients infected with HIV. Single cell transcriptional analysis of the ICOS+CD38+ cTfh after influenza vaccination demonstrated more frequent expression of the GC Tfh transcripts CMAF, BCL6, and CD200 than was found in the ICOS−CD38− cTfh, and bulk transcriptional analysis indicated higher expression of IL-21. These data suggested that ICOS and CD38 mark the rare population of cTfh that are more recent émigrés from the lymph nodes. To evaluate this possibility, we analyzed Tfh in an immune compartment that is more proximal to the lymph nodes than peripheral blood: thoracic duct lymph (TDL). As expected, TDL Tfh had a higher frequency of ICOS+CD38+ cells than cTfh in matched peripheral blood. In addition, TDL had a population of CXCR5+PD1bright Tfh, previously only identified in lymphoid tissues. CXCR5+PD1bright Tfh in the TDL had a significantly increased frequency of ICOS+CD38+ cells. These data provide a critical missing link in the understanding of cTfh and suggest that the ICOS+CD38+ cTfh subset warrants further exploration as peripheral surrogates of the active immune responses within secondary lymphoid tissues.

Deregulated methionine adenosyltransferase α1, c-Myc, and Maf proteins together promote cholangiocarcinoma growth in mice and humans(‡).

c-Myc induction drives cholestatic liver injury and cholangiocarcinoma (CCA) in mice, and induction of Maf proteins (MafG and c-Maf) contributes to cholestatic liver injury, whereas S-adenosylmethionine (SAMe) administration is protective. Here, we determined whether there is interplay between c-Myc, Maf proteins, and methionine adenosyltransferase α1 (MATα1), which is responsible for SAMe biosynthesis in the liver. We used bile duct ligation (BDL) and lithocholic acid (LCA) treatment in mice as chronic cholestasis models, a murine CCA model, human CCA cell lines KMCH and Huh-28, human liver cancer HepG2, and human CCA specimens to study gene and protein expression, protein-protein interactions, molecular mechanisms, and functional outcomes. We found that c-Myc, MATα1 (encoded by MAT1A), MafG, and c-Maf interact with one another directly. MAT1A expression fell in hepatocytes and bile duct epithelial cells during chronic cholestasis and in murine and human CCA. The opposite occurred with c-Myc, MafG, and c-Maf expression. MATα1 interacts mainly with Mnt in normal liver, but this switches to c-Maf, MafG, and c-Myc in cholestatic livers and CCA. Promoter regions of these genes have E-boxes that are bound by MATα1 and Mnt in normal liver and benign bile duct epithelial cells that switched to c-Myc, c-Maf, and MafG in cholestasis and CCA cells. E-box positively regulates c-Myc, MafG, and c-Maf, but it negatively regulates MAT1A. MATα1 represses, whereas c-Myc, MafG, and c-Maf enhance, E-box-driven promoter activity. Knocking down MAT1A or overexpressing MafG or c-Maf enhanced CCA growth and invasion in vivo. There is a novel interplay between MATα1, c-Myc, and Maf proteins, and their deregulation during chronic cholestasis may facilitate CCA oncogenesis. (Hepatology 2016;64:439-455).

Differential expression patterns of MafB and c-Maf in macrophages in vivo and in vitro

The large Maf transcription factors c-Maf and MafB are expressed in macrophage-lineage hematopoietic cells, but the expression patterns of MafB and c-Maf in macrophage subtypes and tissue-resident macrophages have not been fully analyzed. First, we analyzed MafB and c-Maf protein expression in tissue-resident macrophages. Mouse lymph nodes, spleens, lungs, and kidneys were subjected to immunohistochemistry using anti-MafB and anti-c-Maf. Both MafB and c-Maf signals were observed in lymph node macrophages. In the splenic macrophages the MafB signal was detected by anti-MafB, but the c-Maf signal was not detected. No expression of c-Maf or MafB was detected in macrophages in the lung and kidney. Flow cytometry analysis revealed a similar pattern of GFP expression in Mafb/GFP knock-in heterozygous mice. To analyze these different expression patterns in greater detail, we examined the expression of MafB and c-Maf by quantitative RT-PCR in different cytokine- or LPS-induced macrophages in vitro. MafB expression was induced by IL-10 or IL-4 with IL-13 and was reduced by LPS or GM-CSF. By contrast, c-Maf expression was induced by IL-10 and reduced by IL-4 with IL-13 or GM-CSF. These results indicate that MafB and c-Maf have different expression patterns in macrophages, suggesting differences in function.

