IL-4 Gene Locus Research Articles

Role of CNSs Conserved Distal Cis-Regulatory Elements in CD4 + T Cell Development and Differentiation.

Naïve CD4+ T cells differentiate into diverse subsets of effector cells and perform various homeostatic and immune functions. The differentiation and maintenance of these different subsets are controlled through the upregulation and silencing of master genes. Mechanistic studies of the regulation of these master genes identified conserved and distal intronic regulatory elements, which are accessible subsets of conserved non-coding sequences (CNSs), acting as cis-regulatory elements in a lineage-specific manner that controls the function of CD4+ T cells. Abnormal CNS activity is associated with incorrect expression of master genes and development of autoimmune diseases or immune suppression. Here, we describe the function of several conserved, distal cis-regulatory elements at the Foxp3, Rorc, Il-4, Il-10 and Il-17 gene locus were shown to play important roles in CD4+ T cells differentiation. Together, this review briefly outlines currently known CNSs, with a focus on their regulations and functions in complexes modulating the differentiation and maintenance of various CD4+ T cells subsets, in health and disease contexts, as well as during the conversion of T regulatory cells to T helper 17 cells. This article will provide a comprehensive view of CNSs conserved distal cis-regulatory elements at a few loci that control aspects of CD4+ T cells function.

Results of Frequency Analysis Distribution of Polymorphism Rs1800895 592C>A In IL10 Gene among Patients with Chronic Polypoid Rhinosinusitis.

Chronic polyposis rhinosinusitis is a disease characterized by chronic inflammation of the nasal mucosa and sinuses. In the tissues of polyps and intranasal secretions, an increase in the concentration of various inflammatory mediators, in particular interleukins is observed due to an increase in their de novo synthesis by effector cells. The research objective to study the distribution of genotypic forms of the IL10 gene locus and evaluate their role in the development and clinical course of chronic polypoid rhinosinusitis. In accordance with the purpose of the study and to fulfill the assigned tasks, clinical studies were carried out in 140 patients with CPRS and with chronic rhinosinusitis, who were examined and treated at the ENT department of the multidisciplinary clinic of the Tashkent Medical Academy in 2017-2019. The examined patients met the following criteria: the presence of polyposis tissue in the nasal cavity that obstructs the common nasal passage completely or by at least 50%; complaints of prolonged difficulty in nasal breathing; according to the patient, the disease significantly reduces the quality of his life; absence of acute inflammatory pathology; written informed consent for surgical treatment and morphological examination of the surgical material. The revealed reliably high frequency of this genotype in group 1 of patients with CPRS, compared with group 2 of CPRS, also confirms our assumption about the protective role of the genotypic variant of the A/A polymorphism rs1800895 592 C>A in the IL10 gene, in relation to the development of CPRS in patients (6.45 % versus 5.0%, respectively, at χ2=0.02; Р=0.45; RR=1.17; OR=1.18; 95% CI: 8.557-8.942). This fact is probably due to the fact that in carriers of the A/A genotype of the IL10 gene, the anti-inflammatory activity of epithelial matrix metalloproteinases is significantly increased, compared with patients carrying other genotypic variants. The results obtained showed that, with this pathology of the genotypic variant of the A/A polymorphism rs1800895 592 C>A in the IL10 gene, there is a risk of developing CPRS in patients (6.45% versus 5.0%, respectively). This fact is probably due to the fact that in carriers of the A/A genotype of the IL10 gene, the anti-inflammatory activity of epithelial matrix metalloproteinases is significantly increased, compared with patients carrying other genotypic variants.

Il-10 Gene Locus DNA Methylation in Regulatory B Cells.

