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Autophagy promotes efficient T cell responses to restrict high-dose Mycobacterium tuberculosis infection in mice

Nature Microbiology volume 9, pages 684–697 (2024)Cite this article

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Abstract

Although autophagy sequesters Mycobacterium tuberculosis (Mtb) in in vitro cultured macrophages, loss of autophagy in macrophages in vivo does not result in susceptibility to a standard low-dose Mtb infection until late during infection, leaving open questions regarding the protective role of autophagy during Mtb infection. Here we report that loss of autophagy in lung macrophages and dendritic cells results in acute susceptibility of mice to high-dose Mtb infection, a model mimicking active tuberculosis. Rather than observing a role for autophagy in controlling Mtb replication in macrophages, we find that autophagy suppresses macrophage responses to Mtb that otherwise result in accumulation of myeloid-derived suppressor cells and subsequent defects in T cell responses. Our finding that the pathogen-plus-susceptibility gene interaction is dependent on dose has important implications both for understanding how Mtb infections in humans lead to a spectrum of outcomes and for the potential use of autophagy modulators in clinical medicine.

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Fig. 1: High-dose Mtb infection model uncovers protective role of autophagy in myeloid cells in vivo.
Fig. 2: Autophagy genes in macrophages/DCs are required to control high-dose Mtb infection in mice.
Fig. 3: Autophagy does not limit Mtb replication in alveolar macrophages.
Fig. 4: Myeloid autophagy suppresses neutrophil inflammation and promotes T cell response during high-dose Mtb infection.
Fig. 5: Autophagy deficiency in myeloid cells leads to accumulation of MDSC-like cells during high-dose Mtb infection.
Fig. 6: Autophagy myeloid deficiency leads to apoptosis of alveolar macrophage and reduced T cell proliferation after high-dose Mtb infection.

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Data availability

scRNA-seq data have been deposited to the NCBI Gene Expression Omnibus (GEO) database and are accessible through accession number GSE201410. RNA-seq data have been deposited to the NCBI GEO database and are accessible through accession number GSE245206. The raw data are provided in the Supplementary Table 1. The method used for analysing sequencing data is detailed in Methods. Source data are provided with this paper.

Code availability

Standard pipelines were used for the analysis of bulk and scRNA-seq datasets, as indicated in Methods. No custom code was developed for the analysis of data presented in this manuscript.

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Acknowledgements

We thank J. Ernst at UCSF for providing P25/Nur77-GFP/CD45.1 mice; J. Murphy at WEHI and D. Lenschow at WashU for sharing Mlkl−/− mice, and S. Tan at Tufts for sharing pCherry3 plasmid; S. Li and L. Ge at Tsinghua for discussion on autophagy analysis; and S. Andhey at WashU for helpful discussion about scRNA-seq data. The research was supported by the National Key R&D Program of China (2023YFC2306300 to Y.-T.W.), NIH grant R01 (AI132697 to C.L.S.), Burroughs Wellcome Fund Investigators in the Pathogenesis of Infectious Disease (to C.L.S.), the Philip and Sima Needleman Center for Autophagy Therapeutics and Research (to C.L.S.), NIH grant U19 (AI142784 to C.L.S. and H.W.V.), National Natural Science Foundation of China (32370804 to Y.-T.W.), Tsinghua-Peking Joint Center for Life Sciences (to Y.-T.W), Tsinghua University Dushi Program (20231080015 to Y.-T.W.), and Tsinghua University School of Medicine (to Y.-T.W.). Authors also received support from NIH grant T32 AI007172 (to M.E.M.), T32 AI007172 (to J.A.V.W.) and T32 GM007067 to (S.V.H.); and a Potts Memorial Foundation postdoctoral fellowship (to R.L.K.), Stephen I. Morse Fellowship (to S.K.N.), and Alexander & Gertrude Berg Fellowship (to N.D.). This work was supported in part by the Bursky Center for Human Immunology and Immunotherapy Programs at Washington University Immunomonitoring Laboratory. We also thank the Tsinghua University Branch of the China National Center for Protein Sciences (Beijing) and Tsinghua University Core Facilities of the Center for Biomedical Analysis, Technology Center for Protein Research, and the Cell Function Analysing Facility for technical support; and the Genome Technology Access Center at the McDonnell Genome Institute at Washington University School of Medicine.

