IL-10 Locus Research Articles

PW03-013 - Behçet's disease: genotype-phenotype correlations

Although the therapy for Behcet's disease (BD) has improved since infliximab was approved for refractory retinochoroiditis therapy in Japan, the exact pathogenesis of BD remains unclear. Our recent genome-wide association study has identified the IL10 and IL23R-IL12RB2 loci as susceptibility genes for BD, in addition to HLA-A*26 and B*51. rs1495965 is located in the intergenic region between IL23R and IL12RB2 and rs1800872 is located in the promoter region of IL10. IL-10 is an anti-inflammatory cytokine that may have multiple effects in immunoregulation and inflammation. It is thought that regulatory T cell function related to IL-10 is an important factor in the disease pathogenesis.

BET bromodomain inhibition suppresses TH17-mediated pathology

Interleukin (IL) 17-producing T helper (T(H)17) cells have been selected through evolution for their ability to control fungal and bacterial infections. It is also firmly established that their aberrant generation and activation results in autoimmune conditions. Using a characterized potent and selective small molecule inhibitor, we show that the bromodomain and extra-terminal domain (BET) family of chromatin adaptors plays fundamental and selective roles in human and murine T(H)17 differentiation from naive CD4(+) T cells, as well as in the activation of previously differentiated T(H)17 cells. We provide evidence that BET controls T(H)17 differentiation in a bromodomain-dependent manner through a mechanism that includes the direct regulation of multiple effector T(H)17-associated cytokines, including IL17, IL21, and GMCSF. We also demonstrate that BET family members Brd2 and Brd4 associate with the Il17 locus in T(H)17 cells, and that this association requires bromodomains. We recapitulate the critical role of BET bromodomains in T(H)17 differentiation in vivo and show that therapeutic dosing of the BET inhibitor is efficacious in mouse models of autoimmunity. Our results identify the BET family of proteins as a fundamental link between chromatin signaling and T(H)17 biology, and support the notion of BET inhibition as a point of therapeutic intervention in autoimmune conditions.

Protein Phosphatase 2A Enables Expression of Interleukin 17 (IL-17) through Chromatin Remodeling

Protein phosphatase 2A (PP2A) is a heterotrimeric serine/threonine phosphatase involved in essential cellular functions. T cells from patients with systemic lupus erythematosus (SLE) express high levels of the catalytic subunit of PP2A (PP2Ac). A mouse overexpressing PP2Ac in T cells develops glomerulonephritis in an IL-17-dependent manner. Here, using microarray analyses, we demonstrate that increased expression of PP2Ac grants T cells the capacity to produce an array of proinflammatory effector molecules. Because IL-17 is important in the expression of glomerulonephritis, we studied the mechanism through which PP2Ac dysregulation facilitates its production. We report that PP2Ac is involved in the regulation of the Il17 locus by enhancing histone 3 acetylation through a mechanism that involves activation of interferon regulatory factor 4. Increased histone 3 acetylation of the Il17 locus is shared between T cells of PP2Ac transgenic mice and patients with SLE. We propose that, by promoting the inflammatory capacity of T cells, PP2Ac dysregulation contributes to the pathogenesis of SLE.

Cytokine Gene Polymorphisms and Alzheimer's Disease in Brazil

Background: Single-nucleotide polymorphisms in genes encoding immunological mediators can affect the biological activity of these molecules by regulating transcription, translation, or secretion, modulating the genetic risk of inflammatory damage in Alzheimer's disease (AD). Nonetheless, the Brazilian contingent is highly admixed, and few association trials performed herein with AD patients have considered genetic ancestry estimates as co-variables when investigating markers for this complex trait. Methods: We analyzed polymorphisms in 10 inflammatory genes and compared the genotype distribution across outpatients with late-onset AD and noncognitively impaired subjects from Midwest Brazil under a strict criterion, and controlling for ancestry heritage and ApoE genotype. Results: Our findings show an almost 40% lower chance of AD (p = 0.004) among homozygotes of the IL10 -1082A allele (rs1800896). Dichotomization to ApoE and mean ancestry levels did not affect protection, except among those with greater European or minor African heritage. Conclusion: The IL10 locus seems to affect the onset of AD in a context sensitive to the genetic ancestry of Brazilian older adults.