Epigenetic studies are becoming increasingly common in the immunology field thanks to the support of cutting edge technology and to their potential of providing a large amount of data at the single cell level. Moreover, epigenetic modifications were shown to play a role in autoimmune/inflammatory disorders, paving the way for the possibility of using the results of epigenetic studies for therapeutic purposes. In recent years, epigenetic marks such as DNA methylation, histone modifications and nucleosome positioning were shown to regulate B cell fate and function during an immune response, but very little has been done in the context of one of the most recently discovered B cell subsets, that is regulatory B cells. Although no consensus has yet been found on the identity of these immunosuppressive B cells, the role of the IL-10 cytokine is consolidated, both in the murine and human setting. In this chapter we will focus on the analysis of the methylation profile of a gene of interest and we will specifically describe cloning and pyrosequencing bisulphite sequencing PCR (BSP). Given the specific context, we will provide tips and tricks for the analysis of the il-10 gene locus. Nonetheless, the methods presented are valid for the study of any gene of interest.

Long Noncoding RNA AW112010 Promotes the Differentiation of Inflammatory T Cells by Suppressing IL-10 Expression through Histone Demethylation.

Long noncoding RNAs (lncRNAs) have been demonstrated to play important regulatory roles in gene expression, from histone modification to protein stability. However, the functions of most identified lncRNAs are not known. In this study, we investigated the role of an lncRNA called AW112010. The expression of AW112010 was significantly increased in CD4+ T cells from C57BL/6J mice activated in vivo with myelin oligodendrocyte glycoprotein, Staphylococcal enterotoxin B, or in vitro with anti-CD3 anti-CD28 mAbs, thereby demonstrating that activation of T cells leads to induction of AW112010. In contrast, anti-inflammatory cannabinoids such as cannabidiol or δ-9-tetrahydrocannabinol decreased the expression of AW112010 in T cells. Interestingly, the expression of AW112010 was high in in vitro-polarized Th1 and Th17 cells but low in Th2 cells, suggesting that this lncRNA may regulate inflammation. To identify genes that might be regulated by AW112010, we used chromatin isolation by RNA purification, followed by sequencing. This approach demonstrated that AW112010 regulated the transcription of IL-10. Additionally, the level of IL-10 in activated T cells was low when the expression of AW112010 was increased. Use of small interfering RNA to knock down AW112010 expression in activated T cells led to increased IL-10 expression and a decrease in the expression of IFN-γ. Further studies showed that AW112010 interacted with histone demethylase KDM5A, which led to decreased H3K4 methylation in IL-10 gene locus. Together, these studies demonstrate that lncRNA AW112010 promotes the differentiation of inflammatory T cells by suppressing IL-10 expression through histone demethylation.

Transcriptional regulation of IL10 gene in human macrophages

Abstract IL-10 is an anti-inflammatory cytokine with a crucial role in immune homeostasis. Dysregulated production of IL-10 has been linked to certain autoimmune and inflammatory diseases. IL-10 is strongly induced by TLR ligands and suppressed by IFN-γ as part of the synergistic activation of inflammatory genes. However, the molecular mechanisms that regulate expression of the IL10 gene, especially in human macrophages, are incompletely understood. By using epigenomic analysis of ATAC-seq and ChIP-seq analysis we have identified human macrophage-specific enhancers in the IL10 gene locus. In these putative enhancers, we observed that chromatin accessibility and histone acetylation are increased by LPS stimulation. We also found that p300 was frequently recruited to these macrophage-specific cis-regulatory elements (CREs) in LPS-stimulated macrophages. One of these potential LPS-induced enhancers, CRE+6, is located 6kb upstream of the IL10 transcriptional start site and contains transcription factor binding motifs and autoimmune disease-associated SNPs. CRE+6 showed constitutive binding of the macrophage-lineage-determining factor PU.1 and LPS-inducible binding of AP-1 and STAT3. LPS stimulation increased eRNA transcription from CRE+6. The functional relevance of CRE+6 eRNA was confirmed by eRNA-targeted knockdown experiments. Strikingly, the mechanism by which IFN-γ suppresses IL10 expression appeared to be decommissioning of enhancers, as IFN-γ strongly suppressed TLR-induced positive enhancer marks and eRNA transcription. Our study implicates that crucial enhancer elements cooperate with the core promoter in LPS- and IFN-γ-mediated regulation of IL-10 gene transcription in human macrophages.

