Summary Toxoplasma gondii is a universally-distributed pathogen that infects over 1 billion people worldwide. Host resistance to this protozoan parasite depends on the potent production of IL-12 and IFN-γ. Although Toll-like receptor 11 (TLR11) plays a major role in controlling Th1 immunity to this pathogen in mice, this innate immune receptor is not functional in humans, and the mechanisms of TLR11-independent sensing of T. gondii remain elusive. Here, we show that both mucosal innate and adaptive immune responses to T. gondii rely on the indirect stimulation of dendritic cells by normal gut microflora. We identify a novel function for commensal bacteria in which they provide an immunostimulatory effect on dendritic cells during parasitic infection. Our results reveal that indirect immunostimulation by gut commensals provides protection against T. gondii in the absence of TLR11.

Summary The Toll-like receptor adaptor protein MyD88 is essential for the regulation of intestinal homeostasis in mammals. In this study, we determined that Myd88-deficient mice are susceptible to colonic damage that is induced by dextran sulfate sodium (DSS) administration due to uncontrolled dissemination of intestinal commensal bacteria. The DSS-induced mortality of Myd88-deficient mice was completely prevented by antibiotic treatment to deplete commensal bacteria. By using cell type-specific Myd88-deficient mice, we established that B cell-intrinsic MyD88 signaling plays a central role in the resistance to DSS-induced colonic damage via the production of IgM and complement-mediated control of intestinal bacteria. Our results indicate that the lack of intact MyD88 signaling in B cells, coupled with impaired epithelial integrity, enables commensal bacteria to function as highly pathogenic organisms, causing rapid host death.

Toll-like receptor (TLR) activation relies on biochemical recognition of microbial molecules and localization of the TLR within specific cellular compartments. Cell surface TLRs largely recognize bacterial membrane components, and intracellular TLRs are exclusively involved in sensing nucleic acids. Here we show that TLR11, an innate sensor for the Toxoplasma protein profilin, is an intracellular receptor that resides in the endoplasmic reticulum. The 12 membrane-spanning endoplasmic reticulum-resident protein UNC93B1 interacts directly with TLR11 and regulates the activation of dendritic cells in response to Toxoplasma gondii profilin and parasitic infection in vivo. A deficiency in functional UNC93B1 protein abolished TLR11-dependent IL-12 secretion by dendritic cells, attenuated Th1 responses against T. gondii, and dramatically enhanced susceptibility to the parasite. Our results reveal that the association with UNC93B1 and the intracellular localization of TLRs are not unique features of nucleic acid-sensing TLRs but is also essential for TLR11-dependent recognition of T. gondii profilin and for host protection against this parasite. Toll-like receptors (TLRs)2 are a family of type I transmembrane proteins with ectodomains containing leucine-rich repeats that are involved in sensing varied microbial products, including lipids, peptidoglycans, proteins, and nucleic acids (1). TLR activation relies on the ability to sense molecules that are unique to microorganisms (2-4). TLRs involved in sensing bacterial membrane components such as LPS and lipoproteins are expressed and function on the cell surface (5, 6). Host and viral nucleic acids share an additional mechanism for self/nonself discrimination that relies on the localization of nucleic acid-recognizing TLR3, TLR7, and TLR9 within endosomal compartments (7-9). The intracellular localization of these TLRs is important not only for the recognition of viral DNA and RNA but also for the prevention of activation by host-derived nucleic acids (10 -13). Importantly, although the intracellular localization of TLRs can be achieved by distinct targeting sequences, all nucleic acid-recognizing receptors access their ligands in the same intracellular location (14 -16). It has been demonstrated that nucleotide-recognizing TLRs reside in the ER prior to stimulation, and a recently identified protein, UNC93B1, plays a major role in regulating the activation of these TLRs (17, 18). The missense mutation H412R in the UNC93B1 protein completely abolishes the signaling initiated by TLR3, TLR7, and TLR9 (17). Concomitantly, the functions of TLR2, TLR4, and TLR5, which are involved in sensing bacterial membrane and protein components, are not impaired in the absence of functional UNC93B1 protein (17). These data strongly support a surface localization of these "bacterial" innate immune receptors (7). Based on these observations, it has been postulated that nucleic acid-recognizing TLRs are uniquely positioned within intracellular compartments, whereas other TLRs involved in sen...

