Journal
JOURNAL OF IMMUNOLOGY
Volume 203, Issue 4, Pages 1021-1030Publisher
AMER ASSOC IMMUNOLOGISTS
DOI: 10.4049/jimmunol.1801228
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- National Institute of Allergy and Infectious Diseases [R01AI095307]
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Azithromycin is effective at controlling exaggerated inflammation and slowing the long-term decline of lung function in patients with cystic fibrosis. We previously demonstrated that the drug shifts macrophage polarization toward an alternative, anti-inflammatory phenotype. In this study we investigated the immunomodulatory mechanism of azithromycin through its alteration of signaling via the NF-kappa B and STAT1 pathways. J774 murine macrophages were plated, polarized (with IFN-gamma, IL-4/-13, or with azithromycin plus IFN-gamma) and stimulated with LPS. The effect of azithromycin on NF-kappa B and STAT1 signaling mediators was assessed by Western blot, homogeneous time-resolved fluorescence assay, nuclear translocation assay, and immunofluorescence. The drug's effect on gene and protein expression of arginase was evaluated as a marker of alternative macrophage activation. Azithromycin blocked NF-kappa B activation by decreasing p65 nuclear translocation, although blunting the degradation of I kappa B alpha was due, at least in part, to a decrease in IKK beta kinase activity. A direct correlation was observed between increasing azithromycin concentrations and increased IKK beta protein expression. Moreover, incubation with the IKK beta inhibitor IKK16 decreased arginase expression and activity in azithromycin-treated cells but not in cells treated with IL-4 and IL-13. Importantly, azithromycin treatment also decreased STAT1 phosphorylation in a concentration-dependent manner, an effect that was reversed with IKK16 treatment. We conclude that azithromycin anti-inflammatory mechanisms involve inhibition of the STAT1 and NF-kappa B signaling pathways through the drug's effect on p65 nuclear translocation and IKK beta.
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