The role of miR-574-5p in the pathogenesis of rheumatoid arthritis

Abstract

Rheumatoid arthritis (RA) is a common chronic inflammatory joint disease, which leads to cartilage and bone damage as well as disability. To date, the complex aetiology of the disease is not completely understood. RA is characterized by swelling of the joints and infiltration of a variety of immune cells. The inflammation is mainly mediated by the communication of the cells within the affected joints via cytokines and other proinflammatory mediators. The discovery of small extracellular vesicles (sEVs) and their ability to mediate cell-to-cell communication has gained the interest of many research groups in the last decade. sEVs transport their cargo from delivering to recipient cells which has been demonstrated to play major roles in the pathogenesis of several diseases. The focus of sEV research was thereby mainly their involvement in cancer progression and the pathogenesis of inflammatory diseases such as RA. Progressive bone destruction by osteoclasts in the joints affected by RA may be the most severe alteration, as it is irreversible and can lead to complete loss of joint function. Therefore, the main objective of this study was to reveal new insights on the impact of sEV-delivered micro RNAs (miRs) in the context of osteoclast differentiation. MiRs are small non-coding RNAs which are one of the most important post-transcriptional repressors. However, alternative functions of miRs as post-transcriptional activators of gene expression and as ligands for endosomal ssRNA receptors like Toll-like receptor (TLR) 7 or TLR8 have been demonstrated recently. TLRs are receptors of the innate immune system. Their signalling leads to the translocation of the transcription factor kappa-light-chain-enhancer of activated B-cells (NF-κB) and increased transcription of target genes. In this thesis it was revealed that sEVs isolated from the synovial fluid of RA patients induce osteoclast differentiation in vitro in a time- and concentration dependent manner. Analysis of miR content revealed a high abundance of miR-574-5p in these sEVs in comparison to other miRs, such as miR-146a-5p or miR-155-5p. Synovial fibroblasts (SFs), which represent cells in synovial joints that play a major role in the progression of the disease, were identified as one of the sources of extracellular miR 574-5p present in the synovial fluid. In order to study the role of sEV-delivered miR-574 5p on osteoclastogenesis an overexpression (oe) tool was created, which provides an enrichment of miR-574-5p in sEVs. Stimulation of monocytic osteoclast precursor cells revealed that miR-574-5p oe sEVs induced osteoclast differentiation in a time dependent manner. This effect could be addressed to an activation of TLR7 and TLR8. To proof the binding of miR-574-5p to TLR8 a microscale thermophoresis assay (MST) was performed, which revealed a strong binding with a KD of 30.8 ± 5.2 nM. Furthermore, the inhibition of TLR7 and TLR8 abrogated the impact of miR 574 5p on osteoclast formation, indicating that this effect is mediated by TLR7/8 activation. Comparable results to those of the miR-574-5p oe sEVs were obtained with the synthetic TLR7/8 ligand Resiqiumod (R848). Here also a concentration- and time dependent effect on osteoclastogenesis was observed, supporting the general hypothesis that TLR7/8 activation can lead to an enhanced osteoclastogenesis. In addition, it was shown that the mRNA levels of several TLR7/8 target genes, such as interleukin 23 (IL-23), interferon alpha (IFNα) and microsomal prostaglandin E synthase-1 (mPGES-1) were upregulated in response to miR-574-5p oe sEVs. These effects could also be prevented by the inhibition of TLR7 and TLR8. Overall, it was demonstrated in this thesis that sEV-delivered miR-574-5p enhances osteoclastogenesis by activating TLR7 and TLR8 in the context of RA. These findings provide the opportunity for novel treatment approaches for RA in patients in the future by targeting this mechanism.