IL-10-producing Bcells are characterized by a specific methylation signature.

Among the family of regulatory Bcells, the subset able to produce interleukin-10 (IL-10) is the most studied, yet its biology is still a matter of investigation. The DNA methylation profiling of the il-10 gene locus revealed a novel epigenetic signature characterizing murine Bcells ready to respond through IL-10 synthesis: a demethylated region located 4.5 kb from the transcription starting site (TSS), that we named early IL10 regulatory region (eIL10rr). This feature allows to distinguish Bcells that are immediately prone and developmentally committed to IL-10 production from those that require a persistent stimulation to exert an IL-10-mediated regulatory function. These late IL-10 producers are instead characterized by a delayed IL10 regulatory region (dIL10rr), a partially demethylated DNA portion located 9 kb upstream from the TSS. A demethylated region was also found in human IL-10-producing Bcells and, very interestingly, in some B-cell malignancies, such as chronic lymphocytic leukemia and mantle cell lymphoma, characterized by an immunosuppressive microenvironment. Our findings define murine and human regulatory Bcells as an epigenetically controlled functional state of mature Bcell subsets and open a new perspective on IL-10 regulation in Bcells in homeostasis and disease.

Locus-Specific Reversible DNA Methylation Regulates Transient IL-10 Expression in Th1 Cells.

IL-10 is a pleiotropic cytokine with multifaceted functions in establishing immune homeostasis. Although expressed by Th1 and Th2 cells, conventional Th1 cells produce marginal levels of IL-10 compared with their Th2 counterparts. In this study, we investigated the epigenetic mechanisms of Il-10 gene expression in Th1 cells. Bioinformatics EMBOSS CpG plot analysis and bisulfite pyrosequencing revealed three CpG DNA methylation sites in the Il-10 gene locus. Progressive DNA methylation at all of the CpG regions of interest (ROIs) established a repressive program of Il-10 gene expression in Th1 cells. Interestingly, Th1 cells treated with IL-12 and IL-27 cytokines, thereby mimicking a chronic inflammatory condition in vivo, displayed a significant increase in IL-10 production that was accompanied by selective DNA demethylation at ROI 3 located in intron 3. IL-10-producing T cells isolated from lymphocytic choriomeningitis virus-infected mice also showed enhanced DNA demethylation at ROI 3. Binding of STAT1 and STAT3 to demethylated ROI 3 enhanced IL-10 expression in an IL-12/IL-27-dependent manner. Accordingly, CD4+ T cells isolated from STAT1- or STAT3-knockout mice were significantly defective in IL-10 production. Our data suggest that, although stably maintained DNA methylation at the promoter may repress IL-10 expression in Th1 cells, locus-specific reversible DNA demethylation may serve as a threshold platform to control transient Il-10 gene expression.

YB-1 orchestrates onset and resolution of renal inflammation via IL10 gene regulation.

The Y‐box‐binding protein (YB)‐1 plays a non‐redundant role in both systemic and local inflammatory response. We analysed YB‐1‐mediated expression of the immune regulatory cytokine IL‐10 in both LPS and sterile inflammation induced by unilateral renal ischaemia–reperfusion (I/R) and found an important role of YB‐1 not only in the onset but also in the resolution of inflammation in kidneys. Within a decisive cis‐regulatory region of the IL10 gene locus, the fourth intron, we identified and characterized an operative YB‐1 binding site via gel shift experiments and reporter assays in immune and different renal cells. In vivo, YB‐1 phosphorylated at serine 102 localized to the fourth intron, which was paralleled by enhanced IL‐10 mRNA expression in mice following LPS challenge and in I/R. Mice with half‐maximal expression of YB‐1 (Yb1 +/−) had diminished IL‐10 expression upon LPS challenge. In I/R, Yb1 +/− mice exhibited ameliorated kidney injury/inflammation in the early‐phase (days 1 and 5), however showed aggravated long‐term damage (day 21) with increased expression of IL‐10 and other known mediators of renal injury and inflammation.In conclusion, these data support the notion that there are context‐specific decisions concerning YB‐1 function and that a fine‐tuning of YB‐1, for example, via a post‐translational modification regulates its activity and/or localization that is crucial for systemic processes such as inflammation.

