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

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

IL-12 Mediates T-bet–Expressing Myeloid Cell–Dependent Host Resistance against Toxoplasma gondii

To defend against intracellular pathogens such as Toxoplasma gondii, the host generates a robust type 1 immune response. Specifically, host defense against T. gondii is defined by an IL-12-dependent IFN-γ response that is critical for host resistance. Previously, we demonstrated that host resistance is mediated by T-bet-dependent ILC-derived IFN-γ by maintaining IRF8+ conventional type 1 dendritic cells during parasitic infection. Therefore, we hypothesized that innate lymphoid cells are indispensable for host survival. Surprisingly, we observed that T-bet-deficient mice succumb to infection quicker than do mice lacking lymphocytes, suggesting an unknown T-bet-dependent-mediated host defense pathway. Analysis of parasite-mediated inflammatory myeloid cells revealed a novel subpopulation of T-bet+ myeloid cells (TMCs). Our results reveal that TMCs have the largest intracellular parasite burden compared with other professional phagocytes, suggesting they are associated with active killing of T. gondii. Mechanistically, we established that IL-12 is necessary for the induction of inflammatory TMCs during infection and these cells are linked to a role in host survival

Innate Immune Detection of Flagellin Positively and Negatively Regulates <i>Salmonella</i> Infection

<div><p><i>Salmonella enterica</i> serovar Typhimurium is a flagellated bacterium and one of the leading causes of gastroenteritis in humans. Bacterial flagellin is required for motility and also a prime target of the innate immune system. Innate immune recognition of flagellin is mediated by at least two independent pathways, TLR5 and Naip5-Naip6/NlrC4/Caspase-1. The functional significance of each of the two independent flagellin recognition systems for host defense against wild type <i>Salmonella</i> infection is complex, and innate immune detection of flagellin contributes to both protection and susceptibility. We hypothesized that efficient modulation of flagellin expression <i>in vivo</i> permits <i>Salmonella</i> to evade innate immune detection and limit the functional role of flagellin-specific host innate defenses. To test this hypothesis, we used <i>Salmonella</i> deficient in the anti-sigma factor <i>flgM</i>, which overproduce flagella and are attenuated <i>in vivo</i>. In this study we demonstrate that flagellin recognition by the innate immune system is responsible for the attenuation of <i>flgM⁻ S.</i> Typhimurium, and dissect the contribution of each flagellin recognition pathway to bacterial clearance and inflammation. We demonstrate that caspase-1 controls mucosal and systemic infection of <i>flgM⁻ S.</i> Typhimurium, and also limits intestinal inflammation and injury. In contrast, TLR5 paradoxically promotes bacterial colonization in the cecum and systemic infection, but attenuates intestinal inflammation. Our results indicate that <i>Salmonella</i> evasion of caspase-1 dependent flagellin recognition is critical for establishing infection and that evasion of TLR5 and caspase-1 dependent flagellin recognition helps <i>Salmonella</i> induce intestinal inflammation and establish a niche in the inflamed gut.</p></div

Flagellin detection accounts for the caspase-1 increased susceptibility during acute mucosal infection in streptomycin-pretreated mice.

<p>Bacterial burden of WT C57BL/6 (n = 10) and caspase-1−/− (n = 16) mice infected with 1000 cfu flagellin<i>⁻ Salmonella</i> in the cecum (A), spleen (B), liver (C), MLN (D). Bacterial burden WT C57BL/6 (n = 15) and TLR5−/− (n = 16) mice infected with 1000 cfu flagellin<i>⁻ Salmonella</i> in the cecum (E), spleen (F), liver (G), MLN (H). Figures A–D are the combined data from two independent experiments. Figures E–H are the combined data from three independent experiments. Mann-Whitney test * = p<0.05. *** = p<0.001.</p

<i>Salmonella</i> reside within F4/80+ cells the cecum.

