Cytokines are secreted proteins that regulate cellular growth and differentiation. These factors are especially important in regulating immune and inflammatory responses, regulating lymphoid development and differentiation. Cytokines also have critical functions in regulating immune homeostasis, tolerance, and memory. Not surprisingly, cytokines are critical in the pathogenesis of autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease and psoriasis. Moreover, targeting cytokines and cytokine signaling are successful new strategies in treating these diseases, underscoring the need to better understand the molecular basis of cytokine action as it relates to the pathogenesis of immune-mediated disease. The first step in cytokine signaling is activation of receptor associated Janus kinases or Jaks. The next step in signaling is the activation of a family of transcription factors called Stats (signal transducers and activators of transcription). Stat5A and Stat5B are important for development and homeostasis of T cells, B cells and neutrophils. Other STATs, STAT3, STAT4 and STAT6 are important for the differentiation of CD4 helper T cells. T cells orchestrate immune responses through their production of cytokines, shaping the character of the response during host defense and in autoimmune settings. Subsets of helper T (Th) cells include Th1 Th2, Th17 and regulatory T (Treg) cells We previously found that deficiency of Stat5A/B in CD4+ T cells abrogated Treg development in vivo and blocked conversion of peripheral cells to become iTregs. IL-17 is a major inflammatory cytokine, which appears to contribute to the pathogenesis of many autoimmune and autoinflammatory disorders including rheumatoid arthritis, spondyloarthropathies, multiple sclerosis and inflammatory bowel diseases (IBD). We have found that Stat3 is essential for Th17 differentiation and that Stat3 binds to the Il17 gene. Additionally, through analysis of patients with hyperimmunoglobulin E or Job's syndrome, who have dominant negative mutations of STAT3, we showed that Stat3 function is also essential for human Th17 differentiation. We extended these studies by further investigating the role of Stat3 in a model of inflammatory bowel disease and found that absence of Stat3 prevents the development of IBD, as well as proliferation and survival of CD4+ T cells. STAT3 was also an important factor in determining the balance between Th17 vs. Treg differentiation. Using chromatin immunoprecipitation and massive parallel sequencing, we found that STAT3 bound many genes involved in Th17 differentiation, ranging from cytokines and cytokine receptors to transcription factors and genes that regulate cell growth and apoptosis. Interleukin 23 (IL-23) is required for autoimmune inflammation mediated by IL-17-producing helper T cells (Th-17 cells) and has been linked to many human immune disorders including spondyloarthopathy and inflammatory bowel disease. Indeed, polymorphisms of Il23R, JAK2 and STAT3 are linked with these diseases. Bechet's disease is a disease of unknown etiology characterized by recurrent inflammatory attacks affecting the orogenital mucosa, eyes and skin. We collaborated to perform a genome-wide association study on individuals with Bechet's disease and identified an association with polymorphisms of the IL10 and the IL23R genes. The disease-associated IL10 variant was associated with diminished expression. To better understand how Th17 cells arise and contribute to autoimmunity, we revisited factors that drive Th17 differentiation. Although crucial for Th17 cells in vivo, IL-23 has been thought to be incapable of driving initial differentiation. Rather, IL-6 and transforming growth factor (TGF)-β1 have been proposed to be the factors responsible for initiating specification. However, we found that Th17 differentiation can occur in the absence of TGF-βsignaling. Neither IL-6 nor IL-23 alone efficiently generated Th17 cells;however, these cytokines in combination with IL-1βeffectively induced IL-17 production in naive precursors, independently of TGF-β. We found that epigenetic modification of the Il17a, Il17f and Rorc promoters proceeded without TGF-β1, allowing the generation of cells that co-expressed RORγt (encoded by Rorc) and T-bet. T-bet+RORγt+ Th17 cells are generated in vivo during experimental allergic encephalomyelitis, and adoptively transferred Th17 cells generated with IL-23 without TGF-β1 were pathogenic in this disease model and re-emphasize the importance of IL-23. Interleukin 2 (IL-2), a cytokine linked to human autoimmune disease, which we found to limit IL-17 production. We found that deletion of the gene encoding STAT3 in T cells abrogated IL-17 production and attenuated autoimmunity associated with IL-2 deficiency. In dissecting the mechanism of IL-2s ability to inhibit IL-17, we found that whereas STAT3 induced IL-17 and the transcription factor RORγt and inhibited the transcription factor Foxp3, IL-2 inhibited IL-17 independently of Foxp3 and RORγt. Using Chip-seq technology, we found that STAT3 and STAT5 bound to multiple common sites across the locus encoding IL-17. The induction of STAT5 binding by IL-2 was associated with less binding of STAT3 at these sites and the inhibition of associated active epigenetic marks. 'Titration'of the relative activation of STAT3 and STAT5 modulated the specification of cells to the IL-17-producing helper T cell (T(H)17 cell) subset. Thus, the balance rather than the absolute magnitude of these signals determined the propensity of cells to make a key inflammatory cytokine. This is consistent with recent work showing that Treg cells can promote Th17 differentiation by serving as sinks for IL-2. To gain further insight into the molecular basis of helper T cell differentiation, we have also initiated studies mapping genomewide chromatin modifications, examining the role of Stats in the process. Specifically, we analyzed histone modifications in Th1, Th2, Th17, and Treg cells by Chip-Seq technology. We have also assessed the importance of Stat4 and STAT6 in chromatin modifications in Th1 and Th2 cells Using chromatin immunoprecipitation and massive parallel sequencing, we quantitated the full complement of STAT-bound genes, concurrently assessing global STAT-dependent epigenetic modifications and gene transcription by using cells from cognate STAT-deficient mice. STAT4 and STAT6 each bound over 4000 genes with distinct binding motifs. Both played critical roles in maintaining chromatin configuration and transcription of a core subset of genes through the combination of different epigenetic patterns. Globally, STAT4 had a more dominant role in promoting active epigenetic marks, whereas STAT6 had a more prominent role in antagonizing repressive marks. Clusters of genes negatively regulated by STATs were also identified, highlighting previously unappreciated repressive roles of STATs. Therefore, STAT4 and STAT6 play wide regulatory roles in T helper cell specification. In related work, we applied miRNA-, mRNA-, and ChIP-Seq to characterize the microRNome during lymphopoiesis within the context of the transcriptome and epigenome. One action of IL-17 is to induce production of neutrophils and to promote their recruitment to sites of inflammation. Another cytokine, granulocyte macrophage-colony stimulating factor (GM-CSF) controls homeostatic and emergency development of granulocytes. We collaborated to analyze mice with deletion of both Stat5a/b genes and found that it is a key regulator of the CCN3/NOV gene, which is a positive regulator of hematopoietic cell self-renewal and neutrophil differentiation.

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National Institute of Arthritis and Musculoskeletal and Skin Diseases
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