No change in mRNA expression of immune-related genes in peripheral blood mononuclear cells challenged with Theileria annulata in Murrah buffalo (Bubalus bubalis)

Water buffaloes (Bubalus bubalis) act as carrier to Theileria annulata and show less clinical sign of tropical theileriosis as compared to indigenous and exotic cattle. Differential expression of immune-related genes such as major histocompatibility complex, class II, DQ alpha 1 (MHC-DQα), signal-regulatory protein alpha (SIRPA), prion protein (PRNP), Toll-like receptor 10 (TLR10), c-musculoaponeurotic fibrosarcoma oncogene homolog (cMAF) and V-maf avian musculoaponeurotic fibrosarcoma oncogene homolog B (MAFB) genes influence host resistance to this disease in exotic, crossbred and indigenous cattle. In the present study we examined the differential mRNA expression of the abovesaid immune-related genes in response to T. annulata infection in buffaloes. Peripheral blood mononuclear cells (PBMCs) harvested from blood samples of buffaloes were challenged with ground-up tick supernatant carrying T. annulata sporozoites in vitro. After 48h of in vitro challenge qPCR was employed to measure the relative mRNA expression of MHC-DQα, SIRPA, PRNP, TLR10, cMAF and MAFB genes in infected and control PBMCs. In the current study, the selected genes showed no change in mRNA expression after T.annulata infection which indicates that they have little role in providing host resistance to theileriosis in buffaloes.

Genetic programming for self-renewal

Gene Regulation Instead of repopulating themselves from tissue-resident stem cell pools like most types of differentiated cells, tissue macrophages maintain themselves by self-renewing. The underlying genetic programs that allow for this, however, are unknown. Soucie et al. now report that in macrophages at homeostasis, a pair of transcription factors (MafB and c-Maf) bind to and repress the enhancers of genes regulating self-renewal. When macrophages need to replenish their stocks, for example in response to injury, they transiently decrease MafB and c-Maf expression so they can self-renew. A parallel pathway also operates to control the self-renewal of embryonic stem cells. Science , this issue p. [10.1126/science.aad5510][1] [1]: /lookup/doi/10.1126/science.aad5510

Lineage-specific enhancers activate self-renewal genes in macrophages and embryonic stem cells.

Differentiated macrophages can self-renew in tissues and expand long term in culture, but the gene regulatory mechanisms that accomplish self-renewal in the differentiated state have remained unknown. Here we show that in mice, the transcription factors MafB and c-Maf repress a macrophage-specific enhancer repertoire associated with a gene network that controls self-renewal. Single-cell analysis revealed that, in vivo, proliferating resident macrophages can access this network by transient down-regulation of Maf transcription factors. The network also controls embryonic stem cell self-renewal but is associated with distinct embryonic stem cell-specific enhancers. This indicates that distinct lineage-specific enhancer platforms regulate a shared network of genes that control self-renewal potential in both stem and mature cells.

Identification of Biomarkers Associated with MAF-Mediated Resistance to Proteasome Inhibitors in t(14;16) Multiple Myeloma

