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Unique role for ATG5 in neutrophil-mediated immunopathology during M. tuberculosis infection

Abstract

Mycobacterium tuberculosis, a major global health threat, replicates in macrophages in part by inhibiting phagosome–lysosome fusion, until interferon-γ (IFNγ) activates the macrophage to traffic M. tuberculosis to the lysosome. How IFNγ elicits this effect is unknown, but many studies suggest a role for macroautophagy (herein termed autophagy), a process by which cytoplasmic contents are targeted for lysosomal degradation1. The involvement of autophagy has been defined based on studies in cultured cells where M. tuberculosis co-localizes with autophagy factors ATG5, ATG12, ATG16L1, p62, NDP52, BECN1 and LC3 (refs 2, 3, 4, 5, 6), stimulation of autophagy increases bacterial killing6,7,8, and inhibition of autophagy increases bacterial survival1,2,4,6,7. Notably, these studies reveal modest (~1.5–3-fold change) effects on M. tuberculosis replication. By contrast, mice lacking ATG5 in monocyte-derived cells and neutrophils (polymorponuclear cells, PMNs) succumb to M. tuberculosis within 30 days4,9, an extremely severe phenotype similar to mice lacking IFNγ signalling10,11. Importantly, ATG5 is the only autophagy factor that has been studied during M. tuberculosis infection in vivo and autophagy-independent functions of ATG5 have been described12,13,14,15,16,17,18. For this reason, we used a genetic approach to elucidate the role for multiple autophagy-related genes and the requirement for autophagy in resistance to M. tuberculosis infection in vivo. Here we show that, contrary to expectation, autophagic capacity does not correlate with the outcome of M. tuberculosis infection. Instead, ATG5 plays a unique role in protection against M. tuberculosis by preventing PMN-mediated immunopathology. Furthermore, while Atg5 is dispensable in alveolar macrophages during M. tuberculosis infection, loss of Atg5 in PMNs can sensitize mice to M. tuberculosis. These findings shift our understanding of the role of ATG5 during M. tuberculosis infection, reveal new outcomes of ATG5 activity, and shed light on early events in innate immunity that are required to regulate disease pathology and bacterial replication.

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Figure 1: ATG5, in contrast to other autophagy factors, is essential to control M. tuberculosis infection.
Figure 2: Loss of Atg5 in LysM+ cells leads to earlier and more severe lung inflammation during M. tuberculosis infection.
Figure 3: Depletion of PMNs allows for survival of Atg5fl/fl-Lysm-cre mice during M. tuberculosis infection.
Figure 4: Loss of Atg5 in PMNs, but not alveolar macrophages or dendritic cells, can cause susceptibility to M. tuberculosis.

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Acknowledgements

C.L.S. is supported by a Beckman Young Investigator Award from the Arnold and Mabel Beckman Foundation. J.M.K. is supported by a National Science Foundation Graduate Research Fellowship DGE-1143954 and the NIGMS Cell and Molecular Biology Training Grant GM007067. J.P.H. is supported by a National Science Foundation Graduate Research Fellowship DGE-1143954. H.W.V., S.P. and A.K. are supported by U19 AI109725. We would like to acknowledge D. Kreamalmeyer for assistance with the mouse colonies and T. Malek and L. D. Sibley for providing the 1A8 hybridoma.

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Authors and Affiliations

Authors

Contributions

J.M.K. designed and performed experiments, analysed data, and wrote the manuscript. J.P.H. and L.A.W. performed experiments. S.P., A.K. and J.D. generated mouse strains. H.W.V. provided all mouse strains, analysed data and wrote the manuscript. C.L.S. designed experiments, analysed data and wrote the manuscript.

Corresponding author

Correspondence to Christina L. Stallings.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Survival of mice with defects in autophagy genes other than Atg5.

Per cent survival of mice following infection with 100 colony-forming units (c.f.u.) of aerosolized M. tuberculosis. a, Survival of C57Bl/6 (open squares), Ulk1−/− (blue triangles), Ulk2−/− (inverted pink triangles), Atg4b−/− (red diamonds), and p62−/− (green circles) mice. b, Survival of Atg14lfl/fl-Lysm-cre (purple diamonds), Atg12fl/fl-Lysm-cre (red inverted triangles), Atg16l1fl/fl-Lysm-cre (green triangles), Atg7fl/fl-Lysm-cre (pink diamonds), Atg3fl/fl-Lysm-cre (brown circles) and corresponding floxed control mice. Floxed control mice are shown in open shapes, LysM–Cre-expressing mice are shown in closed shapes. c, Survival of C57Bl/6 (open squares), Atg16l1HM1 (open circles). Samples represent biological replicates. See Supplementary Fig. 6 for sample sizes.

Source data

Extended Data Figure 2 Analysis of autophagy in bronchoalveolar macrophages.

Western blot analysis of p62, LC3, and actin levels in ex vivo macrophages isolated from bronchoalveolar lavages of uninfected mice. For gel source data, see Supplementary Fig. 1.

