Op0101 mechanical Loading-Induced Bhlhe40 Promotes Inflammatory Arthritis

Abstract

BackgroundForce induced microdamage to joint tissue is hypothesized to trigger inflammatory events in the joint leading to arthritis. Patients with inflammatory arthritis, such as rheumatoid arthritis (RA) and spondyloarthritis (SpA), are found to have inflammation in “mechanical hotspots” and mechanical loading in mouse models of these diseases is pro-arthritogenic1,2. To date, the molecular mechanism involved in converting force to a biological signal that promotes arthritis is not known.ObjectivesThis study aims to identify stretch induced genes in synovial fibroblasts, and the effect of these “mechano-sensitive” genes on arthritis.MethodsHuman synovial fibroblasts were stretched in vitro for 4hrs using the FlexCell system and analysed by microarray. Top stretch induced genes were measured in RA, SpA and healthy synovial tissue by qPCR. Patient synovium was further analysed by immunohistochemistry. Bhlhe40 deficient mice were subjected to collagen induced arthritis (CIA) and KBxN serum transfer arthritis (STA). FACS was performed on ankle synovium. uCT was performed on whole ankles, with morphological changes scored by blinded readers, and calcaneus erosions by customs scripts in FIJI.Results600 genes were found to be differentially expressed in stretched synovial fibroblasts (fold change > +/-1.5, adjusted p<0.05). 25% of these genes were found to be transcription factors, which included BHLHE40. BHLHE40 mRNA was elevated in the synovial tissue of RA/SpA vs healthy subjects (1.56 fold change), and BHLHE40 protein was widely detectable in synovial fibroblasts and macrophages (Figure 1). Bhlhe40 deficient mice were completely protected against CIA (incidence: 0% vs 40%, n=30 per group), but Bhlhe40 did not block the generation of anti-collagen antibodies. Bhlhe40 deficient mice were partially protected against STA (peak clinical score at day 7; 5.2 vs 6.8, n=15 per group), with reduced synovial macrophage (CD11b+Ly6G-F4/80+) and neutrophil (CD11b+Ly6G+) frequency observed in the arthritic Bhlhe40 deficient mice compared to wildtype controls. Bhlhe40 had no impact on bone erosions with STA.Figure 1.BHLHE40 is widely expressed in human synovium. Synovium obtained from total knee replacement. FFPE samples were stained for synovial macrophages (HLADR+) and fibroblasts (FAP+). Images acquired with the Zeiss LSM 780.ConclusionBHLHE40 was identified as a force-induced gene in synovial fibroblasts and was found to be upregulated in patients with inflammatory arthritis. Importantly, Bhlhe40 strongly promotes joint inflammation in murine models of arthritis and uncouples systemic autoimmunity from joint tissue inflammation. Thus, we have identified BHLHE40 as a novel regulator of mechanical loading-associated inflammation.References[1]Cambré, I. et al. Mechanical strain determines the site-specific localization of inflammation and tissue damage in arthritis. Nat. Commun.9, 4613 (2018).[2]Jacques, P. et al. Proof of concept: enthesitis and new bone formation in spondyloarthritis are driven by mechanical strain and stromal cells. Ann. Rheum. Dis.73, 437–445 (2014).Disclosure of InterestsNone declared

Biomechanics in the onset and severity of spondyloarthritis: a force to be reckoned with

“…BHLHE40 strongly promoted joint inflammation in murine models of arthritis and uncoupled systemic autoimmunity from joint tissue inflammation. 14…”

“…The biological mechanism origin for triggering entheseal inflammation in SpA is consistent with a conceptual model which involves a threshold effect; a lower severity of stimulus may trigger, in a genetically susceptible individual, a substantially exaggerated response. 14 Entheseal microtrauma may perpetuate TNF and IL 17 overexpression and maintain enthesitis. 4 This leads to an overexuberant response contributing to ectopic bone formation and eventual ankylosis.…”