<p>C57BL/6 WT (n = 5) (A+C) or Caspase-1−/− (n = 5) (B+D) mice were infected with 1000 cfu of <i>flgM⁻ Salmonella</i> containing a stable GFP expressing plasmid. Intestinal epithelial cells were stained using TROMA (A+B), and phagocytes were stained using F4/80 (C+D) antibody. Red = TROMA-1 (A, B) and F4/80 (C, D); green = GFP (<i>Salmonella</i>). Shown are representative images from 10 mice examined.</p

Composite analysis of Salmonella WT, flagellin- and flgM<i>⁻</i> infections in WT, caspase-1−/− and TLR5−/− mice.

<p>We combined the CFU data for all experiments using WT, flagellin deficient and <i>flgM⁻ S</i>. Typhimurium SL1344, and the data was analyzed using two-way ANOVA and Bonferroni’s multiple comparisons post tests. * = P<0.05, ** = P<0.01, and *** = P<0.001. Cecum (A), Spleen (B), Liver (C), and MLN (D).</p

Caspase-1 is required for attenuation of <i>flgM</i>-deficient <i>Salmonella</i>.

<p>C57BL/6 WT (n = 9) and caspase 1−/− (n = 8) mice were infected with 1000 CFU <i>flgM⁻ Salmonella.</i> Bacterial burden in the cecum (A). Spleen (B). Liver (C). MLN (D). Representative histology of the cecum of WT mice (E) or caspase-1−/− (F), and liver of WT mice (G) or caspase-1−/− (H). (I) Histological scores for changes in the cecal pathology as described in Fig. 4. Figures A–D represent data from two independent experiments. Liver images (200X magnification), cecal images (100X magnification). Mann-Whitney test, * = p<0.05, ** = p<0.01, *** = p<0.001.</p

<i>flgM⁻ Salmonella</i> has an attenuated <i>in vivo</i> phenotype.

<p> C57BL/6 mice were infected with 1000 CFU of either WT SL1344 (n = 12) or <i>flgM⁻</i> (n = 15) <i>Salmonella</i>. (A) Bacterial burden in the cecum. (B) Spleen. (C) Liver. (D) MLN. Representative histology of the cecum (E+F) and liver (G+H) infected with WT (E+G) or <i>flgM⁻ Salmonella</i> (F+H). (I) Histological scores for changes in the cecum. Submucosal expansion (S); mucosal neutrophilic infiltrate (M); lymphoplasmacytosis (L); goblet cells (G); epithelial integrity (E). Liver images (400X magnification), cecal images (200X magnification). Figures A–D are the combined data from three independent experiments. Mann-Whitney test. ** = p<0.01, *** = p<0.001.</p

Wildtype <i>Salmonella</i> efficiently evades flagellin detection during acute mucosal infection in streptomycin-pretreated mice.

<p>Bacterial burden of WT C57BL/6 (n = 11) and caspase-1−/− (n = 8) mice infected with 1000 cfu WT SL1344 <i>Salmonella</i> in the cecum (A), spleen (B), liver (C), MLN (D). Bacterial burden of WT C57BL/6 (n = 12) and TLR5−/− (n = 13) mice infected with 1000 cfu wildtype <i>Salmonella</i> in the cecum (E), spleen (F), liver (G), MLN (H). Figures A–D are the combined data from two independent experiments. Figures E–H are the combined data from three independent experiments. Mann-Whitney test. * = p<0.05. ** = p<0.01. *** = p<0.001.</p

The innate immune receptor TLR5 is dispensable in the attenuation of <i>flgM⁻</i>.

<p>C57BL/6 WT (n = 14) and TLR5−/− (n = 16) mice were infected with 1000 CFU <i>flgM⁻ Salmonella</i>. Bacterial burden in the cecum (A). Spleen (B). Liver (C). MLN (D). Representative histology of the cecum for WT mice (E) or TLR5−/− (F), and liver of WT mice (G) or TLR5−/− (H). Liver images (200X magnification), cecal images (100X magnification). Figures A–D represent data from three independent experiments. Mann-Whitney test * = p<0.05. ** = p<0.01.(I) Histological scores for changes in the cecal pathology as described in Fig. 4. Figures A–D represent data from three independent experiments. Mann-Whitney test *** = p<0.001.</p