Contained Mycobacterium tuberculosis infection induces concomitant and heterologous protection

Abstract

Progress in tuberculosis vaccine development is hampered by an incomplete understanding of the immune mechanisms that protect against infection with Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis. Although the M72/ASOE1 trial yielded encouraging results (54% efficacy in subjects with prior exposure to Mtb), a highly effective vaccine against adult tuberculosis remains elusive. We show that in a mouse model, establishment of a contained and persistent yet non-pathogenic infection with Mtb ("contained Mtb infection", CMTB) rapidly and durably reduces tuberculosis disease burden after re-exposure through aerosol challenge. Protection is associated with elevated activation of alveolar macrophages, the first cells that respond to inhaled Mtb, and accelerated recruitment of Mtb-specific T cells to the lung parenchyma. Systems approaches, as well as ex vivo functional assays and in vivo infection experiments, demonstrate that CMTB reconfigures tissue resident alveolar macrophages via low grade interferon-γ exposure. These studies demonstrate that under certain circumstances, the continuous interaction of the immune system with Mtb is beneficial to the host by maintaining elevated innate immune responses.

Fig 1. CMTB mice have an altered inflammatory milieu.

(A) Mice were inoculated intradermally in the ear with 10,000 CFU of H37Rv Mtb and bacterial burden in the superficial cervical lymph nodes measured 10, 42, and 365 days following infection by CFU assay. Data are a representative experiment of 2 independent experiments with 3–6 mice/timepoint. (B) CMTB was established as described in (A) and the levels of the indicated cytokines in peripheral blood were measured by multiplexed immunoassay (Luminex) at the indicated time points. ND = below detection limit; NQ = below quantification limit. Dashed line indicates the quantification limit. Out of 38 cytokines/chemokines assayed, only CXCL10, IFNG, and IL6 exhibited CMTB-induced concentration changes (at B-H corrected p < 0.2). For statistical calculations, values below the detection limit were set to zero and values below the quantification limit were set to half of the quantification limit. (C) Relative proportions of CD3⁺CD8⁺TB10.4-tetramer-positive cells in the lung vasculature (CD45.2+) and parenchyma (CD45.2-) determined 42 days after the establishment of CMTB by flow cytometry. Localization to the vasculature or parenchyma was determined by i.v. labeling with an anti-CD45.2 antibody 10 minutes prior to blood collection. Data are representative of 2 experiments with n = 4–5 mice per experiment. Statistical significance was determined by Student’s t-test. Error bars depict the mean and SD. (See S16 Fig for gating strategy).

Fig 2. Contained TB infection provides sustained protection against aerosol challenge with Mtb and heterologous challenges.

(A) 8 weeks after the establishment of CMTB, mice were challenged with 50–100 CFU of Kanamycin-resistant H37Rv via aerosol. Bacterial burdens in the lungs and spleen were measured by CFU at days 14, 42 and 100 following aerosol infection using plates containing Kanamycin. (Mtb was never detected in the spleen of CMTB mice at day 14.) Plot shows representative data from one of two independent experiments (n = 4–5 mice per group). Statistical significance was determined by Student’s t-test. (B) CMTB was established and after 2 weeks CMTB and control mice were treated for 6 weeks with Isoniazid and Rifampicin. Mice were challenged via aerosol with 50–100 CFU of H37Rv Mtb and bacterial burden in the lung and spleen measured at 6 weeks. Data are representative of 2 independent experiments with 4–5 mice per group. Error bars depict mean and SD. Statistical significance was determined by Student’s t-test. (C) CMTB and control mice (20 per group) were infected with an average of 1 CFU of Mtb H37Rv via aerosol. Bacterial burdens in the lung were measured by CFU. Data are representative of two independent experiments. Mice with no detectable bacteria were omitted from the plot. Statistical significance was determined using the Mann-Whitney test. Error bars depict the mean and SEM. (D) Fractions of control and CMTB mice with detectable bacteria in one or two lobes of the lung in the experiments shown in (C). Uninfected mice are excluded from the plot. Significance was assessed using the exact test for the difference of means in two Poisson distributions. (E) CMTB and control mice were challenged i.v. with 10⁵ CFU of Listeria monocytogenes and bacterial burden in the spleen measured 48 hours following infection by CFU. Statistical significance was determined by Student’s t-test. Data are representative of two independent experiments with 4–5 mice per condition. (F) CMTB and control mice were challenged i.v. with 1x10⁶ B16-F10 melanoma cells. Disease burden was quantified by counting the number of metastases visible in the lung 10 days following challenge (left panel, black spots). Data are representative data of three independent experiments with 4–5 mice per group. Significance was assessed by Student’s t-Test. Error bars depict the mean and SD.

Fig 3. The protective phenotype in CMTB mice is associated with elevated tissue inflammation and an early immune response.

