Background/Purpose: The aim of the study was to characterize the expression of TLR7 in rheumatoid arthritis (RA) peripheral blood (PB) cells and to examine the pathogenic role of TLR7 ligation in RA.

Methods: Expression of TLR7 was determined in RA and normal (NL) PB monocytes and in vitro differentiated macrophages by real-time RT-PCR and/or flow cytometry. Next the endogenous TLR7 ligand was identified in RA synovial fluid and its ability to induce monocyte migration was determined by in vitro chemotaxis.

Results: Since we have previously shown that expression of TLR7 was elevated in RA synovial lining and sublining macrophages, we asked whether expression of TLR7 was increased in RA PB and synovial fluid macrophages compared to NL PB monocytes and differentiated macrophages. We show that expression of TLR7 was elevated 18 and 24 fold in RA synovial fluid macrophages compared to RA and NL PB differentiated macrophages respectively by real-time RT-PCR. Levels of TLR7 were 6 and 3 fold higher in RA monocytes compared to RA differentiated macrophages and NL monocytes. Interestingly, mRNA and endosomal expression of TLR7 are reduced when RA PB monocytes differentiate to macrophages. Ligation of TLR7 by a synthetic agonist in RA blood monocytes is responsible for production of high levels of TNF-a (3 ng/ml) and as such we demonstrate that levels of TLR7 and TNF-a in 35 RA monocytes strongly correlate with each other (R²=0.44, p=1.37×10^-5) and disease activity score (TLR7 and DAS28 correlation; R²=0.67, p=1.57×10^-9) suggesting a pathogenic role for TLR7 ligation in RA disease. In light of elevated levels of TLR7 in RA synovial fluid macrophages, we looked for TLR7 endogenous ligands in RA synovial fluid. We discovered that single strand (ss)RNA extracted from RA synovial fluid is a potential TLR7 endogenous ligand, since blockade of TLR7 ligation by TLR7 antagonist in RA monocytes greatly downregulates synovial fluid ssRNA mediated TNF-a transcription. To determine whether TLR7 ligation affects cell trafficking in the RA joint, monocyte chemotaxis was examined in response to a synthetic agonist to TLR7. We show that TLR7 ligation was chemotactic for monocytes beginning at 0.1 ng/ml of TLR7 agonist. Next, studies were performed to determine if the TLR7 ligation affects RA synovial fluid mediated monocyte extravasation. We document that blockade of TLR7 ligation or degradation of synovial fluid ssRNA is equally effective in reducing synovial fluid induced monocyte trafficking and that the combined therapy does not have an enhanced effect suggesting that ligation of joint ssRNA to TLR7 modulates monocyte extravasation through an overlapping pathway and it further points out that RA synovial fluid ssRNA is a potential TLR7 ligand.

Conclusion: We identify, for the first time, TLR7 endogenous ligand in RA joint and we also document a novel role for TLR7 ligation in RA monocyte migration.

ACR Meeting Abstracts - https://acrabstracts.org/abstract/tlr7-ligation-contributes-to-monocyte-migration-in-rheumatoid-arthritis/