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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 sensing bacterial chemical structures are located on the cell surface (1, 6, 7).
Despite progress in understanding the mechanisms involved in recognizing bacterial and viral pathogens, relatively little is known regarding the molecular and cellular bases of sensing parasites. We have established that TLR11, another member of the TLR family, is involved in sensing the protozoan parasite Toxoplasma gondii and other phylogenetically related pathogens via recognition of the unconventional profilin-like proteins in these parasites (19, 20). Recognition of protozoan profilin results in the activation of dendritic cells and the initiation of IL-12-dependent host resistance. This chain of events was established in both TLR11-deficient mouse models (19, 21, 22) and with profilin-deficient T. gondii (20). Lack of either parasite profilin or TLR11 on DCs leads to impaired innate recognition of T. gondii in vivo and in vitro (19, 20). Little is known, however, concerning the cellular mechanisms involved in TLR11-dependent recognition of protozoan profilin.
In the present work, we demonstrate that UNC93B1, previously reported to be a regulator of nucleic acid-sensing TLRs, interacts directly with TLR11 and is essential for proper activation of the TLR11 signaling pathway. Our experiments revealed that C57BL/6-Unc93b13d (3d) mice (deficient in functional UNC93B1 as a result of H412R missense mutation (17), failed to activate TLR11 in response to T. gondii infection and were very susceptible to this protozoan pathogen. We also established that TLR11, a microbial protein-recognizing TLR, has an intracellular but not a surface localization. These results establish that an intracellular localization is not a unique feature of nucleic acid-recognizing TLRs but is essential for the activation of protein-sensing TLR11 and the regulation of host defenses against the highly virulent protozoan parasite T. gondii.
C57BL/6 mice were obtained from the University of Texas Southwestern Medical Center Mouse Breeding Core Facility. 3d mice were obtained from the Mutant Mouse Regional Resource Centers. Myd88−/− and Tlr11−.− mice were generously provided by Drs. S. Akira and S. Ghosh, respectively. All animals used were age- and sex-matched and maintained in the SPF barrier facility at the University of Texas Southwestern Medical Center at Dallas. All experiments were performed using protocols approved by the Institutional Animal Care and Use Committees of the University of Texas Southwestern Medical Center.
Mice were infected intraperitoneally with an average of 20 T. gondii (ME49 strain) cysts. On days 3 and 5, animals were bled for cytokine analysis; on day 7 after infection, the animals were necropsied, and their spleens were harvested for analyses of intracellular cytokine expression. To analyze proinflammatory cytokine secretion, splenic DCs were purified with CD11c magnetic beads (Miltenyi), or in some experiments, the DCs were sort-purified after staining with an anti-CD11c antibody (BD Biosciences). The cytokine-inducing activities of T. gondii profilin, CpG, and LPS were assayed by incubating the DCs overnight with serial dilutions of the test sample, followed by the measurement of IL-12/23p40 in the culture supernatant by ELISA.
During in vivo cytokine reconstitution experiments, mice received daily intraperitoneal injections of 150 ng/mouse recombinant IL-12 (eBioscience) in 200 μl of PBS or vehicle alone starting from day 1 to day 7 of acute infection. Parental profilin-sufficient (ΔTgPRFe/TgPRFi) and profilin-deficient (ΔTgPRFe/TgPRFi + ATc for 72 h) parasites were grown as described previously (20). The IL-12 inducing activity of T. gondii was assayed by incubating DCs overnight with the parasite followed by measurement of IL-12/23p40 by intracellular staining with anti-IL-12p40 antibody (BD Biosciences).
Splenocytes depleted of DCs were incubated with profilin- sufficient or -deficient tachyzoites (2:1 ratio). Transwells (0.4 μm; Costar) were inserted and seeded with sort-purified DCs without treatment. At 20 h after stimulation, culture medium was collected, and IL-12p40 levels were measured by ELISA.
