Background/Purpose:  CD163 is a hemoglobin scavenger receptor and innate pattern recognition receptor, and a marker of activated monocytes and macrophages. It is also expressed on monocytes from children with active systemic juvenile idiopathic arthritis, and associated with emergence of macrophage activation syndrome. However, the regulation of CD163 expression during monocyte and macrophage polarization is poorly understood. MicroRNA post-transcriptionally regulate gene expression, often serving as feedback loops to “fine tune” gene expression programs. We hypothesize that microRNA dysregulated in active SJIA modulate expression of CD163 in polarized macrophage populations

Methods:  Human THP-1 monocytic cells and primary monocytes and monocyte-derived macrophages from healthy donors were polarized in vitro. CD163 expression was determined by quantitative RT-PCR or on a single-cell level by RNA flow cytometry using the PrimeFlow kit. Cells were also transfected with specific microRNA mimics or antagomirs to miR-125a-5p, miR-181a or miR-181c. Direct binding of miR-181 family members to the CD163 3’ untranslated region (UTR) was determined using a luciferase reporter system and through RNA immunoprecipitation (RIP). RNA-sequencing was used to identify differentially regulated genes in macrophages overexpressing miR-125a-5p or control mimics.

Results:  Significantly increased CD163 mRNA levels were detected only in monocytes and macrophages polarized towards M2c conditions by IL-10. Similarly, single-cell analysis showed markedly increased CD163 surface protein and mRNA levels only with IL-10 treatment. Several candidate microRNA which are overexpressed in monocytes from children with active systemic JIA may regulate CD163. Here, we find that overexpression of miR-125a-5p and miR-181c significantly reduced CD163 mRNA expression in IL-10 polarized macrophages. In vitro, both miR-125a and miR-181 family members were elevated in polarizing conditions that restrict CD163 expression, and inhibition of these with antagomirs increased CD163 mRNA levels. Interestingly, these microRNAs regulated CD163 expression through distinct mechanisms. Computationally, miR-181 is predicted to bind directly to the 3’ UTR of CD163 mRNA. Using both a luciferase reporter system and RIP assay we found direct interaction between miR-181 family members and the CD163 mRNA. In contrast, CD163 does not have a predicted miR-125a binding site. RNA-seq based genome wide target analysis identified “Cytokine-cytokine receptor interactions” as the most significantly repressed gene pathway upon miR-125a overexpression, and specifically decreased levels of IL10RA and TGFbR2. This finding suggests that miR-125a exerts global effects on macrophages to modulate polarization.

Conclusion:  Taken together, these data show that microRNAs regulate the IL-10-induced expression of CD163 in human macrophages through distinct mechanisms, directly targeting of CD163 mRNA and indirectly inhibiting cytokine receptor expression. These findings highlight the diverse ways microRNA regulatory networks affect macrophage polarization and phenotype in active SJIA.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/microrna-associated-with-active-systemic-juvenile-idiopathic-arthritis-regulate-cd163-expression-in-polarized-macrophages-through-two-distinct-mechanisms/