Abstract 398: Retinoic Acid Exposure Promotes Phenotypic Characteristics Of Alternative Activation In Macrophages

Abstract

Introduction: Macrophages are key players in atherosclerosis. Classically-activated (M1) macrophages show a pro-inflammatory phenotype, whereas alternatively-activated (M2) are anti-inflammatory. The M1/M2 balance in atherosclerosis determines the fate of the lesions and strategies to increase M2-like macrophages entail promising therapeutic candidates. Retinoic acid (RA), the transcriptionally active form of vitamin A, delays atherosclerosis progression in mice and has been investigated as a modulator of macrophage polarization. Hypothesis: RA modulates macrophage polarization by inducing M2-like responses. Methods: Bone marrow-derived macrophages from three wild-type C57BL/6J mice were differentiated (M0) and stimulated with LPS/IFNγ (M1) or IL-4 (M2) for 24h, followed by 6h exposure to 1 μM RA or vehicle (DMSO). RNAseq and gene enrichment analysis were performed. Efferocytosis assays were performed by co-culture of apoptotic Jurkat cells with treated macrophages and subsequent analysis by flow cytometry. Autophagy flux and lysosomal protein breakdown rate were also analyzed. Results: RNAseq showed that RA induced changes in 1,485 genes in M2 macrophages, while 821 and 227 genes were significantly regulated in M0 and M1, respectively. Given the lower impact of RA on M1, we focused our subsequent analyses on M0 and M2. Arginase 1 , a well-known marker of M2 activation, was significantly upregulated by RA at both the mRNA and the protein levels in M0 and M2. Scavenger receptors pathway was regulated in M2 exposed to RA, and efferocytosis assays showed that this group had significantly the highest efferocytotic capacity. Autophagy and Lysosome pathways were also significantly over-represented in M2 treated with RA, although autophagy flux and protein breakdown rate were not differentially affected by RA in M2 or M0 macrophages. Conclusions: Transcriptomic analyses showed a higher response of M2 macrophages to RA, compared to M0 and M1. Functional assays revealed that RA increased efferocytosis in M2 macrophages, a hallmark of alternative activation. Our results suggest that RA skews macrophages' polarization and it might promote an M2-like phenotype, especially in macrophages exposed to IL4, enhancing alternative activation.