Programmed death-1 (PD-1)-deficient mice are extraordinarily sensitive to tuberculosis

Abstract

The programmed death-1 (PD-1) costimulatory receptor inhibits T and B cell responses and plays a crucial role in peripheral tolerance. PD-1 has recently been shown to inhibit T cell responses during chronic viral infections such as HIV. In this study, we examined the role of PD-1 in infection with Mycobacterium tuberculosis, a common co-infection with HIV. PD-1-deficient mice showed dramatically reduced survival compared with wild-type mice. The lungs of the PD-1-/- mice showed uncontrolled bacterial proliferation and focal necrotic areas with predominantly neutrophilic infiltrates, but a lower number of infiltrating T and B cells. Proinflammatory cytokines, such as TNF-alpha, IL-1, and especially IL-6 and IL-17 were significantly increased in the lung and sera of infected PD-1-/- mice, consistent with an aberrant inflammation. Microarray analysis of the lungs infected with M. tuberculosis showed dramatic differences between PD-1-/- and control mice. Using high-stringency analysis criteria (changes of twofold or greater), 367 transcripts of genes were differentially expressed between infected PD-1-/- and wild-type mice, resulting in profoundly altered inflammatory responses with implications for both innate and adaptive immunity. Overall, our studies show that the PD-1 pathway is required to control excessive inflammatory responses after M. tuberculosis infection in the lungs.

Fig. 1.

PD-1–deficient mice show dramatically reduced survival after M. tuberculosis H37Rv aerosol infection. PD-1^−/− mice are shown as black circles (320 CFU/mouse) or as triangles (50 CFU/mouse). Wild-type mice (squares) survived for >200 d postinfection, after both doses used (320 CFU/mouse shown). Data for 10–15 mice per groups are shown from two independent experiments. Survival curves were compared using log rank test (P < 0.0001).

Fig. 2.

PD-1–deficient mice have increased bacterial loads in the lungs after H37Rv aerosol infection, compared with wild-type mice. (A) cfu values determined in the lungs of wild-type and PD-1^−/− mice, 2, 4, and 8 wk after low-dose H37Rv aerosol infection (50 CFU). Mean values of five mice at each timepoint are shown ± SD. (B) CFU values from organs of control and PD-1^−/− mice at 4 wk postinfection with 320 CFU. Data are representative of three independent experiments. *P < 0.05.

Fig. 3.

Histology of the lungs of PD-1^−/− and control mice at 4 weeks postinfection with M. tuberculosis H37Rv aerosol. (A) Lungs of the PD-1^−/− mice develop large focal necrotic areas. HE stain at 40× magnification. (B Upper) Necrotizing inflammation and neutrophilic infiltration in the lungs of PD-1^−/− mice (HE stain at 400× magnification). (B Lower) Scattered bacteria in the lungs of C57BL/6 mice (Left); massive bacterial load in the lungs of PD-1^−/− mice (Right), visualized by AFB stain (400×, bacteria are shown in red). Mice were infected with 320 CFU/mouse.

Fig. 4.

Altered recruitment of inflammatory cells into the lungs of PD-1^−/− mice infected with M. tuberculosis H37Rv aerosol. (A) Lower numbers of CD4⁺ T cells in the lungs of infected PD-1^−/− mice at 4 wk postinfection. (B) CD8⁺ T cell numbers are similar to control mice. (C) B cell numbers are reduced in the lungs of PD-1^−/− mice at 4 wk postinfection (P < 0.05). Recruitment of myeloid cells such as macrophages (D, P < 0.05) and neutrophils (E) is increased in the lungs of PD-1^−/− mice starting at 3 wk postinfection. Mice were infected with 135 CFU/mouse. FACS analysis was performed on total lung cells; data are shown as average ± SD of three mice per group at each timepoint.

Fig. 5.

Increased cytokine levels measured in the sera of PD-1^−/− mice infected with low-dose M. tuberculosis aerosol. Proinflammatory cytokines TNF-α (A, P < 0.001), IL-1 (B, P < 0.05), and IL-6 (C, P < 0.001) were elevated in the sera of infected PD-1^−/− mice at 4 wk postinfection. (D) Significantly increased IL-17 levels in infected PD-1^−/− mice (P < 0.001). Higher levels of IFN-γ (E) and IL-12 (F) in the sera of infected PD-1^−/− mice. (G) Chemokine KC (CXCL1) is selectively increased in infected PD-1^−/− mice after 19 d (P < 0.001). (H) IL-10 levels are not significantly different between PD-1^−/− and control mice. Mice were infected with H37Rv aerosol 50 CFU/mouse (A–E) or 135 CFU/mouse (F–H). Serum samples were analyzed from five mice at each timepoint; mean values are shown ± SD. *, statistical significance between groups.

Fig. 6.

Increased inflammatory and reduced effector cytokine levels in the lungs of PD-1^−/− mice infected with M. tuberculosis aerosol. IL-1 (A, P < 0.05) and IL-17 (B, P < 0.01) are increased in the lungs of infected PD-1^−/− mice at 3 wk postinfection. (C) Increased levels of chemokine KC (CXCL1) in PD-1^−/− mice (P < 0.05). (D) Higher IL-10 levels in infected PD-1^−/− lungs. Effector cytokine levels IFN-γ (E) and IL-12 (F, P < 0.01) are reduced in the lungs of PD-1^−/− mice, compared with control mice at 3 wk postinfection. (G) Lung cells from infected PD-1^−/− mice re-stimulated in vitro with H37Rv sonicate show less stimulation compared with C57BL/6 mice, measured by IFN-γ production (P < 0.001). Mice were infected with H37Rv aerosol 106 CFU/mouse (A–F), or 135 CFU/mouse (G). Data are shown as mean ± SD of four mice per group at each timepoint. *, statistical significance between groups.