IL-4 Haploinsufficiency Specifically Impairs IgE Responses against Allergens in Mice.

Polymorphisms in genes involved in IL-4 responses segregate with allergic disease risk and correlate with IgE levels in humans, and IL-4 promotes IgE and IgG1 Ab production against allergens in mice. We report that mice with only one intact Il4 gene copy are significantly impaired in their ability to make specific IgE responses against allergens, whereas IgG1 responses to allergens remain unaffected. Il4-hemizygosity also resulted in a modest but detectable drop in IL-4 production by CD4+ T cells isolated from lymph nodes and prevented IgE-dependent oral allergen-induced diarrhea. We conclude that a state of haploinsufficiency for the Il4 gene locus is specifically relevant for IL-4-dependent IgE responses to allergens with the amount of IL-4 produced in the hemizygous condition falling close to the threshold required for switching to IgE production. These results may be relevant for how polymorphisms in genes affecting IL-4 responses influence the risk of IgE-mediated allergic disease in humans.

IL-10 Production By CLL Cells Is Enhanced in the Anergic IGHV Mutated Subset and Associates with Reduced DNA Methylation of the IL-10 Locus

The B-cell receptor (BCR) of chronic lymphocytic leukemia (CLL) cells with unmutated (U-CLL) or mutated (M-CLL) immunoglobulin gene heavy-chain variable (IGHV) regions shows a variable degree of anergy, associated with low surface IgM (sIgM) levels and signaling capacity, more evident in M-CLL. DNA methylation is also different between M-CLL and U-CLL. Patients from both subsets are immunosuppressed from the early stages, possibly influenced by the ability of activated CLL cells to produce IL-10, a property of B10 cells.The aim of this study was to investigate associations between capacity of circulating CLL cells to differentiate into B10 cells and features of anergy. In parallel, the methylation status of the IL-10 gene locus was probed in order to investigate associations between IL-10 gene methylation and production. CLL cells were isolated and cultured +/- TLR9 activation with CpG (ODN-2006). Cytokine secretion was measured in supernatants (Luminex) and IL10 transcript was measured (RT-qPCR) from cell pellets. IL-10 production by CLL cells was measured by intracellular flow cytometry following stimulation with CpG for 24 hours. Epigenetic profiling was performed using MassARRAY and the 450K Array.We found that activation of CLL cells by CpG consistently induced production and secretion of IL-10 protein, while no or low amounts of pro-inflammatory cytokines were detected. Production of IL-10 was significantly higher in M-CLL than in U-CLL. Levels also correlated with lower sIgM levels and signaling capacity, both features of anergy. A linear correlation was present between secretion, intracellular production and transcript levels of IL-10, suggesting no aberrant post-transcriptional controls.In the absence of CpG activation, IL-10 transcript levels were also detected at low levels in M-CLL, while they were even lower in U-CLL. To identify a potential basis for the differential expression, DNA methylation analysis of IL-10 gene locus was performed. While the promoter region displayed similarly low levels of methylation in both U-CLL and M-CLL, methylation differences were detected immediately downstream of the promoter within the first intron (approximately +200 to +500 bp, differentially methylated region 1, DMR1) and in the gene body (approximately +1300 to +1800 bp, DMR2). Both DMR1 and DMR2 were markedly more hypomethylated in M-CLL than U-CLL, with the greatest difference detected at cg17067005 within DMR1. Each of these regions display histone H3 lysine 27 acetylation (H3K27ac) in B cells (GM12878 cells), thus are likely to represent functional DNA elements. Analysis of IL-10 transcript levels with methylation demonstrated a mutually exclusive pattern between expression and methylation of both DMR1 and DMR2.These data document a strong link between capacity to differentiate into B10-like cells and anergy, and suggest an epigenetic component in the regulation of IL-10 production in CLL cells. This capacity may contribute to immunosuppression. While U-CLL appears less able to produce IL-10 on a per cell basis, higher tumor load may compensate, accounting for clinical immunosuppression in both subsets. DisclosuresNo relevant conflicts of interest to declare.

