Evidence inconsistent with a negative influence of T helper 2 cells on protection afforded by a dominant T helper 1 response against Mycobacterium tuberculosis lung infection in mice

Abstract

Mice incapable of generating an efficient Th2 response because of functional deletion of the genes for signal transducer and activation of transcription 6 (Stat6), interleukin-4 receptor alpha chain (IL-4Ralpha), or IL-4 plus IL-13 (IL-4/IL-13) were no more resistant than wild-type (WT) mice to airborne infection with virulent Mycobacterium tuberculosis. WT mice were able to control infection and hold it at a stationary level following 20 days of log linear M. tuberculosis growth. Likewise, infection was kept under control and was held at the same stationary level in IL-4/IL-13(-/-) mice but progressed to a slightly higher level in Stat6(-/-) and IL-4Ralpha(-/-) mice. The onset of stationary-level infection in WT mice was associated with the expression of Th1-mediated immunity, as evidenced by an approximately 100- to 1,000-fold increase in the lungs in the synthesis of mRNA for IL-12, gamma interferon (IFN-gamma), and inducible nitric oxide synthase (NOS2) that was sustained for at least 100 days. IL-12 is essential for the induction of Th1 immunity, IFN-gamma is a key Th1 cytokine involved in mediation of immunity, and NOS2 is an inducible enzyme of macrophages and is needed by these cells to express immunity. In response to infection, the lungs of Stat6(-/-) mice showed increases in synthesis of mRNA for IL-12, IFN-gamma, and NOS2 similar to that seen in WT mice. In IL-4/IL-13(-/-) mice, however, synthesis of mRNA for IFN-gamma and NOS2 reached higher levels than in WT mice. These results argue against the notion that a Th2 response is partly or wholly responsible for the inability of Th1-mediated immunity to resolve infection with a virulent strain of M. tuberculosis.

FIG. 1.

Growth of 2 × 10² CFU of M. tuberculosis strain H37Rv, administered by aerosol, in the lungs, livers, and spleens of WT and IL-4/IL-13^−/− mice. In the lungs of both types of mice, H37Rv grew progressively for approximately 20 days, after which infection was controlled and held at a stationary level of approximately 6.5 logs until day 120, when the experiment was terminated. There was no significant difference in the growth of H37Rv in the livers and spleens of WT and IL-4/IL-13^−/− mice. Data are means ± standard deviations of results from five mice per group per time point.

FIG. 2.

Growth of M. tuberculosis strain H37Rv in the lungs, livers, and spleens of WT and Stat6^−/− mice. There was no significant difference between WT and Stat6^−/− mice in the kinetics of growth of the pathogen in their organs up to day 50 of infection. At day 120, however, the level of infection in the lungs of Stat6^−/− mice was 0.75 log higher (P = 0.0068) than that in the lungs of WT mice. The level of infection was also significantly higher in the spleens of Stat6^−/− mice on day 120 (P = 0.0392), although not in the livers. Data are means ± standard deviations of results from five mice per group per time point.

FIG. 3.

Growth of M. tuberculosis strain H37Rv in the lungs, livers, and spleens of WT and IL-4Rα^−/− mice. There was no significant difference between WT and Stat6^−/− mice in the kinetics of growth of H37Rv in their organs up to day 50 of infection. However, at day 120, the lungs of mutant mice contained 0.75 log more CFU of H37Rv (P = 0.0338) than the lungs of WT mice. Data are means ± standard deviations of results from five mice per group per time point.

FIG. 4.

Changes in the copy number (per total lung RNA) of mRNA for IFN-γ and NOS2 over time of infection with 2 × 10² CFU of M. tuberculosis strain H37Rv administered via the respiratory route. The copy numbers of mRNA for both proteins increased approximately 1,000-fold between days 15 and 25 of infection, and this increase was sustained until day 100. Data are means ± standard deviations of results from three individual experiments.

FIG. 5.

Changes in copy number of mRNA (per total lung RNA) for IL-12p35, IL-12p40, and IL-4 during the course of M. tuberculosis strain H37Rv infection. The copy number for IL-12p35 increased approximately 100-fold in the lungs of both types of mice over the first 25 days of infection and remained at this elevated level until day 100. The copy number of IL-12p40 mRNA increased about 500-fold in the lungs of WT mice and about 1,000-fold in the lungs of Stat6^−/− mice during the first 25 days of infection. In both cases, the copy number of IL-12p40 remained elevated until day 100. The copy number of IL-4 mRNA increased less than 10-fold in the lungs of Stat6^−/− mice and WT mice over the first 25 days of infection but remained elevated through day 100. Data are means ± standard deviations of results of three individual experiments. Similar results were obtained with repeat experiments.

FIG. 6.

Changes in copy number (per total lung RNA) of mRNA for IFN-γ and NOS2 in the lungs of WT and IL-4/IL-13^−/− mice over 50 days of M. tuberculosis strain H37Rv infection. The copy number of IFN-γ mRNA increased approximately 1,000-fold in WT mice and double mutant mice during the first 30 days of infection and increased even further at day 50 in the lungs of mutant mice. Likewise, the NOS2 mRNA copy number increased approximately 1,000-fold in the lungs of WT and mutant mice during the first 30 days of infection and continued to increase until day 50. Data are means ± standard deviations of results from three mice per time point. Additional experiments gave similar results.

FIG. 7.

Photomicrographs of sections of lungs of WT and Stat6^−/− mice at day 50 of infection stained for NOS2 by immunocytochemistry. The low-power micrographs show that infection-induced lesions in WT (a) and Stat6^−/− (b) lungs were similar, being composed of accumulations of macrophages that stained positively for NOS2 (brown) in close proximity to large aggregates of lymphoid cells (dark blue). At higher magnification (c), the cells that stained brown for NOS2 in Stat6^−/− lesions each showed a large pale nucleus typical of epithelioid macrophages, whereas the nuclei of nearby lymphocytes were compact and densely stained. Many of the macrophages can be seen to contain acid-fast bacilli. The lesions of WT mice displayed similar characteristics under high-power magnification.