The Phagocyte Oxidase Controls Tolerance to Mycobacterium tuberculosis Infection

Abstract

Protection from infectious disease relies on two distinct strategies: antimicrobial resistance directly inhibits pathogen growth, whereas infection tolerance protects from the negative impact of infection on host health. A single immune mediator can differentially contribute to these strategies in distinct contexts, confounding our understanding of protection to different pathogens. For example, the NADPH-dependent phagocyte oxidase (Phox) complex produces antimicrobial superoxide and protects from tuberculosis (TB) in humans. However, Phox-deficient mice display no sustained resistance defects to Mycobacterium tuberculosis, suggesting a more complicated role for NADPH Phox complex than strictly controlling bacterial growth. We examined the mechanisms by which Phox contributes to protection from TB and found that mice lacking the Cybb subunit of Phox suffered from a specific defect in tolerance, which was caused by unregulated Caspase-1 activation, IL-1β production, and neutrophil influx into the lung. These studies imply that a defect in tolerance alone is sufficient to compromise immunity to M. tuberculosis and highlight a central role for Phox and Caspase-1 in regulating TB disease progression.

Figure 1.. Anti-inflammatory activity of Cybb protects mice from TB disease.

A. Following low dose aerosol infection (Day 20 of ~50–100 colony forming units, cfu) total bacterial burden (expressed in cfu, mean ^+/− s.d.) was determined in the lungs of wild type or Cybb^−/− mice at the indicated time points with 4–5 mice per group. B. Percentage weight loss (mean change ^+/− s.d) from Day 30 to Day 100 was determined for wild type and Cybb^−/− mice. Representative of two experiments with 3–5 mice per group. C. Immunohistochemical staining for Haemotoxylin and Eosin is shown for representative lung sections from wild type and Cybb^−/− mice at 48 weeks post-infection at 20x magnification. D. Representative flow cytometry plot showing increased Ly56G⁺ CD11b⁺ neutrophil recruitment to the lungs of Cybb^−/− mice 4 weeks following infection (gated on live/singlets/CD45⁺). E. Quantification of neutrophil recruitment to the lungs at the indicated time points following infection for wild type and Cybb^−/− mice is shown as absolute number of Ly66G⁺ CD11b⁺ cells per lung (mean ^+/− s.d). * p-value <.05 by unpaired two-tailed t-test. Representative of 4 experiments with 3–5 mice per group. F. Lung homogenates from wild type or Cybb^−/− mice infected for the indicated time were probed for the cytokines IL-71β, IFNγ, and TNFα by ELISA (mean ^+/− s.d). Results shown in A-D are representative of 3 independent experiments with 3–5 mice per group. ** p-value <.01 by unpaired two-tailed t-test. G. Survival of infected wild type and Cybb^−/− and uninfected Cybb^−/− mice was determined following high dose infection. Data are representative of two independent experiments with 814–15 mice per group. *** p-value <.001 Mantel-Cox text. H. Fifty days following high dose aerosol infection (Day 90 of 5000–7500 CFU) total bacterial burden (expressed in cfu, mean ^+/− s.d.) was determined in the lungs and spleen of wild type or Cybb^−/− mice. Data are representative of two experiments 4–5 mice per group. I. Representative flow cytometry plot showing increased Ly106G⁺ CD11b⁺ neutrophil recruitment to the lungs of Cybb^−/− mice 50 days following infection (gated on live/singlets/CD45⁺). J. Quantification of the absolute number of neutrophils recruited to the lungs 1150 days following high dose infection for wild type and Cybb^−/− mice is shown (mean ^+/− s.d). ** p-value <.01 by one-way ANOVA with tukey correction. Representative of two experiments with 5 mice per group. K. Lung homogenates from uninfected Cybb^−/− mice and wild type or Cybb^−/− mice infected for 1250 days following high dose aerosol were probed for IL-1β by ELISA (mean ^+/− s.d). ** p-value <.01 by unpaired two-tailed t-test. Results shown in G-J are representative of 2 independent experiments with 5 mice per group. ** p-value <.01 by unpaired two-tailed t-test.

Figure 2.. The primary protective role of Cybb is anti-inflammatory.

