Mouse transcriptome reveals potential signatures of protection and pathogenesis in human tuberculosis

Abstract

Although mouse infection models have been extensively used to study the host response to Mycobacterium tuberculosis, their validity in revealing determinants of human tuberculosis (TB) resistance and disease progression has been heavily debated. Here, we show that the modular transcriptional signature in the blood of susceptible mice infected with a clinical isolate of M. tuberculosis resembles that of active human TB disease, with dominance of a type I interferon response and neutrophil activation and recruitment, together with a loss in B lymphocyte, natural killer and T cell effector responses. In addition, resistant but not susceptible strains of mice show increased lung B cell, natural killer and T cell effector responses in the lung upon infection. Notably, the blood signature of active disease shared by mice and humans is also evident in latent TB progressors before diagnosis, suggesting that these responses both predict and contribute to the pathogenesis of progressive M. tuberculosis infection.

Figure 1. Human TB blood transcriptional signature is preserved in blood of TB susceptible mice.

Blood modules of co-expressed genes derived using WGCNA from human TB datasets in Singhania et al. 2018 are shown for blood RNA-seq datasets from TB patients from London (n=21 biologically independent samples), South Africa (n=16 biologically independent samples) (both compared to London controls; n=12 biologically independent samples) and Leicester (n=53 biologically independent samples) (compared to Leicester controls; n=50 biologically independent samples) (Supplementary Table 2); human blood modules were tested in blood RNA-seq datasets obtained from different genetic strains of mice (C57BL/6J, resistant; C3HeB/FeJ, susceptible) infected with low and high doses of M. tuberculosis laboratory strain H37Rv or the M. tuberculosis clinical isolate HN878 (n=4 biologically independent samples per group for H37Rv infection and n=5 biologically independent samples per group for HN878 infection from one experiment per M. tuberculosis infection as depicted in Supplementary Fig. 1a), compared to their respective uninfected controls (Supplementary Table 3). Fold enrichment scores derived using QuSAGE are depicted, with red and blue indicating modules over- or under-abundant, compared to the controls. Colour intensity of the dots represents the degree of perturbation, indicated by the colour scale. Size of the dots represents the relative degree of perturbation, with the largest dot representing the highest degree of perturbation within the plot. Only modules with fold enrichment scores with FDR p-value < 0.05 were considered significant and depicted here (left and middle panels). Module name indicates biological processes associated with the genes within the module (Supplementary Table 1). C’, complement. PRR, pathogen recognition receptor. Cell-type associated with genes within each module were identified using the mouse cell-type-specific signatures from Singhania et al. 2019 (right panel). Cell-type enrichment was calculated using a hypergeometric test, with only FDR p-value < 0.05 considered significant and depicted here (right panel). Colour intensity represents significance of enrichment..

Figure 2. Mouse lung disease modules tested in lungs from diverse mouse TB models.

a, Mouse lung disease modules derived in Singhania et al. 2019 (L1-L38) tested in mouse lung TB samples from different genetic strains of mice (C57BL/6J, resistant; C3HeB/FeJ, susceptible) infected with low and high doses of M. tuberculosis laboratory strain H37Rv or the M. tuberculosis clinical isolate HN878 (n=3 biologically independent samples per group for low dose HN878 infection of C3HeB/FeJ, and n=5 biologically independent samples per group for all other groups as depicted in Supplementary Fig. 1a), compared to their respective uninfected controls (Supplementary Table 4). Red and blue indicate modules over- or under-abundant, compared to the controls. Colour intensity of the dots represents the degree of perturbation, indicated by the colour scale. Size of the dots represents the relative degree of perturbation, with the largest dot representing the highest degree of perturbation within the plot. Only modules with fold enrichment scores with FDR p-value < 0.05 were considered significant and depicted here. GCC, glucocorticoid; K-channel, potassium channel; TM, transmembrane; Ubiq, ubiquitination. b-e, Box plots depicting the module eigengene expression, i.e. the first principal component for all genes within the module, are shown for uninfected (Uninf) and M. tuberculosis infected (Low dose; High dose) C57Bl/6 and C3HeB/FeJ mice, for modules (b) Type I IFN/Ifit/Oas (L5); (c) IL-17 pathway/granulocytes (L11), Inflammation/IL-1 signaling/Myeloid cells (L12), Myeloid cells/Il1b/Tnf (L13); (d) Immunoglobulin h/k enriched (L25); (e) Cytotoxic/T cells/ILC/Tbx21/Eomes/B cells (L35) and Ifng/Gbp/Antigen presentation (L7).