Extended Data Figure 3 Atg5fl/fl bone-marrow-derived macrophages are hypomorphic for ATG5.

Western blot analysis of ATG5 (ATG5–ATG12 conjugate, 56 kDa) and actin in uninfected-bone-marrow-derived macrophages. For gel source data, see Supplementary Fig. 1.

Extended Data Figure 4 Loss of Atg5 or Atg16l1 in LysM+ cells does not lead to increased c.f.u. at 2 w.p.i. log pulmonary c.f.u. at 2 w.p.i.

Samples represent biological replicates; error bars represent mean ± s.e.m. See Supplementary Fig. 7 for sample sizes and results from all statistical comparisons.

Source data

Extended Data Figure 5 Cytokine levels in uninfected lungs.

Concentration of cytokines in lungs (homogenized in 1 ml PBS plus 0.05% Tween 80) from uninfected mice. Levels of IFNγ, IL-6, MIP-1α, IL-17, and G-CSF were below the limit of detection. C57Bl/6 (grey solid bars), Atg5fl/fl (blue striped bars), Atg5fl/fl-Lysm-cre (blue solid bars), Atg16l1fl/fl (green striped bars), Atg16l1fl/fl-Lysm-cre (green solid bars). Statistical differences were determined by one-way ANOVA and Bonferonni’s multiple comparison test. n.s., not significant. Samples represent biological replicates; error bars represent mean ± s.e.m. See Supplementary Fig. 8 for sample sizes and results from all statistical comparisons.

Source data

Extended Data Figure 6 Number of inflammatory cells in lungs of mice at 2 and 3 w.p.i. (related to Fig. 2).

a, Gating strategy for analysis of inflammatory cells in lungs at 2 and 3 w.p.i. Single lung cells were gated based on CD11b, CD11c, Ly6G, Ly6C and autofluorescence (auto). The parental gate is shown above each contour plot. Representative data are shown from an Atg5fl/fl mouse at 2 w.p.i. b, c, C57Bl/6 (grey solid bars), Atg5fl/fl (blue striped bars), Atg5fl/fl-Lysm-cre (blue solid bars), Atg16l1fl/fl (green striped bars), Atg16l1fl/fl-Lysm-cre (green solid bars). Mean number of alveolar macrophages, PMNs, recruited macrophages, and inflammatory monocytes in lungs at 2 w.p.i. (b) and 3 w.p.i. (c). d, e, Flow cytometry data presented in b and c and in Fig. 2 are the compilation of results from five experiments. In some experiments, different amounts of lung were collected for analysis, making it difficult to compare the average number of each cell type between strains, unless the data are normalized (as done in Fig. 2c, d—percentage of total cells). Therefore, to compare the raw number of cells detected in each cell population, each mouse analysed at 2 w.p.i. (d) and 3 w.p.i. (e) has been graphed individually. Each line represents a different mouse, with dots indicating the number of total cells, alveolar macrophages, PMNs, recruited macrophages and inflammatory monocytes. Statistical differences were determined by one-way ANOVA and Bonferonni’s multiple comparison test (b, c); *P < 0.05; n.s., not significant. Samples represent biological replicates; error bars represent mean ± s.e.m. See Supplementary Fig. 9 for sample sizes and results from all statistical comparisons.

Source data

Extended Data Figure 7 Number of inflammatory cells in lungs of mice at 3 w.p.i. (related to Fig. 4).

Number of alveolar macrophages, PMNs, recruited macrophages, and inflammatory monocytes in lungs at 3 w.p.i. C57Bl/6 (grey solid bars), Atg5fl/fl (blue striped bars), Atg5fl/fl-Lysm-cre (blue solid bars), ‘healthy’ Atg5fl/fl-MRP8-cre (purple striped bars), and ‘susceptible’ Atg5fl/fl-MRP8-cre (purple solid bars). Statistical differences were determined by one-way ANOVA and Bonferonni’s multiple comparison test; *P < 0.05; n.s., not significant. Samples represent biological replicates; error bars represent mean ± s.e.m. See Supplementary Fig. 10 for sample sizes and results from all statistical comparisons.

Source data

Extended Data Figure 8 Analysis of autophagy in bone marrow PMNs.

Western blot analysis of p62, LC3, and actin in bone marrow PMNs from uninfected mice. Each lane represents an individual mouse. Two replicates of the Atg5fl/fl-Lysm-cre and Atg16l1fl/fl-Lysm-cre mice are shown. For gel source data, see Supplementary Fig. 1.

Supplementary information

Supplementary Figure 1

This file contains source gel data showing uncropped western blot images and ladder markers. Boxes indicate how each image was cropped for final figures. (PDF 651 kb)

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Kimmey, J., Huynh, J., Weiss, L. et al. Unique role for ATG5 in neutrophil-mediated immunopathology during M. tuberculosis infection. Nature 528, 565–569 (2015). https://doi.org/10.1038/nature16451

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