Development of Classification Criteria for Juvenile Axial Spondyloarthritis and Treatment Recommendations for Axial Spondyloarthritis in Adults In collaboration with Pam Weiss, we worked to develop and validate a disease activity index for juvenile SpA (JSpADA). The JSpADA index discriminates between subjects with active versus inactive disease and is responsive to worsening in disease activity over time. Prospective validation studies are planned. In addition, our collaboration continues with the planned development of classification criteria that identify children with JSpA who have axial arthritis (sacroiliitis). This is an international effort engaging pediatric rheumatology and criteria development experts from the American College of Rheumatology (ACR) and the European League Against Rheumatism (EULAR). We contributed to a large ACR-sponsored project to provide evidence-based recommendations for the treatment of adult patients with ankylosing spondylitis (AS) and nonradiographic axial spondyloarthritis. These recommendations serve as guidelines for managing common clinical situations, although additional work is needed to establish best practices for treatment over time and monitoring disease progression. The Microbiome and Spondyloarthritis Considerable evidence suggests gut microbiota play a role in the development and severity of spondyloarthritis. We demonstrated previously that HLA-B27 and human 2-microglobulin (h2m) affect the gut microbiome in an animal model of human SpA. This preliminary work provided the basis for an in-depth study asking whether specific microbial communities are altered by HLA-B27 and are linked to the development and severity of colitis in this animal model. We examined effects of HLA-B27 on 3 genetic backgrounds that alter the penetrance of disease, from the complete absence of inflammation to severe early onset disease, and have surprising results linking the effects of different microbial communities to development of SpA in a background/environment specific fashion. Preliminary results have been presented at rheumatology and microbiome meetings. These studies will enable us to build and test hypotheses to understand how normal commensal microbiota influence SpA pathogenesis. Functional Genomics of ERAP1 To evaluate ERAP1-HLA-B27 interactions, ERAP1 expression was knocked down in monocytic cells. This led to the accumulation of aberrant forms of HLA-B27 on the cell surface, including complexes with long peptides as well as properly folded heavy chain-peptide-2m complexes. In contrast, no differences in the accumulation of other HLA-B alleles (B51 and B18) were observed. These results suggest that normal levels and/or activity of ERAP1 are necessary to maintain the integrity of cell surface HLA-B27 complexes in monocytes, and that certain alleles may be more susceptible to the effects of ERAP1. To further assess ERAP1-HLA-B27 functional interactions in disease, we produced gene-edited rats that lack ERAP1 expression. Cohorts of HLA-B27 transgenic (B27-Tg), HLA-B7 transgenic (B7-Tg), and wild type rats, with (ERAP1+/+, ERAP1+/-) and without (ERAP1-/-) ERAP1 were generated and monitored over 6 months to determine the frequency and severity of gastrointestinal inflammation and arthritis. Preliminary results show that ERAP1 deficiency confers protection from the development of arthritis in B27-Tg rats, while the clinical severity of gastrointestinal inflammation was unaffected although histology scores were increased slightly. Effects of ERAP1 deficiency on normal and abnormal features of HLA-B27 and their consequences are also being evaluated. These experiments suggest that ERAP1 loss-of-function is protective for arthritis in SpA, and are expected to shed light on mechanisms by which HLA-B27 contributes to disease pathogenesis. Preliminary results have been published in abstract form and have been presented at national and international meetings. Induced Pluripotent Stem Cells Aberrant bone formation in AS is poorly understood, but involves cell types that are not readily accessible for study. To circumvent this obstacle a major goal of our research has been to develop induced pluripotent stem cells (iPSCs) that can be differentiated into mesenchymal stem cells (MSCs) and their derivatives including bone-forming osteoblasts. We have demonstrated the feasibility of reprogramming patient fibroblasts into iPSCs using non-integrating Sendai virus-encoded factors, and have differentiated these cells into multiple disease-relevant cell lineages including MSCs, mineralizing osteoblasts, adipocytes, and myeloid cells. Genome-wide gene expression analysis shows that the expression of several AS risk genes is enriched in MSCs, with some also prominent in iPSCs, supporting the utility of this approach. The ability to generate iPSCs provides a powerful tool to explore the functional genomics of AS risk genes that may impact bone formation and other aspects of disease pathogenesis. Mechanisms of IL-1 Production Mutations in the NLRP3 inflammasome cause a group of autoinflammatory diseases known as cryopyrin-associated periodic syndromes (CAPS), the most severe form of which is NOMID. Inappropriate overproduction of IL-1 is a key pathogenic mechanism. We identified the predominant cell type (CD14hi/CD16low) responsible for IL-1 overproduction in peripheral blood in patients with NOMID, and demonstrated that these cells die rapidly following stimulation in a process known as pyronecrosis, a pro-inflammatory form of cell death. Current studies suggest that IL-1 release and cell death are dependent on mitochondrial STAT3, which appears to alter the mitochondrial membrane potential. Genetic Contributions to Juvenile Arthritis MyD88 is a critical adaptor protein for TLR and IL-1 receptor signaling. Germline loss-of-function mutations in MyD88 cause severe immunodeficiency, while somatic gain-of-function mutations have been linked to certain lymphomas. We discovered a de novo germline MYD88 mutation in a child with early onset destructive polyarticular juvenile idiopathic arthritis (JIA). Cells from the patient overexpress chemokines and cytokines at baseline, and fibroblasts hyper-respond to IL-1. Culture supernatants exhibit strikingly enhanced neutrophil chemotactic activity. The MyD88 mutation is sufficient to increase NF-B activity and cause overexpression of chemokines and cytokines in monocytic cells. Thus, this germline mutation produces gain-of-function effects in MyD88 in hematopoietic as well as non-hematopoietic cells that are likely to contribute to the development of destructive arthritis. This work has been presented in abstract form at a national meeting, and studies are underway to understand how the mutation leads to a complex arthritis phenotype. Nevertheless, these results support a role for single gene defects contributing to the pathogenesis of JIA.