(A) Tissues were isolated 10 or 42 days following the establishment of CMTB or from control mice (plotted at day 0). Absolute levels of cytokines and chemokines significantly altered by CMTB during at least one time-point in the lung or spleen (of 38 assayed) are plotted. Cytokine/chemokine amounts were normalized to total protein (n = 5 mice per time-point). A multiple t-test approach with Benjamini-Hochberg correction was used to test for significance (p < 0.05 for all analytes shown). (B) CD11b+ cells isolated from the lungs of CMTB or control mice were stimulated for 6 hours with the indicated TLR agonists and secreted TNF measured by ELISA (LPS 10 ng/mL, PAM3 300 ng/mL, R848 100 ng/mL). Data are representative of three independent experiments with cells from 3–5 mice pooled per condition. Points indicate technical replicates and significance was assessed by Student’s t-Test. Error bars depict the mean and SD. (C) Haemotoxylin and eosin stained lung sections from control and CMTB mice at the indicated time-points following aerosol challenge. Regions of infiltration by immune cells (filled arrow heads) and intrapulmonary hemorrhages (open arrow heads) are indicated. The corresponding quantitative pathology assessments are presented in S1 Table. Representative images from 5 mice per condition are displayed at 2×. (D) Quantification of relative number of CD11b⁺CD11c⁺CD64⁺Siglec-F^- monocyte derived macrophages in the lung parenchyma in control and CMTB mice at 14 days following aerosol infection with 100 CFU of Mtb H37Rv. Data are representative of two independent experiments with mice (n = 4–5) per condition. Statistical significance was determined by Student’s t-Test. Error bars depict the mean with SD. (See S19 Fig for gating strategy).). (E) Representative cytometry plot and corresponding quantification of CD3⁺CD8⁺TB10.4⁺ cells in the vasculature (CD45.2-PE⁺) and lung parenchyma (CD45.2-PE^-) in control and CMTB mice at 14 days following aerosol infection with 100 CFU of Mtb H37Rv. Statistical significance was determined by Student’s t-Test. Error bars depict the mean with SD. (F) Expression of MHC II on alveolar macrophages (CD11b^intCD11c⁺CD64⁺Siglec-F⁺) from control and CMTB mice isolated from the lung at 10 days following aerosol challenge. (See S19 Fig for gating.) Data are representative of two independent experiments with 5 mice per condition. Statistical significance was determined by Student’s t-Test. Error bars depict the mean with SD.

Fig 4. Alveolar macrophages are reprogrammed by CMTB.

(A) Flow cytometry analysis of AMs from control and CMTB mice (See S19 Fig for gating). Expression of the indicated markers on AMs was quantified by MFI. Significance was determined by Student’s t-Test. Error bars depict the mean and SEM. (Data are pooled from 4 independent experiments with 3–5 mice per experiment). (B) AMs from CMTB mice and controls were isolated by FACS and the transcriptome assessed by RNA sequencing. The plot shows significance vs. fold-change in transcript expression between CMTB and control AMs. Red dots represent transcripts that were significantly differentially expressed between conditions (|log₂(fold-change)| > 1 and FDR < 0.05). Each point represents the average of 3 biological replicates each of which consists of BAL pooled from 10 mice. (C) Infected and uninfected AMs from CMTB mice and controls were isolated by FACS 24 hours after aerosol challenge with ~3000 CFU of mEmerald-expressing Mtb. Red dots represent transcripts that were significantly differentially expressed between conditions (log₂(fold-change) > 1 and FDR < 0.05). Each dot represents the average of two biological replicates, each of which consists of BAL pooled from 10 mice. (D) (left panel) Transcripts differentially expressed (as defined in C) in AMs from control mice compared to AMs from CMTB mice. Transcripts responding to infection are color-coded by whether they are differentially expressed in: red = CMTB only, black = control only, orange = both conditions. (right panel) VENN diagram showing the number of differentially expressed transcripts in each group. (E) Enrichment scores for the 10 most enriched pathways in CMTB AMs defined by GSEA of transcripts altered by Mtb infection at 24 hours in AMs from control and CMTB mice are shown. Red (green) bars indicate significant enrichment with FDR < 0.01 (0.05). White bars indicate FDR > 0.05. (F) Heatmap showing the fold changes of unique, significantly enriched leading edge genes from the GSEA. Key transcripts are highlighted. (G) Control and CMTB mice were infected with ~3000 CFU of mEmerald-expressing Mtb and the distribution of infected cell types determined by flow cytometry at 10 days following challenge. Left panel: Quantification of the distribution of infection across cell types. Right panel: Fraction of AMs infected. (See S19 Fig). Data are representative of three replicate experiments with 4–5 mice per condition. Significance was determined by Student’s t-Test. Error bars depict the mean and SD. (H) AMs from CMTB mice and controls were harvested by bronchoalveolar lavage and infected with H37Rv ex vivo at an MOI of 1. Bacterial burden was measured by CFU at 2 hours and 5 days following infection. Data are representative of two independent experiments with 4–5 mice per condition. Significance was measured by Student’s t-test. Error bars depict the mean and SD.

Fig 5. Immune activation in CMTB mice depends on systemic IFNg signaling.

(A) MHC II expression on circulating monocytes in control and CMTB mice 6 weeks following the establishment of CMTB. (See S17 Fig for gating strategy.) Significance was measured by Student’s t-test. Error bars depict the mean and SD. (B) MHC II expression on circulating monocytes in WT:Ifngr1^-/- mixed bone marrow chimeras with and without CMTB. (See S20 Fig for gating strategy.) (C) MHC II expression on alveolar macrophages in WT:Ifngr1^-/- mixed bone marrow chimeras with and without CMTB. Dashed line indicates MFI of MHC II in unlabeled cells. (See S21 Fig for gating strategy).