Transcription factor Oct1 (Pou2f1) and its coactivator OCA-B (Pou2af1/OBF-1) are critical for CD4 memory T cell formation and function in vivo and in vitro (P1132)

Abstract Stable epigenetic changes occurring during T cell activation are crucial for the generation of immunological memory. Our prior work indicates that a widely expressed transcription factor, Oct1, is required to generate a poised gene expression state at the Il2 locus in resting but previously activated primary CD4 T cells. A known Oct1 coactivator, OCA-B, is not expressed in naïve CD4 T cells or within 6 hr after activation, but is expressed 48 hr after T cell activation. We find that OCA-B is also required to generate a poised gene expression state at Il2. Using in vivo pathogen infection models together with Oct1 and OCA-B loss of function alleles, we find that both Oct1 and OCA-B are critical for the generation of functional CD4 memory T cells. The mechanism underlying this phenotype involves Oct1 and OCA-B recruitment of a specific histone lysine demethylase, KDM3A/Jmjd1a, to maintain chromatin at target loci in a configuration that allows target genes to be rapidly and strongly induced upon antigen re-encounter.

Stepwise Epigenetic and Phenotypic Alterations Poise CD8+ T Cells To Mediate Airway Hyperresponsiveness and Inflammation

The functional plasticity of CD8(+) T cells in an atopic environment, encompassing a spectrum from IFN-γ- to IL-13-producing cells, is pivotal in the development of allergic airway hyperresponsiveness and inflammation, and yet remains mechanistically undefined. We demonstrate that CD8(+) T cell IL-13 induction proceeded through a series of distinct IL-4/GATA3-regulated stages characterized by gene expression and epigenetic changes. In vivo, CD8(+) T cells exposed to an environment rich in IL-4 displayed epigenetic changes at the GATA3 and IL-13 promoter indicative of transcriptional activation and IL-13 production. In vitro, IL-4 triggered the stepwise molecular conversion of CD8(+) T cells from IFN-γ to IL-13 production. During the initial stage, IL-4 suppressed T-bet and induced GATA3 expression, characterized by enhanced activating histone modifications and RNA polymerase II (Pol II) recruitment to the GATA3 locus. Notably, recruitment of GATA3 and RNA Pol II to the IL-13 promoter was also detected at this initial stage. However, enhanced IL-13 transcription only occurred at a later stage after TCR stimulation, indicating that IL-4-induced GATA3 recruitment poises the IL-13 locus for TCR-mediated transcription. Thus, both in vivo and in vitro, an atopic (IL-4) environment poises CD8(+) T cells via stepwise epigenetic and phenotypic mechanisms for pathogenic conversion to IL-13 production, which is ultimately triggered via an allergen-mediated TCR stimulus.

Cutting Edge: An Inactive Chromatin Configuration at the IL-10 Locus in Human Neutrophils

To identify the molecular basis of IL-10 expression in human phagocytes, we evaluated the chromatin modification status at their IL-10 genomic locus. We analyzed posttranslational modifications of histones associated with genes that are active, repressed, or poised for transcriptional activation, including H3K4me3, H4Ac, H3K27Ac, and H3K4me1 marks. Differently from autologous IL-10-producing monocytes, none of the marks under evaluation was detected at the IL-10 locus of resting or activated neutrophils from healthy subjects or melanoma patients. By contrast, increased H3K4me3, H4Ac, H3K4me1, and H3K27Ac levels were detected at syntenic regions of the IL-10 locus in mouse neutrophils. Altogether, data demonstrate that human neutrophils, differently from either monocytes or mouse neutrophils, cannot switch on the IL-10 gene because its locus is in an inactive state, likely reflecting a neutrophil-specific developmental outcome. Implicitly, data also definitively settle a currently unsolved issue on the capacity of human neutrophils to produce IL-10.