Posttranscriptional control of allergic airway inflammation by HuR (IRM11P.638)

Abstract RNA binding protein HuR regulates Th2 differentiation by controlling mRNA stability of IL-4, IL-13 and GATA-3. We hypothesized HuR is required for initiation of allergic airway inflammation. We generated a distal lck-Cre ROSA HuRfl/fl mouse model to conditionally ablate HuR prior to T cell activation. HuR KO mice had 50% knockdown efficiency of YFP+ CD4+ T cells. We used ova challenge model of airway inflammation to ascertain effects of HuR KO in T cells. YFP+ HuR KO T cells migrated normally to lungs during ova challenge and had significant reductions in BAL IL-13, cellular inflammation and serum IgE. Additionally, they had completely abolished lung cellular inflammation to levels similar to non-immunized mice. We conducted in vitro activation experiments to determine mechanisms of suppression of airway inflammation. HuR KO CD4+ T cells were sorted into YFP+ (HuR lo) and YFP- (HuR WT) pools and activated. YFP+ T cells had profound decreases in IL-4, IL-5, IL-13 but not IFNλ at mRNA and protein levels. Gata-3 and IL-4 transcription were significantly reduced, as was expression of IL-2Ra (CD25). YFP+ KO T cells have reductions in p-stat5 signaling which is required for robust Th2 differentiation by activation of the IL-4 gene locus. Remarkably, though only 50% of CD4+ T cells have ablated HuR, this was sufficient to completely suppress Th2 differentiation in airway inflammation. These data reveal a pivotal role for HuR in controlling Th2 cytokines and airway inflammation.

Notch Signaling Enhances FcεRI-Mediated Cytokine Production by Mast Cells through Direct and Indirect Mechanisms

Th2-type cytokines and TNF-α secreted by activated mast cells upon cross-linking of FcεRI contribute to the development and maintenance of Th2 immunity to parasites and allergens. We have previously shown that cytokine secretion by mouse mast cells is enhanced by signaling through Notch receptors. In this study, we investigated the molecular mechanisms by which Notch signaling enhances mast cell cytokine production induced by FcεRI cross-linking. FcεRI-mediated production of cytokines, particularly IL-4, was significantly enhanced in mouse bone marrow-derived mast cells by priming with Notch ligands. Western blot analysis showed that Notch signaling augmented and prolonged FcεRI-mediated phosphorylation of MAPKs, mainly JNK and p38 MAPK, through suppression of the expression of SHIP-1, a master negative regulator of FcεRI signaling, resulting in the enhanced production of multiple cytokines. The enhancing effect of Notch ligand priming on multiple cytokine production was abolished by knockdown of Notch2, but not Notch1, and FcεRI-mediated production of multiple cytokines was enhanced by retroviral transduction with the intracellular domain of Notch2. However, only IL-4 production was enhanced by both Notch1 and Notch2. The enhancing effect of Notch signaling on IL-4 production was lost in bone marrow-derived mast cells from mice lacking conserved noncoding sequence 2, which is located at the distal 3' element of the Il4 gene locus and contains Notch effector RBP-J binding sites. These results indicate that Notch2 signaling indirectly enhances the FcεRI-mediated production of multiple cytokines, and both Notch1 and Notch2 signaling directly enhances IL-4 production through the noncoding sequence 2 enhancer of the Il4 gene.

