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
BackgroundOsteoarthritis (OA) is a complex, multifactorial and heterogeneous joint disease of unknown etiology. OA research has largely focused on articular cartilage degeneration with little attention given to other joint tissues, including the synovium. The synovium lines the joint capsule, produces synovial fluid for lubrication, and is emerging as a contributor to OA pathogenesis. In OA, the synovium exhibits increased vascularization, inflammation, hyperplasia and fibrosis. Synovial cells that contribute to these pathological events during early and advanced stages of OA are not well characterized. The emergence of RNA sequencing (RNAseq) at the resolution of a single cell or nucleus allows for the identification of distinct cells that may contribute to OA pathogenesis.ObjectivesTo delineate the synovium’s role in OA pathogenesis we sought to identify if distinct cell subtypes exist in the synovium of early (KL1) versus late stages (KL3/4) of radiographic knee OA using single nucleus RNA sequencing.MethodsSynovia from patients with early (KL=I; n=5) and late (KL =III/IV; n=4) stage radiographic knee OA were subjected to single nucleus (sn)RNAseq and to bioinformatics analyses. Canonical cell-specific markers were used to identify cell types from the unsupervised clustering analysis and prominent cell types were re-clustered. Differentially expressed gene (DEG) lists between the subclusters were determined based on top gene expression within a cell type between early and late OA synovium. Cell surface markers identified from the DEGs were validated by immunohistochemistry. Pathway and gene ontology enrichment analysis were performed on fibroblast subclusters to identify prominent pathways and transcription factors that were upstream regulators. Ongoing in vivo and in vitro methods are being used to assess these transcription factors in both fibroblast cell culture and an OA mouse model.ResultsFibroblasts and macrophages constituted 75% of the cells from early and late-stage synovium and re-clustering analysis resolved 8 fibroblast and 6 transcriptionally distinct macrophage subclusters (Figure 1). Cluster based nuclei proportion differences identified fibroblast clusters 1, 2, 4 and 6 and macrophage clusters 1, 2 and 5 to contribute to early-stage samples while fibroblast clusters 0, 3 and 5 and macrophage clusters 0, 3 and 4 to late-stages. Downstream analyses focused on fibroblasts and putative cell surface markers from fibroblast subclusters were identified from DEGs and confirmed by immunohistochemistry. The fibroblast subclusters were subjected to pathway analyses which identified clusters 0 and 1 to be the most prominent clusters which both shared common ECM related pathways. Upstream transcription factors that regulate ECM related genes were identified for both subclusters 0 and 1. Current efforts are focussed on selecting and targeting transcription factor(s) for both in vitro and in vivo analyses by utilizing siRNA’s in fibroblast culture and a cre-lox mouse system to identify the mechanisms associated with the synovial pathology during OA.ConclusionSnRNAseq analysis identified distinct subclusters of fibroblasts and macrophages to exist in human OA knee synovia. Certain subclusters were more representative of the early stage while others were more representative of the late stage of the disease. Further validation studies are being performed to assess the functional roles of these subclusters and whether targeting them would attenuate disease progression.KT and EG share equal first author contribution.DE and Mk share equal senior author contribution.AcknowledgementsCanadian Institute of Health Research.Disclosure of InterestsNone Declared.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.