Helios Induces Epigenetic Silencing of Il2 Gene Expression in Regulatory T Cells

Regulatory T cells (Tregs) play a critical role in maintaining immune tolerance and preventing autoimmune disease. Tregs express the transcription factor Foxp3, which acts as a master regulator of their differentiation and controls their capacity to suppress T cell responses. Tregs have an intrinsically anergic phenotype and do not produce IL-2 or proliferate upon stimulation ex vivo. Recent studies identified that Helios, a member of the Ikaros family of transcription factors, is expressed in Tregs. However, its specific function is not fully understood. In this study, we show that Helios regulates IL-2 production in Tregs by suppressing Il2 gene transcription. Loss of Helios in Tregs breaks their anergic phenotype and results in derepression of the Il2 locus, allowing Tregs to display increased baseline proliferation and to produce IL-2 following stimulation. Conversely, forced expression of Helios in CD4(+)Foxp3(-) T cells results in a loss of their normal ability to produce IL-2. Helios acts by binding to the Il2 promoter and inducing epigenetic modifications that include histone deacetylation. We also show that loss of Helios in Tregs results in decreased Foxp3 binding to the Il2 promoter, indicating that Helios promotes binding of Foxp3 to the Il2 promoter. Interestingly, the loss of Helios in Tregs also causes a decrease in suppressive capacity. Our results identify Helios as a key regulator of Il2 expression in Tregs, contributing to the maintenance of the anergic phenotype.

Ontogeny and localization of the cells produce IL-2 in healthy animals

IL-2 is a growth factor for activated T cells and is required for maintenance of naturally arising regulatory T cells (nTregs). Mice defective in IL-2/IL-2 receptor signaling pathways have impaired nTregs and suffer from lymphoproliferative disorders, suggesting that IL-2 is present and functional in healthy animals. However, the cellular source of IL-2 is currently unknown. To determine which cells produce IL-2 in healthy animals, we established mice carrying cre gene knock in at the il-2 locus (termed IL-2cre). When IL-2cre mice were crossed with EGFP reporter mice, EGFP was exclusively expressed by a fraction of CD4 T cells present in both lymphoid and non-lymphoid tissues. Live imaging of IL-2cre mice that carry the luciferase reporter showed concentrated localization of luciferase+ cells in Peyer’s patches. These cells were not observed in new born mice but appeared within 3days after birth. Reduction of antigen receptor repertoire by transgene expression reduced their number, indicating that recognition of environmental antigens is necessary for generation of these IL-2 producers in healthy animals. A substantial fraction of EGFP+ cells also produce IL-10 and IFN-γ, a characteristic profile of type 1 regulatory T cells (Tr1). The data suggest that a group of Tr1 cells have addition roles in immune homeostasis by producing IL-2 along with other cytokines and help maintaining Tregs.

Aberrant histone modifications in peripheral blood mononuclear cells from patients with Henoch–Schönlein purpura

Henoch-Schönlein purpura (HSP), the most common type of leukocytoclastic vasculitis, is caused by T cell-mediated autoimmune reactions. In this study, we analyze histone modification patterns in peripheral blood mononuclear cells (PBMCs) of HSP patients, and investigate the expression levels of inflammatory cytokines (IFN-γ, IL-2, IL-4, IL-6 and IL-13), transcription factors (T-bet, GATA-3 and TIM-1) and chemokines (CXCL4 and CXCL10) in HSP patients. Our results show that histone H3 acetylation and methylation are significantly enhanced in PBMCs from HSP patients. We also demonstrate specifically that marked increases in histone H3 acetylation and H3 lysine 4 trimethylation occur at the IL-4 loci in these patients. In addition, the expression levels of IL-4, IL-6, IL-13, GATA-3, TIM-1 and CXCL4 are also increased. These findings suggest that abnormal histone modifications are present in the PBMCs of patients with HSP, possibly contributing to the activation of pathological immune responses associated with HSP.

