Expression of the nitric oxide synthase 2 gene is not essential for early control of Mycobacterium tuberculosis in the murine lung

Abstract

The interleukin-12 and gamma interferon (IFN-gamma) pathway of macrophage activation plays a pivotal role in controlling tuberculosis. In the murine model, the generation of supplementary nitric oxide by the induction of the nitric oxide synthase 2 (NOS2) gene product is considered the principal antimicrobial mechanism of IFN-gamma-activated macrophages. Using a low-dose aerosol-mediated infection model in the mouse, we have investigated the role of nitric oxide in controlling Mycobacterium tuberculosis in the lung. In contrast to the consequences of a systemic infection, a low dose of bacteria introduced directly into the lungs of mice lacking the NOS2 gene is controlled almost as well as in intact animals. This is in contrast to the rapid progression of disease in mice lacking IFN-gamma or a key member of the IFN signaling pathway, interferon regulatory factor 1. Thus while IFN-gamma is pivotal in early control of bacterial growth in the lung, this control does not completely depend upon the expression of the NOS2 gene. The absence of inducible nitric oxide in the lung does, however, result in increased polymorphonuclear cell involvement and eventual necrosis in the pulmonary granulomas of the infected mice lacking the NOS2 gene.

FIG. 1

The course of M. tuberculosis infection in mice lacking components of the IFN-γ-mediated macrophage activation pathway. Mice were infected aerogenically with 100 virulent bacteria, and the numbers of bacteria detected in the lungs of C57BL/6 (solid circles), IRF-1-KO (empty circles) and NOS2-KO mice (triangles) were determined. Data represent the mean log₁₀ number of viable bacteria per organ (n = 4) ± the standard error. ∗, means which were statistically different from the C57BL/6 values as determined by Student's t test (P ≤ 0.05). †, mice were euthanized due to morbidity. The graph reflects one experiment representative of two similar experiments.

FIG. 2

The course of M. tuberculosis infection in mice lacking the IFN-αβ signal transduction receptor. Mice were infected aerogenically with 100 virulent bacteria, and the numbers of bacteria detected in the lungs of B6/129 control mice (circles) and IFN-αβ R-KO mice (squares) were determined. Data represent the mean log₁₀ number of viable bacteria per organ (n = 4) ± the standard error. ∗, means which were statistically different from the B6/129 values as determined by Student's t test (P ≤ 0.05). The graph reflects one experiment representative of two similar experiments.

FIG. 3

Representative photomicrographs (from two experiments) of formalin-fixed sections from the lungs of C57BL/6 (A), NOS2-KO (B), and IRF-1-KO (C) mice aerogenically infected 30 days previously. The granuloma is primarily mononuclear in nature in the lungs of C57BL/6 mice, compared to the neutrophilic accumulation in the lungs of NOS2-KO (B, inset) and IRF-1-KO (C, inset) mice. In both the NOS2-KO and the IRF-1-KO mice, purulent debris can be seen breaking into the airways (B and C, main images). Magnification for main panels, ×200; for insets, ×1,000.