Cytokine Signaling, Immunoregulation and Autoimmune and Immunodeficiency Diseases

National Institute Of Arthritis And Musculoskeletal And Skin Diseases

Investigators

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Abstract

Cytokines are secreted proteins that regulate immune and inflammatory responses, but also control hematopoiesis, metabolism, tissue repair, and growth and development of many tissues. As such, cytokines are essential for host defense against bacteria, viruses and fungi. They are also fundamental to the pathogenesis of autoimmune diseases such as rheumatoid arthritis, juvenile arthritis (JIA), Sjogren’s syndrome, systemic lupus erythematosus (SLE), inflammatory bowel disease (IBD), psoriasis and psoriatic arthritis (PsA), atopic dermatitis (AD), as well as asthma and allergies. In addition, cytokines are both products and regulators of lymphocytes. A major goal of the project is to understand mechanisms of cytokine signaling, the impact of these factors on lymphocytes and to better understand the regulation of disease-relevant cytokines. Our hope is that a better understanding of the molecular basis of cytokine action and cytokine regulation in lymphocytes will provide insights into the pathogenesis of immune-mediated disorders and continue to identify new therapeutic targets. This has been a highly successful strategy in our work over the last three decades. A major aspect of this project is the investigation of the function of cytokines that signal via a family of cytokine-activated DNA binding proteins called signal transducers and activators of transcription (STATs). We have made important insights into the role of STATs in lymphoid differentiation and regulation and have employed various cells from humans with Stat mutations and mice engineered with these mutations to elucidate the roles of these transcription factors in normal immune function and in models of infectious disease and autoimmunity. Our goal is to more deeply understand how cytokines exert their effect, mediate host defense against viruses and other pathogens, and provoke autoimmune and allergic disease by investigating the role of STAT family and other transcription factors in gene expression and epigenomic modifications. Our overarching goal is to link cell signaling, gene regulation, epigenetic control, and chromatin dynamics with mechanisms of human disease in hopes of improving therapeutic strategies. Summary Discovery of patient mutations of Janus kinase (JAKs) and STATs reveal new disease mechanisms, insights into pathophysiology, and opportunities for new therapeutics as we have generated in the past. NIH investigators have previously identified patients with loss-of-function mutations of STAT1 that exhibit viral and bacterial susceptibility; accordingly, we have generated mice with STAT loss of function variants. Mice lacking STAT1 in T cells control parasitic infection. However, a consequence of infection is lethal immunopathology associated with a type 1 (interferon gamma) response, reduced IL-10 generation and increased IL-13 production. Conversely, STAT1 activating mutations underlie heterogenous disorders including immunodeficiency and autoimmunity (including SLE, scleroderma and related disorders). To better understand immunopathogenic mechanisms of disease, we developed mice that express an activating STAT1 mutant in a tissue-selective, conditional manner. We found that these mice have increased susceptibility to viral infection, including SARS-CoV2. These mice exhibit a cytokine storm-like phenotype due to impaired function of STAT4 and regulation interferon gamma. We found that exogenous interferon gamma can reverse aspects of the pathology. Allergies and asthma are genetically linked but also associated with environmental factors. The type 2 cytokines, interleukin (IL)-4, IL-13 and IL-5, which are major factors that contribute to allergy and asthma reside within a multigene cluster. Both type 2 innate lymphoid (ILC2) and adaptive T helper 2 (TH2) lymphocytes selectively produce type 2 cytokines. Using transcription factor footprint and chromatin accessibility, we systemically catalogued regulatory elements (REs) denoted as SHS-I/II, KHS-I/II, +6.5kbIl13, 5HS-I(a, b, c, d, e), 5HS-II and 5HS-III(a, b, c) across the extended Il4-Il13-Il5 locus in mice. Physical proximities amongst REs were coordinately remodeled in three-dimensional space after cell activation, leading to divergent compartmentalization of Il4, Il13 and Il5 with varied combinations of REs. Deletions of REs revealed no single RE solely accounted for selective regulation of a given cytokine in vivo. Instead, individual RE differentially contribute to proper genomic positioning of REs and target genes. RE deletions resulted in context-dependent dysregulation of cytokine expression and immune response in tissue. Thus, signal-dependent remodeling of three-dimensional configuration underlies divergent cytokine outputs from the type 2 loci. These regulatory elements help explain both genetic contributions of disease and environmental stimuli. Other projects in our laboratory described in other reports help explain mechanisms that govern chromatin architecture regulated by CTCF, the cohesion complex and NIPBL. STAT6 (signal transducer and activator of transcription 6) is a critical transcription factor that plays a central role in the pathophysiology of allergic inflammation. We have identified multiple families with profound allergic immune dysregulation including: atopic dermatitis, gastrointestinal disease, asthma, food allergies, and anaphylaxis. These patients carry monoallelic rare activating STAT6 variants and sustained STAT6 phosphorylation, increased STAT6 target gene expression, and TH2 skewing. We have generated mice to better understand the evolution of disease, response to infection and other challenges and insights into immunopathologic mechanisms. Disabling pansclerotic morphea (DPM) is a rare systemic inflammatory disorder, characterized by poor wound healing, fibrosis, cytopenias, hypogammaglobulinemia, and squamous-cell carcinoma. The cause is unknown, and mortality is high. We identified patients with activating mutants of STAT4. Ruxolitinib, a JAK inhibitor is efficacious in treatment of these patients. We also generated mice with activating mutations of STAT4. STAT5 encompasses two genes, STAT5A and STAT5B. Our previous work established that STAT5 is a master regulator of energy and amino acid metabolism and is critical for hematopoiesis. We further established that STAT5 regulates the activity of key metabolically relevant biochemical pathways including AKT, mTOR and MYC. In collaboration with NIH investigators, we identified loss of function and activating mutations of STAT5 in cancer. The same tyrosine residue in the STAT5B SH2 domain was altered by two mutations, STAT5BY665H and STAT5BY665F. We generated mice expressing these mutant alleles with the STAT5BY665F allele resulting in accumulation of CD8+ effector and memory and CD4+ regulatory T cells. Conversely, the loss of function allele showed diminution of these populations. Discontinuous transcription is evolutionarily conserved and a fundamental feature of gene regulation; yet, the exact mechanisms underlying transcriptional bursting are unresolved. Analyses of bursting transcriptome-wide have focused on the role of cis-regulatory elements, but other factors that regulate this process remain elusive. We applied mathematical modeling to infer bursting dynamics transcriptome-wide and identified that Mediator complex subunit 26 (MED26) primarily regulates frequency, MYC regulates burst size, while cohesin and Bromodomain-containing protein 4 (BRD4) can modulate both. Notably, chemotherapeutic agents induce interferon signaling and transcriptional bursting and gene expression. Typically, IFN engages the non-canonical STAT1 homodimeric complex and the phosphorylated interferon stimulated gene factor complex (ISGF3). By contrast, the unphosphorylated ISGF3 contributes gene expression. Currently, we are also investigating patient-derived STAT variants to dissect the impact on transcriptional dynamics.