MyD88-deficient mice display a profound loss in resistance to Mycobacterium tuberculosis associated with partially impaired Th1 cytokine and nitric oxide synthase 2 expression

Abstract

Mycobacterium tuberculosis possesses agonists for several Toll-like receptors (TLRs), yet mice with single TLR deletions are resistant to acute tuberculosis. MyD88(-/-) mice were used to examine whether TLRs play any role in protection against aerogenic M. tuberculosis H37Rv infection. MyD88(-/-) mice failed to control mycobacterial replication and rapidly succumbed. Moreover, expressions of interleukin 12, tumor necrosis factor alpha, gamma interferon, and nitric oxide synthase 2 were markedly decreased in the knockout animals. These results argue that resistance to M. tuberculosis must depend on MyD88-dependent signals mediated by an as-yet-undetermined TLR or a combination of TLRs.

FIG. 1.

MyD88^−/− mice are more susceptible than WT mice to aerogenic M. tuberculosis infection. (A) MyD88^−/− mice (diamonds) and C57BL/6 × 129sv (F₁) WT animals (squares), used to control for the possible influence of contaminating 129 genes, were infected in groups of 5 to 7 mice with 20 to 50 CFU of M. tuberculosis and monitored for survival. Data are representative of two separate experiments. Bacterial burdens (means ± standard errors) in the lungs (B) or livers (C) were also measured in three mice per group at the times indicated. Asterisks indicate statistically significant (P ≤ 0.05) differences determined by unpaired t test after log transformation between CFU values in MyD88^−/− and WT animals. Data are representative of the results from two experiments.

FIG. 2.

MyD88^−/− mice infected with M. tuberculosis exhibit more bacilli, exacerbated pathology, and reduced NOS2 expression in lungs, as well as delayed granuloma formation in liver. Formalin-fixed, paraffin-embedded lung (A to D, G, and H) and liver (E and F) tissue sections from mice 3 weeks after infection with aerosol M. tuberculosis were stained by the Kinyoun acid-fast method to detect mycobacteria (red staining) (A and B) or with hematoxylin and eosin stain (C to F). Note the absence of granulomas in panel F. NOS2 was visualized immunohistochemically in serial sections of these same tissues (G and H). Sections shown are representative of multiple fields from the organs of at least three animals per group. Original magnifications are ×63 (A and B), ×5 (C and D), ×10 (E and F), and ×20 (G and H).

FIG. 3.

Expression of IL-12, IFN-γ, and TNF-α, as well as NOS2, is impaired in MyD88^−/− mice following aerogenic M. tuberculosis infection. Real-time RT-PCR was used to quantitate IL-12p40 (A), IFN-γ (B), TNF-α (C), and NOS2 (F) mRNA expression in the lungs of infected WT (gray bars) and MyD88^−/− (black bars) mice. In parallel experiments, splenocytes from infected WT and MyD88^−/− mice were isolated and restimulated with purified protein derivative, and the supernatants were analyzed 3 days later for IL-12p40 (D) and IFN-γ (E) by enzyme-linked immunosorbent assay. Each bar is the mean (± standard deviation) of data from three mice. Asterisks indicate a P value of ≤0.05 determined by unpaired t test. Data are representative of results from three experiments.