Early synovial B-cell infiltration as a candidate mechanism of pathogenesis in the OA of obesity

Abstract

Purpose: Obesity is a major risk factor for the development of osteoarthritis (OA), the greatest cause of disability in the US. With the incidence of both obesity and OA on the rise, the comorbid disease burden is only worsening, yet there are no disease modifying therapies approved for the use in OA to date. Recent studies, from our lab and others, have discovered that synovial inflammation and immune cell infiltration are associated the accelerated progression of OA in obese mice and humans. These findings led us to hypothesize that obesity-induced pathobiological change in the synovium is the seminal driver of accelerated OA degeneration of joints. To test this hypothesis, we performed a transcriptomic analysis of synovial tissue from lean and obese mice, at a time point well before any histologic joint degeneration is evident. Methods: Male C57BL/6J mice were fed a low fat or high fat diet (HF) for 16 weeks, at which point the synovial membrane was removed for RNA isolation. RNA samples were then sequenced using Illumina’s HiSeq2500v4, and the next generation sequencing (NGS) data were processed and aligned. Following alignment, the significantly differentially expressed genes (DEG) were evaluated for cell type enrichment using CTen, as well as enriched canonical pathways and diseases and functions using Ingenuity Pathway Analysis (IPA). To analyze the various cell populations in the synovial membrane, we performed flow cytometry on cells isolated from lean and obese synovium. Results: Before any histologic evidence of cartilage degeneration, significant pro-inflammatory changes were observed in the obese synovium, suggesting these changes may be crucial to disease pathogenesis. Cell type enrichment analysis identified the macrophage as a significant mediator of early synovial pathogenic change (Fig. 1a). Correlating with this, pathway analysis of RNAseq data revealed significant induction of numerous inflammatory pathways including activation of NF-κB, stimulation of macrophages, and inflammation of the joint (Fig. 1b). Additionally, we discovered a potentially novel mediator of the early pathogenesis of the OA of obesity; the B-cell. Cell type enrichment analysis identified a significant B-cell signature in the obese synovial membranes (Fig. 1a). IPA analysis provides further evidence for the role of the B-cell, as pathways related to the B-cell including B-cell receptor signaling, quantity of B lymphocytes (Fig. 1c, d), and PI3K signaling in B lymphocytes were significantly activated. To confirm the presence of the B-cell in the synovial membrane, we performed flow cytometry on cells isolated from lean and obese synovium. This analysis identified an increased population of B-cells residing in the synovial membrane of obese mice, providing further evidence for the transcriptome analysis (Fig. 1 e, f). Conclusions: Significantly, this study is the first direct evidence that synovial disease occurs long before discernable joint degeneration, supporting the concept that the OA of obesity is preceded by a disease state in the synovial membrane. Transcriptome analysis identified a significant inflammatory change in the synovium of obese mice, with the macrophage being recognized as a prominent mediator of disease. Additionally, both cell type enrichment analysis and pathway analysis identified the B-cell as a potentially novel mediator of the OA of obesity. These findings warrant deeper study of the B-cell in the OA of obesity, where we hypothesize it plays a role as an antigen presenting cell for activation of other immune cells which in aggregate are responsible for supporting a proinflammatory environment leading to joint degeneration.