Polymorphisms in the interleukin-10 gene and chronic periodontitis in patients with atherosclerotic and aortic aneurysmal vascular diseases

BackgroundChronic periodontitis (CP), atherosclerotic and aortic aneurysmal vascular diseases (VD) are chronic inflammatory conditions with multifactorial etiologies, including involvement of predisposing genetic factors. In a previous study, polymorphisms in the gene for the anti-inflammatory interleukin-1 receptor antagonist were associated with CP in patients with VD.ObjectiveThis study investigates whether polymorphisms in the gene for the anti-inflammatory interleukin-10 (IL10) could be related to CP in the same manner.MethodsSeventy-two patients with VD of whom 35 had CP were genotyped for single nucleotide polymorphisms (SNPs) in the IL10 −592 (rs1800872), −819 (rs1800871), and −1,082 (rs1800896) gene by Taqman rtPCR method and by DNA sequencing.ResultsThe C alleles and C/C genotypes of IL10 −592 and IL10 −819 frequencies were significantly higher, while the frequencies of the IL10 −592 (C/A) and IL10 −819 (C/T) heterozygote genotypes were significantly lower in the VD group with CP compared to those without CP. The IL10 haplotype ATA frequency (−1,082, −819, −592) showed a trend to a significant difference between the two groups indicating protection against CP.ConclusionsTaken together, our findings suggest an independent association of genetic polymorphisms in the IL-10 gene locus with CP in patients with VD. Development of CP and the implications on vascular disease emphasize the importance of early detection and adequate treatment of periodontitis among these patients.

Primary murine CD4+ T cells fail to acquire the ability to produce effector cytokines when active Ras is present during Th1/Th2 differentiation.

Constitutive Ras signaling has been shown to augment IL-2 production, reverse anergy, and functionally replace many aspects of CD28 co-stimulation in CD4+ T cells. These data raise the possibility that introduction of active Ras into primary T cells might result in improved functionality in pathologic situations of T cell dysfunction, such as cancer or chronic viral infection. To test the biologic effects of active Ras in primary T cells, CD4+ T cells from Coxsackie-Adenovirus Receptor Transgenic mice were transduced with an adenovirus encoding active Ras. As expected, active Ras augmented IL-2 production in naive CD4+ T cells. However, when cells were cultured for 4 days under conditions to promote effector cell differentiation, active Ras inhibited the ability of CD4+ T cells to acquire a Th1 or Th2 effector cytokine profile. This differentiation defect was not due to deficient STAT4 or STAT6 activation by IL-12 or IL-4, respectively, nor was it associated with deficient induction of T-bet and GATA-3 expression. Impaired effector cytokine production in active Ras-transduced cells was associated with deficient demethylation of the IL-4 gene locus. Our results indicate that, despite augmenting acute activation of naïve T cells, constitutive Ras signaling inhibits the ability of CD4+ T cells to properly differentiate into Th1/Th2 effector cytokine-producing cells, in part by interfering with epigenetic modification of effector gene loci. Alternative strategies to potentiate Ras pathway signaling in T cells in a more regulated fashion should be considered as a therapeutic approach to improve immune responses in vivo.

Conditional Deletion of Histone Deacetylase 1 in T Cells Leads to Enhanced Airway Inflammation and Increased Th2 Cytokine Production

Chromatin modifications, such as reversible histone acetylation, play a key role in the regulation of T cell development and function. However, the role of individual histone deacetylases (HDACs) in T cells is less well understood. In this article, we show by conditional gene targeting that T cell-specific loss of HDAC1 led to an increased inflammatory response in an in vivo allergic airway inflammation model. Mice with HDAC1-deficient T cells displayed an increase in all critical parameters in this Th2-type asthma model, such as eosinophil recruitment into the lung, mucus hypersecretion, parenchymal lung inflammation, and enhanced airway resistance. This correlated with enhanced Th2 cytokine production in HDAC1-deficient T cells isolated from diseased mice. In vitro-polarized HDAC1-deficient Th2 cells showed a similar enhancement of IL-4 expression, which was evident already at day 3 of Th2 differentiation cultures and restricted to T cell subsets that underwent several rounds of cell divisions. HDAC1 was recruited to the Il4 gene locus in ex vivo isolated nonstimulated CD4(+) T cells, indicating a direct control of the Il4 gene locus. Our data provide genetic evidence that HDAC1 is an essential HDAC that controls the magnitude of an inflammatory response by modulating cytokine expression in effector T cells.

