Induced Pluripotent Stem Cells Reveal Ankylosing Spondylitis Risk Gene Expression in Bone Progenitors Aberrant bone formation in AS is poorly understood, and involves cell types that are not readily accessible for study. To circumvent this obstacle one goal of our research has been to develop patient-derived induced pluripotent stem cells (iPSCs) that can be differentiated into mesenchymal stem cells (MSCs) and their derivatives including bone-forming osteoblasts. We demonstrated the feasibility of reprogramming skin fibroblasts obtained from shave skin biopsies into iPSCs using non-integrating 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 using RNA-Seq 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. Preliminary studies suggest that osteoblasts derived from patients with AS mineralize to a greater extent than cells from healthy controls. Experiments designed to understand the mechanism underlying this effect are underway. 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. The Interaction Between HLA-B27 and ERAP1 in Spondyloarthritis Genetic studies point toward loss-of-function of ERAP1 as a protective factor for the development of AS in HLA-B27 positive individuals. The mechanism underlying this effect is not known. To further assess HLA-B27-ERAP1 interactions in disease, we produced a functional ERAP1 knockout in rats using genome editing. Preliminary results show that ERAP1 deficiency confers partial protection from the development of experimental spondyloarthritis caused by HLA-B27 expression in rats. ERAP1 deficiency reduced the prevalence of arthritis and orchitis by two thirds, while gastrointestinal inflammation was not reduced. ERAP1 deficiency increases presentation of longer peptides and also promotes the formation of disulfide-linked dimers of HLA-B27 on the cell surface. In contrast, ERAP1 deficiency reduces the accumulation of misfolded disulfide-linked dimers and oligomers in the endoplasmic reticulum (ER), and mitigates the development of ER stress. Overall, ERAP1 deficiency improves HLA-B27 folding and reduces misfolding, while it impairs the folding of another allele (HLA-B7). Current studies are focused on determining whether reduced ER stress and its consequences explain effects of ERAP1 deficiency on the spondyloarthritis phenotype, and whether this pathway can be targeted to reduce the incidence and severity of experimental spondyloarthritis in rats. The Role of Autophagy in the Degradation of Misfolded HLA-B27 Heavy Chains The propensity of HLA-B27 to misfold can generate cellular stress when sufficient heavy chains accumulate in the ER. Cells can eliminate misfolded heavy chains through a quality control pathway known as ER-associated degradation (ERAD), but this appears to be insufficient to prevent HLA-B27 from accumulating and generating ER stress in rat macrophages. We found that autophagy is also involved in the degradation of HLA-B27 heavy chains, although it is also insufficient to prevent ER stress. However, further augmentation of this pathway with the pharmacologic inhibitor of mTOR rapamycin promptly increases the degradation of misfolded HLA-B27. Moreover, ubiquitination of HLA-B27 heavy chains in the ER was inefficient compared to another allele (HLA-B7) that does not misfold, identifying this as a potential cause of inefficient ERAD. This work has been submitted for publication and is under revision. Genetic or pharmacologic manipulation of the autophagy pathway may be exploited to alleviate the consequences of HLA-B27 misfolding. The Microbiome and Spondyloarthritis Gut microbiota are thought to play a critical role in the development of spondyloarthritis. We reported previously that HLA-B27 expression alters the gut microbiome in a rat model of human spondyloarthritis. We asked whether specific microbial communities altered by HLA-B27 were linked with the development of colitis in rats with experimental spondyloarthritis. Surprisingly, we found that gut microbial communities altered by HLA-B27 were very different in 3 strains of rats housed in two different locations. Thus, genetic and environmental factors play a large role in determining effects of HLA-B27. These preliminary results have been presented at rheumatology and microbiome meetings and submitted for publication. We are currently investigating common functions of the diverse microbiota, and plan to build on these studies to better understand how commensal microbiota affect spondyloarthritis pathogenesis. Genetic Contributions to Early Onset Severe Juvenile Arthritis MyD88 is a critical adaptor protein for TLR and IL-1 receptor signaling. 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 destructive polyarticular juvenile arthritis, and have found that the mutation increases MyD88 oligomerization and NF-B activation. Cells expressing mutant MyD88 overexpress chemokines and cytokines, and some hyper-respond to IL-1. Culture supernatants exhibit strikingly enhanced neutrophil chemotactic activity. Thus, this germline MyD88 mutation produces gain-of-function effects in hematopoietic as well as non-hematopoietic cells that are likely to contribute to the development of arthritis. This work has been presented in abstract form at national meetings, and is under review for publication. Future studies will focus on how the mutation leads to a complex arthritis phenotype. These results support a role for single gene defects contributing to the pathogenesis of JIA.
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