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Author notes

  1. These authors contributed equally: Siwei Feng, Michael E. McNehlan.

Authors and Affiliations

  1. Center for Infectious Disease Research, Department of Basic Medical Sciences, School of Medicine, Tsinghua University, Beijing, China

    Siwei Feng, Xiaoyan Cui & Ya-Ting Wang

  2. Department of Molecular Microbiology, Center for Women’s Infectious Disease Research, Washington University School of Medicine, St. Louis, MO, USA

    Michael E. McNehlan, Rachel L. Kinsella, Chanchal Sur Chowdhury, Sthefany M. Chavez, Sumanta K. Naik, Samuel R. McKee, Jacob A. Van Winkle, Neha Dubey, Amanda Samuels, Skyler V. Hendrix, Reilly Woodson, Darren Kreamalmeyer, Asya Smirnov, Ya-Ting Wang & Christina L. Stallings

  3. Department of Pathology and Immunology, Washington University School of Medicine, St Louis, MO, USA

    Amanda Swain, Maxim N. Artyomov & Herbert W. Virgin

  4. Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Herbert W. Virgin

  5. SXMU-Tsinghua Collaborative Innovation Center for Frontier Medicine, Shanxi Medical University, Taiyuan, Shanxi, China

    Ya-Ting Wang

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Contributions

Y.-T.W. and C.L.S. designed the project, analysed the data and wrote the manuscript. S.F. performed experiments, analysed the data, made figures and assisted with manuscript writing. Y.-T.W., M.E.M., R.L.K., C.S.C., S.K.N., S.R.M., N.D. and A. Samuels performed experiments. A. Swain and M.N.A helped design and perform the scRNA-seq experiment. J.A.V.W., S.M.C., X.C., S.V.H., R.W., D.K. and A. Smirnov assisted with experiments and data analysis. H.W.V advised on project design. All authors read and edited the manuscript.

Corresponding authors

Correspondence to Ya-Ting Wang or Christina L. Stallings.

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Competing interests

H.W.V. is a founder of Casma Therapeutics and the Vaccine Company. The work reported here was not funded by either company. H.W.V. also holds shares in Vir Biotechnology, which did not fund this work. All other authors declare no competing interests.

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Nature Microbiology thanks Maziar Divangahi and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Analysis of Atg16l1 gene deletion efficiency from primary cells and weight loss of mice infected with high-dose Mtb.

a, PCR assay to confirm deletion of Atg16l1 in myeloid cells from the lung of mice. 2 representative samples of n = 4. b-c, Weight loss of mice after aerosol infection with high-dose Mtb. Data are presented as mean ± s.e.m.

Extended Data Fig. 2 Ex vivo and in vivo analysis of autophagy during Mtb infection in alveolar macrophages.

a, Representative images of LC3-stained alveolar macrophages isolated from naïve mice and then infected ex vivo with mCherry-Mtb for 48 h. LC3 (green), nuclear staining (blue) and Mtb (red). Scale bars, 2 or 3 μm. b,c, Quantitative analysis of the area of LC3 puncta (b) and % of Mtb colocalized with LC3 (c) in mCherry-Mtb+ CD11c+ alveolar macrophages infected ex vivo with mCherry-Mtb. Atg16l1f/f, n = 14 cells for area of LC3 puncta, n = 15 cells for % of LC3+ puncta Mtb area; Atg16l1f/f-CD11c-cre, n = 21 cells for area of LC3 puncta, n = 26 cells for % of LC3+ puncta Mtb area. d, Representative images of LC3-stained alveolar macrophages from mice at 14 dpi with high-dose mCherry-Mtb. LC3 (green), nuclear staining (blue) and Mtb (red). Scale bars, 2 μm. e,f, Quantitative analysis of the area of LC3 puncta (e) and % of Mtb colocalized with LC3 (f) in mCherry-Mtb+ CD11c+ alveolar macrophages from BALF of mice at 14 dpi of high-dose Mtb infection. Atg16l1f/f, n = 27 cells for area of LC3 puncta, n = 31 cells for % of LC3+ puncta Mtb area; Atg16l1f/f-CD11c-cre, n = 29 cells for area of LC3 puncta, n = 31 cells for % of LC3+ puncta Mtb area. Data representative (Means ± s.e.m.) of n = 3 biological repeats. P values calculated by two-tailed Mann-Whitney tests. * for P < 0.05, and **** P < 0.0001. ns = not significant.

