Heme catabolism by heme oxygenase-1 confers host resistance to Mycobacterium infection

Abstract

Heme oxygenases (HO) catalyze the rate-limiting step of heme degradation. The cytoprotective action of the inducible HO-1 isoform, encoded by the Hmox1 gene, is required for host protection against systemic infections. Here we report that upregulation of HO-1 expression in macrophages (M) is strictly required for protection against mycobacterial infection in mice. HO-1-deficient (Hmox1(-/-)) mice are more susceptible to intravenous Mycobacterium avium infection, failing to mount a protective granulomatous response and developing higher pathogen loads, than infected wild-type (Hmox1(+/+)) controls. Furthermore, Hmox1(-/-) mice also develop higher pathogen loads and ultimately succumb when challenged with a low-dose aerosol infection with Mycobacterium tuberculosis. The protective effect of HO-1 acts independently of adaptive immunity, as revealed in M. avium-infected Hmox1(-/-) versus Hmox1(+/+) SCID mice lacking mature B and T cells. In the absence of HO-1, heme accumulation acts as a cytotoxic pro-oxidant in infected M, an effect mimicked by exogenous heme administration to M. avium-infected wild-type M in vitro or to mice in vivo. In conclusion, HO-1 prevents the cytotoxic effect of heme in M, contributing critically to host resistance to Mycobacterium infection.

Fig 1

Expression of HO-1 is required for host resistance to M. avium infection. BALB/c mice were infected Mycobacterium avium 2447 SmT (10⁶ CFU, i.v.). (A) Expression of HO-1 protein in the liver, detected by Western blotting at 1, 15, 30, and 60 days after infection. (B) Hmox1 mRNA expression quantified by quantitative reverse transcription-PCR (qRT-PCR) in the livers of BALB/c mice and densitometry of the Western blot shown in panel A. Data are shown as fold change in infected relative to noninfected mice (dashed line) (n = 3 to 5 mice per time point). (C) HO-1 expression detected by immunofluorescence in a representative liver section from BALB/c mice at 30 days after M. avium infection. Arrows indicate F4/80⁺ cells expressing HO-1. (D) Bacterial loads in the livers, spleens, and lungs of M. avium-infected Hmox1^+/+, Hmox1^+/−, and Hmox1^−/− BALB/c (n = 3 per genotype) or SCID (n = 4 or 5 per genotype) mice. Bars represents mean + standard deviation of log₁₀ CFU/organ. *, P < 0.05; ***, P < 0.001; NS, not significant.

Fig 2

Hmox1-deficient mice produce normal or elevated levels of inflammatory cytokines and chemokines in response to M. avium infection. Cytokine and chemokine concentrations in the plasma, livers, and spleens of SCID.BALB/c Hmox1^+/+ and Hmox1^−/− mice at 30 days after M. avium infection were assessed by a bead-based multiplex immune assay. Data are shown as mean + standard deviation for a group of 3 mice per genotype. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Fig 3

Hmox1-deficient mice do not form granulomas. Liver sections from BALB/c Hmox1^+/+ (A, B, and C) and Hmox1^−/− (D, E, and F) mice stained with hematoxylin and eosin (A, B, D, and E) or Masson's trichrome (collagen fibers stain blue) (C and F) at 60 days after M. avium infection are shown. Arrows indicate granulomas. Bar, 100 μm. Notice the lack of granulomas in Hmox1-deficient mice (D to F) versus wild-type controls (A to C).

Fig 4

Hmox1-deficient mice develop oxidative stress. (A) Quantification of hemoglobin and free heme in the plasma and total iron in the livers of BALB/c Hmox1^+/+ and Hmox1^−/− mice at 60 days after M. avium infection (n = 3 to 5). Bars represent the average + standard deviation for each group. *, P < 0.05; **, P < 0.01. (B) Oxidative stress in the livers of BALB/c mice was monitored by detecting oxidized nucleosides 8-OHdG and 8-OHG, which are markers of oxidative damage to DNA and RNA, respectively, by immunohistochemistry. Note the increased DAB (brown) straining in the cytoplasm (RNA) and nuclei (DNA, arrows) of Hmox1^−/− mice. The sections were counterstained with methyl green. Bar = 50 μm.

Fig 5

Hmox1-deficient mice have increased cell death. (A and B) Cell death in the livers of BALB/c Hmox1^+/+ and Hmox1^−/− mice was evaluated by TUNEL (green) (A) and by detecting cleaved caspase-3 (red) (B) at 60 days after M. avium infection. Nuclei were stained with DAPI (blue). Bar, 100 μm. (C) Quantification of the positive cells in panels A and B. Bars represent the average + standard deviation for each group. *, P < 0.05; ***, P < 0.001.

Fig 6

Heme administration triggers Mϕ cell death and hampers granuloma formation. Bone marrow-derived Mϕ (BMMϕ) from wild-type mice were exposed to heme 1 h prior to M. avium infection. (A) Cell death of BMMϕ visualized by TUNEL (green) at 24 h after M. avium infection. DAPI (blue) was used for total DNA staining. (B) BMMϕ viability was quantified by resazurin staining at 24 h after M. avium infection. Results are shown as mean + standard deviation for three culture wells per condition. (C) Bacterial load was quantified at 4 days after infection as CFU. Results are shown as mean + standard deviation of the log₁₀ increase in CFU/well of three wells per condition. *, P < 0.05; ***, P < 0.001. (D) Number of granulomas in the livers of BALB/c mice treated with heme (40 mg/kg, every other day, i.v.) or vehicle and infected with M. avium for 16 days. Each bar represents the mean + one standard deviation (n = 3). *, P < 0.05. (E) Bacterial loads in the livers, spleens, and lungs of the mice described for panel D. Each bar represents the mean + one standard deviation of log₁₀ CFU/organ. (F) Representative images of hematoxylin- and eosin-stained liver sections. Arrows indicate granulomas. The inset shows one typical granuloma. Bar, = 100 μm.

Fig 7

Expression of HO-1 is required for host resistance to M. tuberculosis infection. (A) The expression of HO-1 was detected by immunohistochemistry in the lungs of BALB/c mice infected aerogenically with a low dose (30 CFU/mouse) of M. tuberculosis for 2 months. Note the expression of HO-1 (brown) within the granuloma. Bar, 200 μm. (B) Bacterial loads in the lungs and spleens of BALB/c mice infected aerogenically with a low dose (30 CFU/mouse) of M. tuberculosis for 2 months. *, P < 0.05; **, P < 0.01. Each symbol indicates an individual mouse analyzed, and horizontal bars indicate mean values. (C) Survival of M. tuberculosis-infected mice. Values in brackets indicate the log₁₀ CFU in the lungs of moribund mice. Hmox1^+/+, n = 5; Hmox1^+/−, n = 4; and Hmox1^−/−, n = 5. (D) Representative lung sections stained with hematoxylin and eosin from infected wild-type (left) and Hmox1^−/− (right) mice at 60 days after infection. Notice the granuloma in formation in the wild-type mouse and the monocytic, less organized infiltrate in the Hmox1^−/− mouse. (E) Representative Ziehl-Neelsen staining of the lung of a moribund Hmox1^−/− mouse (bar, 50 μm). (F) Representative TUNEL staining of the lung of an Hmox1^−/− mouse infected with M. tuberculosis for 2 months.