The inducible nitric oxide synthase locus confers protection against aerogenic challenge of both clinical and laboratory strains of Mycobacterium tuberculosis in mice

Abstract

Murine macrophages effect potent antimycobacterial function via the production of nitric oxide by the inducible isoform of the enzyme nitric oxide synthase (NOS2). The protective role of reactive nitrogen intermediates (RNI) against Mycobacterium tuberculosis infection has been well established in various murine experimental tuberculosis models using laboratory strains of the tubercle bacillus to establish infection by the intravenous route. However, important questions remain about the in vivo importance of RNI in host defense against M. tuberculosis. There is some evidence that RNI play a lesser role following aerogenic, rather than intravenous, M. tuberculosis infection of mice. Furthermore, in vitro studies have demonstrated that different strains of M. tuberculosis, including clinical isolates, vary widely in their susceptibility to the antimycobacterial effects of RNI. Thus, we sought to test rigorously the protective role of RNI against infection with recent clinical isolates of M. tuberculosis following both aerogenic and intravenous challenges. Three recently isolated and unique M. tuberculosis strains were used to infect both wild-type (wt) C57BL/6 and NOS2 gene-disrupted mice. Regardless of the route of infection, NOS2(-/-) mice were much more susceptible than wt mice to any of the clinical isolates or to either the Erdman or H37Rv laboratory strain of M. tuberculosis. Mycobacteria replicated to much higher levels in the organs of NOS2(-/-) mice than in those of wt mice. Although the clinical isolates all exhibited enhanced virulence in NOS2(-/-) mice, they displayed distinct growth rates in vivo. The present study has provided results indicating that RNI are required for the control of murine tuberculous infection caused by both laboratory and clinical strains of M. tuberculosis. This protective role of RNI is essential for the control of infection established by either intravenous or aerogenic challenge.

FIG. 1

Bacterial burdens in lungs of NOS2^−/− (closed circles) and wild-type C57BL/6 (open circles) mice following intravenous infection with M. tuberculosis Erdman (A) or clinical isolate CI3 (B), CI4 (C), or CI7 (D). Mice were infected with 10⁵ to 10⁶ CFU of M. tuberculosis, and lung homogenates were plated at the times indicated to determine the number of viable bacilli. Each point is the mean of three mice, and the bar is the standard error and is representative of two experiments. By 4 weeks postinfection and beyond, the bacterial burden in the lungs of NOS2^−/− mice is significantly greater than that of wild-type C57BL/6 animals for all of the M. tuberculosis strains examined (P < 0.05).

FIG. 2

Bacterial burdens in lungs of NOS2^−/− (closed circles) and wild-type C57BL/6 (open circles) mice following aerosol infection with M. tuberculosis Erdman (A) or clinical isolate CI3 (B), CI4 (C), or CI7 (D). Mice were infected by aerosol with approximately 50 viable bacilli, and lung homogenates were plated at the times indicated to determine the number of viable bacilli. Each point is the mean of three mice (except on day 1 postinfection, when two mice were examined), and the bar is the standard error. M. tuberculosis Erdman and clinical isolate CI3 were evaluated three times with similar results. Isolates CI4 and CI7 were studied once. By 33 days postinfection and beyond, the bacillary load in the lungs of NOS2^−/− mice was significantly greater than that of wild-type C57BL/6 animals for all of the strains of M. tuberculosis tested (P < 0.05)

FIG. 3

Survival of NOS2-deficient mice following infection with 10⁵ to 10⁶ CFU intravenously (A) or with approximately 50 CFU aerogenically (B). The M. tuberculosis strains used for infection included Erdman (open triangle) and clinical isolates CI3 (closed circle), CI4 (open circle), and CI7 (closed triangle). No wild-type C57BL/6 mouse succumbed to infection. There were five mice per group. Similar results were obtained in a second experiment.

FIG. 4

Growth of clinical isolates CI3 (A) and CI7 (B) in C57BL/6 macrophages. Thioglycolate-elicited peritoneal macrophages were left unactivated (solid line) or were activated with IFN-γ and LPS (dashed line) and then infected with one of the clinical isolates of M. tuberculosis at a multiplicity of infection of 2 to 4. The number of intracellular bacteria was determined over 3 days by plating cell lysates onto 7H10 agar plates and enumerating bacterial colonies 21 days later. Each point is the mean of three wells; bars are standard errors. The x axis denotes hours postinfection.