The pEGFPN1 and pmCherryN1 vectors were obtained from Clontech. The pcDNA3.1 vector was obtained from Invitrogen. An open reading frame encoding TLR11 was designed by Synthetic Biology (Integrated DNA Technologies). Tlr11 was cloned between the NheI and SacII sites of pEGFPN1 and pmCherryN1 using standard PCR techniques with the forward primer 5′-GCTAGCATGGGCCGCTACTGGCTGCTGCCCG and the reverse primer 5′-CCGCGGCCCCAGCCTGCTGCGCAGCCAG. TLR11 was myc-tagged and cloned between the NheI and XbaI sites of pcDNA3.1 using the primers 5′-GCTAGCATGGGCCGCTACTGGCTGCTGCCCG and 5′-TCTAGACTACAGATCCTCTTCTGAGATGAGTTTTTGTTCCCCCAGCCTGCTGCGCAGCCA. The TLR9GFP construct was a gift from Dr. Cynthia Leifer (Cornell University). Tlr4 was cloned into XhoI and HindIII sites of pEGFPN1 using the primers 5′-GGACTCAGATCTCGAGATGATGCCTCCCTGGCTCC and 5′-GCAGAATTCGAAGCTTGGCGTAGTCTGGCACATCATAGGGGTAGGTCCAAGTTGCCGTTTCTTG. Unc93b1 was cloned into the XhoI and HindIII sites of pmCherryN1 using the forward primer 5′-GTTTCTCGAGATGAAGGAAGTCCCAACCAGC and the reverse primer 5′-GTTTCTAAGCTTCTGCTCCTCAGGCCCATC. All tagged proteins are C-terminal fusions. All plasmids were prepared using the Endofree Midiprep kit from Clontech.
LPS, cytochalasin D, chloroquine, brefeldin A, and bafilomycin were purchased from Sigma. CpG 1826 was purchased from Invivogen. LysoTracker Red DND-99 and cholera toxin subunit B-Alexa Fluor 647 conjugate were purchased from Invitrogen. T. gondii profilin was expressed and purified as described previously (19). Purified recombinant T. gondii profilin was labeled with Alexa Fluor 488 or Alexa Fluor 532 using Alexa Fluor 488 or 532 protein labeling kits respectively (Invitrogen). Recombinant murine IL-12 was purchased from eBioscience.
HEK293 and RAW264.7 cell lines were obtained from ATCC and maintained in tissue culture flasks in RPMI 1640 medium containing 10% FBS (Hyclone), 10 mm HEPES, 1 mm 2-mercaptoethanol, 1 mm sodium pyruvate, 0.1 mm nonessential amino acids, 0.5 mg/ml l-glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin. HEK293 or RAW264.7 cells were plated on glass coverslips and were transfected using TransIT-293 (Mirus Bio) or Lipofectamine 2000 (Invitrogen), respectively. In some experiments, RAW264.7 cells were pretreated with IFN-γ (10 ng/ml) for 12 h prior to confocal analysis.
For immunofluorescence assays, HEK293 and RAW264.7 cells were grown on glass slides, transfected with the TLR11-myc construct for 36 h, and then fixed with 4% paraformaldehyde. After fixation, slides were rinsed in PBS and 0.1 m glycine. Cells were then permeabilized in PBS and 0.2% Triton X-100 for 20 min and blocked in the same buffer with 2% BSA. Slides were incubated with anti-myc antibody (Abcam) or normal rabbit serum, washed, and incubated for 60 min with Alexa Fluor 488-labeled goat anti-rabbit antibody diluted in blocking buffer. Images were acquired using a Leica SPE microscope equipped with a 63× objective as described previously (23).
The HEK293 cells were cotransfected with bait and prey constructs at 70% confluence using TransIT-293 according to the manufacturer's directions. Thirty-six hours after transfection, the cells were lysed in lysis buffer (20 mm Tris-HCl, pH 8, 137 mm NaCl, 2 mm EDTA, 0.5% Nonidet P-40, 10% glycerol, Complete Mini Protease Inhibitors (Roche Applied Science) and 1 mm PMSF). The cell lysates were centrifuged, and the supernatants were used for immunoprecipitation with 2 μg of anti-GFP or anti-mCherry monoclonal antibody (Clontech) bound to protein A/G beads (Pierce). Equal amounts of eluted protein were electrophoresed by SDS-PAGE, transferred to nitrocellulose, and blotted using the antibodies described above.
