Induction of IL-12p40 and type 1 immunity by Toxoplasma gondii in the absence of the TLR-MyD88 signaling cascade.

Toxoplasma gondii is an orally acquired pathogen that induces strong IFN-γ based immunity conferring protection but that can also be the cause of immunopathology. The response in mice is driven in part by well-characterized MyD88-dependent signaling pathways. Here we focus on induction of less well understood immune responses that do not involve this Toll-like receptor (TLR)/IL-1 family receptor adaptor molecule, in particular as they occur in the intestinal mucosa. Using eYFP-IL-12p40 reporter mice on an MyD88-/- background, we identified dendritic cells, macrophages, and neutrophils as cellular sources of MyD88-independent IL-12 after peroral T. gondii infection. Infection-induced IL-12 was lower in the absence of MyD88, but was still clearly above noninfected levels. Overall, this carried through to the IFN-γ response, which while generally decreased was still remarkably robust in the absence of MyD88. In the latter mice, IL-12 was strictly required to induce type I immunity. Type 1 and type 3 innate lymphoid cells (ILC), CD4+ T cells, and CD8+ T cells each contributed to the IFN-γ pool. We report that ILC3 were expanded in infected MyD88-/- mice relative to their MyD88+/+ counterparts, suggesting a compensatory response triggered by loss of MyD88. Furthermore, bacterial flagellin and Toxoplasma specific CD4+ T cell populations in the lamina propria expanded in response to infection in both WT and KO mice. Finally, we show that My88-independent IL-12 and T cell mediated IFN-γ production require the presence of the intestinal microbiota. Our results identify MyD88-independent intestinal immune pathways induced by T. gondii including myeloid cell derived IL-12 production, downstream type I immunity and IFN-γ production by ILC1, ILC3, and T lymphocytes. Collectively, our data reveal an underlying network of immune responses that do not involve signaling through MyD88.

Toxoplasma gondii dense granule protein GRA24 drives MyD88-independent p38 MAPK activation, IL-12 production and induction of protective immunity.

The apicomplexan Toxoplasma gondii induces strong protective immunity dependent upon recognition by Toll-like receptors (TLR)11 and 12 operating in conjunction with MyD88 in the murine host. However, TLR11 and 12 proteins are not present in humans, inspiring us to investigate MyD88-independent pathways of resistance. Using bicistronic IL-12-YFP reporter mice on MyD88+/+ and MyD88-/- genetic backgrounds, we show that CD11c+MHCII+F4/80- dendritic cells, F4/80+ macrophages, and Ly6G+ neutrophils were the dominant cellular sources of IL-12 in both wild type and MyD88 deficient mice after parasite challenge. Parasite dense granule protein GRA24 induces p38 MAPK activation and subsequent IL-12 production in host macrophages. We show that Toxoplasma triggers an early and late p38 MAPK phosphorylation response in MyD88+/+ and MyD88-/- bone marrow-derived macrophages. Using the uracil auxotrophic Type I T. gondii strain cps1-1, we demonstrate that the late response does not require active parasite proliferation, but strictly depends upon GRA24. By i. p. inoculation with cps1-1 and cps1-1:Δgra24, we identified unique subsets of chemokines and cytokines that were up and downregulated by GRA24. Finally, we demonstrate that cps1-1 triggers a strong host-protective GRA24-dependent Th1 response in the absence of MyD88. Our data identify GRA24 as a major mediator of p38 MAPK activation, IL-12 induction and protective immunity that operates independently of the TLR/MyD88 cascade.

Impact of IFN-y and CD40 signalling on Toxoplasma gondii cyst formation in differentiated Neuro-2a neuroblastoma cells.

Signalling by IFN-y and CD40 is known to trigger anti-microbial activity in macrophages infected with Toxoplasma gondii, but their effects on infected neurons are less well known. Here, we compared how stimulation with IFN-y and an agonistic anti-CD40 mAb impacts infection and cyst formation in the mouse neuroblastoma cell line Neuro-2a relative to bone marrow-derived macrophages. Both IFN-y and CD40 mAb decreased cyst emergence in Neuro-2a cells. In macrophages, these stimuli decreased infection, but had no impact on infection in the neuroblastoma cell line. Resistance to killing in Neuro-2a cells may explain why neurons preferentially harbour parasites during chronic infection in the brain.

Impact of MyD88, Microbiota, and Location on Type 1 and Type 3 Innate Lymphoid Cells during Toxoplasma gondii Infection.

Toxoplasma gondii induces strong IFN-γ-based immunity. Innate lymphoid cells (ILC), in particular ILC1, are an important innate source of this protective cytokine during infection. Our objective was to determine how MyD88-dependent signaling influences ILC function during peroral compared with i.p. infection with T. gondii. MyD88 +/+ and MyD88 -/- mice were orally inoculated with ME49 cysts, and small intestinal lamina propria ILC were assessed using flow cytometry. We observed T-bet+ ILC1, retinoic acid-related orphan receptor γt+ ILC3, and a population of T-bet+retinoic acid-related orphan receptor γt+ double-positive ILC. In MyD88 -/- mice, IFN-γ-producing T-bet+ ILC1 frequencies were reduced compared with wild-type. Treatment of MyD88 -/- mice with an antibiotic mixture to deplete microflora reduced IFN-γ+ ILC1 frequencies. To examine ILC responses outside of the mucosal immune system, peritoneal exudate cells were collected from wild-type and knockout mice after i.p. inoculation with ME49 cysts. In this compartment, ILC were highly polarized to the ILC1 subset that increased significantly and became highly positive for IFN-γ over the course of infection. Increased ILC1 was associated with expression of the Ki67 cell proliferation marker, and the response was driven by IL-12p40. In the absence of MyD88, IFN-γ expression by ILC1 was not maintained, but proliferation remained normal. Collectively, these data reveal new aspects of ILC function that are influenced by location of infection and shaped further by MyD88-dependent signaling.