Mast cells promote pathology and susceptibility in tuberculosis
- Department of Microbiology, The University of Chicago, United States
- Department of Molecular Microbiology, Washington University in St. Louis, United States
- Division of Allergy, Immunology and Rheumatology, Department of Medicine, University of Rochester Medical Center, United States
- Technologico de Monterrey, Escuela de Medicina y Ciencias de la Salud, Mexico
- Laboratory of Immunobiology and Genetics and Department of Pathology, Instituto Nacional de Enfermedades Respiratorias Ismael Cosio Villegas, Mexico
- Southwest National Primate Research Center, Texas Biomedical Research Institute, United States
Figures
Figure 1 with 1 supplement
Chymase-positive mast cells (MCs) are predominant in TB-infected human and macaque lung tissue.
Lung biopsies from healthy individuals (n = 4) or patients with PTB (n = 5) were stained for tryptase MCT (green) or chymase MCC (red). (A) Immunofluorescence microscopy shows MCTS (green) in healthy lung biopsies (HC). MCTCS (red and green merge) are located around the early granulomas, while MCCS (red) surround the late granulomas in TB-infected lung biopsies. (B) Predominance of MCTS in healthy lungs transitioning to MCTCS in early granuloma and becoming MCCS in late granulomas in TB-infected lungs. (C) Immunofluorescence microscopy shows MCTS (green) and MCTCS (merge) in lungs of healthy (HC), LTBI, and PTB macaques. (D) Predominance of MCTS (green) and MCTCS (merge) in PTB compared to LTBI and HC. Statistical analysis was performed using GraphPad v5, unpaired, two-tailed Student’s t-test, ***p < 0.0001, **p < 0.001, *p < 0.05.
Figure 1—figure supplement 1
Predominance of MCTs in human and NHP lung interstitium, blood vessels, and bronchi.
Healthy lung tissue and TB-infected biopsies from human and NHP samples were stained for MCT (green) and MCC (red). Accumulation and localization of (A–C) MCTS, (D–F) MCTCS, and (G–I) MCCS in healthy and TB-infected human lung, and (J–L) MCTS and (M–O) MCTCS in LTBI and TB macaque lungs. Statistical analysis was performed using GraphPad v5, an unpaired, two-tailed Student’s t-test, ***p < 0.0001, **p < 0.001, *p < 0.05.
Figure 2 with 1 supplement
Mast cell (MC) signatures across disease conditions in NHPs.
Data was reanalyzed from the lungs of M. mulatta infected with Mtb CDC1551 (GSE200151). (A) Schematic of the study design across disease conditions (B) UMAP embedding of FCER1A+ MCs, showing the distribution of these cells across the different disease conditions (PTB in pink, HC in green, and LTBI in blue). (C) Heatmap of Hallmark pathway analysis for differentially expressed genes, highlighting the top pathways with the highest FDR values for each condition. UCell module for pathways: IFNγ signaling (D), TNF-α signaling (E), oxidative phosphorylation (F), and Th2 signature (G) across disease conditions, shown on UMAP embeddings. (H) Violin plots of gene expression for key MC markers (CMA1, TPSG1, and LOC699599) across disease conditions. (I) Cell counts of different MC subtypes (MCC, MCT, and MCTC) across disease conditions (PTB, red bars, LTBI, blue bars, and HC, green bars). (J) UMAP plot of the NHP lung granuloma dataset (GSE200151), showing the distribution of cells at 4 weeks (high disease burden) and 10 weeks (low disease burden) in M. fasicularis infected with Mtb Erdman. (K) Gene expression violin plots for key MC markers (CMA1, TPSG1, and LOC699599) from the new dataset across time points. (L) Proportions of different MC subtypes (MCC, MCT, and MCTC). (M) Violin plots of summed module scores for the key pathways (IFNγ signaling, TNF-α signaling, oxidative phosphorylation) across disease burdens, showing pathway activity. Statistical significance was assessed using GraphPad v10, Kruskal–Wallis tests with Dunn’s multiple comparison correction (****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05).
