Tuberculous granulomas are hypoxic in guinea pigs, rabbits, and nonhuman primates

Abstract

Understanding the physical characteristics of the local microenvironment in which Mycobacterium tuberculosis resides is an important goal that may allow the targeting of metabolic processes to shorten drug regimens. Pimonidazole hydrochloride (Hypoxyprobe) is an imaging agent that is bioreductively activated only under hypoxic conditions in mammalian tissue. We employed this probe to evaluate the oxygen tension in tuberculous granulomas in four animal models of disease: mouse, guinea pig, rabbit, and nonhuman primate. Following infusion of pimonidazole into animals with established infections, lung tissues from the guinea pig, rabbit, and nonhuman primate showed discrete areas of pimonidazole adduct formation surrounding necrotic and caseous regions of pulmonary granulomas by immunohistochemical staining. This labeling could be substantially reduced by housing the animal under an atmosphere of 95% O(2). Direct measurement of tissue oxygen partial pressure by surgical insertion of a fiber optic oxygen probe into granulomas in the lungs of living infected rabbits demonstrated that even small (3-mm) pulmonary lesions were severely hypoxic (1.6 +/- 0.7 mm Hg). Finally, metronidazole, which has potent bactericidal activity in vitro only under low-oxygen culture conditions, was highly effective at reducing total-lung bacterial burdens in infected rabbits. Thus, three independent lines of evidence support the hypothesis that hypoxic microenvironments are an important feature of some lesions in these animal models of tuberculosis.

FIG. 1.

Comparison of solid and caseous granulomas as revealed by H&E staining and anti-PIMO adduct labeling. Shown are granulomas from mice (A and B), humans (C and D), guinea pigs (E to H), rabbits (I to L), and macaques (M to P) infected with M. tuberculosis complex. Solid granulomas, lacking necrosis, are seen in human disease (C) and in each of the animal models (A, E, I, and M) and do not label strongly with PIMO (B, F, J, and N). In contrast, caseous necrotic granulomas (D, G, K, and O) have cellular regions that, in the guinea pig, rabbit, and macaque models displaying these lesions, form PIMO adducts (H, K, and P; brown coloration), indicating low oxygen tension. The murine strains lacked caseous granulomas and were not labeled with an anti-PIMO adduct antibody. The inset in panel B shows murine kidney staining with an anti-PIMO antibody from the same mouse. Resected human lung tissue was not stained with PIMO. Effective magnification, ×10.

FIG. 2.

Summary of pimonidazole staining by species and lesion type. (A) Summary of the distribution of granulomas, showing solid and caseous histology in each animal model. (B) Proportion of each granuloma type labeling with anti-PIMO in the animal models. Mo, mouse; Gp, guinea pig; Rb, rabbit; Mk, macaque. Open bars, solid granulomas; filled bars, caseous/necrotic granulomas.

FIG. 3.

Pimonidazole adduct formation is O₂ dependent in the rabbit model. (A) Necrotic granulomas showed reduced staining for activated PIMO adducts (brown coloring) after infected rabbits were housed under a 95% O₂ atmosphere during PIMO exposure. (B) Granuloma stained for activated PIMO adducts after infected rabbits were housed in normal room air. A representative granuloma is shown. Effective magnification, ×100.

FIG. 4.

Measurement of pO₂ in granulomas of M. tuberculosis complex-infected rabbits using a fiber optic oxygen probe. (A) After intubation and ventilation with medical air, a thoracotomy was performed and rib spreaders were used to open the thorax. The oxygen probe (sheathed in a 22-gauge needle) was inserted into visible granulomas or apparently normal portions of the lung. (B) Immunohistochemistry of an M. tuberculosis-induced granuloma for the hypoxia marker PIMO. Shown is a representative granuloma where pO₂ was measured (original magnification, ×50). (C) Representative traces of the response to the fiber optic pO₂-temperature probe upon insertion into normal lung or granulomas within the same rabbit. The probe was maintained in position for ∼1 min in each location. (D) Summary of direct measurements of pO₂ with the fiber optic probe in lungs of uninfected rabbits (norm), grossly normal lungs in infected rabbits (norm inf), granulomas of M. bovis-infected rabbits (Mb gran), and granulomas of M. tuberculosis-infected rabbits (Mtb gran). Two to five granulomatous lesions (diameters, 3 to 5 mm) and two to three macroscopically normal areas were measured in the right medial and caudal lobes of seven infected rabbits. Two to three measurements were made in the same region for three uninfected rabbits. Each time the system was used or a probe was changed, the probe function was confirmed by measurement of pO₂ in humidified 35 to 37°C room air (∼151 mm Hg) and the fourth intercostal muscle (∼25 mm Hg). For each group of measurements, the horizontal line and error bar represent the mean and standard deviation. The difference in pO₂ was significant between normal lung tissue and either type of granuloma (P < 0.001), but there was no significant difference in pO₂ between normal-appearing lungs in uninfected or infected rabbits (P > 0.05) by using one way ANOVA with Tukey's posttest.

FIG. 5.

Metronidazole treatment of M. bovis Ravenel-infected rabbits is as effective as rifampin in reducing the burden of viable bacilli. (A) Number of M. bovis CFU in the right middle lobe per gram of lung tissue of rabbits given 28 days of drug therapy with Mtz (M), Rif (R), or vehicle only (C) (n = 6 per group). A one-way ANOVA analysis returned a P value of <0.0001; the Bonferroni multiple-comparison test returned P values of <0.001 (*) for Mtz and <0.0052 (**) for Rif compared to the control. (B) Number of M. bovis CFU per individual 1- to 1.5-mm granuloma in the same experiment. P < 0.0001 by ANOVA. By the Bonferroni multiple-comparison test, P < 0.0001 (*) for Mtz (n = 17) and P < 0.0004 (**) for Rif (n = 19) compared to the control (n = 16).