OP0012 Microrna Associated with Active Systemic Juvenile Idiopathic Arthritis Regulate CD163 Expression in Polarized Macrophages through Two Distinct Mechanisms
Authors: S. Thornton, T.D. Do, R. Tan, A. Sproles, M. Bennett, M. Medvedovic, M. DeLay, N. Shen, A. Grom, G. Schulert
Journal: Annals of the Rheumatic Diseases (2016)

<h3>Background</h3> CD163 is a hemoglobin scavenger receptor and serves as a marker of activated monocytes and macrophages. It is also expressed on monocytes from children with active systemic juvenile idiopathic arthritis, and highly enriched on hemophagocytic macrophages that characterize the emergence of macrophage activation syndrome. However, the regulation of CD163 expression during monocyte and macrophage polarization is poorly understood. MicroRNA are small non-coding RNA that post-transcriptionally regulate gene expression, often serving as feedback loops to "fine tune" gene expression programs. <h3>Objectives</h3> Determine microRNA regulation of CD163 at the protein and mRNA levels using novel flow cytometry techniques. <h3>Methods</h3> Human THP-1 monocytic cells and primary monocytes and monocyte-derived macrophages (MDMs) from healthy donors were polarized with activating conditions including M1 (LPS and IFNg), M2a (IL-4), M2b (LPS and immune complexes) and M2c (TGFb or IL-10). CD163 expression was determined by quantitative RT-PCR or on a single-cell level using RNA flow cytometry. Transcripts were detected using the PrimeFlow kit, with probes specific for CD163 mRNA and other cell surface markers. Cells were also transfected with specific microRNA mimics. <h3>Results</h3> Significantly increased CD163 mRNA levels were detected only in monocytes and macrophages polarized towards M2c by IL-10 treatment. Similarly, single-cell RNA analysis showed markedly increased CD163 mRNA upon IL-10 treatment, with more modest increases in protein expression. Several candidate microRNA may regulate CD163. As such, our studies indicate that overexpression of miR-125a and miR-181c significantly reduced CD163 mRNA expression in IL-10 polarized macrophages. Notably, both of these microRNAs were significantly increased in monocytes from children with active systemic JIA. <i>In vitro</i>, miR-125a and miR-181c were both elevated in polarizing conditions that restrict CD163 expression. Interestingly, these microRNAs regulated CD163 expression through distinct mechanisms. Computationally, miR-181 is predicted to bind directly to CD163 mRNA. Indeed, using RNA immunoprecipitation (RIP) we found that overexpression of miR-181c significantly increased delivery of CD163 mRNA to the RNA-induced silencing complex. In contrast, CD163 does not have a predicted miR-125a binding site. Using whole transcriptome analysis of macrophages overexpressing miR-125a-5p, significantly reduced levels of mRNA encoding several macrophage receptors, including IL10RA and TGFbR2, was observed. This suggests that miR-125a-5p exerts more global effects to restrict M2c polarization. <h3>Conclusions</h3> 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 illustrate how microRNA "fine-tune" innate immune activation during inflammatory responses, and expand the understanding of the phenotype of activated monocytes and macrophages in systemic JIA. <h3>Disclosure of Interest</h3> S. Thornton: None declared, T. Do: None declared, R. Tan: None declared, A. Sproles: None declared, M. Bennett: None declared, M. Medvedovic: None declared, M. DeLay: None declared, N. Shen: None declared, A. Grom Grant/research support from: Novartis, Novimmune, Consultant for: Novartis, Novimmune, G. Schulert: None declared