Figure 2—figure supplement 1
Immune cell marker expression and pathway analysis across disease conditions.
Data was reanalyzed from the lungs of NHPs (GSE200151). (A) UMAP embeddings of mast cells (MCs) showing expression of cell surface markers: FCER1A, MS4A2, CD48, and ITGAX across identified clusters. (B) Dot plot showing the expression levels of select genes across clusters, where the dot size represents the percentage of cells expressing each gene, and the color intensity represents the average expression level. (C) Violin plot of summed module scores for IFNγ signaling, oxidative phosphorylation (D), TNF-α signaling (E), Th2 signature (F) pathway across different disease conditions: LTBI/HC (green), PTB (pink), and the respective clusters. (G) UMAP embeddings show the expression of key MC markers, including TPSG1, LOC699599, and CMA1 across identified clusters, with color intensity indicating expression levels. Statistical significance was assessed using GraphPad v10, Kruskal–Wallis tests with Dunn’s multiple comparison correction (****p < 0.0001, ***p < 0.001, **p < 0.01).
Figure 3 with 1 supplement
Mast cell (MC)-deficient mice are resistant to Mtb chronic infection.
(A) C57BL/6 and Cg-KitW-sh mice were infected with a low aerosol dose (~100 CFU) of Mtb HN878 and mice were sacrificed at 50, 100, and 150 dpi. (B) Bacterial burden was assessed in lungs and spleens by plating. (C) Lungs were harvested, fixed in formalin, and embedded in paraffin. Hematoxylin and eosin (H&E) staining was carried out for blinded and unbiased analysis of histopathology. (D) Representative images and the area of inflammation measured in each lobe are shown. Scale bars: 2 mm. Original magnification: ×20. Data points represent the mean ± SD of two experiments (n = 8–15 per time point per group). Statistical analysis was performed using GraphPad v5, unpaired, two-tailed Student’s t-test between C57BL/6 and Cg-KitW-sh mice, ***p < 0.0001, **p < 0.001, *p < 0.05.
Figure 3—figure supplement 1
Mast cells (MCs) appear at early Mtb infection.
C57BL/6 mice were infected with a low aerosol dose (~100 CFU) of Mtb HN878 and mice were sacrificed at 5, 21, and 30 dpi. (A) The number of MCs in the lungs was enumerated in Mtb-infected mice. (B) Bacterial burden was assessed by plating. Data points represent the mean ± SD (n = 4–5 per time point). Statistical analysis was performed using GraphPad v5, an unpaired, two-tailed Student’s t-test, ***p < 0.0001, **p < 0.001, *p < 0.05.
Figure 4 with 2 supplements
Mast cell (MC)-deficient mice have dysregulated immune profiles after Mtb infection.
C57BL/6 and Cg-KitW-sh mice were infected with a low aerosol dose (~100 CFU) of Mtb HN878 and mice were sacrificed at 50, 100, and 150 dpi. Number of (A) MCs, (B) dendritic cells (DCs), (C) recruited macrophages (RMs), (D) neutrophils, (E) alveolar macrophages (AMs), and (F) monocytes were enumerated in the lungs of Mtb-infected mice. (G) Cytokine and chemokine production in lung homogenates from mice, collected at 150 dpi, was assessed by multiplex cytokine analysis. Data points represent the mean ± SD of 1 of 2 individual experiments (n = 4–10 per time point per group). Statistical analysis was performed using GraphPad v5, unpaired, two-tailed Student’s t-test between C57BL/6 and Cg-KitW-sh mice, for (A–F), ***p < 0.0001, **p < 0.001, *p < 0.05; and using GraphPad v10, two-way ANOVA Sidak’s multiple comparison test for (G) ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05. Outliers were removed from the subsets using Grubb’s outlier test.