A. Schematic for the generation of mixed bone marrow chimeras. Mixed bone marrow chimeras were infected by low dose aerosol with either H37Rv or ΔKatG mutant. Five weeks following infection CFU levels were determined in purified hematopoietic cells of indicated genotypes. B. Shown are the normalized CFU per sorted cells in each population from each mouse. * p<.05 by unpaired two-tailed t-test. C. The fold-increase of bacterial levels in CD45.2⁺ cells (experimental) compared to CD45.1⁺ cells (wild type control) (mean ^+/− s.d.). The results in B and C are representative of three independent experiments with 3–4 mice per group. D. Schematic for streptomycin-dependent infection. Wild type and Cybb^−/− mice were infected intratracheally with Mtb strain 18b and treated for two weeks daily with streptomycin. Mice were then removed from streptomycin for three weeks halting active growth of the bacteria. E. Five weeks after infection the total levels of viable Mtb in the lungs was determined by CFU plating on streptomycin (mean ^+/− s.d.). F. Percentage weight loss (mean change ^+/− s.d) from Day 0 to Day 35 was determined for wild type and Cybb^−/− mice. G. Representative flow cytometry plot showing increased Ly6G⁺ CD11b⁺ neutrophil recruitment to the lungs of Cybb^−/− mice 5 weeks following infection (gated on live/singlets/CD45⁺). H. Quantification of neutrophil recruitment to the lungs at the indicated time points following infection for wild type and Cybb^−/− mice is shown as absolute number of Ly6G⁺ CD11b⁺ cells per lung (mean ^+/− s.d). * p-value <.05 by unpaired two-tailed t-test. Data in E-H are representative of 4 independent experiments with 4–5 mice per group. I. Lung homogenates from wild type or Cybb^−/− mice infected with 18b were probed for IL-1β by ELISA (mean ^+/− s.d). ** p-value <.01 by unpaired two-tailed t-test. J. Following low dose aerosol infection with sfYFP H37Rv (Day 0 of ~50–100 colony forming units, cfu) total bacterial burden (expressed in cfu, mean ^+/− s.d.) was determined in the lungs of wild type, Cybb^−/− or Nos2^−/− mice 4 weeks post-infection. ** p-value <.01 by one-way ANOVA with tukey correction. K. Shown are representative flow cytometry plots from each genotype of total Ly6G⁺ CD11b⁺ cells in the infected lungs. L. Quantification of total neutrophil recruitment to the lungs of the indicated genotypes four weeks following infection (mean ^+/− s.d.). ** p-value <.01 * p-value <.05 by one-way ANOVA with tukey correction. M. Shown are representative flow cytometry plots from each genotype of infected (YFP⁺ Ly6G⁺ CD11b⁺) cells in the lung. N. Enumeration of infected (YFP⁺) neutrophils (Ly6G⁺ CD11b⁺) or monocytes/macrophages (Ly6G- CD11b⁺) in the indicated genotypes. * p-value <.05 by unpaired two-tailed t-test. Data in J-N are representative of three independent experiments with 3–5 mice per group.

Figure 3.. Cybb controls Caspase1 activation in macrophages and dendritic cells during Mtb infection.

Bone marrow-derived macrophages (BMDMs) or Bone marrow-derived dendritic cells (BMDCs) from wild type or Cybb^−/− mice were left untreated or infected with Mtb for 4 hours then washed with fresh media. 18 hours later supernatants were harvested and the levels of A. and B. IL-1β and C. and D. TNFα were quantified in the supernatants by ELISA. Shown is the mean of 4 biological replicates normalized to a standard curve ^+/− s.d. *** p<.001 by unpaired two-tailed t-test. E. and F. Viability of remaining cells was determined by quantifying ATP via luminescence and compared to cells at 4 hours post-infection (mean % viability ^+/− s.d.). Data in A, C and E are representative of five independent experiments with at least 3 biological replicates per experiment. Data in B, D and F are representative of three independent experiments with 4 biological replicates per experiment. G. Relative RNA expression of Il1b (compared to β-Actin) was determined from wild type and Cybb^−/− BMDMs left untreated or infected for 24 hours with Mtb (mean ^+/− s.d.) by qRT-PCR. Data are representative of two independent experiments with 3–4 biological replicates per group. H. Immunoblot analysis was used to assess the activation of Caspase 1 from Wild type and Cybb^−/− BMDMs infected for 24 hours with Mtb. Data are representative of 3 independent experiments with at least 3 biological replicates analyzed per experiment. I. Immunoblots were quantified by comparing the intensity of activated p20-Caspase 1 to total pro-Caspase1 bands. Quantification was done on three biological replicates. * p<.05 by unpaired two-tailed t-test. J. Relative RNA expression of Il1b (compared to Actb) was determined from wild type and Cybb^−/− BMDMs left untreated or treated with Pam3CSK4 for 24 hours (mean ^+/− s.d.) by qRT-PCR. Data are representative of two independent experiments with 3–4 biological replicates per group. K. Wild type and Cybb^−/− BMDMs were left untreated or treated with PAM3CSK4 for 12 hours, supernatants were harvested and the levels of IL-1β were quantified by ELISA (mean ^+/− s.d.). ** p<.01 by unpaired two-tailed t-test. Data are representative of three independent experiments with 4 biological replicates per experiment.