An inactive chromatin configuration at the IL-10 locus in human neutrophils

Event Abstract Back to Event An inactive chromatin configuration at the IL-10 locus in human neutrophils Nicola Tamassia1*, Maili Zimmermann1, Flavia Bazzoni1, Gioacchino Natoli2 and Marco A. Cassatella1 1 University of Verona, Department of Pathology and Diagnostic, Italy 2 European Institute of Oncology (IEO), Department of Experimental Oncology, Italy The ability of human neutrophils to express IL-10, a key cytokine whose function is to limit and terminate the inflammatory responses by regulating the expression of pro- and anti-inflammatory mediators, is currently controversial in the literature. In our hands, in fact, stimulation of highly purified populations of human neutrophils with diverse pathogen-associated molecular patterns (PAMPs), including Toll-like receptors (TLR) and C-type lectin receptor (CLR) agonists, or damage-associated molecular patterns (DAMPs), including serum amyloid protein A (SAA), do not trigger any mRNA expression or production of IL-10, differently from autologous monocytes and from studies of other groups. To identify the molecular basis of IL-10 expression in human phagocytes, we evaluated the chromatin modification status at their IL-10 genomic locus. By chromatin immunoprecipitation (ChIP) assays, we analyzed posttranslational modifications of histones associated with genes that are active, repressed or poised for transcriptional activation, including H3K4me3, H4Ac, H3K27Ac and H3K4me1 marks. Differently from autologous IL-10-producing monocytes, none of the marks under evaluation was detected at the IL-10 locus of resting or activated neutrophils. By contrast, elevated H3K4me3, H4Ac, H3K4me1 and H3K27Ac levels were detected at syntenic regions of the IL-10 locus in mouse neutrophils. Altogether, data demonstrate that human neutrophils, differently from either monocytes or mouse neutrophils, cannot switch on the IL-10 gene because its locus is in an inactive state, likely reflecting a neutrophil-specific developmental outcome. Implicitly, data also definitively settle a currently unsolved issue on the capacity of human neutrophils to produce IL-10. Keywords: IL-10, Neutrophils, Histone modificaitons, Monocytes, Chromatin Immunoprecipitation Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Innate immunity Citation: Tamassia N, Zimmermann M, Bazzoni F, Natoli G and Cassatella MA (2013). An inactive chromatin configuration at the IL-10 locus in human neutrophils. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00992 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 27 Jun 2013; Published Online: 22 Aug 2013. * Correspondence: Dr. Nicola Tamassia, University of Verona, Department of Pathology and Diagnostic, Verona, 37134, Italy, nicola.tamassia@univr.it Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Nicola Tamassia Maili Zimmermann Flavia Bazzoni Gioacchino Natoli Marco A Cassatella Google Nicola Tamassia Maili Zimmermann Flavia Bazzoni Gioacchino Natoli Marco A Cassatella Google Scholar Nicola Tamassia Maili Zimmermann Flavia Bazzoni Gioacchino Natoli Marco A Cassatella PubMed Nicola Tamassia Maili Zimmermann Flavia Bazzoni Gioacchino Natoli Marco A Cassatella Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

In-vitro characterization of novel and functional regulatory SNPs in the promoter region of IL2 and IL2R alpha in a Gabonese population

BackgroundThe selection pressure imposed by the parasite has a functional consequence on the immune genes, leading to altered immune function in which regulatory T cells (Tregs) induced by parasites during infectious challenges modulate or thwart T effector cell mechanism.MethodsWe identified and investigated regulatory polymorphisms in the immune gene IL2 and its receptor IL2R alpha (also known as CD25) in Gabonese individuals exposed to plentiful parasitic infections.ResultsWe identified two reported variants each for IL2 and its receptor IL2R alpha gene loci. Also identified were two novel variants, -83 /-84 CT deletions (ss410961576) for IL2 and -409C/T (ss410961577) for IL2R alpha. We further validated all identified promoter variants for their allelic gene expression using transient transfection assays. Three promoter variants of the IL2 locus revealed no significant expression of the reporter gene. The identified novel variant (ss410961577C/T) of the IL2R alpha revealed a significant higher expression of the reporter gene in comparison to the major allele (P<0.05). In addition, the rs12722616C/T variant of the IL2R alpha locus altered the transcription factor binding site TBP (TATA box binding protein) and C/EBP beta (CCAAT/enhancer binding protein beta) that are believed to regulate the Treg function.ConclusionsThe identification and validation of such regulatory polymorphisms in the immune genes may provide a basis for future studies on parasite susceptibility in a population where T cell functions are compromised.

The Role of IL-15 in Activating STAT5 and Fine-Tuning IL-17A Production in CD4 T Lymphocytes

IL-15 is an important IL-2-related cytokine whose role in Th17 cell biology has not been fully elucidated. In this study, we show that exogenous IL-15 decreased IL-17A production in Th17 cultures. Neutralization of IL-15 using an Ab led to increases in IL-17A production in Th17 cultures. Both Il15(-/-) and Il15r(-/-) T cell cultures displayed higher frequency of IL-17A producers and higher amounts of IL-17A in the supernatants compared with those of wild-type (WT) cells in vitro. IL-15 down-modulated IL-17A production independently of retinoic acid-related orphan receptor-γt, Foxp3, and IFN-γ expression. Both Th17 cells and APCs produced IL-15, which induced binding of STAT5, an apparent repressor to the Il17 locus in CD4 T cells. Also, in a model of myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis (EAE), Il15(-/-) mice displayed exacerbated inflammation-correlating with increased IL-17A production by their CD4(+) T cells-compared with WT controls. Exogenous IL-15 administration and IL-17A neutralization reduced the severity of EAE in Il15(-/-) mice. Taken together, these data indicate that IL-15 has a negative regulatory role in fine-tuning of IL-17A production and Th17-mediated inflammation.

