Background/Purpose: Cells of the monocyte/macrophage lineage are critical to RA pathogenesis: unravelling mechanisms underlying macrophage inflammatory gene expression should elucidate novel disease-associated pathways and thereby biomarkers and therapeutic targets. MicroRNA (miR) comprise small, non-coding RNA species that are key regulators of mammalian gene expression. We sought to define the expression and regulation of novel miR species that regulate RA macrophage biology.

Methods: Blood (PB; healthy controls & RA patients with defined treatment characteristics) or synovial fluid (SF; RA) CD14+ cells were purified using histopaque centrifugation & autoMACS bead separation. CD14⁺ cells were matured with M-CSF or GM-CSF for up to 7 days, or stimulated with various TLR ligands. For T cell -macrophage interaction, CD4⁺ T cells were cultured with TNF, IL-2, IL-6, prior to 24hr co-culture with M-CSF-matured CD14⁺ cells. miR expression and copy number was variously quantified in cells or tissues via TLDA, validatory qPCR & in situ hybridisation with specific primers and DIG or FITC labelled probes, respectively.

Results: Prior TLDA elicited several dysregulated miRs in RA SF compared to PB CD14⁺ cells, including miR-34a. Fold change and copy number PCR assays confirmed elevated miR-34a in RA SF cells (n=10; p<0.01). Crucially miR-34a was also elevated in PB CD14⁺ cells from biologic-resistant RA patients compared to cDMARD good responders or matched healthy controls (n=19-30), associating miR-34a expression with chronic, treatment-resistance. miR-34a expression correlated with disease activity assessed by swollen joint count. In situ hybridisation demonstrated elevated miR-34a expression in RA compared with non-inflammatory and inflammatory OA synovial tissues (n=6); double fluorescent staining confirmed expression in lining and sub-lining layer CD68+ macrophages, plus adjacent cells of FLS morphology. miR-34a was rapidly upregulated during monocyte maturation induced by adherence, M-CSF or GM-CSF, but was not further enhanced by addition of TLR ligands (LPS, CLO97, PAM3, PolyIC, CpG). Similarly co-culture with RA derived SF (10%) enhanced miR-34a expression in monocytes. Finally, cognate interactions between CD4+ T cells and macrophages further enhanced miR-34a expression in the latter cells.

Conclusion: miR-34a expression is elevated in RA SF macrophages and in PB monocytes of treatment resistant RA patients where it correlates with clinical disease activity measures. miR-34a is upregulated by the maturation rich synovial microenvironment and by interactions with activated T cells. Putative miR-34a molecular targets include NOTCH1 rendering it a plausible novel immune regulator in synovitis.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/mir-34a-in-rheumatoid-arthritis-characterisation-of-elevated-synovial-expression-and-association-with-treatment-resistance/