Virulence of a Mycobacterium tuberculosis clinical isolate in mice is determined by failure to induce Th1 type immunity and is associated with induction of IFN-alpha /beta

Abstract

To understand how virulent mycobacteria subvert host immunity and establish disease, we examined the differential response of mice to infection with various human outbreak Mycobacterium tuberculosis clinical isolates. One clinical isolate, HN878, was found to be hypervirulent, as demonstrated by unusually early death of infected immune-competent mice, compared with infection with other clinical isolates. The differential effect on survival required lymphocyte function because severe combined immunodeficiency (SCID) mice infected with HN878 or other clinical isolates all died at the same rate. The hypervirulence of HN878 was associated with failure to induce M. tuberculosis-specific proliferation and IFN-gamma production by spleen and lymph node cells from infected mice. In addition, 2- to 4-fold lower levels of tumor necrosis factor-alpha (TNF-alpha), IL-6, IL-12, and IFN-gamma mRNAs were observed in lungs of HN878-infected mice. IL-10, IL-4, and IL-5 mRNA levels were not significantly elevated in lungs of HN878 infected mice. In contrast, IFN-alpha mRNA levels were significantly higher in lungs of these mice. To further investigate the role of Type 1 IFNs, mice infected with HN878 were treated intranasally with purified IFN-alpha/beta. The treatment resulted in increased lung bacillary loads and even further reduced survival. These results suggest that the hypervirulence of HN878 may be due to failure of this strain to stimulate Th1 type immunity. In addition, the lack of development of Th1 immunity in response to HN878 appears to be associated with increased induction of Type 1 IFNs.

Figure 1

Survival and bacterial load in lungs of B6D2/F₁ mice infected with M. tuberculosis clinical isolates. Mice were infected by aerosol with 1.6 log₁₀ NHN5 (●) or with 1.4 log₁₀ HN878 (□). (Upper) Growth of mycobacteria in lungs. Results are from one representative experiment. Each time point is the mean of 3–4 mice per group per time point; error bars indicate SD. (Lower) Survival of infected mice. Results are from one representative experiment with 22 mice per group.

Figure 2

Survival and bacterial load in lungs of SCID mice infected with M. tuberculosis clinical isolates. Mice were infected by aerosol with 2.2 log₁₀ NHN5 (●) or with 2.6 log₁₀ HN878 (□). (Upper) Growth of mycobacteria in lungs. Results are from one representative experiment. Each time point is the mean of 4–5 mice per group per time point. (Lower) Survival of infected mice. Results are from one representative experiment with 10 mice per group.

Figure 3

Cell proliferation and IFN-γ production in vitro. Mice were infected by aerosol with either M. tuberculosis NHN5 (filled bar) or HN878 (open bar). Proliferation in response to H37Ra sonicate was measured by [³H]thymidine incorporation (Top and Bottom). IFN-γ release by spleen cells was measured by ELISA (Middle). All results are from one representative experiment with 10 mice per group per time point.

Figure 4

Cytokine mRNA levels (normalized to β-actin mRNA levels) in lungs of mice infected with M. tuberculosis. (A) Cytokine mRNA at 14 and 28 days postinfection with NHN5 (filled symbols) and HN878 (open symbols): IFN-γ mRNA (□, ■) and IL-12 (○, ●). (B) mRNA at 28 days postinfection with NHN5 (filled bar) or HN878 (open bar). Uninfected mouse lungs had very low or undetectable levels of cytokine mRNA (not shown). (C) mRNA at 28 days postinfection with NHN5 (filled bar) or HN878 (open bar) or uninfected (cross-hatched bar). (C Inset) Relative levels of IFN-α mRNA in lungs of mice infected for 14 days with NHN5 (filled bar) or HN878 (open bar). Results are means of three independent experiments expressed as percentage activity relative to HN878-infected lungs. A, B, and C are single representative experiments.

Figure 5

Survival and bacterial load in lungs of B6D2/F₁ mice infected with M. tuberculosis. Mice were infected by aerosol with 2.3 log₁₀ HN878 and treated intranasally with IFN-α/β (■; A and C) or IFN-γ (▴; B and D), or untreated (□). (A and B) Survival of mice (11 mice per experimental group). (C and D) Growth of HN878 in lungs of cytokine-treated (filled symbols) or untreated (open symbols) aerosol-infected mice over 28 days. Results are means of three to four mice per group per time point.