Limited Evidence for Parent-of-Origin Effects in Inflammatory Bowel Disease Associated Loci

BackgroundGenome-wide association studies of two main forms of inflammatory bowel diseases (IBD), Crohn’s disease (CD) and ulcerative colitis (UC), have identified 99 susceptibility loci, but these explain only ∼23% of the genetic risk. Part of the ‘hidden heritability’ could be in transmissible genetic effects in which mRNA expression in the offspring depends on the parental origin of the allele (genomic imprinting), since children whose mothers have CD are more often affected than children with affected fathers. We analyzed parent-of-origin (POO) effects in Dutch and Indian cohorts of IBD patients.MethodsWe selected 28 genetic loci associated with both CD and UC, and tested them for POO effects in 181 Dutch IBD case-parent trios. Three susceptibility variants in NOD2 were tested in 111 CD trios and a significant finding was re-evaluated in 598 German trios. The UC-associated gene, BTNL2, reportedly imprinted, was tested in 70 Dutch UC trios. Finally, we used 62 independent Indian UC trios to test POO effects of five established Indian UC risk loci.ResultsWe identified POO effects for NOD2 (L1007fs; OR = 21.0, P-value = 0.013) for CD; these results could not be replicated in an independent cohort (OR = 0.97, P-value = 0.95). A POO effect in IBD was observed for IL12B (OR = 3.2, P-value = 0.019) and PRDM1 (OR = 5.6, P-value = 0.04). In the Indian trios the IL10 locus showed a POO effect (OR = 0.2, P-value = 0.03).ConclusionsLittle is known about the effect of genomic imprinting in complex diseases such as IBD. We present limited evidence for POO effects for the tested IBD loci. POO effects explain part of the hidden heritability for complex genetic diseases but need to be investigated further.

Gcn5 Is Required for PU.1-Dependent IL-9 Induction in Th9 Cells

Naive CD4+ T cells differentiate into various effector Th subsets depending on the Ags and cytokine microenvironment they encounter. IL-9-secreting Th9 cells are the most recent Th subset to be described. PU.1, one of the transcription factors required for the development of Th9 cells, binds to the Il9 gene. In this study, we show that PU.1 increases histone acetylation at the Il9 locus through direct interactions with histone acetyltransferases. In the absence of PU.1, there is decreased association of Gcn5 and p300/CBP associated factor and increased association of histone deacetylases at the Il9 locus in Th9 cells. Inhibition of histone deacetylase activity augments PU.1-dependent IL-9 production. PU.1 forms a complex with Gcn5, and inhibition of the expression of Gcn5 results in reduced IL-9 production. Moreover, the effects of Gcn5 on IL-9 production are specific as the production of IL-10 and IL-21, two additional cytokines produced by Th9 cells, is not altered after decreased Gcn5 expression. Together, these data define a PU.1-dependent mechanism for altered histone acetylation and expression of the Il9 locus in Th9 cells.

Dysregulation of the serine/threonine phosphatase PP2A contributes to autoimmunity

