Response to Hypoxia and the Ensuing Dysregulation of Inflammation Impacts Mycobacterium tuberculosis Pathogenicity

Abstract

Rationale: Different Mycobacterium tuberculosis (Mtb) strains exhibit variable degrees of virulence in humans and animal models. Differing stress response strategies used by different strains of Mtb could influence virulence. Objectives: We compared the virulence of two strains of Mtb with use in animal model research: CDC1551 and Erdman. Methods: Rhesus macaques, which develop human-like tuberculosis attributes and pathology, were infected with a high dose of either strain via aerosol, and virulence was compared by bacterial burden and pathology. Measurements and Main Results: Infection with Erdman resulted in significantly shorter times to euthanasia and higher bacterial burdens and greater systemic inflammation and lung pathology relative to those infected with CDC1551. Macaques infected with Erdman also exhibited significantly higher early inflammatory myeloid cell influx to the lung, greater macrophage and T cell activity, and higher expression of lung remodeling (extracellular matrix) genes, consistent with greater pathology. Expression of NOTCH4 (neurogenic locus notch homolog 4) signaling, which is induced in response to hypoxia and promotes undifferentiated cellular state, was also higher in Erdman-infected lungs. The granulomas generated by Erdman, and not CDC1551, infection appeared to have larger regions of necrosis, which is strongly associated with hypoxia. To better understand the mechanisms of differential hypoxia induction by these strains, we subjected both to hypoxia in vitro. Erdman induced higher concentrations of DosR regulon relative to CDC1551. The DosR regulon is the global regulator of response to hypoxia in Mtb and critical for its persistence in granulomas. Conclusions: Our results show that the response to hypoxia is a critical mediator of virulence determination in Mtb, with potential impacts on bacillary persistence, reactivation, and efficiency of therapeutics.

Keywords: hypoxia; mycobacterium; tuberculosis.

Figure 1.

Comparative infection of rhesus macaques with Mycobacterium tuberculosis (Mtb) CDC1551 and Erdman strains. (A) A comparison of different Mtb strains shows that Mtb Erdman results in reduced survival compared with Mtb CDC1551 infection in Indian rhesus macaques with Mtb Erdman (n = 6, shown in vermillion) compared with Mtb CDC1551 (n = 8, shown in blue) (P = 0.0281). Log-rank (Mantel-Cox) test was used. (B) All animals were monitored longitudinally for clinical signs of disease, including serum (CRP); unpaired t test (P = 0.0336) was used. (C) CXR score was established by veterinary clinicians for pneumonia; unpaired t test (P = 0.0361). (D and E) Throughout the study, animals were monitored for signs of respiratory distress, including respiratory rate (D) and pulmonary oxygen concentrations (E). Multiple t tests using Holm-Sidak method were used. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. (F) Monocyte (P = 0.0277) and (G) neutrophil (P = 0.0031) counts were measured as ×10³/μl of ethylenediaminetetraacetic acid blood at Week 2 after infection; unpaired t test was used. (H and I) Week 3 postinfection plasma (H) IL-6 (P = 0.0452) and (I) IFN-γ (P = 0.0499) were measured by multiplex; unpaired t test was used. (J–M) Throughout the study, Mtb Erdman–infected nonhuman primates (NHPs) had higher bacterial burden in their BAL (J, not significant) that was higher at necropsy in (K) their lungs (P = 0.1404), (L) individual granulomas (P = 0.0001), and (M) liver (P = 0.0472) compared with Mtb CDC1551–infected NHPs; unpaired t tests were used. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. BPM = beats per minute; CRP = C-reactive protein; CXR = chest X-ray; Sp_O2 = oxygen saturation as measured by pulse oximetry.

Figure 2.

Activation phenotype in alveolar and interstitial macrophages after infection with comparative strains of Mycobacterium tuberculosis (Mtb) in the lungs of rhesus macaques. (A–D) In Erdman-infected nonhuman primates (NHPs) at necropsy, gross lung (A) alveolar (CD163⁺CD206⁺; P = 0.108) and (B) interstitial (CD163⁻CD206⁺; P = 0.0312) as well as (C) granuloma alveolar (P = 0.0048) and (D) interstitial (P < 0.0001) macrophages had significantly increased CD40 expression and activation. (E and F) At necropsy, Erdman-infected NHPs had more MIP1-α (macrophage inflammatory protein 1-alpha) in their (E) BAL supernatant (P = 0.0136) and (F) plasma (P = 0.0613). (G–I) As early as Week 3 after tuberculosis infection, Erdman-infected NHPs had significantly more (G) IL-1B (P = 0.0452), (H) I-TAC (P = 0.0368), and (I) IL-6 (P = 0.0014) in their BAL supernatant. Unpaired t tests were used. *P < 0.05, **P < 0.01, and ****P < 0.0001.

