Environmental/genetic Risk Factors and Pathogenesis of Autoimmune Disease

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Multidisciplinary studies, including immunologic, pathologic, epidemiologic and molecular genetic investigations, are being used to complement findings in each area. Current studies are focusing on: exploring possible environmental risk and protective factors; identifying genetic risk and protective factors by candidate gene and whole genome SNP and sequencing analyses; defining the associations among clinical, laboratory and immunologic features of autoimmune diseases for diagnostic, prognostic and pathogenic purposes; and understanding differences in gene expression and proteomic patterns between monozygotic twins discordant for disease. Evaluation of exposures to silica, organic solvents and other xenobiotics, ultraviolet light, vaccinations, selected drugs and dietary supplements, hormones and pregnancy, tobacco smoke, stressful life events and infectious agents in the development of systemic autoimmune diseases are being conducted via a study of twins and close siblings discordant for systemic autoimmune disease. A group of poorly-understood, life-threatening autoimmune muscle diseases called the myositis syndromes or idiopathic inflammatory myopathies (IIM) are defined by chronic muscle inflammation and weakness and are associated with specific autoantibodies. The major forms of myositis are polymyositis (PM), in which multiple muscles are affected by inflammation, and dermatomyositis (DM), in which patients also develop skin inflammation. Yet there appear to be other types of myositis based on the clinical presentations, pathology and autoantibodies. We are studying both the adult (IIM) and juvenile (JIIM) forms of these diseases to understand possible differences in pathogenesis and risk factors. One area of investigation in which we have made recent advances involves identifying new genetic associations with juvenile and adult IIM. To accomplish this goal, we formed collaborations with investigators around the world, called the Myositis Genetic Consortium (MYOGEN). We performed a genome-wide association study (GWAS) of adult and juvenile myositis patients of European ancestry. Single-nucleotide polymorphisms showing strong associations (P<5 X 10-8) in GWAS were identified in the major histocompatibility complex (MHC) region, specifically the 8.1 ancestral haplotype (AH8.1), for all myositis, as well as for the four clinical and autoantibody phenotypes. Although the HLA DRB1*03:01 allele showed stronger associations with adult and juvenile DM, and HLA B*08:01 with PM and anti-Jo-1 autoantibody-positive myositis, multiple alleles of AH8.1 were required for full risk effects. Our findings establish that alleles of the AH8.1 comprise the primary genetic risk factors associated with the major myositis phenotypes in geographically diverse Caucasian populations. These studies have found that genes associated with other autoimmune diseases are also seen in myositis phenotypes. Additional HLA associations were seen with myositis specific autoantibodies in Immunochip studies, including HLA DRB1*03:01 associated with NT5c1A autoantibodies, DRB1*07:01 with anti-Mi-2 autoantibodies, and DQB1*02:02 with anti-TIF1 autoantibodies in adult DM, while DQB1*02:01 was associated with anti-TIF1 in children. Amino acid positions within certain HLA alleles also more strongly defined these HLA associations. We are continuing these investigations using Immunochip and other approaches and assessing additional phenotypes. We have examined environmental factors as risk factors for myositis and for flares of myositis. Tobacco smoking has been associated with clinical and autoantibody myositis phenotypes in Caucasians. Caucasian ever-smokers were more likely to have PM (adjusted odds ratio, OR=2.24), and anti-Jo-1 autoantibodies (adjusted OR=1.94) and less likely to have anti-p155/140 autoantibodies (adjusted OR=0.36). In Caucasians, ever-smokers with HLA-DRB1*03:01 allele had the highest odds of PM, ILD, and anti-Jo-1 autoantibodies. We have found through national surveillance data, NHANES, that the prevalence of a positive anti-nuclear autoantibody (ANA), as a surrogate for autoimmune disease, has risen in recent years: 11.0% of the population sample had a positive ANA in 1998-1991, and 15.9% are positive in 2011-2012, corresponding to 22 million and 41 million affected individuals in the United States. ANA prevalence notably increased among adolescents ages 12-19 years, in older adults and non-Hispanic whites. Trends in obesity, smoking and alcohol usage did not explain the rise in ANA prevalence over this period of time. Further investigation is warranted to determine factors underlying these increases in ANA prevalence, which may enable the development of preventable measures for autoimmune diseases. We investigated the role of neutrophil extracellular traps (NETs) in the pathogenesis of myositis. NETs are important sources of modified autoantigens and molecules that stimulate the immune system. We found pathogenic neutrophil subsets (low-density granulocytes LDGs) and NETs were elevated in the blood of patients with myositis. LDGs from myositis patients had an enhanced ability to form NETs. LDGs and NETs correlated with myositis disease activity and muscle damage. Anti-MDA5 autoantibodies correlated with circulating and tissue NETs, and directly enhanced NET formation. An enhanced neutrophil gene signature was present in myositis muscle tissue and was associated with muscle injury and interferon (IFN) gene expression, an important cytokine pathway activated in patients with DM and PM. NETs from myositis patients decreased the viability of muscle cells in the test tube. These data suggest that dysregulated neutrophil pathways may have a role in the disease process in myositis patients through their ability to directly injure muscle cells and other affected tissues. We also investigated muscle and peripheral blood IFN gene signature:in adult myositis, type I interferon (IFN) gene expression was highest in DM muscle tissue, moderate in anti-synthetase syndrome,and lowest in inclusion body myositis. Type 2 IFN gene expression was high in DM biopsies, inclusion body myositis and anti-synthetase syndrome, but low in immune-mediated necrotizing myopathy. We also examined peripheral blood IFN in patients with JDM compared to some of the Mendelian autoinflammatory interferonopathies, CANDLE and SAVI, which are caused by genetic mutations and have extremely elevated IFN signatures thought to drive pathology. We found the peripheral blood IFN gene score was significantly higher in JDM patients than in healthy controls, but lower than in CANDLE or SAVI, and the contributing genes of the IFN gene score in JDM overlapped more with SAVI than CANDLE. IFN gene score also correlated moderately with JDM disease activity measures and more strongly with skin activity. Our findings demonstrate peripheral IFN gene expression in JDM overlaps with monogenic interferonopathies, reflecting in both conditions a shared pathway driven by IFN-beta, and indicates potential therapeutic targets for JDM. Examining muscle biopsies for unique transcriptional signatures, we found CAMK1G, EGR3, and CXCL8 are highest expressed in the affected muscle tissue of patients with anti-synthetase syndrome and not in DM, while APOA4 is highest pressed in anti-HMGCR myopathy and MADCAM1 in the biopsies of patients with anti-Mi-2. These data suggest different pathologic mechanisms underlie each of the myositis- specific autoantibodies.