Immune correlates of tuberculosis disease and risk translate across species

Abstract

One quarter of the world's population is infected with Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB). Although most infected individuals successfully control or clear the infection, some individuals will progress to TB disease. Immune correlates identified using animal models are not always effectively translated to human TB, thus resulting in a slow pace of translational discoveries from animal models to human TB for many platforms including vaccines, therapeutics, biomarkers, and diagnostic discovery. Therefore, it is critical to improve our poor understanding of immune correlates of disease and protection that are shared across animal TB models and human TB. In this study, we have provided an in-depth identification of the conserved and diversified gene/immune pathways in TB models of nonhuman primate and diversity outbred mouse and human TB. Our results show that prominent differentially expressed genes/pathways induced during TB disease progression are conserved in genetically diverse mice, macaques, and humans. In addition, using gene-deficient inbred mouse models, we have addressed the functional role of individual genes comprising the gene signature of disease progression seen in humans with Mtb infection. We show that genes representing specific immune pathways can be protective, detrimental, or redundant in controlling Mtb infection and translate into identifying immune pathways that mediate TB immunopathology in humans. Together, our cross-species findings provide insights into modeling TB disease and the immunological basis of TB disease progression.

Fig.1.. Overview of the samples used and the computational analysis.

(A) Workflow of cross-species immune correlates of TB disease. (B) Mouse samples were divided into progressor and controller sample groups based on (Log₁₀ bacterial burden) × (Log₁₀ percentage lung inflammation), using thresholds of < 6.5 and > 8.5, respectively, (C) PCA clustering of the mouse RNA-Seq samples based on the default DESeq2 method (top 500 most variable genes) and (D) PCA clustering of the macaque RNA-Seq samples.

Fig.2.. Conserved differentially expressed genes in controllers and progressors.

The counts of overlapping significantly differentially expressed genes between mouse genes, macaque genes and human genes (7) are shown for (A) genes higher in controller and (B) genes higher in progressor. Shaded areas represent gene sets used for REACTOME enrichment testing, as shown in panels (C) for genes higher in controller and (D) for genes higher in progressor. *Gene sets visualized in Fig.3.

Fig.3.. Differentially expressed genes that are significantly different in TB controllers across species.

(A) Higher in controller vs progressor and naive in mouse, and higher in controller vs progressor in macaque and human; (B) lower in controller vs progressor and naive in both mouse and macaque; (C) higher in progressor vs controller and naive in both mouse and macaque, and higher in progressor vs controller in mouse (includes ACS signature genes and top 10 most significant other genes); (D) lower in progressor vs controller and naive in both mouse and macaque and lower in progressor vs controller in mouse (top 10 most significant shown).

Fig.4.. Expression of the 16-gene ACS signature genes across species.

(A) Ortholog gene expression of 13 out of 16 human ACS signature genes (7) in progressor versus controller, progressor versus naive and controller versus naive, in mouse and macaque. Note that both FCGR1A and FCGR1B are represented by a single gene in mice and macaques, and human ETVTs ortholog in mouse is called Etv6. (B) SCARF1 expression in progressor, controller versus naive in mouse and macaque is shown. (C) SEPT4 expression in progressor, controller and naive is shown in mouse and macaque. All P values shown on the expression swarm plots represent FDR-corrected significance values for differential expression calculated by DESeq2.

Fig.5.. Genes in the 16-gene ACS signature that mediate protective immune responses in the mouse model.

Control or gene deficient mice were infected with Mtb HN878 (100 CFU) by aerosol route. On 30, 60 and 100 days post-infection (dpi), lungs were harvested to assess bacterial burden by plating, or inflammatory cell infiltration by flow cytometry, or histologically by H&E staining in (A) Statt^−/− mice (B) Tap1^−/− mice, (C) GbpChr3^−/− mice (D) Trafdt^−/− mice and (E) Sept4^−/− mice and relevant controls are shown. The data points represent the means (±SEM) of one out of two separate experiments. Significance for bacterial burden analysis for C57BL/6 and gene deficient mice was determined using unpaired two-tailed Student’s t-test at different dpi.

Fig.6.. Genes in the 16-gene ACS signature that mediate detrimental effects during infection.

Wild-type or gene-deficient mice strains were infected with Mtb HN878 (100 CFU) by aerosol route. On 30, 60, 100 and 120 days post-infection (dpi), lungs were harvested to assess bacterial burden by plating, or inflammatory cell infiltration by flow cytometry, or histologically by H&E staining in (A) Batf2/3r^−/− mice, (B) Fcgrt^−/− mice (C) Scarft^−/− mice and relevant controls (D) DESeq2 differential gene expression comparisons identified in infected Scarft^−/− mice compared to wild-type mice at 120 dpi respectively, (E) Gene set enrichment analysis (GSEA) for Reactome pathways significantly enriched among human proteins with high correlation to SCARF1. Enrichment plots are shown for each of the enriched terms, showing the relative rank of genes from the enriched pathways (across the x-axis) and (F) IL-10 and TNFa production was measured in culture supernatants from uninfected and Mtb HN878-infected bone marrow derived macrophages (MOI 1) from C57BL/6J and ScarfT^−/− mice after 2 dpi. The data points represent the means (±SEM) of one out of two separate experiments. Bacterial burden in gene deficient mice compared to control mice and cytokine production from macrophages was done by unpaired two-tailed Student’s t-test at different dpi.