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

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.

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. Atg16l1^f/f, n = 14 cells for area of LC3 puncta, n = 15 cells for % of LC3+ puncta Mtb area; Atg16l1^f/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. Atg16l1^f/f, n = 27 cells for area of LC3 puncta, n = 31 cells for % of LC3+ puncta Mtb area; Atg16l1^f/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. Atg16l1^f/f, n = 7 mice at 14, 21dpi; Atg16l1^f/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. Atg16l1^f/f, n = 15 mice at naïve condition, n = 7 mice at 14 dpi, n = 12 mice at 21 dpi; Atg16l1^f/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. Atg16l1^f/f, n = 6; Atg16l1^f/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. Atg16l1^f/f, n = 6; Atg16l1^f/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 Ly6G^intGr-1^int 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-1^hi) neutrophils (a), Gr-1 int (Gr-1^int) 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. Atg16l1^f/f, n = 9 mice for control treatment, n = 10 mice for 1A8 treatment; Atg16l1^f/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. Atg16l1^f/f, n = 10 mice for control treatment, n = 11 mice for 1A8 treatment; Atg16l1^f/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. Atg16l1^f/f, n = 9 mice for control treatment, n = 10 mice for 1A8 treatment; Atg16l1^f/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. Atg16l1^f/f, n = 6; Atg16l1^f/f-LysM-cre, n = 6 mice. P values calculated by two-tailed Mann-Whitney tests. ns = not significant.

Figure 1.. High-dose Mtb infection model uncovers protective role of autophagy in myeloid cells in vivo.

a-e,g, Survival of mice that harbor myeloid deficiency (LysM-cre) in autophagy genes (Becn1 (a), Atg14 (b), Fip200 (c), Atg16l1 (d), Atg7 (e) versus floxed littermate controls, and Rubicon^−/− versus Rubicon^+/+/Rubicon^+/− (WT/Het) (g) after aerosol infection with high-dose of 1000 colony-forming units (CFU) of M. tuberculosis (Mtb) strain Erdman. P values were calculated using log-rank Mantel-Cox tests. f, Western blot analysis of p62, LC3 and Actin in alveolar macrophages from bronchoalveolar lavage fluid (BALF) of naïve mice. Representative data of n≥5 biological replicates. h-i, Mtb CFU in lungs and spleens at 21 days post infection (dpi) of high-dose Mtb infections. Means ± s.e.m. pooled from ≥3 experiments are graphed. Atg14^f/f, n= 16 mice for lungs and n= 12 mice for spleens; Atg14^f/f-LysM-cre, n= 16 mice for lungs and n= 8 mice for spleens. P values were calculated by two-tailed Mann-Whitney tests. j-k, Survival of mice after aerosol infection with low-dose of Mtb. P values were calculated using log-rank Mantel-Cox tests. l, Mtb CFU in lungs at 21 dpi of low-dose Mtb infections. Means ± s.e.m. pooled from 2 experiments are graphed. Atg14^f/f, Atg14^f/f-LysM-cre, n= 5; Atg5^f/f, Atg5^f/f-LysM-cre, n= 4 mice. P value was calculated by two-tailed Mann-Whitney tests. ns= not significant. ** for P < 0.01, *** P < 0.001, and **** P < 0.0001.

Figure 2.. Autophagy genes in macrophages/DCs are required to control high-dose Mtb infection in mice.

a-e, Survival of mice harboring Becn1 (a, c), Atg14 (b, d), Atg16l1 (e) deletion in Mrp8⁺ or CD11c⁺ cells versus controls after aerosol infection with high-dose Mtb. P by log-rank Mantel-Cox tests. f, Mtb CFU in lungs at 21 dpi with high-dose Mtb infection. Data are mean ± s.e.m., pooled from 2 experiments. Atg14^f/f, n= 11; Atg14^f/f-CD11c-cre, n= 10 mice. P value was calculated using two-tailed Mann-Whitney tests. g, Survival of mice after aerosol infection with high-dose Mtb. P value was calculated by two-tailed Mann-Whitney tests. P < 0.05 was denoted *, and **** for P < 0.0001. ns= not significant.

