Mycobacterium abscessus Clearance by Neutrophils Is Independent of Autophagy

Abstract

Mycobacterium abscessus, a rapidly growing nontuberculous mycobacterium, is increasingly prevalent in chronic lung disease, including cystic fibrosis, and infections are characterized by neutrophil-dominated environments. However, mechanisms of immune control are poorly understood. Azithromycin, a macrolide antibiotic with immunomodulatory effects, is used to treat M. abscessus infections. Recently, inhibition of macrophage bactericidal autophagy was described for azithromycin, which could be detrimental to the host. Therefore, we explored the role of autophagy in mycobactericidal neutrophils. Azithromycin did not affect M. abscessus-induced neutrophil reactive oxygen species formation, phagocytosis, or cytokine secretion, and neutrophils treated with azithromycin killed M. abscessus equally as well as untreated neutrophils from either healthy or cystic fibrosis subjects. One clinical isolate was killed more effectively in azithromycin-treated neutrophils, suggesting that pathogen-specific factors may interact with an azithromycin-sensitive pathway. Chloroquine and rapamycin, an inhibitor and an activator of autophagy, respectively, also failed to affect mycobactericidal activity, suggesting that autophagy was not involved. However, wortmannin, an inhibitor of intracellular trafficking, inhibited mycobactericidal activity, but as a result of inhibiting phagocytosis. The effects of these autophagy-modifying agents and azithromycin in neutrophils from healthy subjects were similar between the smooth and rough morphotypes of M. abscessus However, in cystic fibrosis neutrophils, wortmannin inhibited killing of a rough clinical isolate and not a smooth isolate, suggesting that unique host-pathogen interactions exist in cystic fibrosis. These studies increase our understanding of M. abscessus virulence and of neutrophil mycobactericidal mechanisms. Insight into the immune control of M. abscessus may provide novel targets of therapy.

Keywords: azithromycin; cell trafficking; chloroquine; cystic fibrosis; innate immunity; phagocytosis; reactive oxygen species; wortmannin.

FIG 1

No inhibition of M. abscessus killing by AZM. (A) Human peripheral neutrophils were incubated with AZM (closed bars; 25 μg/ml) for 20 min and infected with M. abscessus smooth (Sm-ATCC) and rough (R-ATCC) morphotypes for 1 h. Surviving bacteria were compared to the initial inoculum to determine % killing; n = 10 independent experiments. (B) Infection with CF M. abscessus clinical isolates. Morphotypes are indicated below; n = 4 to 5 independent experiments. *, P < 0.05.

FIG 2

Wortmannin inhibits neutrophil killing of M. abscessus. Neutrophils were incubated with wortmannin (WT; 10 μM) (A), chloroquine (Cq; 5 μg/ml) (B), and rapamycin (500 nM) (C) for 20 min and infected with smooth (Sm-ATCC) and rough (R-ATCC) isolates of the type strain; n = 7 to 10 independent experiments. (D) Neutrophil killing of Sm-ATCC is compared to a smooth clinical isolate (Sm-CF016) in cells treated with wortmannin (WT); n = 6 to 7. *, P < 0.05; †, P < 0.001.

FIG 3

Wortmannin inhibits neutrophil killing of P. aeruginosa. Neutrophils were incubated with AZM (closed bar; 25 μg/ml) or wortmannin (WT) (hatched bar; 10 μM) for 20 min and infected with P. aeruginosa PAO1 for 1 h; n = 6 independent experiments. Ctl, control nontreated cells; **, P < 0.01.

FIG 4

Azithromycin does not affect intracellular ROS production. (A) Time course of intracellular ROS production; RFU, relative fluorescence units. Neutrophils were incubated with the ROS detection reagent CM-H2DCFA and infected with Sm-ATCC (closed circle) or R-ATCC (closed square). (B) Area under the concentration-time curve (AUC) of ROS production over 120 min in the absence or presence of AZM (closed bars; 25 μg/ml). (C) AUC of ROS production in the absence or presence of chloroquine (Cq, 5 μg/ml; closed bars). (D) AUC of ROS production in the absence or presence of wortmannin (WT, 10 μg/ml; closed bars); n = 3 to 8. *, P < 0.05; **, P < 0.01.

FIG 5

Azithromycin does not affect phagocytosis. Neutrophils were incubated with FITC-labeled Sm-ATCC and R-ATCC (FITC-Mab) for the indicated times. (A) Representative flow cytometry plots showing forward scatter (FSC) and side scatter (SSC) of nonstimulated neutrophils, and gating of Alexa Fluor-450-conjugated anti-CD16. (B) Flow cytometric analysis of FITC-Mab association with CD16⁺ neutrophils, indicated by the boxed region, at 5 and 60 min in the absence and presence of AZM. Examples of smooth (Sm-Mab; upper panels) and rough (R-Mab; lower panels) M. abscessus uptake are shown. (C) Summary of the effect of AZM (closed bars) on neutrophil uptake of M. abscessus; n = 5.

FIG 6

Wortmannin inhibits M. abscessus phagocytosis. Neutrophils were incubated with FITC-Mab (green; Sm-ATCC) for 1 h in the presence of wortmannin (WT; 10 μM) or cytochalasin D (cytoD; 5 μg/ml). Following cytocentrifugation, neutrophils were stained for neutrophil elastase (red). Images are presented with (upper) and without (lower) FITC-Mab staining to clarify localization. Pictures are representative of 5 independent experiments.

FIG 7

Azithromycin does not affect cytokine secretion. Neutrophils were incubated with AZM (closed bars) and infected with Sm-ATCC or R-ATCC for 2 h, and secretion of IL-8 (A) and CCL4/MIP1β (B) was determined by ELISA; n = 3. *, P < 0.05.

FIG 8

CF neutrophil killing of M. abscessus. (A) CF neutrophils were incubated with Sm-ATCC or R-ATCC for the indicated times. (B) CF neutrophils were incubated with AZM (closed bars; 25 μg/ml) or chloroquine (Cq) (gray bars; 5 μg/ml) before infection with clinical smooth and rough morphotypes of CF016 for 1 h; n = 3. (C) CF neutrophils were incubated with wortmannin (WT) (closed bars; 10 μM) before infection; n = 5 to 7. *, P < 0.05; **, P < 0.01.

FIG 9

Different neutrophil processing pathways for M. abscessus and autophagic cargo. Phagocytic uptake and intracellular processing of M. abscessus are independent of classical autophagy. Rapamycin (rapa) enhances autophagic vesicle formation (open arrow), while azithromycin (AZM) and chloroquine (Cq) prevent autophagosome-lysosome fusion; these agents fail to modify neutrophil killing of M. abscessus. Wortmannin (WT) disrupts autophagic vesicle formation (not shown), but also inhibits M. abscessus killing by reducing phagocytosis. Specific vesicles are indicated in italics; M. abscessus (closed ovals) and autophagic targets (gray squares) are depicted.