Fatal Mycobacterium tuberculosis infection despite adaptive immune response in the absence of MyD88

Abstract

Toll-like receptors (TLRs) such as TLR2 and TLR4 have been implicated in host response to mycobacterial infection. Here, mice deficient in the TLR adaptor molecule myeloid differentiation factor 88 (MyD88) were infected with Mycobacterium tuberculosis (MTB). While primary MyD88(-/-) macrophages and DCs are defective in TNF, IL-12, and NO production in response to mycobacterial stimulation, the upregulation of costimulatory molecules CD40 and CD86 is unaffected. Aerogenic infection of MyD88(-/-) mice with MTB is lethal within 4 weeks with 2 log(10) higher CFU in the lung; high pulmonary levels of cytokines and chemokines; and acute, necrotic pneumonia, despite a normal T cell response with IFN-gamma production to mycobacterial antigens upon ex vivo restimulation. Vaccination with Mycobacterium bovis bacillus Calmette-Guerin conferred a substantial protection in MyD88(-/-) mice from acute MTB infection. These data demonstrate that MyD88 signaling is dispensable to raise an acquired immune response to MTB. Nonetheless, this acquired immune response is not sufficient to compensate for the profound innate immune defect and the inability of MyD88(-/-) mice to control MTB infection.

Figure 1

Impaired proinflammatory cytokine and NO production in MyD88^–/– macrophages and DCs. BM-derived macrophages (A, C, and E) and DCs (B, D, and F) (5 × 10⁵ cells/ml) prepared from MyD88^–/– (white bars) and wild-type (black bars) mice were incubated with LPS (100 ng/ml), M. bovis BCG, MTB H37Ra, or MTB H37Rv (all at 2 bacteria per cell). After 24 hours, the production of TNF (A and B), IL-12 p40 (C and D), or nitrite (E and F) was determined in the supernatant by ELISA or Griess reaction. TNF and IL-6 production by pulmonary macrophages stimulated in the same conditions was also measured (G and H). Upregulation of CD40 (I) and CD86 (J) expression by DCs stimulated with LPS or M. bovis BCG were analyzed by FACS. Data are from 1 experiment, representative of 3 independent experiments with n = 2 mice per genotype; mean values ± SD are shown. MFI, mean fluorescence intensity.

Figure 2

MyD88^–/– mice are unable to control an MTB infection. MyD88^–/– (open circles), TNF^–/– (open squares), and wild-type (filled squares) mice were exposed to a low aerogenic dose of MTB H37Rv (200 CFU per mouse i.n.) and monitored for body weight (A; mean values of n = 6–8 mice per group from 1 representative experiment out of 7 independent experiments) and survival (B; n = 13–24 mice pooled from several experiments; P – 0.01 between MyD88^–/– or TNF^–/– mice and wild-type controls, and between TNF- and MyD88-deficient mice). (C) Lung wet weight of TNF^–/– mice (gray bars), MyD88^–/– mice (white bars), and wild-type controls (black bars) were measured 17 and 27 days after infection. The numbers of viable bacteria present in the lungs (D) and spleen (E) of MyD88^–/– (white bars) and wild-type (black bars) mice were determined after 27 days of infection (mean ± SD of n = 3–4 lung or n = 5–7 spleen samples from 1 representative experiment out of 2 or 3 independent experiments, respectively; **P – 0.01).

Figure 3

MyD88^–/– mice exhibit acute necrotic pneumonia with large nodules but defective granuloma formation in response to MTB infection. Lung tissue from MyD88^–/– (A, C, E, and G) and wild-type (B, D, F, and H) mice was analyzed 27 days after MTB H37Rv infection (200 CFU i.n.). Lungs of MyD88^–/– mice showed large and confluent nodules (A) in comparison with those of wild-type mice (B). Microscopic examination showed extensive inflammation and necrosis in infected MyD88^–/– lungs (C–F; H&E) with abundant mycobacteria in the extracellular space (G and H; Ziehl-Neelsen). Low-power micrographs of representative lung sections are shown in C and D (magnification, ×50), and higher magnification shows details in E–H (magnification, ×400).

Figure 4

T lymphocyte and myeloid cell recruitment and activation in the lung of MyD88^–/– infected mice. Infiltrating cells from the lungs of MyD88^–/– and control mice were isolated 27 days after infection and analyzed by flow cytometry for the expression of CD4, CD8, CD44, CD11b, CD11c, Ly-6G, and MHC class II I^A–I^E. Typical dot plots of FACS analysis are shown for CD44 expression in CD4⁺ and CD8⁺ T cells (A; gated on T lymphocytes). Graphic representations of the different leukocyte populations (B) and of the expression of MHC class II on APCs (C) are shown. Results are expressed as absolute numbers of positive cells. Mean ± SD from 2 MyD88^–/– mice are shown and are representative of 2 independent experiments.

