Growth of Yersinia pseudotuberculosis in mice occurs independently of Toll-like receptor 2 expression and induction of interleukin-10

Abstract

Pathogenic Yersinia translocates effector proteins into target cells via a type III secretion system (TTSS), modulating the host immune response. A component of the TTSS translocon, LcrV, has been implicated in preventing inflammation through Toll-like receptor 2 (TLR2) by inducing expression of the anti-inflammatory cytokine interleukin-10 (IL-10). TLR2(-/-) mice were reported to be less susceptible to the enteropathogen Yersinia enterocolitica. To determine whether TLR2 also plays a role in recognition of the enteropathogen Yersinia pseudotuberculosis and whether this results in an immune response that is detrimental to the host, we evaluated the macrophage cytokine response to live Y. pseudotuberculosis and analyzed the susceptibility of TLR2(-/-) mice to enteropathogenic Yersinia. We find that Yersinia induction of macrophage IL-10 occurs independently of TLR2 and LcrV and is blocked by the TTSS. In particular, the TTSS effector protein YopJ, which inhibits production of the inflammatory cytokine tumor necrosis factor alpha (TNF-alpha), also inhibits IL-10 expression. Consistent with these results, IL-10 is undetectable in Y. pseudotuberculosis-infected mouse tissues until advanced stages of infection. In addition, we find that TLR2(-/-) mice (derived independently from those used in previous studies) do not display altered susceptibility to enteropathogenic Yersinia compared to wild-type mice. Tissue levels of IL-10, as well as the inflammatory cytokines TNF-alpha, IL-6, and gamma interferon and the chemokine macrophage chemotactic protein 1, are similar in TLR2(+/+) and TLR2(-/-) mice during enteropathogenic Yersinia infection. Therefore, the absence of TLR2 alone does not affect the cytokine response of macrophages to, or the in vivo growth and survival of, enteropathogenic Yersinia.

FIG. 1.

Characterization of Y. pseudotuberculosis IP2666pIB1 infection in vivo after i.p. inoculation of 2 × 10³ to 6 × 10³ bacteria. (A) Average CFU per g tissue ± SEM. Data represent three independent experiments (n = 10 mice for each time point). (B and C) Average pg cytokine/g spleen (B) and liver (C) ± SEM. Data represent two independent experiments (n = 14 [spleen] and n = 12 [liver] for uninfected mice; n = 6 for each infected-mouse time point). (D) Average pg cytokine/ml serum ± SEM. Data represents two independent experiments (n = 1 for uninfected mice; n = 7 for each infected-mouse time point).

FIG. 2.

TLR2^−/− mice are not resistant to enteropathogenic Yersinia. (A) Analysis of Y. pseudotuberculosis infection at 4 days after i.p. inoculation of 2 × 10³ to 3 × 10³ Y. pseudotuberculosis IP2666pIB1 bacteria. Individual closed diamonds represent data from one organ. Dashes represent the geometric mean. (B) Analysis of Y. enterocolitica infection at 4 days after i.p. inoculation of 10⁴ Y. enterocolitica serogroup O:8 strain 8081 bacteria. Open diamonds indicate no CFU detected upon overnight culturing of organ homogenates. *, CFU present (as determined by culturing homogenates) but below the limit of regular plating detection (see Materials and Methods). (C) Analysis of Y. enterocolitica infection at 5 days after oral inoculation of 4 × 10⁷ Y. enterocolitica 8081 bacteria. Each graph represents data from two independent experiments.

FIG. 3.

TNF-α, IFN-γ, MCP-1, and IL-6 are produced in enteropathogenic Yersinia-infected tissues. The spleens (A to C) and livers (D to F) of uninfected C57BL/6 or TLR2^−/− mice or of mice infected with Y. pseudotuberculosis IP2666pIB1 (A and D) or Y. enterocolitica 8081 (B, C, E, and F) from Fig. 2 were assayed for cytokines. For each graph, data from two independent experiments was pooled and the average pg/g tissue ± SEM is shown. (A to C) n = 14 for uninfected C57BL/6 mice; n = 4 for uninfected TLR2^−/− mice. (D to F) n = 12 for uninfected C57BL/6 mice; n = 4 for uninfected TLR2^−/− mice. (A, C, D, and F) n = 7 for infected C57BL/6 mice; n = 8 for infected TLR2^−/− mice. (B and E) n = 8 for infected mice. Note that each graph has a different y axis scale.

FIG. 4.

IL-10 is produced in Y. pseudotuberculosis-infected tissues regardless of TLR2 expression during late stages of infection. Results are for 101 h after i.p. inoculation of 4.5 × 10³ Y. pseudotuberculosis IP2666pIB1 bacteria. (A) Individual diamonds represent CFU data from one organ. Dashes represent the geometric mean. (B) Spleen and liver homogenates from the mice from panel A were assayed for cytokines. Individual diamonds represent pg IL-10 per g tissue from one organ. Dashes represent average pg/g tissue.

FIG. 5.

Y. pseudotuberculosis lacking LcrV induces macrophage IL-10 independently of TLR2, and this IL-10 expression is suppressed by YopJ. TLR2^+/− (A to C) or TLR2^−/− (C) BMDMs were infected for 2 hours with Y. pseudotuberculosis and grown under Yop-inducing conditions. Data represent averages of five (A and B) or three (C) independent experiments ± SEM. Supernatant was assayed for levels of TNF-α (A) and IL-10 (B and C). Confidence values: P < 0.008 compared to IP2666pIB1 (A and B, *), P < 0.1 compared to pYV⁻ (A, **), and P = 0.03 compared to pYV⁻ (B, **). (D) Results are for 4 days after i.p. inoculation of 2 × 10³ to 4 × 10³ Y. pseudotuberculosis bacteria expressing YopJ [IP2666(pIB1-yopH^Nde)] or lacking YopJ (ΔyopJ). Individual diamonds represent data from one organ. Dashes represent the geometric mean. Data represent two (+YopJ) or three (−YopJ) independent experiments.