Figure 4—figure supplement 1
Mast cell (MC)-deficient mice have no baseline differences in T cell numbers.
Six weeks C57BL/6 mice and Cg-KitW-sh mice were sacrificed to enumerate baseline numbers of (A) CD4+ T cells and (B) CD8+ T cells. Data points represent the mean ± SD (n = 4–5 per time point).
Figure 4—figure supplement 2
Mast cell (MC)-deficient mice have reduced numbers of activated CD4+ and CD8+ T cells in the lung.
C57BL/6 and Cg-KitW-sh mice were infected with a low aerosol dose (~100 CFU) of Mtb HN878 and mice were sacrificed at 50, 100, and 150 dpi. Number of (A) CD4+ CD44+ T cells, (B) CD4+ CD44+ IFNγ+ T cells, (C) CD4+ CD44+ TNF-α+ T cells, (D) CD4+ CD44+ IFNγ+ TNF-α+ T cells, (E) CD8+ CD44+ T cells, (F) CD8+ CD44+ IFNγ+ T cells, (G) CD8+ CD44+ TNF-α+ T cells, and (H) CD8+ CD44+ IFNγ+ TNF-α+ T cells in the lungs of Mtb-infected mice. Data points represent the mean ± SD of 1 of 2 individual experiments (n = 4–10 per time point per group). Statistical analysis was performed using GraphPad v5, an unpaired, two-tailed Student’s t-test between C57BL/6 and Cg-KitW-sh mice, ***p < 0.0001, **p < 0.001, *p < 0.05. Outliers were removed from the subsets using Grubb’s outlier test.
Figure 5 with 1 supplement
Wild-type mice with airway transferred mast cells (MCs) promote bacterial dissemination.
Bone marrow-derived in vitro cultured MCs (5 × 104 cells/mouse) were adoptively transferred into the lung airways of C57BL/6 mice 1 day before infecting with a low aerosol dose (~100 CFU) of Mtb HN878. MCs were replenished in these mice at 21 dpi, and mice were sacrificed at 30 dpi. Frequencies of (A) MCs, (B) neutrophils, and (C) recruited macrophages (RMs) were enumerated in the lungs of Mtb-infected mice. Bacterial burden was assessed in (D) lungs and (E) spleens by plating. (F) Lungs were harvested, fixed in formalin, and embedded in paraffin. Hematoxylin and eosin (H&E) staining was carried out for blinded and unbiased analysis of histopathology. (G) Immunofluorescence microscopy shows more neutrophil infiltration in the lungs of MC-transferred WT mice. (H) Ly6G+ cells per area of lung granuloma measured in each lobe are shown. Scale bars: 2 mm. Original magnification: ×20. Data points represent the mean ± SD, of 1 of 2 individual experiments (n = 4 per group). Statistical analysis was performed using GraphPad v5, an unpaired, two-tailed Student’s t-test between the groups, ***p < 0.0001, **p < 0.001, *p < 0.05.
Figure 5—figure supplement 1
Lung myeloid cell accumulation did not vary in mast cell (MC)-transferred WT Mtb-infected mice.
Bone marrow-derived in vitro cultured MCs (n = 50,000 cells per mouse) were adoptively transferred into the lung airways of WT mice 7 days before infecting with a low aerosol dose (~100 CFU) of Mtb HN878. MCs were replenished in these mice at 15 dpi, and mice were sacrificed at 30 dpi. Numbers of (A) MCs, (B) neutrophils, and (C) recruited macrophages (RMs) were enumerated in the lungs of Mtb-infected mice. Data points represent the mean ± SD of 1 of 2 individual experiments (n = 4 per group).
Additional files
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Source data 1
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- https://cdn.elifesciences.org/articles/102634/elife-102634-data1-v1.xlsx
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Mast cells promote pathology and susceptibility in tuberculosis
eLife 13:RP102634.
https://doi.org/10.7554/eLife.102634.3