Introduction : Multiple myeloma (MM) patients with t(14;16)(p16;q32) leading to high expression of C-MAF mRNA and protein have a poor prognosis and have not benefitted from the recent advances in MM therapy. Our previous work showed that posttranslational modification up-regulated C-MAF (ASH Abstract # 281, ASH 2013) and MAF-B proteins (ASH Abstract #2091, 2014) conferred innate resistance to proteasome inhibitors (PI). In order to identify the molecular mechanism underlying this resistance in high C-MAF and MAFB-expressing patients, the present study investigated the downstream targets regulated by C-MAF in MM cells and analyzed the clinical association of target genes using a C-MAF-loss and gain of function cell line model as well as primary plasma cells from MM patients.Methods : To elucidate the molecular mechanism underlying the resistance of MAF expressing cells to PIs, we silenced the C-MAF gene in 3 MM cell lines that highly expressed C-MAF mRNA and protein using lentiviral shRNA. Annexin V staining and analyzed by FACS were used to determine the effect of manipulating MAF on PI-induced apoptosis and activation of the caspase family. Gene expression prolife (GEP) was utilized to determine downstream target genes regulated by MAF. To identify the proteins associated with C-MAF, we used mass spectrometry (MS)-based quantitative proteomics with stable isotope labeling by amino acids in cell culture (SILAC).Results: Our results demonstrated that apoptotic cell numbers were higher in shMAF cells, compared to shControl cells when cultured in the presence of serial concentrations of bortezomib (BZB) or carfilzomib (CFZ), indicating that silencing MAF expression enhances PI-induced apoptosis. The Annexin V + cells numbers were also higher in shMAF compared to shControl when cells were treated with BZB or CFZ in co-culture with bone marrow stromal cells. Furthermore, knockdown of C-MAF expression augmented the PI-induced activation of the caspase family with an increase in the cleavage fragments of caspases-3, -7, -8, PARP and LaminA/C. In contrast, PI-induced apoptotic numbers were lower in XG1/MAF, a stably induced MAF overexpressing MM cell line, than in XG1/EV, the control cell line carrying an empty vector when cultured in serial concentrations of BZB and CFZ. Similarly, the caspase cleavage fragments, PARP and lamin A/C were decreased in XG1/MAF compared with XG1/EV in response to BZB and CFZ. These results suggest that MAF confers MM resistance to PI by preventing the PI-induction of apoptosis.To further identify the downstream target genes regulated by C-MAF, we analyzed the gene expression between shMAF and shControl MM cells by microarray analysis and identified more than 150 genes differentially expressed. Of them, ATP2C1, CCNE2 , CCND2, ELK4 , IGFBP3, SUPT16 and TUBA1A were significantly (p=5.62e-5) lower in shMAF than shControl cells indicating that these genes might be regulated by C-MAF. Furthermore, expression of these genes in primary CD138 plasma cells were significant higher (p<0.05) in the UAMS molecular MF subgroup than other UAMS molecular subgroups. A number of genes were also expressed significantly lower (p=5.62e-5) in shMAF cells compared to shControl cells. The clinical association of these genes was analyzed and showed that the expression of IL10Ra (p<0.05), PAXBP1PAX3 (p=0.0032), USP1 (p=0.0038), BNIP3 (p<0.05), ACSL (p=0.009) and SP3 (p=0.018) in CD138 plasma cells was associated with poor overall survival in patients from the MF subgroup indicating that these targets may play a central role in MAF regulated proliferation of MM cells.To identify the proteins associated with C-MAF, we used MS-SILAC and identified more than 20 proteins as differentially expressed between MAF positive cell lines and shMAF cell lines . Western blotting and flow cytometry analysis are ongoing to confirm the functional role of these proteins in MAF regulated MM cell growth.Conclusions : Taken together, these results suggest that MAF confers MM resistance to PI by preventing the induction of apoptosis. Global gene and protein expression profiling along with correlation with clinical data, has identified a number of relevant biologically relevant candidates. Experiments are ongoing to confirm the function of these MAF regulated candidates in order to identify potential alternate approaches for the treatment of this poor performing molecular subgroup. DisclosuresQiang:University of Arkansas for Medical Sciences: Employment. Ye:University of Arkansas for Medical Sciences: Employment. Huang:University of Arkansas for Medical Sciences: Employment. Johnson:University of Arkansas for Medical Sciences: Employment. Stein:University of Arkansas for Medical Sciences: Employment. Barlogie:University of Arkansas for Medical Sciences: Employment. Epstein:University of Arkansas for Medical Sciences: Employment. Morgan:MMRF: Honoraria; Weismann Institute: Honoraria; University of Arkansas for Medical Sciences: Employment; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; CancerNet: Honoraria; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Davies:Celgene: Consultancy; Janssen: Consultancy; Millenium: Consultancy; Onyx: Consultancy; University of Arkansas for Medical Sciences: Employment.

The AP-1 Transcription Factor JunB Promotes Multiple Myeloma (MM) Cell Proliferation, Survival and Drug Resistance in the Bone Marrow Microenvironment

