Azithromycin blocks autophagy and may predispose cystic fibrosis patients to mycobacterial infection
Maurizio Renna, … , David C. Rubinsztein, R. Andres Floto
Maurizio Renna, … , David C. Rubinsztein, R. Andres Floto
Published August 1, 2011
Citation Information: J Clin Invest. 2011;121(9):3554-3563. https://doi.org/10.1172/JCI46095.
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Research Article Immunology

Azithromycin blocks autophagy and may predispose cystic fibrosis patients to mycobacterial infection

Abstract

Azithromycin is a potent macrolide antibiotic with poorly understood antiinflammatory properties. Long-term use of azithromycin in patients with chronic inflammatory lung diseases, such as cystic fibrosis (CF), results in improved outcomes. Paradoxically, a recent study reported that azithromycin use in patients with CF is associated with increased infection with nontuberculous mycobacteria (NTM). Here, we confirm that long-term azithromycin use by adults with CF is associated with the development of infection with NTM, particularly the multi-drug-resistant species Mycobacterium abscessus, and identify an underlying mechanism. We found that in primary human macrophages, concentrations of azithromycin achieved during therapeutic dosing blocked autophagosome clearance by preventing lysosomal acidification, thereby impairing autophagic and phagosomal degradation. As a consequence, azithromycin treatment inhibited intracellular killing of mycobacteria within macrophages and resulted in chronic infection with NTM in mice. Our findings emphasize the essential role for autophagy in the host response to infection with NTM, reveal why chronic use of azithromycin may predispose to mycobacterial disease, and highlight the dangers of inadvertent pharmacological blockade of autophagy in patients at risk of infection with drug-resistant pathogens.

Authors

Maurizio Renna, Catherine Schaffner, Karen Brown, Shaobin Shang, Marcela Henao Tamayo, Krisztina Hegyi, Neil J. Grimsey, David Cusens, Sarah Coulter, Jason Cooper, Anne R. Bowden, Sandra M. Newton, Beate Kampmann, Jennifer Helm, Andrew Jones, Charles S. Haworth, Randall J. Basaraba, Mary Ann DeGroote, Diane J. Ordway, David C. Rubinsztein, R. Andres Floto

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Figure 4

Azithromycin blocks autophagosome and phagosome acidification by impairing lysosomal function.

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Azithromycin blocks autophagosome and phagosome acidification by impairi...
(A) Prevention of IFN-γ–induced autophagic flux. RAW 264.7 cells stably expressing mRFP-GFP-LC3 were treated with vehicle alone (as control), BafA1 (200 nM), azithromycin (40 μg/ml), IFN-γ (200 ng/ml), or azithromycin plus IFN-γ. Representative images are shown. Quantification of acidified (mCherry+GFP–; red) and nonacidified (mCherry+GFP+; green) vesicles revealed a significant reduction in basal and IFN-γ–induced autophagosomal acidification by azithromycin treatment. (B) Reduced acidification of lysosomes in primary human macrophages. Double-labeled (FITC and TMR) dextran was loaded into lysosomes before cells were treated for 4 hours with azithromycin (80 μg/ml) or BafA1 (400 nM). As determined by confocal fluorescence analysis, azithromycin significantly reduced lysosomal acidification compared with controls, as did BafA1. Intracellular pH calibrations were performed as described in Supplemental Methods. (C) Azithromycin decreased acidification of phagosomes containing M. abscessus. Patient-derived M. abscessus strains were heat-killed, double-labeled with FAM and Alexa Fluor 633, and incubated for 24 hours with primary human macrophages untreated or treated with azithromycin (20 μg/ml) or BafA1 (100 nM). Representative confocal/DIC images of macrophages with intracellular labeled M. abscessus show increased mycobacterial FAM fluorescence (indicating reduced acidification) after treatment with azithromycin or BafA1. Also shown is quantification by flow cytometry of FAM fluorescence of macrophages that have internalized M. abscessus (i.e., Alexa Fluor 633+). Corresponding phagosomal pH values (see Supplemental Methods) demonstrated significant alkalinization of phagosomes with azithromycin or BafA1 treatment. Scale bars: 10 μm.