STAT1 signaling is required for optimal Th1 cell differentiation in mice

Although IFN-γ alone does not prime type I T helper cell (Th1) differentiation, the loss of IFN-γ signaling leads to impaired Th1 phenotype: IFN-γ receptor-deficient (Ifngr −/−) Th1 cells fail to permanently repress IL-4 expression. They can differentiate into IL-4-producing cells under Th2-inducing conditions. These observations suggest that IFN-γ signaling plays a critical role in silencing Il4 gene in Th1 cells and stabilizing Th1 phenotype. IFN-γ signaling has been further shown to inhibit IL-4 expression by inhibiting STAT6 phosphorylation. This work aims to study the mechanism by which STAT1, the downstream molecule that transduces IFN-γ signaling, may mediate suppression of IL-4 expression in Th1 cells. The results show that STAT1-deficient naive CD4+ T cells express a reduced level of IFN-γ as well as an elevated level of IL-4. These cells exhibit bias to differentiate into Th2 cells under unpolarized conditions. Under Th1-inducing conditions, STAT1-deficient naive CD4+ T cells show impaired Th1 differentiation: Stat1 −/− Th1 cells express reduced levels of IFN-γ and T-bet. These cells also fail to repress the expression of IL-4 and GATA-3 and retain STAT6 signaling. More importantly, Stat1 −/− Th1 cells can be effectively induced to differentiate into IL-4-producing cells under Th2-inducing conditions. Ectopic expression of T-bet in Stat1 −/− Th1 cells dramatically represses their ability to do so and drastically restores IFN-γ expression, suggesting that STAT1 may inhibit IL-4 expression through T-bet. Finally, histone H3 acetylation (H3AC) and histone H3 K4 dimethylation (H3K4dim) were observed in two enhancer regions of Il4 gene locus in Stat1 −/− Th1 cells, suggesting a permissive status of Il4 gene locus in these cells. Thus, this study reveals new mechanisms by which STAT1 signaling may mediate repression of Il4 gene in Th1 cells: upregulating T-bet that subsequently represses GATA3 and IL-4 expression, antagonizing STAT6 signaling, and inhibiting epigenetic modifications in Il4 gene locus.

Selection for Genetic Variation Inducing Pro-Inflammatory Responses under Adverse Environmental Conditions in a Ghanaian Population

BackgroundChronic inflammation is involved in the pathogenesis of chronic age-associated, degenerative diseases. Pro-inflammatory host responses that are deleterious later in life may originate from evolutionary selection for genetic variation mediating resistance to infectious diseases under adverse environmental conditions.Methodology/Principal FindingsIn the Upper-East region of Ghana where infection has remained the leading cause of death, we studied the effect on survival of genetic variations at the IL10 gene locus that have been associated with chronic diseases. Here we show that an IL10 haplotype that associated with a pro-inflammatory innate immune response, characterised by low IL-10 (p = 0.028) and high TNF-α levels (p = 1.39×10−3), was enriched among Ghanaian elders (p = 2.46×10−6). Furthermore, in an environment where the source of drinking water (wells/rivers vs. boreholes) influences mortality risks (HR 1.28, 95% CI [1.09–1.50]), we observed that carriers of the pro-inflammatory haplotype have a survival advantage when drinking from wells/rivers but a disadvantage when drinking from boreholes (pinteraction = 0.013). Resequencing the IL10 gene region did not uncover any additional common variants in the pro-inflammatory haplotype to those SNPs that were initially genotyped.Conclusions/SignificanceAltogether, these data lend strong arguments for the selection of pro-inflammatory host responses to overcome fatal infection and promote survival in adverse environments.