Extended Data Fig. 3 Heightened inflammation in lungs of autophagy deficient mice after high-dose Mtb infection.

a, Concentration of cytokines in high-dose Mtb infected lungs as detected by multiplex cytokine panel. Data (Means ± s.e.m.) pooled from 2 independent experiments. Atg16l1f/f, n = 7 mice at 14, 21dpi; Atg16l1f/f-LysM-cre, n = 9 mice at 14 dpi, n = 7 mice at 21 dpi. P values were calculated by two-tailed t-tests. b, The number of total immune cells in the lung of mice during high-dose Mtb infection. Data (Means ± s.e.m.) pooled from 3 independent experiments. Atg16l1f/f, n = 15 mice at naïve condition, n = 7 mice at 14 dpi, n = 12 mice at 21 dpi; Atg16l1f/f-LysM-cre, n = 16 mice at naïve condition, n = 10 mice at 14 dpi, n = 17 mice at 21 dpi. P values were calculated by two-tailed Mann-Whitney tests. * for P < 0.05, ** for P < 0.01, *** P < 0.001, and **** P < 0.0001. ns = not significant.

Extended Data Fig. 4 Histology analysis of naïve mouse lungs.

a, Representative H&E-stained sections of naïve mouse lungs. Data represent n = 3 mice each group.

Extended Data Fig. 5 Analysis of antigen specific T cells and MHC-II level on innate immune cells.

a,b,c Quantification (a,b) and representative flow plot (c) of Ag85a and ESAT6 positive CD4+ T cells from lungs and mLNs of mice at 14 dpi (a) and 21 dpi (b,c) of high-dose Mtb infection. Atg16l1f/f, n = 6; Atg16l1f/f-LysM-cre, n = 6 mice. d, MHC-II mean fluorescent intensity (MFI) in alveolar macrophages, non-alveolar macrophages, DCs, and monocytes from lungs at 14 dpi of high-dose infection with high-dose Mtb. Atg16l1f/f, n = 6; Atg16l1f/f-LysM-cre, n = 5 mice. Data (Means ± s.e.m.) from 2 independent experiments are graphed. P values calculated by two-tailed Mann-Whitney tests. ns = not significant.

Extended Data Fig. 6 Accumulation of Ly6GintGr-1int neutrophils in autophagy deficient mice is associated with susceptibility and high Mtb burden.

a,d,f, The number and percentage of Gr-1 high (Gr-1hi) neutrophils (a), Gr-1 int (Gr-1int) neutrophils(d), and alveolar macrophages and DCs (f) in lungs of high-dose Mtb infected mice treated with neutrophil depletion antibody (1A8) or isotype control immunoglobulin (control) at 21 dpi of high-dose Mtb infection. Atg16l1f/f, n = 9 mice for control treatment, n = 10 mice for 1A8 treatment; Atg16l1f/f-LysM-cre, n = 9 mice for control treatment, n = 10 mice for 1A8 treatment. b, Mtb CFU in lungs at 21 dpi of high-dose Mtb infections with 1A8 or control antibody treatment. Atg16l1f/f, n = 10 mice for control treatment, n = 11 mice for 1A8 treatment; Atg16l1f/f-LysM-cre, n = 10 mice for control treatment, n = 12 mice for 1A8 treatment. c,e, number of Mtb infected cells, measured as GFP+ cells in the lungs at 21 dpi with 1A8 or control antibody treatment. Atg16l1f/f, n = 9 mice for control treatment, n = 10 mice for 1A8 treatment; Atg16l1f/f-LysM-cre, n = 9 mice for control treatment, n = 10 mice for 1A8 treatment. Data (Means ± s.e.m.) pooled from 3 independent experiments. P values calculated by two-tailed Mann-Whitney tests. * for P < 0.05, ** for P < 0.01, *** P < 0.001, and **** P < 0.0001. ns = not significant.

Extended Data Fig. 7 Non-alveolar macrophages in the lungs did not exhibit increased apoptosis at 21 dpi.

a, Representative histogram and quantification of flow cytometry analysis of FLICA+ non-alveolar macrophages from lungs of mice at 21 dpi of high-dose Mtb infection. Grey histogram indicates isotype control. Data are presented as mean ± s.e.m. Atg16l1f/f, n = 6; Atg16l1f/f-LysM-cre, n = 6 mice. P values calculated by two-tailed Mann-Whitney tests. ns = not significant.

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Feng, S., McNehlan, M.E., Kinsella, R.L. et al. Autophagy promotes efficient T cell responses to restrict high-dose Mycobacterium tuberculosis infection in mice. Nat Microbiol 9, 684–697 (2024). https://doi.org/10.1038/s41564-024-01608-x

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