Figure 3. Histological analysis of mouse lungs from M. tuberculosis infected mice.

Representative photomicrographs of hematoxylin and eosin (H&E) or Ziehl–Neelsen (ZN) stained lung sections from different genetic strains of mice (C57BL/6J, resistant; C3HeB/FeJ, susceptible) infected with low and high doses of M. tuberculosis laboratory strain H37Rv or the M. tuberculosis clinical isolate HN878 (n=2 biologically independent samples per group for H37Rv infection, HN878-infected C57BL/6J mice low dose and HN878-infected C3HeB/FeJ mice high dose, and n=3 biologically independent samples per group for HN878-infected C57BL/6J mice high dose and HN878-infected C3HeB/FeJ mice low dose, from one experiment per M. tuberculosis infection). From top to bottom, scale bar represents 2 mm, 200 μm and 100 μm for H&E staining, 20 μm for ZN staining; arrows locate bacteria.

Figure 4. Gene networks of specific TB modules in human blood from TB patients, and blood and lung from M. tuberculosis infected mice.

Differential expression of genes from human blood modules Inflammasome/Granulocytes (HB3), B cells (HB15) and NK & T cells (HB21) depicting the top 50 “hub” network of genes with high intramodular connectivity found within the mouse data (i.e., mouse genes most connected with all other genes within the module), is shown for data from blood from TB patients (Leicester cohort), and blood and lungs from mice infected with M. tuberculosis, each against their respective controls. An enlarged representative network showing human gene names is shown for human blood (top) and an enlarged representative network showing mouse gene names is shown for blood samples from C3HeB/FeJ mice infected with high dose of HN878 (bottom). Each gene is represented as a circular node with edges representing correlation between the gene expression profiles of the two respective genes. Colour of the node represents log2 foldchange of the gene for human blood TB samples or mouse blood and lung samples from M. tuberculosis infected mice compared to respective controls.

Figure 5. Histological analysis of mouse lungs from M. tuberculosis infected mice for neutrophils, T and B cells.

Representative photomicrographs of lung sections from different genetic strains of mice (C57Bl/6, resistant; C3HeB/FeJ, susceptible) infected with low and high doses of M. tuberculosis laboratory strain H37Rv or the M. tuberculosis clinical isolate HN878 (n=2 biologically independent samples per group for H37Rv infection, HN878-infected C57BL/6J mice low dose and HN878-infected C3HeB/FeJ mice high dose, and n=3 biologically independent samples per group for HN878-infected C57BL/6J mice high dose and HN878-infected C3HeB/FeJ mice low dose, from one experiment per M. tuberculosis infection) depicting neutrophils (2B10, brown) by immunohistochemistry or T (CD3 positive, red) and B (B220 positive, green) cells by immunofluorescence (nuclear staining depicted in blue, DAPI). Scale bar represents 100 μm (top) and 50 μm (bottom) for Neutrophils, 200 μm (top) and 100 μm (bottom) for T & B cells.

Figure 6. Quantitation of specific blood modular signatures against extent of lung pathology in mouse models and human TB.