Protein phosphatase 2A is a ubiquitously expressed serine/threonine phosphatase that regulates a large number of cellular processes. Levels of the catalytic subunit of PP2A (PP2Ac) are tightly controlled. T cells from patients with systemic lupus erythematosus (SLE) express abnormally high levels of PP2Ac. This is promoted by a lupus-associated SNP and by DNA hypomethylation that alters local transcription factor binding. PP2Ac regulates CREB, Elf-1, and SP1, and could thus contribute to several molecular abnormalities described in SLE T cells. In order to determine whether increased levels of PP2Ac in T cells play a role in the development and/or promotion of autoimmune disease, we genetically engineered a mouse to transgenically express high levels of PP2A in T cells. This isolated abnormality allowed us to evaluate the effects of PP2Ac dysregulation in an otherwise normal immune system. PP2Ac transgenic (Tg) mice developed modest splenomegaly and lymphadenopathy, but no overt signs of autoimmunity. Susceptibility of PP2Ac Tg mice to immune-mediated disease was evaluated by antibody-induced glomerulonephritis. Glomerular damage and proteinuria were significantly more severe in PP2Ac Tg mice than in nontransgenic littermates. To determine the mechanism underlying increased kidney damage, we analyzed cytokine production by T cells from PP2Ac Tg and control mice. Production of IL-17 was increased ~10-fold in Tg mice. This phenomenon was documented in vitro and in vivo and was pathologically relevant since blockade of IL-17 abrogated the enhanced susceptibility to glomerulonephritis of Tg mice. These results indicated that increased PP2Ac levels enhance the production of IL-17 in T cells and, through this mechanism, promote immune-mediated organ damage. To determine the molecular pathways through which PP2Ac enhances IL-17 production, we analyzed differentiation of CD4 T cells into effector subsets (that is, Th1, Th2, Th17). Although IL-17A and IL-17F production were constantly higher in PP2Ac Tg T cells, other Th17 characteristics did not differ between Tg and control T cells suggesting that the IL-17-inducing effect occurred independently of Th17 differentiation. Analysis of the Il17 locus demonstrated that PP2Ac overexpression was associated with local permissive epigenetic changes (increased histone 3 acetylation), which explained enhanced IL-17 production. We provide evidence that supports dysregulation of PP2Ac in T cells is able to facilitate autoimmune disease by promoting the production of IL-17. This effect is independent of Th17 differentiation and is explained by the capacity of PP2Ac to modify chromatin accessibility at the Il17 locus by inducing defined epigenetic modifications.

Genetic variants of TNFα, IL10, IL1β, CTLA4 and TGFβ1 modulate the indices of alcohol-induced liver injury in East Indian population

Alcohol induced liver disease or alcoholic liver disease (ALD), a complex trait, encompasses a gamut of pathophysiological alterations in the liver due to continuous exposure to a toxic amount of alcohol (more than 80g per day). Of all chronic heavy drinkers, only 15–20% develops hepatitis or cirrhosis concomitantly or in succession. Several studies revealed that inter-individual as well as inter-ethnic genetic variation is one of the major factors that predispose to ALD. The role of genetic factors in ALD has long been sought for in ethnically distinct population groups. ALD is fast emerging as an important cause of chronic liver disease in India; even in populations such as “Bengalis” who were “culturally immune” earlier. While the genetic involvement in the pathogenesis of ALD is being sought for in different races, the complex pathophysiology of ALD as well as the knowledge of population level diversity of the relevant alcohol metabolizing and inflammatory pathways mandates the need for well designed studies of genetic factors in ethnically distinct population groups. An array of cytokines plays a critical role as mediators of injury, inflammation, fibrosis and cirrhosis in ALD. We, therefore, studied the association of polymorphisms in five relevant cytokine genes with “clinically significant” ALD in an ethnic “Bengali” population in Eastern India. Compared with “alcoholic” controls without liver disease (n=110), TNFα −238AA genotype, IL1β −511CC genotype, TGFβ1 −509CC genotype and IL10 −592AA genotype were significantly overrepresented in ALD patients (n=181; OR=2.4 and 95% CI 1.2–5.5, Pgenotype=0.042, Pallelic=0.008; OR=2.7 and 95% CI 1.2–5.9, Pgenotype=0.018, Pallelic=0.023; OR=4.7 and 95% CI 1.7–13.1, Pgenotype=0.003, Pallelic=0.014; and OR=2.2 and 95% CI 1.1–4.8, Pgenotype=0.04, Pallelic=0.039 respectively). Moreover a cumulative genetic risk analysis revealed a significant trend for developing ALD with an increase in the number of risk alleles on IL10 and TGFβ1 loci among alcoholics. The risk genotype of IL1β and TGFβ1 also influences the total bilirubin, albumin and alanine aminotransferase levels among alcoholic “Bengalis”. The present study is the first case–control study from Eastern India that comprehensively identified polymorphic markers in TNFα, IL10, IL1β and TGFβ1 genes to be associated with ALD in the Bengali population, accentuating the significance of genetic factors in clinical expressions of ALD.

