TLR11-independent inflammasome activation is critical for CD4+ T cell-derived IFN-γ production and host resistance to Toxoplasma gondii

Abstract

Innate recognition of invading intracellular pathogens is essential for regulating robust and rapid CD4+ T cell effector function, which is critical for host-mediated immunity. The intracellular apicomplexan parasite, Toxoplasma gondii, is capable of infecting almost any nucleated cell of warm-blooded animals, including humans, and establishing tissue cysts that persist throughout the lifetime of the host. Recognition of T. gondii by TLRs is essential for robust IL-12 and IFN-γ production, two major cytokines involved in host resistance to the parasite. In the murine model of infection, robust IL-12 and IFN-γ production have been largely attributed to T. gondii profilin recognition by the TLR11 and TLR12 heterodimer complex, resulting in Myd88-dependent IL-12 production. However, TLR11 or TLR12 deficiency failed to recapitulate the acute susceptibility to T. gondii infection seen in Myd88-/- mice. T. gondii triggers inflammasome activation in a caspase-1-dependent manner resulting in cytokine release; however, it remains undetermined if parasite-mediated inflammasome activation impacts IFN-γ production and host resistance to the parasite. Using mice which lack different inflammasome components, we observed that the inflammasome played a limited role in host resistance when TLR11 remained functional. Strikingly, in the absence of TLR11, caspase-1 and -11 played a significant role for robust CD4+ TH1-derived IFN-γ responses and host survival. Moreover, we demonstrated that in the absence of TLR11, production of the caspase-1-dependent cytokine IL-18 was sufficient and necessary for CD4+ T cell-derived IFN-γ responses. Mechanistically, we established that T. gondii-mediated activation of the inflammasome and IL-18 were critical to maintain robust CD4+ TH1 IFN-γ responses during parasite infection in the absence of TLR11.

Fig 1. The inflammasome plays a limited role in host resistance against T. gondii.

(A) Survival of WT, Nlrp3^-/-, Asc^-/-, and Casp1/11^-/- mice that were i.p. infected with 20 cysts of the ME49 strain of T. gondii. (B) Analysis of T. gondii parasite loads by qPCR in WT and inflammasome-deficient mice from PECs, livers, and spleens on day 8 of infection. Results are representative of three-independent experiments involving at least 3 mice per group. Statistical analyses of survival curve was done using Log-Rank (Mantel Cox) Test. All other statistical analyses were performed using one-way ANOVA with a Tukey’s multiple comparison test, *P<0.05, **P<0.01, ***P<0.001. Error bars, standard error mean.

Fig 2. The inflammasome has a limited role in stimulating T. gondii-mediated CD4+ T_H1 derived IFN-γ responses.

(A, E) WT, Nlrp3^-/-, Asc^-/-, and Casp1/11^-/- mice were i.p. infected with 20 cysts of T. gondii. PECs (A) and spleens (E) were harvested and IFN-γ production by CD4+ T cells was analyzed by flow cytometry. (B, C, F, G) Average frequencies (B, F) and absolute quantification (C, G) of CD4+IFN-γ+ cells in the PECs and spleens were analyzed on day 8 following infection. (D, H) Mean fluorescent intensity (MFI) of CD4+ T cell IFN-γ in the PECs (D) and spleens (H) was analyzed on day 8 post-infection. Results are representative of three-independent experiments involving at least 3 mice per group. Statistical analyses were done using one-way ANOVA with a Tukey’s multiple comparison test, *P<0.05, **P<0.01. Error bars, standard error mean.

Fig 3. The inflammasome is required for host resistance against T. gondii in the absence of TLR11.

(A) Survival of WT, Casp1/11^-/-, TLR11^-/-, TLR11xCasp1/11^-/-, and Myd88^-/- (▼) mice that were infected i.p. with 20 cysts of ME49. (B) Analysis of T. gondii parasite loads by qPCR in WT, TLR11^-/-, TLR11xCasp1/11^-/-, and Myd88^-/- mice from PECs and spleens on day 8 of infection. (C) qRT-PCR analysis of relative IFN-γ expression measured in the PECs and spleens of mice infected with 20 cysts of T. gondii on day 8 post-infection. (D) Analysis by ELISA of serum IL-12/23p40 and IFN-γ in WT, TLR11^-/-, TLR11xCasp1/11^-/-, and Myd88^-/- mice infected with T. gondii was performed on day 8 of infection. Survival curve is a combination of two-independent experiments involving at least 3 mice per group. Statistical analyses of survival curve was done using Log-Rank (Mantel Cox) Test. All other results are representative of three-independent experiments involving at least 5 mice per group. Statistical analyses were done using one-way ANOVA with a Tukey’s multiple comparison test, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Error bars, standard error mean.

Fig 4. In the absence of TLR11, inflammasome activation is required for robust T_H1 responses during T. gondii infection.

(A, E) WT, TLR11^-/-, TLR11xCasp1/11^-/-, and Myd88^-/- mice were infected i.p. with 20 cysts of T. gondii. PECs (A) and spleens (E) were harvested and IFN-γ production by CD4+ T cells was analyzed by flow cytometry. (B, C, F, G) Average frequencies (B, F) and absolute quantification (C, G) of CD4+IFN-γ+ cells in the PECs and spleens were analyzed on day 8 following infection. (D, H) MFI of CD4+ T cell IFN-γ was analyzed on day 8 post-infection. Results are representative of three-independent experiments involving at least 3 mice per group. Statistical analyses were done using one-way ANOVA with a Tukey’s multiple comparison test, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Error bars, standard error mean.

Fig 5. IL-18 is sufficient to augment T_H1-derived IFN-γ responses in the absence of TLR11.

(A, C) WT and TLR11xCasp1/11^-/- mice were infected with 20 cysts of T. gondii. TLR11xCasp1/11^-/- mice were administered 200 ngs of IL-18 i.p. on days 0, 1, 2, and 3. PECs (A) and spleens (C) were harvested and IFN-γ production by CD4+ T cells was analyzed by flow cytometry. (B, D) Absolute quantification of CD4+IFN-γ+ cells in the PECs (B) and spleens (D) were analyzed on day 8 following infection. Results are representative of three-independent experiments involving at least 3 mice per group. Statistical analyses were done using one-way ANOVA with a Tukey’s multiple comparison test, *P<0.05. Error bars, standard error mean.