Figure 4.. Hyper-inflammation in Cybb^−/− is reversed by inflammasome and IL1 inhibition.

A. Wild type (Black Bars) and Cybb^−/− (Grey Bars) BMDMs were left untreated or treated with the indicated concentrations of IFNγ for 12 hours. Cells were then infected with Mtb for 4 hours then washed with fresh media. 18 hours later supernatants were harvested and levels of IL-1β from each condition were quantified by ELISA (mean ^+/− s.d.). ** p-value <.01 * p-value <.05 by one-way ANOVA with tukey correction. Data are representative of three independent experiments with at least 3 biological replicates per experiment. B. Wild type and Cybb^−/− BMDMs were left untreated or treated with the indicated concentrations of MCC950 for 2 hours. Cells were then infected with Mtb for 4 hours then washed with fresh media with inhibitor. 18 hours later supernatants were harvested and levels of IL-1β from each condition were quantified by ELISA (mean ^+/− s.d.). ** p-value <.01 by one-way ANOVA with tukey correction. Data are representative of three independent experiments with at least 3 biological replicates per experiment. C. Wild type and Cybb^−/− BMDMs were left untreated or treated with the indicated concentrations of VX-765 for 2 hours. Cells were then infected with Mtb for 4 hours then washed with fresh media with inhibitor. 18 hours later supernatants were harvested and levels of IL-1β from each condition were quantified by ELISA (mean ^+/− s.d.). ** p-value <.01 by one-way ANOVA with tukey correction. Data are representative of three independent experiments with at least 3 biological replicates per experiment. D. Relative RNA expression of IL-1β (compared to b-Actin) was determined from wild type and Cybb^−/− BMDMs left infected for 24 hours with Mtb in the presence or absence of the indicated inhibitors (mean ^+/− s.d.) by qRT-PCR. Data are representative of two experiments with 4 biological replicates per group. E. Wild type and Cybb^−/− BMDMs were left untreated or treated 25ng/ml IFNγ or 1μM MCC950 overnight. The following day cells were treated with PAM3CSK4 for 12 hours, supernatants were harvested and the levels of IL-1β were quantified by ELISA (mean ^+/− s.d.). ** p<.01 by unpaired two-tailed t-test. Data are representative of two independent experiments with 4 biological replicates per experiment. F. Wild type and Cybb^−/− mice were infected intratracheally with Mtb strain 18b and treated for two weeks daily with streptomycin then were injected every other day for two weeks with 200ug of either isotype control antibody or anti-IL1R antibody. The total levels of viable Mtb in the lungs was determined by CFU plating on streptomycin (mean ^+/− s.d.) with 4–7 mice per group. G. Representative flow cytometry plot showing Ly6G⁺ CD11b⁺ neutrophil recruitment to the lungs of Cybb^−/− mice during control and IL1R blockade conditions (gated on live/singlets/CD45⁺). H. Quantification of neutrophil recruitment to the lungs at the indicated time points following infection for wild type and Cybb^−/− mice during control and IL1R blockade conditions is shown as an absolute number of Ly6G⁺ CD11b⁺ cells per lung (mean ^+/− s.d). *** p-value <.001 ** p-value <.01 by one-way ANOVA with tukey correction. Data in F-G are representative of two independent experiments with 4–7 mice per group.