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. Understanding the molecular basis of cytokine action provides important insights into the pathogenesis of immune-mediated disease and offers new therapeutic targets. 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 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 showed 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. 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 proliferation and survival. 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. We restricted deficiency in the IL-23 receptor to defined cell populations in vivo to investigate the requirement for IL-23 signaling in the development and function of T(H)-17 cells in autoimmunity, inflammation and infection. In the absence of IL-23, Th-17 development was stalled at the early activation stage. This was associated with less proliferation;consequently, fewer effector Th-17 cells were produced in the lymph nodes and hence available to emigrate to the bloodstream and tissues Behet'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 Behet's disease. We identified an association with polymorphisms of the IL10 and the IL23R genes. The disease-associated IL10 variant was associated with diminished expression. Another factor that regulates IL-17 production is IL-1, which is regulated by the inflammasome. Mutations of one component of the inflammasome, NLRP3, underlie the diseases Neonatal Onset Multisystem Disease, Muckle-Wells and Familial Cold Autoinflammatory syndrome. (cryopyrinopathies). In effort to generate a mouse model of these cryopyrinopathy disorders, mice with mutations of NLRP3 were created. Like patients with mice with NLRP3 mutations have severe, overwhelming inflammatory disease. This disease is characterized by exagerated Th17 differentiation;however, the disease also occurs in the absence of adaptive immune cells. In a separate study, we also searched for sources of IL-17 and IL-22 in innate immune cells and found that lymphoid tissue inducer cells are important producers of these cytokines. To better understand the process 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. In another study, we collaborated to show that deletion of the kinase Lyn in myeloid cells resulted in enhanced Th2 differentiation that was driven by increased IL-4 production by basophils. Neutrophils play a vital role in the immune defense, as evidenced by the severity of neutropenia causing life-threatening infections. Granulocyte macrophage-colony stimulating factor (GM-CSF) controls homeostatic and emergency development of granulocytes. However, little is known about the contribution of the downstream mediating transcription factors signal transducer and activator of transcription 5A and 5B (STAT5A/B). To elucidate the function of this pathway, we generated mice with complete deletion of both Stat5a/b genes in hematopoietic cells. In homeostasis, peripheral neutrophils were markedly decreased in these animals. Moreover, during emergency situations, such as myelosuppression, Stat5a/b-mutant mice failed to produce enhanced levels of neutrophils and were unable to respond to GM-CSF. To investigate the target genes of STAT5A/B activated by cytokines in HSCs and progenitors, we performed microarray analyses using Lineage- Sca-1+ c-Kit+ (KSL) cells in the presence and absence of STAT5A/B. CCN3/NOV is a positive regulator of human HSC self-renewal and development of committed blood cells. Without stimulation, the Ccn3/Nov signal level was low in control KSL cells similar to Stat5a/b-null KSL cells. To determine which cytokine activates the Ccn3/Nov gene we analyzed Lineage- c-Kit+ (KL) and 32D cells using qPCR and ChIP assays. While stimulation with a cocktail lacking IL-3 prevented the induction of Ccn3/Nov in control KL cells, IL-3 alone could induce Ccn3/Nov mRNA in control KL and 32D cells. ChIP assays using 32D cells revealed IL-3-induced binding of STAT5A/B to a GAS site in the Ccn3/Nov gene promoter.
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