Figure 3.. Autophagy does not limit Mtb replication in alveolar macrophages.

a,g, Mtb CFU in the right lungs of mice at 7 dpi (a) and 14 dpi (g) of high-dose Mtb infections. Atg16l1^f/f, n= 9 mice at 7 dpi, n= 7 at 14 mice dpi; Atg16l1^f/f-LysM-cre, n= 10 mice. b,h, Number of total mCherry+ cells in the left lungs at 7 dpi (b) and 14 dpi (h) after high-dose infection with mCherry-Mtb. Data (Means ± s.e.m.) from 2 independent experiments are graphed. Atg16l1^f/f, n= 9 mice for macrophages, neutrophils at 7 dpi, n= 5 mice for DCs, monocytes at 7 dpi, n= 7 mice for all cell types at 14 dpi; Atg16l1^f/f-LysM-cre, n= 9 mice for macrophages, neutrophils at 7 dpi, n= 5 mice for DCs, monocytes at 7 dpi, n= 8 mice for all cell types at 14 dpi. c,i, mCherry mean fluorescent intensity (MFI) in mCherry+ cells at 7 dpi (c) and 14 dpi (i) after high-dose infection with mCherry-Mtb. Data (Means ± s.e.m.) from 2 independent experiments are graphed. Atg16l1^f/f, Atg16l1^f/f-LysM-cre, n= 9 mice at 7 dpi; Atg16l1^f/f, n= 7 mice at 14 dpi; Atg16l1^f/f-LysM-cre, n= 8 mice at 14 dpi. d, Quantification of the number and area of LC3 puncta in Mtb+ cells and bystander cells from mice infected with. Data (Means ± s.e.m.) representative of two independent experiments of 3 biological replicates each. Atg16l1^f/f, n= 29 cells for bystander cells, n= 25 cells for Mtb+ cells; Atg16l1^f/f-LysM-cre, n= 16 cells for bystander cells, n= 33 cells for Mtb+ cells. e, Representative images (of n>3 mice) of LC3-stained alveolar macrophages from mice at 7 dpi with high-dose mCherry-Mtb. LC3 (green), nuclear staining (blue) and Mtb (red). Scale bars, 5 μm. f, % of Mtb coated with LC3 in Mtb infected alveolar macrophages from mice at 7 dpi with high-dose mCherry-Mtb. Atg16l1^f/f, Atg16l1^f/f-LysM-cre, n= 31 mice. Data (Means ± s.e.m.) representative of two independent experiments of 3 biological replicates each. 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.

Figure 4.. Myeloid autophagy suppresses neutrophil inflammation and promotes T cell response during high-dose Mtb infection.

a,b,c,d,e,f,j, The number and percentage of immune cells in the lung of mice during high-dose Mtb infection. Data (Means ± s.e.m.) pooled from 3 independent experiments. Atg16l1^f/f, n= 15 mice at naïve condition, n= 7 mice at 14 dpi, n= 12 mice for alveolar macrophages, total neutrophils, Mtb+ neutrophils, T cells at 21 dpi, n= 9 mice for non-alveolar macrophages, monocytes at 21 dpi; Atg16l1^f/f-LysM-cre, n= 16 mice at naïve condition, n= 10 mice at 14 dpi, n= 17 mice for alveolar macrophages, total neutrophils, Mtb+ neutrophils, T cells at 21 dpi, n= 9 mice for non-alveolar macrophages, monocytes at 21 dpi. P values were calculated by two-tailed Mann-Whitney tests. g, Representative H&E-stained sections of mouse lungs at 14 dpi and 21 dpi of high-dose Mtb of 4 biological replicates. h,i, Survival of high-dose Mtb infected Caspase1/11 (h) or MLKL (i) deficient mice that harboring ATG14 myeloid deficiency versus floxed littermate controls. P by log-rank Mante-Cox tests. k, Mtb CFU in mediastinal lymph nodes (mLN) lungs after high-dose Mtb infections. Data represent Means ± s.e.m. Atg16l1^f/f, n= 6 mice at 8 dpi, n= 7 mice at 11, 14 dpi; Atg16l1^f/f-LysM-cre, n= 5 mice at 8, 14 dpi, n= 6 mice at 11 dpi. P by two-tailed Mann-Whitney tests. l,m, The percentage of P25 CD4+ T cells that have undergone proliferation measured by CellTrace dilution, number of P25 CD4+ T cells, and the percentage of Nur77-GFP negative P25 CD4+ T cells in the mediastinal lymph nodes (mLN, l) and lungs (m) after high-dose Mtb infection. Data (Means ± s.e.m.) from 2 independent experiments are graphed. Atg16l1^f/f, n= 6 mice at 8 dpi, n= 7 mice at 11, 14 dpi; Atg16l1^f/f-LysM-cre, n= 5 mice at 8, 14 dpi, n= 6 mice at 11 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.

