Azithromycin alters macrophage phenotype

Objectives: To investigate the in vitro effects of azithromycin on macrophage phenotype. Utilizing a mouse macrophage cell line (J774), we examined the effect of azithromycin on the properties that define classical macrophage activation (M1) and alternative macrophage activation (M2). Methods: J774 cells were cultured in the presence of azithromycin and stimulated with classical activation [interferon-gamma (IFN gamma)] and alternative activation [interleukin (IL)-4 and IL-13] cytokines along with lipopolysaccharide (LPS). Macrophages were analysed for inflammatory cytokine production, surface receptor expression, inducible nitric oxide synthase (iNOS) protein expression and arginase activity. Results: Azithromycin altered the overall macrophage phenotype. Azithromycin-treated J774 macrophages demonstrated a significantly reduced production of the pro-inflammatory cytokines IL-12 and IL-6, increased production of the anti-inflammatory cytokine IL-10 and decreased the ratio of IL-12 to IL-10 by 60%. Receptor expression indicative of the M2 phenotype (mannose receptor and CD23) was increased, and receptor expression typically up-regulated in M1 cells (CCR7) was inhibited. The presence of azithromycin increased arginase (M2 effector molecule) activity 10-fold in cells stimulated with IFN gamma and LPS, and iNOS protein (M1 effector molecule) concentrations were attenuated by the drug. Conclusions: These data provide evidence that azithromycin affects the inflammatory process at the level of the macrophage and shifts macrophage polarization towards the alternatively activated phenotype. This recently defined M2 phenotype has been described in conditions in which pulmonary inflammation and fibrosis are major determinants of clinical outcome, but the concept of antibiotics altering macrophage phenotype has not yet been critically evaluated.