Mast cells promote pathology and susceptibility in tuberculosis

Abstract

Tuberculosis (TB), caused by the bacterium Mycobacterium tuberculosis (Mtb), infects approximately one-fourth of the world's population. We reported an increased accumulation of mast cells (MCs) in the lungs of macaques with active pulmonary TB (PTB), compared with those with latent TB infection (LTBI). MCs respond in vitro to Mtb exposure via degranulation and by inducing proinflammatory cytokines. In the current study, we demonstrate an increased production of chymase by MCs in granulomas of humans and macaques with PTB. Single-cell (sc) RNA sequencing analysis revealed distinct MC transcriptional programs between LTBI and PTB, with PTB associated MCs enriched in interferon gamma, oxidative phosphorylation, and MYC signaling. In a mouse model, MC deficiency led to improved control of Mtb infection that coincided with reduced accumulation of lung myeloid cells and diminished lung inflammation at chronic stages of infection. Airway transfer of MCs into wild-type Mtb-infected mice showed increased neutrophils, decreased recruited macrophages, and elevated Mtb dissemination to the spleen. Together, these findings highlight MCs as active drivers of TB pathogenesis and potential targets for host-directed therapies for TB.

Keywords: Mast cells; Tuberculosis; inflammation; innate cell; lung.

Figure 1.. Chymase positive 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 MC_T (green) or chymase MC_C (red). (A) Immunofluorescence microscopy shows MC_TS (green) in healthy lung biopsies (HC). MC_TCS (red and green merge) are located around the early granulomas, while MC_CS (red) surround the late granulomas in TB infected lung biopsies. (B) Predominance of MC_TS in healthy lungs transitioning to MC_TCS in early granuloma and becoming MC_CS in late granulomas in TB infected lungs. (C) Immunofluorescence microscopy shows MC_TS (green) and MC_TCS (merge) in lungs of healthy (HC), LTBI and PTB macaques. (D) Predominance of MC_TS (green) and MC_TCS (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.

Figure 2.. MC signatures across disease conditions in NHPs.

Data was re-analyzed 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+ mast cells, 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. (D-G) 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, LOC699599) across disease conditions. (I) Cell counts of different MC subtypes (MC_C, MC_T, MC_TC) 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, LOC699599) from the new dataset across time points. (L) Proportions of different MC subtypes (MC_C, MC_T, MC_TC). (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 Kruskal-Wallis tests with Dunn’s multiple comparison correction (**p < 0.01, ***p < 0.001, ****p < 0.0001).

Figure 3:. MC-deficient mice are resistant to Mtb chronic infection.

(A) C57BL/6 and CgKitW^sh 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 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: 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 CgKitW^sh mice, **** p < 0.0001, *** p < 0.001, * p< 0.05.

Figure 4:. MC-deficient mice have dysregulated immune profile after Mtb infection.

C57BL/6 and CgKitW^sh 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) MCs, (B) DCs (C) RMs, (D) neutrophils (E)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 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 CgKitW^sh mice, *** p < 0.0001, ** p < 0.001, * p< 0.05. Outliers were removed from the subsets using Grubb’s outlier test.

Figure 5:. Wild-type mice with airway transferred 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 7 days before infecting with a low aerosol dose (~100CFU) of Mtb HN878. MCs were replenished in these mice at 15 dp and mice were sacrificed at 30 dpi. Frequencies of (A) MCs, (B) neutrophils, and (C) 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. 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: 2mm. Original magnification: ×20. Data points represent the mean ± SD. Statistical analysis was performed using an unpaired, 2-tailed Student’s t test between the groups, **** p < 0.0001, *** p < 0.001, * p< 0.05.