TLR9 regulates Th1 responses and cooperates with TLR2 in mediating optimal resistance to Mycobacterium tuberculosis

Abstract

To investigate the role of Toll-like receptor (TLR)9 in the immune response to mycobacteria as well as its cooperation with TLR2, a receptor known to be triggered by several major mycobacterial ligands, we analyzed the resistance of TLR9(-/-) as well as TLR2/9 double knockout mice to aerosol infection with Mycobacterium tuberculosis. Infected TLR9(-/-) but not TLR2(-/-) mice displayed defective mycobacteria-induced interleukin (IL)-12p40 and interferon (IFN)-gamma responses in vivo, but in common with TLR2(-/-) animals, the TLR9(-/-) mice exhibited only minor reductions in acute resistance to low dose pathogen challenge. When compared with either of the single TLR-deficient animals, TLR2/9(-/-) mice displayed markedly enhanced susceptibility to infection in association with combined defects in proinflammatory cytokine production in vitro, IFN-gamma recall responses ex vivo, and altered pulmonary pathology. Cooperation between TLR9 and TLR2 was also evident at the level of the in vitro response to live M. tuberculosis, where dendritic cells and macrophages from TLR2/9(-/-) mice exhibited a greater defect in IL-12 response than the equivalent cell populations from single TLR9-deficient animals. These findings reveal a previously unappreciated role for TLR9 in the host response to M. tuberculosis and illustrate TLR collaboration in host resistance to a major human pathogen.

Figure 1.

Role for both TLR9 and TLR2 in the MyD88-dependent IL-12p40 response of splenic DCs to M. tuberculosis. (A) Purified CD11c⁺ spleen cells from WT or MyD88^−/− mice were exposed to live (MOI = 1:1 [M. tuberculosis 1] or 3:1 [M. tuberculosis 3]) or heat-killed (MOI = 1:1 H-K M. tuberculosis 1]) M. tuberculosis for 24 h. (B) WT DCs were treated with 5 μg/ml cytochalasin D or vehicle (DMSO) for 30 min and then incubated with live M. tuberculosis (MOI = 1:1) or 10 μg/ml LPS for 24 h. (C) DCs from WT or TLR2^−/− mice were treated with 5 μg/ml chloroquine or vehicle (saline) for 30 min and then stimulated with live M. tuberculosis (MOI = 1:1) for 24 h. (D) DCs from WT, MyD88^−/−, TLR2^−/−, TLR9^−/−, and TLR2/9^−/− were exposed to live M. tuberculosis (MOI = 1:1) or TLR agonists as described in A. In all experiments, supernatants were harvested and IL-12p40 was determined by ELISA. Results are means ± SE of triplicate measurements. Experiments shown are representative of at least three performed. *, P < 0.05 between experimental and control groups in A, B, and C. **, P < 0.05 between TLR2^−/− versus TLR9^−/− values in D.

Figure 2.

Role of TLR9 and TLR2 in proinflammatory cytokine production by M. tuberculosis–stimulated macrophages. BMM from WT, MyD88^−/−, TLR2^−/−, TLR9^−/−, and TLR2/9^−/− were stimulated with M. tuberculosis (MOI = 1:1), 15 μg/ml CpG, 5 μg/ml PGN, or 100 ng/ml LPS for 24 h. (A) IL-12p40, (B) TNF, and (C) IL-6 production was measured in the culture supernatants by ELISA. Results are means ± SE of triplicate measurements. Experiments shown are representative of two performed. *, P < 0.05 between WT versus KO values.

Figure 3.

Interaction of TLR9 and TLR2 in host resistance to aerosol M. tuberculosis infection. (A) WT, TLR2-, TLR9-, TLR2/9-, and MyD88-deficient mice were aerogenically infected with 50–100 CFUs/mouse (n = 6 animals per group), and survival was monitored. The results shown are representative of two independent experiments. Statistical analysis revealed that the MyD88-, TLR9-, and TLR2/9-deficient mice were significantly more susceptible (P < 0.01) than WT animals and that the survival curve of the TLR2/9^−/− mice is significantly different from that of the TLR9 (P = 0.027), TLR2 (P = 0.0053), or MyD88 (P = 0.002) animal groups. (B) Lungs from infected animals were harvested at 21 and 42 d after infection, and mycobacterial loads were determined. Results are mean ± SE of measurements from four animals. The experiment shown is representative of two performed. *, differences in CFUs between the KO versus WT groups that are statistically significant (P < 0.05).

Figure 4.

Increased susceptibility of TLR9^−/− mice to high dose M. tuberculosis infection. (A) WT, MyD88-, and TLR9-deficient mice were aerogenically infected with 500 CFUs/mouse (n = 5 animals per group) instead of the usual 50–100 CFU challenge, and survival was monitored. The results shown are representative of two independent experiments. Statistical analysis revealed that the MyD88- and TLR9-deficient mice were significantly more susceptible (P < 0.001) than WT animals and that the survival curve of the TLR9 mice is significantly different (P = 0.0042) from that of the MyD88 animal group. (B) Lungs from infected animals were harvested at 14 and 21 d after infection, and mycobacterial loads were determined. Results are mean ± SE of measurements from four animals. *, a statistically significant difference (P < 0.05) in CFUs between TLR9^−/− versus WT mice.

Figure 5.

Lungs from TLR2/9^−/− mice display exacerbated pulmonary pathology and increased acid-fast bacilli. Formalin-fixed, paraffin-embedded pulmonary tissue sections from day 42 infected mice were stained with hematoxylin and eosin (A–J). Note the increased inflammation in TLR2^−/− and TLR2/9^−/− lungs (A and E), with the more extreme pathology in the latter group. Acid-fast bacilli in lung tissue were stained with the Ziehl-Neelsen method (K–O). Representative sections from infected WT (A, F, and K), MyD88^−/− (B, G, and L), TLR2^−/− (C, H, and M), TLR9^−/− (D, I, and N), and TLR2/9^−/− (E, J, and O) mice are shown. Original magnification is 5 (A–E), 20 (F–J), and 10 (K–O).

Figure 6.

Influence of TLR9 on the generation of IFN-γ–producing CD4⁺ T cells and Th1-associated cytokines in M. tuberculosis–infected mice. (A) Lung cells isolated from day 30 infected mice were stimulated with anti-CD3 mAb, and intracellular IFN-γ production was determined by flow cytometry after gating lymphocyte populations by forward and side scatter parameters. The FACS profiles of anti-CD4– and anti-CD8–stained lymphocytes in A are from pooled cells from two mice and are representative of results from four animals per group. The majority (85–95%) of the IFN-γ plus CD4⁻ cells shown in the dot plots in the top panel were determined to be CD8⁺ T cells (unpublished data). Based on its nonspecific staining with multiple antibodies, the CD4 dim IFN-γ⁺ population in the lung preparations from infected MyD88 KO mice is likely to represent dead cells, consistent with their abundance in sections of the same tissue (Fig. 5, B and G). Percentage of CD4⁺ and CD8⁺ T cells that stain positively for IFN-γ calculated from the experiments shown in A. Relative expression of mRNAs for IFN-γ (C) and IL-12p40 (D) determined in lungs at 30 d after infection. Results are mean ± SE of measurements from three animals. (E) Purified splenic CD4⁺ T cells from the same mice described in A were cocultured with BMDCs infected with different MOIs for 72 h. IFN-γ was assayed by ELISA in culture supernatants. The means ± SE of measurements from triplicate wells are presented. The experiment shown was performed twice with similar results. *, significantly different values (P < 0.05) between WT and KO cells.