Box plots depicting the module Eigengene expression for human blood modules Interferon/PRR (HB12) and Interferon/C’/Myeloid (HB23) (a, b), Inflammasome/Granulocytes (HB3) and Innate immunity/PRR/C’/ Granulocytes (HB8) (c, d), B cells (HB15) and NK & T cells (HB21) (e, f), are shown for mouse blood samples from uninfected (Uninf; n = 5 biologically independent samples per group) and M. tuberculosis H37Rv or HN878 infected (L, low dose; H, high dose) C57Bl/6 and C3HeB/FeJ mice (n=3 biologically independent samples per group for low dose HN878 infection of C3HeB/FeJ, and n=5 biologically independent samples per group for all other groups as depicted in Supplementary Fig. 1a) (a, c, e); and for human blood samples from the London TB cohort divided in Healthy Control (no X-ray; n=12 biologically independent samples) and TB patients grouped according to the radiographic extent of disease as No disease (n=21 biologically independent samples), Minimal (n=7 biologically independent samples), Moderate (n = 6 biologically independent samples) or Advanced (n=8, biologically independent samples, described in Berry et al. 2010) (b, d, f). Lung lesion global score (a), neutrophil (c) and lymphocyte (e) scores from H&E stained lung sections are also shown for uninfected (Uninf, n=5 biologically independent samples per group) and M. tuberculosis H37Rv or HN878 infected (L, low dose; H, high dose) C57Bl/6 and C3HeB/FeJ mice (n=2 biologically independent samples per group for H37Rv infection, HN878-infected C57BL/6J mice low dose and HN878-infected C3HeB/FeJ mice high dose, and n=3 biologically independent samples per group for HN878-infected C57BL/6J mice high dose and HN878-infected C3HeB/FeJ mice low dose, from one experiment per M. tuberculosis infection).

Figure 7. Quantitation of specific blood modular signatures in blood of healthy controls, LTBI, LTBI-progressors and active TB patients.

Box plots depicting the module Eigengene expression for human blood modules Interferon/PRR (HB12) and Interferon/C’/Myeloid (HB23) (a), Inflammasome/Granulocytes (HB3) and Innate immunity/PRR/C’/ Granulocytes (HB8) (b), B cells (HB15) and NK & T cells (HB21) (c), are shown for human blood samples from the Leicester TB cohort divided in Control (IGRA^-ve TB contacts who remained healthy; n=50 biologically independent samples), LTBI (IGRA^+ve TB contacts who remained healthy; n=49 biologically independent samples), LTBI_Progressor (TB contacts who developed TB, time point just before the contact was diagnosed with active TB; n=6 biologically independent samples) and Active_TB (patients with active disease; n=53 biologically independent samples) (left panels) or divided in Control – LTBI (IGRA^-ve and IGRA^+ve TB contacts who remained healthy) or TB patients grouped according to the radiographic extent of disease as Minimal, Moderate and Advanced (right panels; Supplementary Table 7).

Figure 8. Quantitation of IFN, neutrophil and lymphocyte-specific gene expression in blood of healthy controls, LTBI, LTBI-progressors and active TB patients.

Box plots depicting the log₂ expression values of selected genes from type I IFN-associated modules HB12 and HB23 (a), neutrophil-associated modules HB3 and HB8 (b) and NK & T cell module HB21 (c) are shown for human blood samples from the Leicester TB cohort divided in Control (IGRA^-ve TB contacts who remained healthy; n=50 biologically independent samples), LTBI (IGRA^+ve TB contacts who remained healthy; n=49 biologically independent samples), LTBI_Progressor (TB contacts who developed TB, time point just before the contact was diagnosed with active TB; n=6 biologically independent samples) and Active_TB (patients with active disease; n=53 biologically independent samples) (left panels) or divided in Control – LTBI (IGRA^-ve and IGRA^+ve TB contacts who remained healthy) and TB patients grouped according to the radiographic extent of disease as Minimal, Moderate and Advanced (right panels; Supplementary Table 7). Box plots are also shown for human blood samples of LTBI (non-progressors; n=217 biologically independent samples) and LTBI_Progressor (individuals who developed TB, time point 1 to 180 days before diagnosis; n=17 biologically independent samples) from an independent cohort (GSE79362, Zak et al. 2016) (middle panels).