Background/Purpose: Joint destructions progress throughout the course of rheumatoid arthritis (RA) without complete remission. Optineurin (OPTN) is an autophagy receptor with multiple functions related to homeostasis of cells and has been reported to negatively regulate osteoclast differentiation in vivo. However, the role of OPTN in RA, especially in joint destruction, has been unclear. In this study, we clarify the role of OPTN in the pathogenesis of joint destructions in RA using synovial fibroblasts (RASFs).

Methods: RASFs were obtained from RA patients who fulfilled the ACR 2010 criteria during the knee replacement surgery. RASFs with passages of 4 to 8 were used in this study. RASFs were incubated with pro-inflammatory cytokines and the expression of OPTN was analyzed using quantitative realtime PCR (RT-qPCR) and western blot. RASFs were transfected with siRNA targeting OPTN and the downregulated efficacy was confirmed by RT-qPCR and western blot. The expression of RANKL and osteoprotegerin (OPG) on OPTN-reduced RASFs were analyzed by flow cytometry and RT-qPCR after treatment with TNF-a or IFN-g. CD14+ monocytes isolated from healthy subjects were cocultured with OPTN-reduced RASFs or control RASFs in the presence of M-CSF. RANKL+M-CSF added in cocultured cells were used as positive control. Cocultured cells were stained with Tartrate-Resistant Acid Phosphatase (TRAP). TRAP+ cells with number of nuclei≥3 were considered as osteoclasts. Protein levels of IkBa were analyzed to evaluate the activation of NF-kB signaling. RNA sequencing was performed to analyze further pro-inflammatory/pro-destructive genes regulated by OPTN knockdown. Data within a group were compared using Student’s unpaired t-test and data between groups were compared using the analysis of variance (GraphPad Prism). Differences were considered significant if p< 0.05.

Results: OPTN levels were upregulated after TNF-a or IFN-g stimulation at both mRNA and protein levels (n = 5, p< 0.05). The cell surface expression of RANKL was significantly increased following treatment with TNF-a or IFN-g and the effect was further pronounced in OPTN-reduced RASFs compared with that in control RASFs (n = 5, p< 0.05). The mRNA levels of RANKL were also increased in OPTN-reduced RASFs (n = 5, p< 0.05) while OPG levels remained unchanged. CD14+ monocytes cocultured with OPTN-reduced RASFs differentiated more into TRAP+ multinucleated cells (osteoclasts) compared to those cocultured with control RASFs (n = 4, p< 0.05). IkBa degradation following TNF-a treatment was prolonged in OPTN-reduced RASFs. RNA sequencing detected dysregulation of genes related to cartilage degradation, joint inflammation and bone formation, including MMP-3, CHST15, HAS1 and GATA-3, and the results were confirmed by RT-qPCR.

Conclusion: OPTN may play a protective role in RA with its upregulation when immersing with pro-inflammatory cytokines. Absence of OPTN might worsen RA by generating joint destructive state including increased RANKL expression on RASFs and subsequent osteoclast differentiation.

ACR figure

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/autophagy-receptor-optineurin-in-synovial-fibroblasts-plays-a-protective-role-against-joint-destruction-in-rheumatoid-arthritis/