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MicroRNAs (miRNAs) constitute fine tuners of gene expression and are implicated in a variety of diseases spanning from inflammation to cancer. miRNA expression is deregulated in rheumatoid arthritis (RA), however, their specific role in key arthritogenic cells such as the synovial fibroblast (SF) remains elusive. We have shown in the past that the expression of the miR-221/222 cluster is upregulated in RA SFs. Here, we demonstrate that miR-221/222 activation is downstream of major inflammatory cytokines, such as TNF and IL-1β, which promote miR-221/222 expression independently. miR-221/222 expression in SFs from the huTNFtg mouse model of arthritis correlates with disease progression. Targeted transgenic overexpression of miR-221/222 in SFs of the huTNFtg mouse model led to further expansion of synovial fibroblasts and disease exacerbation. miR-221/222 overexpression altered the transcriptional profile of SFs igniting pathways involved in cell cycle progression and ECM regulation. Validated targets of miR-221/222 included p27 and p57 cell cycle inhibitors, as well as Smarca1 (a chromatin remodeling component). In contrast, complete genetic ablation of miR-221/222 in arthritic mice led to decreased proliferation of fibroblasts, reduced synovial expansion and attenuated disease. scATAC-seq data analysis revealed increased miR-221/222 gene activity in the pathogenic and activated clusters of the intermediate and lining compartment. Taken together, our results establish an SF-specific pathogenic role of the miR-221/222 cluster in arthritis and suggest that its therapeutic targeting in specific subpopulations should inform the design of novel fibroblast-targeted therapies for human disease.

Synovial fibroblasts (SFs) are specialized cells of the synovium that provide nutrients and lubricants for the maintenance of proper function of diarthrodial joints. Chronic TNF signals are known to trigger activation of SFs and orchestration of arthritic pathology via proinflammatory effector functions, secretion of cartilage degrading proteases and promotion of osteolysis. We performed single-cell (sc) profiling of SF’s transcriptome by RNA-sequencing (scRNA-seq) and of chromatin accessibility by scATAC-seq in normal mouse SFs and SFs derived from early and advanced TNF-driven arthritic disease. We describe here distinct subsets of SFs in the homeostatic synovium, serving diverse functions such as chondro- and osteogenesis, tissue repair and immune regulation. Strikingly, development of spontaneous arthritis by transgenic TNF overexpression primes the emergence of distinct pathology-associated SF subtypes. We reveal 7 constitutive and 2 disease-specific SF subtypes. The latter emerge in the early stage, expand in late disease and are localized in areas at the interface between the invasive pannus and the articular bone. The associated transcription profiles are characterized by enhanced inflammatory responses, promigratory behaviour, neovascularization and collagen metabolic processes. Temporal reconstruction of transcriptomic events indicated which specific sublining cells may function as progenitors at the root of trajectories leading to intermediate subpopulations and culminating to a destructive lining inflammatory identity. Integrated analysis of chromatin accessibility and transcription changes revealed key transcription factors such as Bach and Runx1 to drive arthritogenesis. Parallel analysis of human arthritic SF data showed highly conserved core regulatory and transcriptional programs between the two species. Therefore, our study dissects the dynamic SF landscape in TNF-mediated arthritis and sets the stage for future investigations that might address the functions of specific SF subpopulations to understand joint pathophysiology and combat chronic inflammatory and destructive arthritic diseases.

Background The transmembrane-TNF transgenic mouse, TgA86, has been shown to develop spontaneously peripheral arthritis with signs of axial involvement. To assess similarity to human spondyloarthritis, we performed detailed characterization of the axial, peripheral, and comorbid pathologies of this model. Methods TgA86 bone pathologies were assessed at different ages using CT imaging of the spine, tail vertebrae, and hind limbs and characterized in detail by histopathological and immunohistochemical analysis. Cardiac function was examined by echocardiography and electrocardiography and bone structural parameters by μCT analysis. The response of TgA86 mice to either early or late anti-TNF treatment was evaluated clinically, histopathologically, and by μCT analysis. Results TgA86 mice developed with 100% penetrance spontaneous axial and peripheral pathology which progressed with time and manifested as reduced body weight and body length, kyphosis, tail bendings, as well as swollen and distorted hind joints. Whole-body CT analysis at advanced ages revealed bone erosions of sacral and caudal vertebrae as well as of sacroiliac joints and hind limbs and, also, new ectopic bone formation and eventually vertebral fusion. The pathology of these mice highly resembled that of SpA patients, as it evolved through an early inflammatory phase, evident as enthesitis and synovitis in the affected joints, characterized by mesenchymal cell accumulation, and neutrophilic infiltration. Subsequently, regression of inflammation was accompanied by ectopic bone formation, leading to ankylosis. In addition, both systemic bone loss and comorbid heart valve pathology were evident. Importantly, early anti-TNF treatment, similar to clinical treatment protocols, significantly reduced the inflammatory phase of both the axial and peripheral pathology of TgA86 mice. Conclusions The TgA86 mice develop a spontaneous peripheral and axial biphasic pathology accompanied by comorbid heart valvular dysfunction and osteoporosis, overall reproducing the progression of pathognomonic features of human spondyloarthritis. Therefore, the TgA86 mouse represents a valuable model for deciphering the role of transmembrane TNF in the pathogenic mechanisms of spondyloarthritis and for assessing the efficacy of human therapeutics targeting different phases of the disease.