Figure 5.. Autophagy deficiency in myeloid cells leads to accumulation of MDSC like cells during high-dose Mtb infection.

a. Representative flow cytometry plots of live CD45⁺MerTK⁻CD64⁻ cells gated for Ly6G^high and Ly6G^int neutrophils from mice lungs. Data is representative of >3 mice. b, Survival of high-dose Mtb infected mice that were treated with neutrophil depletion antibody (1A8) or isotype control immunoglobulin (control) every other day from 8–28 dpi. P values calculated by log-rank Mantel-Cox tests. Atg14^f/f, n= 13 mice for control and 1A8 treatment; Atg14^f/f-LysM-cre, n= 12 mice for control and 1A8 treatment; Atg16l1^f/f, n= 16 mice for control treatment, n= 14 mice for 1A8 treatment; Atg16l1^f/f-LysM-cre, n= 16 mice for control treatment, n= 15 mice for 1A8 treatment. c, The number of T cells in lungs of high-dose Mtb infected mice treated with 1A8 or control antibody at 21 dpi. Data (Means ± s.e.m.) pooled from 3 independent experiments. Atg16l1^f/f, n= 10 mice for control treatment, n= 8 mice for 1A8 treatment; Atg16l1^f/f-LysM-cre, n= 8 mice for control treatment, n= 9 mice for 1A8 treatment. P values calculated by two-tailed Mann-Whitney tests. d, Gene set enrichment analysis (GSEA) of bulk RNAseq of lungs from Atg14^f/f-LysM-cre versus Atg14^f/f mice at 14 dpi of high-dose Mtb infection. The green curve represents the density of the genes identified in RNA-seq analysis where normalized enrichment score (NES), P_adj, and false discovery rate (q-value) are indicated. e,f, UMAP plot of scRNAseq results of all immune cells (e) and neutrophils (f) across all samples (lungs from naïve and 21 dpi of high-dose Mtb infected Atg14^f/f, Atg14^f/f-CD11c-cre, Atg14^f/f-LysM-cre mice, n=4 samples pooled per group). Colored according to identified clusters, and dot plot showing the expression level of markers gene S100a8. g, Distribution of neutrophil clusters from scRNAseq analysis of lungs of naïve and 21 dpi of high-dose Mtb infected (Mtb) mice. h, GSEA of activated-PMN-MDSC and PMN-MDSC signatures (Fig. S6) in scRNAseq neutrophil cluster 1 and 4 (PMN1, PMN4). The green curve represents the density of the genes identified in scRNA-seq analysis. i, Dot plot showing highly expressed genes in PMN1 and PMN4, and expression of these genes in PMN-MDSCs of mouse cancer models and human cancer patients, and active TB patients. Dot size represents prevalence of the transcript, and shade indicates expression level. The red arrows indicate genes identified among signature genes in active TB patients. j, Level of surface molecules on neutrophils from lungs of mice at 14 dpi of high-dose Mtb infection, measured by flow cytometory analysis. Representative data (mean ± s.e.m.) of 2 experiments. Atg16l1^f/f, n= 4; Atg16l1^f/f-LysM-cre, n= 5 mice. P values were calculated by two-tailed Mann-Whitney tests. * for P < 0.05, ** for P < 0.01, *** P < 0.001. k, GSEA of neutrotime early gene and late gene signatures in scRNAseq neutrophil 4 (PMN4). The green curve represents the density of the genes identified in scRNA-seq analysis. l, Representative flow cytometry plots of live CD45⁺ cells from mice blood and bone marrow gated for Ly6G^high and Ly6G^int neutrophils. Data is representative of 3 mice from 2 experiments. * for P < 0.05, ** for P < 0.01, *** P < 0.001, and **** P < 0.0001. ns= not significant.

Figure 6.. Autophagy myeloid deficiency leads to apoptosis of alveolar macrophage and reduced T cell proliferation after high-dose Mtb infection.

a,f, UMAP plots of macrophages/monocytes/DCs clusters (MMD, d), and T cell clusters (h) from scRNAseq analysis colored by clusters. b,g, Distribution of MMD (b), and T cells (g) sub-clusters. f, significantly elevated and downregulated HALLMARK pathways found within all CD11c⁺ (Itgax) cells (see Fig. S8) from infected Atg14^f/f-LysM-cre lungs compared with infected Atg14^f/f controls as assessed by GSEA analysis. d,e, Representative histogram and quantification of flow cytometry analysis of Caspase3/7 activity (FLICA+) in alveolar macrophages from the lungs at 14 dpi (d) and 21 dpi (e) of high-dose Mtb infection. Data (mean ± s.e.m.) pooled from 2 independent experiments. Atg16l1^f/f, n= 6; Atg16l1^f/f-LysM-cre, n= 6 mice. P values calculated by two-tailed Mann-Whitney tests. * for P < 0.05. ns= not significant.