Toll-like receptors play a central role in the innate recognition of pathogens and the activation of dendritic cells (DCs). Here, we established that, in addition to TLR11, TLR12 recognizes the profilin protein of the protozoan parasite Toxoplasma gondii and regulates IL-12 production by DCs in response to the parasite. Similar to TLR11, TLR12 is an endolysosomal innate immune receptor that colocalizes and interacts with UNC93B1. Biochemical experiments revealed that TLR11 and TLR12 directly bind to T. gondii profilin and are capable of forming a heterodimer complex. We also established that the transcription factor IRF8, not NF-kB, plays a central role in the regulation of the TLR11- and TLR12-dependent IL-12 response of DCs. These results suggest a central role for IRF8-expressing CD8+ DCs in governing the TLR11- and TLR12-mediated host defense against T. gondii.

Primary infection with Toxoplasma gondii stimulates production of high levels of IL-12 and IFN-γ by cells of the innate immune system. These two cytokines are central to resistance to T. gondii. Signaling through the Toll-like receptor (TLR) adaptor protein MyD88 is indispensible for activating early innate immune responses. Recent studies have established that TLR11 plays a dominant role in sensing T. gondii. At the same time, TLR11 is represented in humans only by a pseudogene, and the major question of how innate and adaptive immune responses occur in the absence of TLR11 remains unanswered. In this article similarities and differences in sensors and effector molecules that determine host resistance to the parasite in humans and mice are discussed.

Regulatory Foxp3+ T cells are a critical cell population that suppresses T cell activation in response to microbial and viral pathogens. We identify a cell-intrinsic mechanism by which effector CD4+ T cells overcome the suppressive effects of Treg cells in the context of three distinct infections, Toxoplasma gondii, Listeria monocytogenes, and vaccinia virus. The acute responses to the parasitic, bacterial, and viral pathogens resulted in a transient reduction in frequency and absolute number of Treg cells. The infection-induced partial loss of Treg cells was essential for the initiation of potent Th1 responses and for host protection against the pathogens. The observed disappearance of Treg cells was a result of insufficiency in IL-2 caused by the expansion of pathogen-specific CD4+ T cells with a limited capacity of IL-2 production. Exogenous IL-2 treatment during the parasitic, bacterial, and viral infections completely prevented the loss of Treg cells, but restoration of Treg cells resulted in a greatly enhanced susceptibility to the pathogens. These results demonstrate that the transient reduction in Treg cells induced by pathogens via IL-2 deprivation is essential for optimal T cell responses and for host resistance to microbial and viral pathogens.

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The gut metabolic landscape is complex and is influenced by the microbiota, host physiology, and enteric pathogens. Pathogens have to exquisitely monitor the biogeography of the gastrointestinal tract to find a suitable niche for colonization. To dissect the important metabolic pathways that influence virulence of enterohemorrhagicEscherichia coli(EHEC), we conducted a high-throughput screen. We generated a dataset of regulatory pathways that control EHEC virulence expression under anaerobic conditions. This unraveled that the cysteine-responsive regulator, CutR, converges with the YhaO serine import pump and the fatty acid metabolism regulator FadR to optimally control virulence expression in EHEC. CutR activates expression of YhaO to increase activity of the YhaJ transcription factor that has been previously shown to directly activate the EHEC virulence genes. CutR enhances FadL, which is a pump for fatty acids that represses inhibition of virulence expression by FadR, unmasking a feedback mechanism responsive to metabolite fluctuations. Moreover, CutR and FadR also augment murine infection byCitrobacter rodentium, which is a murine pathogen extensively employed as a surrogate animal model for EHEC. This high-throughput approach proved to be a powerful tool to map the web of cellular circuits that allows an enteric pathogen to monitor the gut environment and adjust the levels of expression of its virulence repertoire toward successful infection of the host.