Chymase positive mast cells 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 unpaired, 2-tailed Student’s t test, **** p < 0.0001, *** p < 0.001, * p< 0.05.

Mast cells in lung in active TB show an inflammatory and metabolically active transcriptome as compared to HC or LTBI.

Single-cell (sc) RNA-seq Data was re-analyzed from the lung of non-human primates (Esauolva et al). (A) UMAP embedding of the FCER1A+ mast cells, revealed four transcriptionally distinct clusters; the distribution of the MCs across disease condition is indicated by the color PTB (pink), HC (green) and LTBI (blue). (B) Showing the average expression of MCs specific marker genes (FCER1A, MS4A2 and CD48) and a macrophage gene ITGAX in contrast. (C) Dot plot indicating expression of top differentially expressed genes detected for each cell cluster identified. The dot color represents the expression level, and the dot size represents the percentage of cells in each cluster expressing a particular gene. (D) Hallmark Pathway analysis of the differential genes, only top pathways with highest FDR in each cluster is plotted. The color indicates the –log10 FDR; E-H) UMAP plots with the U cell module score (averaged score of all genes) of the pathways and their corresponding violin plots. Cluster 2 from LTBI/HC were compared to the rest of the PTB groups using a Kruskal-Wallis test with Dunn’s multiple correction. **** p < 0.0001, *** p < 0.001, ns: not significant.

MC deficient mice are resistant to Mtb chronic infection.

(A) C57BL/6 and CgKitWsh mice were infected with a low aerosol dose (∼100CFU) 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. H&E staining was caried out for blinded and unbiased analysis of histopathology. (D) Representative images and the area of inflammation measured in each lobe is shown. Scale bars: 2mm. 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 unpaired, 2-tailed Student’s t test between C57BL/6 and CgKitWsh mice, **** p < 0.0001, *** p < 0.001, * p< 0.05.

MC deficient mice have dysregulated immune profile after Mtb infection.

C57BL/6 and CgKitWsh mice were infected with a low aerosol dose (∼100CFU) of Mtb HN878 and mice were sacrificed at 50, 100 and 150 dpi. Number of (A) mast cells, (B) dendritic cells, (C) recruited macrophages, (D) alveolar macrophages, (E) neutrophils and (F) monocytes were enumerated in the lungs of Mtb infected mice. (G) Cytokine and chemokines production in lung homogenates from mice, collected at 150dpi, was assessed by multiplex cytokine analysis. Data points represent the mean ± SD of 1 of 2 individual experiments (n = 5-8 per time point per group). Statistical analysis was performed using unpaired, 2-tailed Student’s t test for (A) to (F) and Two-way ANOVA Sidak’s multiple comparison test for (G) between C57BL/6 and CgKitWsh mice, *** p < 0.0001, ** p < 0.001, * p< 0.05. Outliers were removed from the subsets using Grubb’s outlier test.

Predominance of MCTs in human and NHPs lung interstitium, blood vessels and bronchi.

Healthy lung tissue and TB infected biopsies from human and NHP samples were stained for MCT (green), MCC (red). Accumulation and localization of (A) MCTS, (B) MCCS, and (C) MCTCS in healthy and TB infected human lung, and (D) MCTS and (E) MCTCS in LTBI and TB macaque lungs. Statistical analysis was performed using unpaired, 2-tailed Student’s t test, *** p < 0.0001, ** p < 0.001, * p< 0.05.

MC deficient mice have reduced numbers of activated CD4+ and CD8+ T cells in lung.

C57BL/6 and CgKitWsh mice were infected with a low aerosol dose (∼100CFU) 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 = 5-8 per time point per group). Statistical analysis was performed using unpaired, 2-tailed Student’s t test between C57BL/6 and CgKitWsh mice, *** p < 0.0001, ** p < 0.001, * p< 0.05. Outliers were removed from the subsets using Grubb’s outlier test.