The molecular interactions between TLRs and their ligands have been characterized extensively. In addition to the identification of TLR ligands, major efforts have been placed on understanding the cellular localization of TLRs (6). It has been established that the receptors involved in sensing nucleic acids, including TLR3, TLR7, and TLR9, are localized intracellularly (5, 9, 24,–26). In contrast, TLRs that are known to be involved in the recognition of lipoproteins (TLR2), LPS (TLR4), and the microbial protein flagellin (TLR5) are expressed largely on the cell surface (27,–34). These results led to a model postulating that an intracellular localization is a unique feature of nucleic acid-sensing TLRs and is essential for preventing activation by endogenous nucleic acids (1). TLR11, another member of the innate immune TLR family, is activated by the protozoan parasite protein profilin (19). Because TLR11 and TLR5 are activated by microbial proteins, it has been suggested that TLR11 is localized to the cell surface similarly to TLR5 (1). However, no experimental studies have investigated the biology of TLR11 localization or activation. To determine the localization of TLR11, we generated a GFP-tagged TLR11 construct and analyzed TLR11 localization. To our surprise, the microscopy studies revealed that TLR11 was not detected on the surface of cells. Instead, TLR11 demonstrated a predominantly intracellular localization (Fig. 1A), co-localizing with an ER-specific marker (Fig. 1, B and C, and supplemental Fig. S1). We further confirmed an intracellular localization of TLR11 in mouse macrophages by transfection with TLR11-GFP and co-staining plasma membrane with recombinant cholera toxin subunit B (Fig. 1D). When the transfected macrophages were incubated with the TLR11 ligand profilin, we observed redistribution of TLR11 toward endolysosomes within 1 h of stimulation (Fig. 1, E and F). The intracellular localization of TLR11 was also revealed by immunofluorescence assays of TLR11-myc-transfected macrophages and HEK293 cells (supplemental Fig. S2).
The activation of nucleic acid-sensing TLRs occurs in the endolysosomal compartment and can be abrogated by treatment with bafilomycin A1 or chloroquine (25, 26, 35, 36), two chemical agents that prevent acidification within the endolysosomes (37, 38). Thus, we next explored the effects of bafilomycin A1 and chloroquine on the activation of endogenous TLR11 triggered by T. gondii profilin. As a readout, IL-12/23p40 production by splenic DCs following profilin stimulation was analyzed. We observed that similar to CpG activation of DCs, both of the tested chemicals prevented T. gondii profilin-induced IL-12/23p40 secretion (Fig. 1G). In agreement with previous reports (25, 26, 30), neither of the tested compounds affected LPS-induced cytokine secretion, which is dependent on the cell surface expression of TLR4 (Fig. 1H). These results suggest that despite its microbial protein-responsiveness, TLR11 activation is sensitive to chemical compounds that interfere with endosomal acidification. Furthermore, a brief incubation of all examined cells with Alexa Fluor 488-labeled profilin, including primary DCs, revealed a rapid accumulation of parasitic protein within endosomal compartments (supplemental Figs. S3–S5). Furthermore, by applying Alexa Fluor 488-labeled profilin, we observed co-localization of TLR11 and T. gondii profilin intracellularly, but not on the cell surface (supplemental Fig. S6). Taken together, these data strongly argued that T. gondii profilin activates intracellular TLR11 and initial contact between the innate immune receptor and its ligand occurs within the endosomal compartment.