MEK/ERK and NF-kB signaling pathways have been reported to play a key role in multiple myeloma (MM) survival, proliferation and drug resistance. These pathways regulate the activity of numerous transcription factors. For example, the activator protein-1 (AP-1) transcription factor has been implicated in a multitude of physiologic processes, but also tumorigenesis. However, the function of AP-1 in MM is largely unknown. Our data show a vast variety of AP-1 (c-Jun, JunB, JunD, c-Maf and c-Fos) expression levels in MM cells. Importantly, co-culture of MM cells with bone marrow stromal cells (BMSCs), i.e. isotypic primary BMSCs as well as BMSC lines KM-105 and HS-27A, rapidly and strongly induces expression of JunB, but not other AP-1 members. Previous studies have shown that JunB exerts opposite functions depending on the cellular origin and the physiopathological context. For example, it serves as a gatekeeper in acute and chronic myeloid leukemia, but as a positive regulator in Hodgkin9s lymphomas and anaplastic large cell lymphomas. The relevance of JunB activity in MM growth, survival and drug resistance is elusive. First, our data demonstrate that induction of JunB is predominantly mediated by soluble factors secreted by BMSCs rather than direct MM-BMSC contact. Indeed, using cytokine arrays, we identified IL-6 among the most potent factors that trigger JunB expression. Mechanistically, JunB upregulation occurs at both transcriptional as well as translational level. Pharmacologic inhibition was used next in order to identify upstream signaling pathways, which mediate BMSC- induced JunB upregulation in MM cells. Our data show that activation of MEK/ERK or NF-kB is required for induction of JunB expression and AP-1 transcriptional activity. To delineate the specific functional role of JunB in MM pathogenesis, we transduced MM cells with pLKO.1-JunB shRNA or pLKO.1-scrambled shRNA (SCR). After puromycin- selection, effects of JunB knockdown on MM proliferation, survival and drug resistance were analyzed by 3H-thymidine incorporation, flow cytometry and western blot. Indeed, we observed significant inhibition of proliferation in MM/ JunB shRNA (decreased to ~ 25 – 40 %, p Furthermore, 4-hydroxytamoxifen (4-OHT) treatment of MM cell lines stably transduced with pMSCV-JunB-ER-IRES-GFP but not pMSCV-IRES-GFP induced significant AP-1 luciferase activity (~ 3.3 fold, p 1000 MM patient samples of different prognostic groups were compared to samples from healthy donors using the gene set enrichment analysis (GSEA). Our results further support a key role for JunB in MM pathogenesis. In summary, our data demonstrate for the first time an important role of JunB/AP-1 in MM tumorigenesis and strongly propose it as a novel therapeutic target in MM. Disclosures No relevant conflicts of interest to declare.

Multiple functions of Maf in the regulation of cellular development and differentiation.

SummaryCellular muscular aponeurotic fibrosarcoma (c‐Maf) is a member of the large macrophage‐activating factor family. C‐Maf plays important roles in the morphogenetic processes and cellular differentiation of the lens, kidneys, liver, T cells and nervous system, and it is particularly important in pancreatic islet and erythroblastic island formation. However, the exact role of c‐Maf remains to be elucidated. In this review, we summarize the research to clarify the functions of c‐Maf in the cellular development and differentiation. The expression of c‐Maf is higher in pancreatic duct cells than in pancreatic islet cells. Therefore, we suggest that pancreatic duct cells may be converted to the functional insulin‐secreting cells by regulating c‐Maf. © 2015 National Natural Science Foundation of China. Diabetes/Metabolism Research and Reviews Published by John Wiley & Sons Ltd.

Twist1 and Twist2 Contribute to Cytokine Downregulation following Chronic NOD2 Stimulation of Human Macrophages through the Coordinated Regulation of Transcriptional Repressors and Activators.

Proper regulation of microbial-induced cytokines is critical to intestinal immune homeostasis. Acute stimulation of nucleotide-binding oligomerization domain 2 (NOD2), the Crohn's disease-associated sensor of bacterial peptidoglycan, induces cytokines. However, chronic NOD2 stimulation in macrophages decreases cytokines upon pattern recognition receptor (PRR) restimulation; cytokine attenuation to PRR stimulation is similarly observed in intestinal macrophages. The role for the transcriptional repressors Twist1 and Twist2 in regulating PRR-induced cytokine outcomes is poorly understood and has not been reported for NOD2. We found that Twist1 and Twist2 were required for optimal cytokine downregulation during acute and, particularly, chronic NOD2 stimulation of human macrophages. Consistently, Twist1 and Twist2 expression was increased after chronic NOD2 stimulation; this increased expression was IL-10 and TGF-β dependent. Although Twist1 and Twist2 did not coregulate each other's expression, they cooperated to enhance binding to cytokine promoters after chronic NOD2 stimulation. Moreover, Twist1 and Twist2 contributed to enhance expression and promoter binding of the proinflammatory inhibitor c-Maf and the transcriptional repressor Bmi1. Restoring c-Maf and Bmi1 expression in Twist-deficient macrophages restored NOD2-induced cytokine downregulation. Furthermore, with chronic NOD2 stimulation, Twist1 and Twist2 contributed to the decreased expression and cytokine promoter binding of the transcriptional activators activating transcription factor 4, C/EBPα, Runx1, and Runx2. Knockdown of these transcriptional activators in Twist-deficient macrophages restored cytokine downregulation after chronic NOD2 stimulation. Finally, NOD2 synergized with additional PRRs to increase Twist1 and Twist2 expression and Twist-dependent pathways. Therefore, after chronic NOD2 stimulation Twist1 and Twist2 coordinate the regulation of both transcriptional activators and repressors, thereby mediating optimal cytokine downregulation.

