Abstract
Evidence links aryl hydrocarbon receptor (AHR) activation to rheumatoid arthritis (RA) pathogenesis, although results are inconsistent. AHR agonists inhibit pro-inflammatory cytokine expression in macrophages, pivotal cells in RA aetiopathogenesis, which hints at specific circuits that regulate the AHR pathway in RA macrophages. We compared microRNA (miR) expression in CD14+ cells from patients with active RA or with osteoarthritis (OA). Seven miR were downregulated and one (miR-223) upregulated in RA compared to OA cells. miR-223 upregulation correlated with reduced Notch3 and Notch effector expression in RA patients. Overexpression of the Notch-induced repressor HEY-1 and co-culture of healthy donor monocytes with Notch ligand-expressing cells showed direct Notch-mediated downregulation of miR-223. Bioinformatics predicted the AHR regulator ARNT (AHR nuclear translocator) as a miR-223 target. Pre-miR-223 overexpression silenced ARNT 3’UTR-driven reporter expression, reduced ARNT (but not AHR) protein levels and prevented AHR/ARNT-mediated inhibition of pro-inflammatory cytokine expression. miR-223 counteracted AHR/ARNT-induced Notch3 upregulation in monocytes. Levels of ARNT and of CYP1B1, an AHR/ARNT signalling effector, were reduced in RA compared to OA synovial tissue, which correlated with miR-223 levels. Our results associate Notch signalling to miR-223 downregulation in RA macrophages, and identify miR-223 as a negative regulator of the AHR/ARNT pathway through ARNT targeting.
Highlights
Targets; this causes their degradation or translation repression depending on the degree of base-pair complementarity between the miR and its target mRNA5
We found no differences in aryl hydrocarbon receptor (AHR) mRNA levels between OA and RA patients in our cohort (Supplementary Fig. S7b), which suggests that the functional impairment of the AHR/ ARNT pathway is a result of reduced ARNT protein levels
We combined gene expression and advanced bioinformatics analyses to demonstrate that (i) miR-223 is selectively upregulated in CD14+ cells from synovial fluid (SF) of RA patients with active disease; (ii) Notch activation represses miR-223 expression in myeloid cells; (iii) miR-223 targets ARNT, a necessary co-receptor for AHR-dependent transcriptional activity; (iv) miR-223 prevents AHR-mediated inhibition of pro-inflammatory cytokines; and (v) AHR target genes and ARNT protein itself are downmodulated in RA compared to OA synovial tissues
Summary
Targets; this causes their degradation or translation repression depending on the degree of base-pair complementarity between the miR and its target mRNA5. MiR participate in immune cell differentiation, maturation and function[6], and their aberrant expression is associated with pathogenesis in several osteoarticular diseases. The miR profile in RA patients differs from that in patients with OA9, a non-autoimmune disease characterised by low inflammation levels. The relevance of these miR in RA aetiopathogenesis is poorly understood. AHR stimulation inhibits TNF-α , IL-6 and IL-1β expression in lipopolysaccharide (LPS)-stimulated macrophages[21,22,23]. These disparate results hint at cell type-specific circuits that regulate the AHR/ARNT pathway in specific contexts. We analysed the role of miR-223 as a part of these regulatory circuits in RA-derived macrophages
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