A Protective Role for Human IL-10–Expressing CD4+ T Cells in Colitis

IL-10 is an immunoregulatory cytokine expressed by numerous cell types. Studies in mice confirm that different IL-10-expressing cell subsets contribute differentially to disease phenotypes. However, little is known about the relationship between cell- or tissue-specific IL-10 expression and disease susceptibility in humans. In this study, we used the previously described human (h)IL10BAC transgenic model to examine the role of hIL-10 in maintaining intestinal homeostasis. Genomically controlled hIL-10 expression rescued Il10(-/-) mice from Helicobacter-induced colitis and was associated with control of proinflammatory cytokine expression and Th17 cell accumulation in gut tissues. Resistance to colitis was associated with an accumulation of hIL-10-expressing CD4(+)Foxp3(+) regulatory T cells specifically within the lamina propria but not other secondary lymphoid tissues. Cotransfer of CD4(+)CD45RB(lo) cells from Il10(-/-)/hIL10BAC mice rescued Rag1(-/-) mice from colitis, further suggesting that CD4(+) T cells represent a protective source of hIL-10 in the colon. In concordance with an enhanced capacity to express IL-10, CD4(+)CD44(+) T cells isolated from the lamina propria exhibited lower levels of the repressive histone mark H3K27Me3 and higher levels of the permissive histone mark acetylated histone H3 in both the human and mouse IL10 locus compared with the spleen. These results provide experimental evidence verifying the importance of T cell-derived hIL-10 expression in controlling inflammation within the colonic mucosa. We also provide molecular evidence suggesting the tissue microenvironment influences IL-10 expression patterns and chromatin structure in the human (and mouse) IL10 locus.

Silencing of the Il2 gene transcription is regulated by epigenetic changes in anergic T cells

Anergy is induced in T cells as a consequence of a partial or suboptimal stimulation. Anergic T cells become unresponsive and fail to proliferate and produce cytokines. We had previously shown that in anergic CD4(+) T cells, Ikaros participates in the transcriptional repression of the Il2 gene by recruiting histone deacetylases that cause core histone deacetylation at the Il2 promoter. Here we show that deacetylation at the Il2 promoter is the initial step in a process that leads to the stable silencing of the Il2 gene transcription in anergic T cells. We have found that anergy-induced deacetylation of the Il2 promoter permits binding of the histone methyl-transferase Suv39H1, which trimethylates lysine-9 of histone H3 (Me3H3-K9). Furthermore, the establishment of the Me3H3-K9 mark allows the recruitment of the heterochromatin protein HP1, allowing the silenced Il2 loci to reposition close to heterochromatin-rich regions. Our results indicate that silencing of Il2 transcription in anergic T cells is attained through a series of epigenetic changes that involve the establishment of repressive marks and the subsequent nuclear repositioning of the Il2 loci, which become juxtaposed to transcriptionally silent regions. This mechanism may account for the stable nature of the inhibition of IL-2 production in anergic cells.

Conserved Intergenic Elements and DNA Methylation Cooperate to Regulate Transcription at the il17 Locus

Naive CD4(+) T cells can differentiate into distinct lineages with unique immune functions. The cytokines TGFβ and IL-6 promote the development of Th17 cells that produce IL-17, an inflammatory cytokine not expressed by other T helper lineages. To further understand how IL-17 production is controlled, we studied an ~120-kb genomic region containing the murine il17a and il17f genes and seven evolutionarily conserved, intergenic noncoding sequences. We show that the +28-kb noncoding sequence cooperates with STAT3, RORγt, and Runx1 to enhance transcription from both il17a and il17f promoters. This enhancer and both promoters exhibited Th17 lineage-specific DNA demethylation, accompanied by demethylation of lysine 27 of histone H3 (H3K27) and increased H3K4 methylation. Loss of DNA methylation tended to occur at STAT3 consensus elements, and we show that methylation of one of these elements in the il17a promoter directly inhibits STAT3 binding and transcriptional activity. These results demonstrate that TGFβ and IL-6 synergize to epigenetically poise the il17 loci for expression in Th17 cells, and suggest a general mechanism by which active STAT3 may be epigenetically excluded from STAT3-responsive genes in non-Th17 lineages.