Novel role for inflammasome-dependent IL-18 in promoting Th2 responses during Leishmania infections (IRC7P.420)

Abstract Leishmaniasis is a global health disease and is categorized under neglected tropical disease by WHO. Despite tremendous amount of economic burden and morbidity and mortality associated with Leishmania infections, studies to understand these parasitic bugs are nevertheless lacking. As a result, specific targeted treatment options or vaccines to treat Leishmaniasis are still missing. Herein, we sought to understand how inflammasomes are involved in host responses during Leishmania major infection. One of the hallmarks of inflammasome is the activation of caspase-1 and ultimate release of bioactive IL-1β and IL-18. Interestingly, we found that mice lacking components of the NLRP3 inflammasome were resistant to L. major infections in comparison to their WT counterparts. Using in vitro and in vivo approaches, we showed that L. major activates NLRP3 inflammasome. We further demonstrated that NLRP3 inflammasome induced IL-18, but not IL-1β, promoted Th2 responses during L. major infection. Mechanistically, IL-18 enhanced expression of Th2 transcription factors GATA3 and cMAF in T cells. Finally, we were able to show that IL-18 neutralization in WT mice protected from L. major infections. In conclusion, our study uncovered a novel role for inflammasome-induced IL-18 in promoting Th2 responses and Leishmaniasis, which can potentially be targeted for therapeutic benefits during L. major infections.

The aryl hydrocarbon receptor/microRNA-212/132 axis in T cells regulates IL-10 production to maintain intestinal homeostasis.

Aryl hydrocarbon receptor (Ahr), a transcription factor, plays a critical role in autoimmune inflammation of the intestine. In addition, microRNAs (miRNAs), small non-coding oligonucleotides, mediate pathogenesis of inflammatory bowel diseases (IBD). However, the precise mechanism and interactions of these molecules in IBD pathogenesis have not yet been investigated. We analyzed the role of Ahr and Ahr-regulated miRNAs in colonic inflammation. Our results show that deficiency of Ahr in intestinal epithelial cells in mice exacerbated inflammation in dextran sodium sulfate-induced colitis. Deletion of Ahr in T cells attenuated colitis, which was manifested by suppressed Th17 cell infiltration into the lamina propria. Candidate miRNA analysis showed that induction of colitis elevated expression of the miR-212/132 cluster in the colon of wild-type mice, whereas in Ahr (-/-) mice, expression was clearly lower. Furthermore, miR-212/132(-/-) mice were highly resistant to colitis and had reduced levels of Th17 cells and elevated levels of IL-10-producing CD4(+) cells. In vitro analyses revealed that induction of type 1 regulatory T (Tr1) cells was significantly elevated in miR-212/132(-/-) T cells with increased c-Maf expression. Our findings emphasize the vital role of Ahr in intestinal homeostasis and suggest that inhibition of miR-212/132 represents a viable therapeutic strategy for treating colitis.

Glycogen synthase kinase-3 controls IL-10 expression in CD4(+) effector T-cell subsets through epigenetic modification of the IL-10 promoter.

The serine/threonine kinase glycogen synthase kinase-3 (GSK3) plays an important role in balancing pro- and anti-inflammatory cytokines. We have examined the role of GSK3 in production of IL-10 by subsets of CD4+ T helper cells. Treatment of naive murine CD4+ T cells with GSK3 inhibitors did not affect their production of IL-10. However, treatment of Th1 and Th2 cells with GSK3 inhibitors dramatically increased production of IL-10. GSK3 inhibition also led to upregulation of IL-10 among Th1, Th2, and Th17 subsets isolated from human blood. The encephalitogenic potential of GSK3 inhibitor treated murine Th1 cells was significantly reduced in adoptive transfer experiments by an IL-10-dependent mechanism. Analysis of the murine IL-10 promoter in response to inhibition of GSK3 in Th1 cells showed modification to a transcriptionally active state indicated by changes in histone H3 acetylation and methylation. Additionally, GSK3 inhibition increased expression of the transcription factors c-Maf, Nfil3, and GATA3, correlating with the increase in IL-10. These findings are important in the context of autoimmune disease since they show that it is possible to reprogram disease-causing cells through GSK3 inhibition.