Background/Purpose: Expression of Toll-like receptor 7 (TLR-7) is highly elevated in rheumatoid arthritis (RA) and osteoarthritis (OA) compared to normal (NL) synovial tissue lining and sublining macrophages. Interestingly in RA blood monocytes, TLR7 expression closely correlates with disease activity score (DAS28) and TNF-α transcription levels. We recently uncovered a novel endogenous TLR7 ligand; miR-Let7b, that is predominately packaged in RA synovial fluid macrophages. To document the impact of miR-Let7b ligation to TLR7 in RA pathogenesis, studies were performed using RA cells and preclinical models.

Methods: To understand the mechanism by which synovial fluid miR-Let7b promotes RA pathology, ligation of TLR7 was investigated on inflammatory response provoked by myeloid and T cells. Next, the impact of ectopic expression of miR-Let7b was examined in naïve mice and collagen induced arthritis (CIA) preclinical models.

Results: We found that TLR7 endogenous ligands, single strand (ss)RNAs, were undetectable in NL and RA plasma while these ligands were highly expressed in RA synovial fluid (SF). Consistent with the distribution of RA SF ssRNA, we show that miR-Let7b is markedly elevated in RA SF compared to OA SF (14 fold lower), RA (260 fold lower) and NL plasma (450 fold lower). We reveal that exosomes released from RA SF macrophages are an important source of miR-Let7b storage (78 copies; shown as 1 fold) and that cell death mediated by apoptosis (660 copies, 8 fold) or necrosis (74820 copies, 950 fold) can further potentiate the discharge of exosomal miR-Let7b into RA SF. Moreover, we determined that stimulation with miR-Let7b or TLR7 agonist can markedly accentuate TNF-α and IL-6 transcription and production (50-100 fold increase respectively) in RA peripheral blood (PB) in vitro differentiated macrophages. In contrast, neither TLR7 agonist nor miR-Let7b stimulation had any effect on IL-10 transcription. We also found that in PB mononuclear cells (T cells plus myeloid cells), TH-17 cells are strongly polarized by TLR7 ligation, in part due to secretion of IL-6 and IL-1β from RA myeloid cells. We next show that ligation of TLR7 by local injection of miR-Let7b, results in significantly elevated joint inflammation. Consistently, when CIA mice were ectopically treated with miR-Let7b, joint swelling was markedly potentiated, while the control ankle circumference remained at a plateau phase. We uncovered that levels of monokines secreted from CIA ankle joints, including TNF-α, CCL2, CCL5 and IL-1β were exacerbated by local administration of miR-Let7b in comparison to the control mice. Extending our observations in RA cells, we document that miR-Let7b treatment in CIA mice can amplify joint TH-17 cells/IL-17 differentiation. Our results indicate that RA disease activity is augmented by miR-Let7b ligation to TLR7 which activates effector myeloid and T cells function in these patients.

Conclusion: Overall our findings suggest that ligation of miR-Let7b to TLR7 perpetuates RA inflammation and blockade of this pathway may be an attractive target for RA therapy.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/impact-of-tlr7-ligation-in-ra-pathology/