Azithromycin Treatment Alters Gene Expression in Inflammatory, Lipid Metabolism, and Cell Cycle Pathways in Well-Differentiated Human Airway Epithelia

Abstract

Prolonged macrolide antibiotic therapy at low doses improves clinical outcome in patients affected with diffuse panbronchiolitis and cystic fibrosis. Consensus is building that the therapeutic effects are due to anti-inflammatory, rather than anti-microbial activities, but the mode of action is likely complex. To gain insights into how the macrolide azithromycin (AZT) modulates inflammatory responses in airways, well-differentiated primary cultures of human airway epithelia were exposed to AZT alone, an inflammatory stimulus consisting of soluble factors from cystic fibrosis airways, or AZT followed by the inflammatory stimulus. RNA microarrays were conducted to identify global and specific gene expression changes. Analysis of gene expression changes revealed that the AZT treatment alone altered the gene profile of the cells, primarily by significantly increasing the expression of lipid/cholesterol genes and decreasing the expression of cell cycle/mitosis genes. The increase in cholesterol biosynthetic genes was confirmed by increased filipin staining, an index of free cholesterol, after AZT treatment. AZT also affected genes with inflammatory annotations, but the effect was variable (both up- and down-regulation) and gene specific. AZT pretreatment prevented the up-regulation of some genes, such as MUC5AC and MMP9, triggered by the inflammatory stimulus, but the up-regulation of other inflammatory genes, e.g., cytokines and chemokines, such as interleukin-8, was not affected. On the other hand, HLA genes were increased by AZT. Notably, secreted IL-8 protein levels did not reflect mRNA levels, and were, in fact, higher after AZT pretreatment in cultures exposed to the inflammatory stimulus, suggesting that AZT can affect inflammatory pathways other than by altering gene expression. These findings suggest that the specific effects of AZT on inflamed and non-inflamed airway epithelia are likely relevant to its clinical activity, and their apparent complexity may help explain the diverse immunomodulatory roles of macrolides.

Antimycobacterial Drugs Modulate Immunopathogenic Matrix Metalloproteinases in a Cellular Model of Pulmonary Tuberculosis

“…The mechanisms of MMP inhibition in respiratory epithelial cells are not yet dissected, but since they are regulated by NF-B and AP-1 (3,62,63), modulation of promoter binding is likely. Our data are consistent with decreased MMP-9 secretion observed in airway epithelial cells treated with azithromycin and costimulated by inflammatory soluble factors from cystic fibrosis airways (64).…”

CFTR, Mucins, and Mucus Obstruction in Cystic Fibrosis

“…Interestingly, AZT pre-treatment of cells exposed to inflammatory stimuli does not prevent up-regulation of most inflammatory mediators, but prevents up-regulation of the MUC5AC gene (Ribeiro et al 2009), suggesting that AZT may directly impact expression of that mucin gene. In H292 cells, AZT and clarithromycin have inhibitory effects on MUC5AC mRNA levels induced by HNP-1 or lipopolysaccharide (LPS), whereas telithromycin, a semisynthetic erythromycin, has an inhibitory effect on MUC5AC production induced by LPS, but not by HNP-1.…”

Macrolides: from in vitro anti-inflammatory and immunomodulatory properties to clinical practice in respiratory diseases

“…Hence, the immunomodulatory effects of AZM are associated with the induction of alternative and regulatory macrophage activation characteristics during infection. In summary, AZM has been hitherto demonstrated to decrease neutrophil influx, increase monocyte and CD11b cell influx into the airway compartment, induce macrophage activation and reduce production of proinflammatory cytokines, without any effect on bacterial clearance [8,9,22].…”