miR-24 Regulates Macrophage Polarization and Plasticity

OBJECTIVE: MicroRNAs (miRNA) are ubiquitous regulators of human biology and immunity. Previously, we have demonstrated an inhibitory role for miR-24 in the phagocytosis of Escherichia coli and Staphylococcus aureus bioparticles and the induction of cytokine secretion in response to lipopolysaccharide (LPS) of the same origin; also, we have identified divergent and convergent miRNA responses to LPS from the periodontopathic pathogens Aggregatibacter actinomycetemcomitams (Aa) and Porphyromonas gingivalis (Pg), and revealed cigarette smoke extract as an environmental modifier of Pg LPS structure (Pg CSE) impacting macrophage miRNA responses. This study was designed to investigate the role of miR-24 on macrophage polarization and plasticity. METHODS: Primary human macrophages were differentiated from CD14(+) monocytes isolated from peripheral blood mononuclear cells (PBMCs) by MACS positive selection and transfected with miR-24 miRNA mimics, inhibitors, or negative control mimic; followed by stimulation with cytokines and/or LPS under various conditions representing key stages of macrophage activation. Macrophage activation and polarization was assessed using assays for cytokine production (ELISA) and protein expression (flow cytometry, immunoblot). MiR-24 expression was assessed by RT-PCR. RESULTS: Stimulation of macrophages with LPSs of Aa, Pg, and Pg CSE origin resulted in dissimilar levels of cytokine expression and differential expression of miR-24. Overexpression of miR-24 inhibited cytokine secretion in response to LPS. Priming of macrophages with interferon gamma (IFN-γ) did not overcome this inhibitory effect, but classical activation of macrophages with IFN-γ plus TNF-α, TNF-β, or IL-17, modulated the pattern of miR-24 mediated suppression in a cytokine-specific fashion. Overexpression of miR-24 enhanced CD206 upregulation during alternative macrophage activation and inhibited its downregulation in macrophage transitioning from alternative to classical activation states. Overexpression of miR-24 resulted in reduced expression of the Class 1A PI 3-kinase subunit p110 delta (p110δ). CONCLUSION: Pathogen- and environment-specific modifications in LPS alter the expression of cytokines and miR-24 in human macrophages. MiR-24 is a negative regulator of macrophage classical activation by LPS and promotes alternative activation under conditions of polarization and plasticity. MiR-24 mediated inhibition of LPS-induced cytokine secretion is dependent upon macrophage activation state at the point of stimulation, and this may be due to the degree to which p110δ is involved in the intracellular signaling pathway/s that transduce receptor ligation into cytokine induction. While important differences were observed in the effect of miR-24 on macrophages, these data indicate that overexpression of miR-24 would be predominantly anti-inflammatory