Figure 5

Reduced DTH response, but normal IFN-γ secretion, to mycobacterial antigens stimulation in MyD88^–/– mice. (A) Cutaneous DTH response was performed 3 weeks after vaccination with M. bovis BCG (10⁵ CFU s.c.; v) by assessing the footpad swelling in response to PPD injection as described. Paw swelling is defined as the difference in thickness between the left paw injected with PPD and the right paw injected with saline. DTH responses of nonvaccinated MyD88^–/– and MyD88^+/+ mice are shown as control (nv). Data are expressed as mean ± SD (n = 7–8) and are from 1 representative experiment out of 2. **P – 0.01. (B) For the T cell response, spleen cells from M. bovis BCG–vaccinated MyD88-deficient and control mice were harvested 4 weeks after inoculation (10⁵ CFU s.c.) and were restimulated in vitro in the presence of soluble BCG antigens (SupBCG, 10 μg/ml) or unrelated antigen (HKLM, 100 bacteria per cell). Naive MyD88^–/– mice and wild-type mice were used as negative control. IFN-γ production was quantified in the supernatants after 48 hours of incubation. Results are mean ± SD from n = 2 mice per genotype and are representative of 3 independent experiments. (C) Intracellular IFN-γ staining of CD4⁺ or CD8⁺ splenocytes from BCG-infected MyD88-deficient and control mice 4 weeks after infection, restimulated for 18 hours in the presence of soluble BCG antigens (SupBCG, 10 μg/ml) or unrelated antigen (HKLM, 100 bacteria per cell). Typical dot blots are shown for CD4⁺ T cells (numbers indicate percentage of IFN-γ–positive CD4⁺ cells).

Figure 6

BCG vaccination confers an initial protection to MTB-challenged MyD88^–/– mice. MyD88^–/– mice and control mice were either immunized by s.c. injection of M. bovis BCG (10⁵ CFU) or left naive and 5 weeks later challenged by MTB H37Rv aerogenic infection (200 CFU per mouse i.n.). The mice were monitored for body weight (A) or survival (B) or were sacrificed at 4 weeks after MTB challenge and analyzed for lung weight (C) and bacterial counts (D) as in Figure 2 (A: n = 7–8 mice per group from 1 representative experiment out of 2; B: n = 11–13 mice per group from 2 experiments; C and D: n = 4 per group from 1 representative experiment out of 3; *P – 0.05; **P – 0.01).

Figure 7

BCG vaccination prevents necrotic pneumonia by aerogenic MTB in MyD88^–/– mice. Lung tissue from BCG-vaccinated MyD88^–/– (A, C, and E) and wild-type (B, D, and F) mice was analyzed at 4 weeks after MTB H37Rv infection (200 CFU i.n.). Lungs of MyD88^–/– mice display small nodules (A), similar to vaccinated wild-type mice (B). Microscopic examination reveals a striking increase of mononuclear cells including abundant lymphocytes in the lungs of vaccinated and infected MyD88^–/– and wild-type mice (C–F; H&E). Low-power micrographs of representative lung sections are shown in C and D (magnification, ×50), and higher magnification shows details in E and F (magnification, ×400).

Figure 8

Cell recruitment in the lungs of vaccinated and MTB-challenged MyD88^–/– mice. MyD88^–/– and control mice were immunized by s.c. injection of M. bovis BCG (10⁵ CFU) and 5 weeks later challenged by MTB aerogenic infection (200 CFU) as in Figure 6. Leukocytes from infected lung were isolated 4 weeks after infection and analyzed by flow cytometry for CD44 expression in CD4- and CD8-positive T cells (A and B); for CD11b-, CD11c-, and Ly-6G–positive cells (B); and for MHC class II I^A–I^E expression (C) as in Figure 4. Results are expressed as absolute numbers of positive cells. Mean ± SD from 2 MyD88^–/– mice are shown and are representative of 2 independent experiments.

Figure 9

The high pulmonary levels of cytokines and chemokines in MTB-infected MyD88^–/– mice is reduced upon vaccination. Cytokine and chemokine concentrations were determined in lung homogenates from MyD88^–/– and control mice immunized by s.c. injection of M. bovis BCG (10⁵ CFU) and challenged 5 weeks later by MTB aerogenic infection (200 CFU) as in Figure 6. IL-1β (A), IFN-γ (B), TNF (C), MIP-1α (D), MCP-1 (E), and RANTES (F) were quantified by SearchLight protein array. Results are expressed as mean ± SD from 4 mice per group (*P – 0.05; **P – 0.01).