Early Disease Characteristics of ERA Reveals Elevated TGF-beta1 We have preliminary data describing clinical and laboratory findings in an inception cohort of ERA subjects. Thirty-seven subjects satisfying the ILAR criteria for ERA from 5 pediatric rheumatology centers were studied at presentation. Clinical features at baseline are similar to comparison cohorts derived from the literature and from a larger inception cohort we defined using the CARRAnet Registry. Median time from disease onset was 5.4 months;patients were predominantly male (78%) with a median age of 12 years. HLA-B27 was present in 59%, ANA in 25%. Markers of inflammation (ESR/CRP) were not elevated. The median active joint count was 1.0 and entheses count 4, with 78% of subjects having enthesitis. Only 1 patient had x-ray evidence of sacroiliitis and 1 had acute anterior uveitis. The most notable cytokine abnormality was an elevation in serum TGF-beta1 (p=0.0075), while IL-22 and GM-CSF were decreased (p<0.04), relative to a cohort of healthy controls. Other Th17 and Th1 cytokines were no different between patients and controls. TGF-beta1 levels were higher in patients with a history of gastrointestinal (GI) complaints compared to both healthy controls and patients without GI symptoms. Consistent with TGF-beta1 elevation, our previous studies have shown evidence of a TGF-beta1 gene expression signature in the peripheral blood of these subjects. This study informs us on early disease characteristics of ERA, and identifies TGF-beta1 as a potential marker not previously reported in ERA. Other reports of elevated TGF-beta1 in chronic gut inflammation, together with recent evidence for elevated fecal calprotectin in ERA, emphasize the need to consider the microbiome and GI abnormalities in juvenile spondyloarthritis. Worse Pain and Health Status in ERA In collaboration with Pam Weiss (CHOP), we have a manuscript in press reporting that children with ERA have with higher pain intensity and poorer health status compared to children with other forms of JIA. Dr. Weiss used the CARRAnet Registry to compare 2,571 subjects with various forms of JIA. Multivariate analyses showed that a higher active joint count, current NSAID, biologic or corticosteroid use were associated with worse scores on all patient-reported measures. ERA and older age were associated with higher pain intensity and poorer health status, and enthesitis, sacroiliac tenderness, and use of NSAIDs were associated independently with pain intensity in ERA. These results suggest that current treatments for various forms of JIA may not be equally effective for the manifestations of ERA such as enthesitis and sacroiliac tenderness. Whole Body MRI and Enthesitis in Spondyloarthritis The degree to which clinical enthesitis, an important characteristic of SpA, correlates with inflammation remains unknown. Thirteen patients and four healthy controls enrolled in our spondyloarthritis natural history protocol had a complete physical exam and whole body (WB) MRI (STIR) without contrast to assess clinical and imaging evidence of enthesitis, respectively. Patients met ILAR criteria for ERA (62% of subjects <16 years of age) or PsA (15% of subjects <16 years of age), ASAS criteria for axial SpA (46% of all subjects) or New York criteria for AS (1% or all subjects). All patients had at least one tender enthesis on exam, with a total of 108 clinically active entheses among the patients. There were four active entheses by exam in the healthy controls. Two patients and none of the controls had enhancement at entheseal sites on WB MRI, which included two greater trochanters and one anterior superior iliac crest. There was poor agreement between WB MRI and clinical exam when considering all entheseal sites (kappa = 0), or even specific sites (kappa range -0.23 to 0.24), and only one entheseal site was positive on both clinical exam and WB MRI. In healthy controls, all four entheses positive on exam were negative on WB MRI. The overall poor agreement between physical exam and imaging suggests that non-contrast WB MRI may have limited sensitivity to detect enthesitis in SpA. HLA-B27 Misfolding and ER Stress in the Transgenic Rat Model of SpA HLA-B27 misfolding generates ER stress and activates the unfolded protein response (UPR) in cells from HLA-B27 transgenic rats, which promotes the production of certain cytokines including IL-23 and IFN-β. SpA-like disease in these rats is strongly associated with expansion and activation of IL-23-responsive Th17 T cells in the colon, joints, and draining lymph nodes. We are currently using this model to evaluate the role of HLA-B27 misfolding as an upstream innate immune stimulus in causing this disease. Cell autonomous effects of HLA-B27 generated through misfolding and ER stress raise the possibility that it may contribute to the SpA phenoptype through downstream effects. For example, upregulation of HLA-B27 by interferons and TNF-αcan cause UPR activation, and may alter the biological response to these cytokines. We have begun to investigate potential effects on bone homeostasis in SpA, where bone loss is juxtaposed with abnormal bone formation. We have now shown that HLA-B27 promotes TNF-α-induced osteoclast (OC) development from rat monocytes 2.5 fold (P <0.05) compared to wild type (WT) rats or rats expressing HLA-B7 (and human β2m). This effect is specific, as RANKL-induced OC development is not affected. Increased production of IL-1alpha from HLA-B27-expressing cells is both necessary and sufficient to account for stimulatory effect. Interestingly, IFN-beta production is also increased, which serves to limit the stimulatory effect of IL-1alpha. IFN-beta neutralization increases the magnitude of the IL-1alpha effect on OC formation to over 3-fold. Additional data indicate that HLA-B27 misfolding and ER stress are responsible for generating this biological effect. Importantly, while the net effect under these conditions is pro-osteoclastogenic and may contribute to the bone loss seen in these rats, other factors that tip the IL-1alpha/IFN-beta balance could shift this to a net anti-osteoclastogenic effect. ERAP1 and the Immunobiology of HLA-B27 ERAP1 variants confer susceptibility to AS, but only in HLA-B27 positive individuals. ERAP1 is an ER aminopeptidase involved in trimming peptides that are presented by MHC class I molecules. Some data suggest that protective variants have reduced function, however, their role in pathogenesis and specifically their effect on HLA-B27 remain unexplored. We have established human and rat lentiviral shRNA constructs that knockdown ERAP1 expression. In a human monocyte/macrophage cell line (U937) transfected with a genomic HLA-B27 construct (U937-B27), we have found that ERAP1 knockdown (KD) to levels 30-50% of normal increases expression of folded HLA-B27 complexes and some loss of free heavy chain from the cell surface. However, ERAP1 KD attenuates the upregulation of cell surface HLA-B27 seen with IFN-gamma and TNF-alpha stimulation. HLA-B27 upregulation also leads to UPR activation and increases apoptosis, while ERAP1 KD limits apoptosis. Current studies are aimed at determining whether apoptosis is due to HLA-B27 misfolding and UPR activation, and how this is affected by ERAP1. Collaborative work with Granfors and Penttinen suggest that these cells have altered PKR/STAT1 signaling pathways that result from heavy chain misfolding, and that these cells may be useful to study adaptation to ER stress. Parallel studies will be performed in HLA-B27 transgenic rat cells and ultimately cells derived from SpA patients who express HLA-B27, to better understand the basis for the ERAP1-HLA-B27 gene-gene interaction in disease.

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Project End
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4
Fiscal Year
2012
Total Cost
$2,651,151
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Name
National Institute of Arthritis and Musculoskeletal and Skin Diseases
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