Ikaros Is a Regulator of Il10 Expression in CD4+ T Cells

IL-10 is a regulatory cytokine critical for controlling inflammatory responses. Here we show that Ikaros, a zinc finger DNA-binding protein, plays an important role in the regulation of Il10 in murine CD4(+) T cells. Upon initial stimulation of the TCR, T cells deficient in Ikaros express significantly lower levels of IL-10 compared with wild-type T cells. In addition, under Th2 skewing conditions, which induce IL-10 production by wild-type T cells, Ikaros null T cells are unable to properly differentiate, producing only low levels of IL-10. Expression of a dominant-negative isoform of Ikaros in wild-type Th2 cells represses IL-10 production but does not significantly alter expression levels of the genes encoding the transcription factors GATA-3 and T-bet. Furthermore, expression of Ikaros in Ikaros null T cells restores expression of the Th2 cytokines IL-10 and IL-4 while reducing production of the Th1 cytokine, IFN-gamma. Coexpression of Ikaros and GATA-3 further increases IL-10 production, showing that these two factors have an additive effect on activating Il10 expression. Finally, we show that Ikaros binds to conserved regulatory regions of the Il10 gene locus in Th2 cells, supporting a direct role for Ikaros in Il10 expression. Thus, we provide evidence for Ikaros as a regulator of Il10 and Ifng gene expression and suggest a role for Ikaros in directing lineage-specific cytokine gene activation and repression.

P08: Effects of inflammation-related cytokine gene polymorphisms on gastric cancer risk among atomic bomb survivors

RERF epidemiology studies found that risks of certain cancers among atomic-bomb (A-bomb) survivors, including gastric cancer, increased with past exposure dose to atomic radiation, and that these risks remain high even today. RERF immunology studies implied that radiation exposure greatly affected host immune systems of A-bomb survivors, further providing for the possibility that altered immune response in this population, specifically inflammatory response and immunological host defense, might be involved in the development of various cancers. In fact, we have reported that the enhancement of persistent inflammation with increased age was further accelerated among people exposed to A-bomb radiation. In this study, we examined relationship between gastric cancer risk and radiation dose based on 43 SNPs of inflammation-related genes ( IL1B, IL6, IL8, IL10, BAT1, NFKBIL1, LTA, TNF, and LST1 ), in a case-control study of 238 cases and 1757 controls within a subcohort of the RERF Adult Health Study. We found statistically significant association between IL10 , LTA , and TNF SNPs and risk of gastric cancer, but not between other SNPs and risk of gastric cancer. Three SNPs on the LTA-TNF loci were associated with elevated risk of gastric cancer. Additionally, our genome analysis identified a single haplotype block within the IL10 gene locus, which was indicated by four htSNPs (generating two major alleles, IL10-ATTA and IL10-GGCG ). We found that risk of gastric cancer significantly varied by IL-10 haplotype in both non-exposed and exposed individuals, and that risk-enhancing effects of radiation were evident in all the haplotypes. Our findings offer further evidence that variation in the IL10 and TNF loci affects gastric cancer risk.

Ikaros regulates IL-10 expression in CD4+ T cells (38.27)

Abstract IL-10 is a regulatory cytokine critical for controlling inflammatory responses. However, little is known about the molecular mechanisms controlling Il10 gene expression, although GATA-3 is known to be involved in Il10 expression by Th2 cells. Here we show that Ikaros, a zinc finger DNA-binding protein, plays an important role in the regulation of IL-10 in T cells. Upon initial stimulation by T cell receptor, cells deficient in Ikaros (Ikaros null) express significantly lower levels of IL-10 compared to wild type T cells. In addition, under Th2 skewing conditions, which induce high levels of IL-10 production by wild type cells, Ikaros null T cells are unable to properly differentiate and produce only low levels of IL-10. The use of a retroviral vector system to express a dominant negative isoform of Ikaros in wild type Th2 cells represses IL-10 production but does not significantly alter the levels of transcription factors Gata3 and Tbet. Further, expression of Ikaros in Ikaros null T cells restores IL-10 expression. We found that Ikaros binds to conserved regulatory regions of the Il10 gene locus in Th2 cells, supporting a direct role for Ikaros in Il10 expression. In addition, we have evidence to suggest a coordinate role for Ikaros and Gata3 in the regulation of Il10. A direct interaction of these proteins is being examined. Thus, we suggest of novel role of Ikaros in regulating Il10 gene expression.