Long-term control of Mycobacterium bovis BCG infection in the absence of Toll-like receptors (TLRs): investigation of TLR2-, TLR6-, or TLR2-TLR4-deficient mice

Abstract

Live mycobacteria have been reported to signal through both Toll-like receptor 2 (TLR2) and TLR4 in vitro. Here, we investigated the role of TLR2 in the long-term control of the infection by the attenuated Mycobacterium, Mycobacterium bovis BCG, in vivo. We sought to determine whether the reported initial defect of bacterial control (K. A. Heldwein et al., J. Leukoc. Biol. 74:277-286, 2003) resolved in the chronic phase of BCG infection. Here we show that TLR2-deficient mice survived a 6-month infection period with M. bovis BCG and were able to control bacterial growth. Granuloma formation, T-cell and macrophage recruitment, and activation were normal. Furthermore, the TLR2 coreceptor, TLR6, is also not required since TLR6-deficient mice were able to control chronic BCG infection. Finally, TLR2-TLR4-deficient mice infected with BCG survived the 8-month observation period. Interestingly, the adaptive response of TLR2- and/or TLR4-deficient mice seemed essentially normal on day 14 or 56 after infection, since T cells responded normally to soluble BCG antigens. In conclusion, our data demonstrate that TLR2, TLR4, or TLR6 are redundant for the control of M. bovis BCG mycobacterial infection.

FIG. 1.

Reduced TNF and nitrite production in TLR2-deficient macrophages stimulated with BCG or mycobacterial preparations. Bone marrow-derived macrophages from TLR2- and/or TLR4-deficient mice and controls were incubated with LPS (0.1 μg/ml), BLP (0.5 μg/ml), heat-killed BCG (HKBCG; 10 μg/ml), BCG killed by lyophilization (BCG Lyoph; 10 μg/ml), and a lyophilized preparation of BCG culture supernatant (Sup BCG; 10 μg/ml) or were infected with BCG (two bacteria per cell) or dispersed BCG (two bacteria per cell) for 24 h. TNF (A) and nitrite (B) were measured in the supernatants by ELISA and Griess reaction, respectively. The results are means ± the standard deviations (SD) from n = two mice and are from one representative experiment out of three independent experiments.

FIG. 2.

Expression of CD40 and CD86 in BCG-infected TLR2- and/or TLR4-deficient macrophages. CD40 (A) and CD86 (B) expression in TLR2- and/or TLR4-deficient macrophages after 18 h stimulation with LPS or BLP or after infection with BCG was evaluated. Unstimulated (thin line) and stimulated (thick line) cells were analyzed. The results are from cells pooled from two mice and are representative of three independent experiments.

FIG. 3.

Control of BCG infection in TLR2-deficient mice. Spleen (A), liver (B), and lung (C) relative weights of TLR2-deficient (•) and control (⋄) mice infected with BCG (2 × 10⁶ CFU/mouse, given i.v.) are expressed as the percentage of body weight (BW; mean + the SD). Viable bacteria were assessed by counting CFU in spleen (D), liver (E), and lung (F) homogenates. The data are expressed as mean CFU values per whole organ + the SD. (n = four to six mice per point; P > 0.05).

FIG. 4.

Normal hepatic granuloma formation in BCG-infected TLR2-deficient mice. Hepatic granulomas from control (A and C) and TLR2-deficient (B and D) mice 28 days after BCG infection (2.10⁶ CFU/mouse, given i.v.) were examined at magnifications of ×200 and ×400. (E) A histogram compilation of the hepatic granuloma counts on day 28 to 140 postinfection is shown (mean + the SD, n = four to six mice; P > 0.05).

FIG. 5.

Cellular recruitment and activation in BCG-infected TLR2-deficient mice. Liver immunostaining was done for CD3 (A and B), F4/80 (C and D), MHC class II (E and F), and iNOS (G and H) samples at 28 days after BCG infection in control (A, C, E, and G) and TLR2-deficient (B, D, F and H) mice. Four mice per group were examined, and representative sections are shown. Magnification, ×400.

FIG. 6.

Delayed lung inflammation in BCG-infected TLR2-deficient mice. Lung sections from control (A and C) and TLR2-deficient (B and D) mice infected with BCG (2 × 10⁶ CFU/mouse, given i.v.) were examined at 28 days (A and B; magnification, ×100) and 56 days (C and D; magnification, ×200) postinfection.

FIG. 7.

Control of BCG infection in TLR6-deficient mice. Unimpaired TNF (A) and nitrite (B) production in TLR6-deficient macrophages stimulated with BCG or mycobacterial preparations as in Fig. 1. The results are means ± the SD from n = two mice and are from one representative experiment out of three independent experiments. Spleen (C), liver (D), and lung (E) relative weights of TLR6-deficient (▪) and control (⋄) mice infected with BCG (2 × 10⁶ CFU/mouse, given i.v.) are expressed as the percentage of body weight (BW; mean ± the SD, n = four mice per point; P > 0.05). Viable bacteria were assessed by counting CFU in spleen (F), liver (G), and lung (H) homogenates. The data are expressed as mean CFU values per whole organ ± the SD (n = four mice per point; P > 0.05).

FIG. 8.

Control of BCG infection and T-cell priming in TLR2-TLR4-deficient mice. Viable bacterial load was assessed in spleen (A), liver (B), and lung (C) homogenates of TLR2-TLR4-deficient (▪) and control (□) mice infected with BCG (2 × 10⁶ CFU/mouse, given i.v.). The data are expressed as mean CFU values per whole organ ± the SD (n = four mice per point; P > 0.05). (D) Splenocytes isolated from BCG-infected control and TLR2-TLR4-deficient mice 14 days after infection (2 × 10⁶ CFU/mouse, given i.v.) or from uninfected control mice were restimulated in vitro in the presence of concanavalin A (ConA; 2.5 μg/ml), soluble BCG antigens (Sup BCG, 100 μg/ml), live BCG (two bacteria/cell), or an unrelated antigen, HKLM (100 bacteria/cell). IFN-γ production was quantified in the supernatants after 48 h of incubation. The results are means ± the SD (from n = four mice per genotype). (E) Cutaneous DTH response was performed 8 months after infection of TLR2-TLR4-deficient and control mice with BCG (2 × 10⁶ CFU, given i.v.) by assessing the footpad swelling in response to PPD injection. 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 noninfected TLR2-TLR4 wild-type mice are shown as controls. The data are expressed as means ± the SD (n = 3).