Azithromycin blocks autophagy and may predispose cystic fibrosis patients to mycobacterial infection - PubMed (original) (raw)
. 2011 Sep;121(9):3554-63.
doi: 10.1172/JCI46095. Epub 2011 Aug 1.
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
Affiliations
- PMID: 21804191
- PMCID: PMC3163956
- DOI: 10.1172/JCI46095
Azithromycin blocks autophagy and may predispose cystic fibrosis patients to mycobacterial infection
Maurizio Renna et al. J Clin Invest. 2011 Sep.
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.
Figures
Figure 1. Azithromycin blocks autophagy by impairing autophagic flux.
(A) At Papworth Hospital, the numbers of adult CF patients on long-term azithromycin (AZM; red) and with new sputum cultures positive for NTM (gray) rose over the last 5 years. (B) Analysis of the 2007–2008 CF patient cohort. In patients currently or previously infected with NTM, long-term azithromycin use (current or within 6 months of first NTM culture) was significantly more common that in uninfected patients (P = 0.0009; 2-tailed uncorrected χ2). (C) In GFP-LC3+ COS7 cells, 24-hour treatment with azithromycin increased the number of cells containing 10 or more GFP-LC3+ vesicles more so than rapamycin (200 nM). (D) In HeLa cells, azithromycin caused a dose-dependent increase in LC3-II levels (EC50, 4.5 μg/ml). exp., exposure. (E) In the presence of 400 nM BafA1, azithromycin treatment of HeLa cells did not increase LC3-II compared with BafA1 alone. (F) Azithromycin treatment (16 μg/ml) of HeLa cells, while increasing LC3-II, did not alter mTOR-dependent signaling (monitored by changes in phosphorylation of S6 and p70S6 kinase); however, it did increase p62 levels. (G) Azithromycin dose-dependently increased LC3-II and p62 levels in primary human macrophages. (H) Similar effect of azithromycin, assessed by LC3-II and p62 levels, in primary macrophages derived from clinically stable CF patients and healthy controls (n = 3 per group). *P < 0.05; **P < 0.005.
Figure 2. Azithromycin blocks autophagic clearance and autophagosome acidification.
(A) Azithromycin reduced basal (black) and rapamycin-induced (gray) clearance of mutant (A53T) α-synuclein in stable inducible PC12 cells. The A53T α-synuclein transgene was induced with doxycycline for 48 hours and then switched off (by antibiotic removal) before cells were treated with vehicle alone, rapamycin (200 nM), and/or azithromycin for a further 24 hours. (B) Azithromycin reduced clearance of mutant (Q74) huntingtin. Aggregates of GFP-Q74 huntingtin were reduced by rapamycin treatment, but increased when autophagy was blocked by either BafA1 or azithromycin (4–16 μg/ml). Con, control. *P < 0.05; **P < 0.005. Scale bar: 10 μm.
Figure 3. Azithromycin blocks acidification of autophagosomes without impairing lysosomal function.
(A) Azithromycin did not impair autophagosome-lysosome fusion. Confocal microscopy of HeLa cells coexpressing LC3-mCherry with the lysosomal marker lgp120-GFP revealed that although rapamycin, BafA1, and azithromycin (16 μg/ml) treatment increased the total number of LC3+ vesicles per cell, the colocalization of LC3+ vesicles with lgp120 (LC3+/lgp120+) was not significantly different from controls after azithromycin treatment, but increased with rapamycin and decreased with BafA1 treatment. (B) Azithromycin impaired autophagosome acidification. HeLa cells stably expressing the mCherry-GFP-LC3 construct were treated with either 8–16 μg/ml azithromycin or BafA1 for 24 hours and analyzed by confocal microscopy. Compared with the control, azithromycin caused a significant reduction in the number of acidified LC3+ vesicles, but increased total LC3+ vesicles per cell. Number of vesicles was normalized to control cells. **P < 0.005. Scale bars: 10 μm.
Figure 4. Azithromycin blocks autophagosome and phagosome acidification by impairing lysosomal function.
(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.
Figure 5. Azithromycin blocks phagosomal degradation and phagosome-lysosome fusion.
(A) OVA-coated beads were incubated with primary human macrophages (1-hour pulse, 23-hour chase). Internalized beads were released, and the amount of OVA coating was quantified by flow cytometry after incubation with fluorescent anti-OVA antibody. Treatment with azithromycin (20 μg/ml) significantly reduced OVA degradation compared with untreated cells, to levels close to those achieved by BafA1 (100 nM) or leupeptin/pepstatin. (B) Human primary macrophages were incubated (1-hour pulse, 4-hour chase) with TMR- and biotin-conjugated dextran to load lysosomes and then fed with IgG-coated streptavidin-conjugated fluorescent latex beads (30-minute pulse, 2-hour chase) with no treatment or in the presence of azithromycin (80 μg/ml) or BafA1 (400 nM). Beads were recovered by cell disruption, and the degree of bound dextran fluorescence was quantified by flow cytometry. Shown are a representative histogram and average geometric mean fluorescence of triplicate samples.
Figure 6. Azithromycin blocks intracellular killing of mycobacteria.
(A and B) Human macrophages were infected with luminescent mycobacteria — M. bovis BCG (A) and M. abscessus (B) — for 2 hours, washed, and exposed to the indicated concentrations of azithromycin (μg/ml) and/or 200 nM rapamycin for 24 hours. Viable intracellular mycobacteria were then assessed by measuring cell-associated luminescence after cell lysis. (C) IFN-γ and TNF-α enhanced intracellular killing of M. abscessus in human macrophages, and this was blocked by azithromycin pretreatment. Results were normalized to levels obtained without cytokine addition. (D) Patient-derived M. abscessus strains were incubated with peripheral blood from healthy subjects pretreated for 24 hours with vehicle alone or increasing concentrations of azithromycin. After 72 hours of incubation at 37°C, viable mycobacteria were quantified after sample lysis by counting CFUs. Addition of azithromycin, while reducing mycobacterial growth (white), prevented any additional autophagy-dependent killing achieved by coincubation with IFN-γ (200 U/ml) or rapamycin (100 nM). (E) Induction of macrolide resistance in M. abscessus. M. abscessus-lux was grown for 6 days in liquid culture (i.e., Middlebrook 7H9 plus ADC enrichment) in the presence of 0.1 μg/ml azithromycin or vehicle alone, washed, and resuspended in RPMI with 10% FCS with the indicated concentrations of azithromycin. Viable mycobacteria were assessed by measuring luminescence after 24 hours. *P < 0.05; **P < 0.005.
Figure 7. Azithromycin promotes M. abscessus infection in vivo.
(A) C57BL/6 mice were infected by aerosol challenge with azithromycin-resistant M. abscessus. Treatment with azithromycin (100 mg/kg) by gavage for 5 days per week resulted in failure of M. abscessus clearance from lungs. (B) Representative lung histology demonstrating that azithromycin treatment led to persistent lung infection associated with extensive granulomas (arrows) and peribronchiolar inflammation at day 30. Ziehl-Neelsen staining confirmed the presence of large numbers of intracellular mycobacteria in azithromycin-treated, but not control, animals. Scale bars: 200 μm (histology); 10 μm (Ziehl-Neelsen). (C) Extent and severity of lung lesions in _M. abscessus_–infected mice at days 15 and 30 of treatment with vehicle alone or azithromycin. (D) Assessment of intracellular cytokine profiles for lung macrophages, dendritic cells, and CD4+ and CD8+ effector T cells during infection in the presence or absence of chronic azithromycin treatment.