Figure 3.

Lung T cells were more proliferative after CDC1551 infection in nonhuman primates (NHPs), but more antigen-specific effector T cells were observed after Erdman infection. (A) Proliferating CD4⁺ T cells were quantified by Ki67 expression in multiple tissues at necropsy. (B) Lung cells collected from Erdman-infected NHPs were stimulated overnight with Mycobacterium tuberculosis (Mtb) cell wall (MtbCW) and produced significantly more cytokines than lung cells from CDC1551-infected NHPs when compared in bulk (P = 0.0013 unpaired t test) or (C) when IFN-γ (P < 0.0001) and TNF-α (tumor necrosis factor-α; P = 0.0033) were compared separately by multiple t tests. (D and E) When polyfunctional CD4⁺ T cell responses were considered, the repertoire of lung T cells producing cytokines in response to Mtb cell wall was more diverse in (D) Erdman- than in (E) CDC1551-infected rhesus macaques. Unpaired t-tests were used. *P < 0.05, **P < 0.01, and ***P < 0.001. Br LN = bronchial lymph node; GrB = granzyme B.

Figure 4.

Comparable pathology was observed, but B cell activity was increased after Mycobacterium tuberculosis (Mtb) Erdman and CDC1551 infection in granulomas. (A) Percentage of lung involvement determined by board-certified veterinary pathologists (not significant; P = 0.0704). (B and C) Hematoxylin and eosin (H&E) representative figures show infected lung from CDC1551-infected nonhuman primate (NHP) necropsied at an early (B) and a late (C) time point. (D and E) H&E representative figures show infected lung from an Erdman-infected NHP necropsied at an early (D) and a late (E) time point. (F) B cells were quantified in granulomas as a proportion of lymphocytes. (G) Erdman-infected rhesus macaques had significantly more activation of their granuloma B cells with increased expression of CD69 (cluster of differentiation 69) (P = 0.0016). (H and I) Erdman-infected rhesus macaques had significantly more expression of proliferative marker Ki67 expressed on (H) CD4⁺ (P = 0.0006) and (I) CD8⁺ (P = 0.0154) T cells in their granulomas. This analysis compared up to three granulomas each from n = 6 Erdman- and n = 8 CDC1551-infected rhesus macaques. Unpaired t tests were used. Scale bars, 1 mm. *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 5.

RNAseq gene-expression analysis revealed increased lung remodeling and T-helper cell type 2 (Th2) signaling in the lungs of Erdman-infected rhesus macaques. (A) RNAseq gene-expression analysis showing 52 differentially expressed genes were identified in the lungs of Erdman- versus CDC1551-infected macaques that revealed an increase in lung remodeling pathways, platelet-related genes, M2 and Th2 signaling (IL-4), and NOTCH4 (neurogenic locus notch homolog 4) signaling pathways. (B) Plasma IL-4 at Week 3 (P = 0.0102) measured by multiplex. Unpaired t tests were used. *P < 0.05.

Figure 6.

Differential patterns of hypoxia-related genes in Mycobacterium tuberculosis (Mtb) Erdman and CDC1551 were observed in vitro after hypoxia and reaeration. Bacteria were cultured under hypoxic conditions, and their responses were characterized by RNAseq. (A and B) Major differences were detected in the magnitude and kinetics of the expression of the DosR (dormancy survival regulator)-regulon, a set of ∼50 Mtb genes, which the pathogen uses to respond to hypoxia, (A) after hypoxia and (B) after the reaeration phase. (C) Quantitative real-time RT-PCR measuring the expression amounts of dosR in Erdman and CDC1551 samples at 5 days of hypoxia and at 6 hours of normoxia showed that the amounts of dosR were significantly higher in Erdman than in CDC1551 (P = 0.001). Unpaired t tests were used. **P < 0.01.