We have demonstrated previously (19) that soluble profilin isolated from T. gondii is fully capable of eliciting TLR11-mediated induction of IL-12 by DCs. Nevertheless, the manner in which DCs might “see” profilin in a live parasite is not inherently obvious, as profilin is not known to be expressed on the surface of apicomplexan pathogens. Therefore, we next asked whether direct infection of DCs might be necessary for activation of intracellular TLR11 in the context of exposure to live tachyzoites rather than soluble antigen. To address this, sort purified splenic DCs were incubated with YFP-expressing tachyzoites alone or in the presence of the indicated amount of cytochalasin D, a drug that disrupts the parasite actin cytoskeleton and prevents invasion (39). Cytochalasin D at 1 μm concentration efficiently prevented DC infection by tachyzoites as was evident by flow-cytometry analysis (Fig. 2A), but had only a minor effect on IL-12/23p40 production from DCs (Fig. 2B). Moreover, when intracellular staining for IL-12/23p40 was performed, both infected and noninfected DCs were found to express the cytokine (Fig. 2C). Similar results were obtained in vivo with mice infected with YFP-expressing tachyzoites (Fig. 2D). Finally, when purified DCs were placed in the upper portion of a Transwell plate and DC-depleted splenocytes were placed in the lower well, IL-12 production was unimpeded despite physical separation of the parasite and the DCs (Fig. 2E). Importantly, depletion of profilin in the parasite (20) completely abolished DC IL-12 secretion in response to the live parasites (Fig. 2E). Taken together, the data indicate that neither direct contact with nor infection by T. gondii is required for the TLR11-dependent production of IL-12 by DCs and suggest that soluble T. gondii profilin activates intracellular TLR11.
Using a forward genetic screening approach, the 3d (“triple defect” of nucleic acid sensing) mutation was detected, and homozygote mice were found to be incapable of responding to ligands for TLR3, TLR7, and TLR9 (17). The 3d phenotype is caused by a mutation in Unc93b1, a gene encoding a 12-membrane-spanning protein that resides chiefly in the ER (17). UNC93B1 associates with TLR9, TLR3, and TLR7 via a direct interaction with their transmembrane domains (18, 40, 41). UNC93B1-deficient cells cannot signal via intracellular TLR3, TLR7, and TLR9, but the functions of cell surface-localized TLRs remain intact in 3d mice (17). To investigate whether TLR11 interacts with UNC93B1 in a way similar to the nucleic acid-sensing TLRs, we co-expressed UNC93B1 and TLR11 in transiently transfected HEK293 cells. As a positive control, UNC93B1 was co-expressed with TLR9, and as a negative control, we utilized TLR4, which does not interact with UNC93B1. To achieve identical co-immunoprecipitation conditions, all TLRs were tagged with GFP, and UNC93B1 was expressed as a fusion protein with the mCherry reporter protein. Immunoprecipitation experiments with anti-GFP antibody revealed that similarly to TLR9, TLR11 co-precipitated the UNC93B1 protein, as detected using the anti-mCherry antibody (Fig. 3A). In contrast and as expected based on previous reports, the immunoprecipitation experiments failed to detect an association between TLR4 and UNC93B1 (Fig. 3A). Control Western blots using anti-GFP or anti-mCherry GFP antibodies demonstrated comparable levels of expression among all of the tested TLRs and UNC93B1 in the transfected cells (Fig. 3A and supplemental Fig. S7). Taken together, these biochemical experiments strongly suggest that TLR11 interacts with UNC93B1. We further expanded these observations by confocal microscopy analysis of cells expressing TLR11 and UNC93B1 tagged with the fluorescent proteins GFP and mCherry, respectively (Fig. 3, B–D). In all of the co-transfected cells, TLR11 co-localized with UNC93B1 (Fig. 3D).
To test whether UNC93B1 is required for TLR11-mediated reactivity to T. gondii profilin, splenic DCs were freshly isolated from WT or 3d mutant mice, and their reactivity to TLR11, TLR9, and TLR4 agonists was analyzed. Whereas WT DCs secreted high levels of IL-12/23p40 following stimulation with various doses of T. gondii profilin, DCs isolated from 3d mice were indistinguishable from the nontreated DCs (Fig. 4A). As expected, the UNC93B1-deficient DCs failed to secrete the proinflammatory cytokine IL-12/23p40 when stimulated with the TLR9 agonist but produced normal levels of IL-12/23p40 when stimulated with LPS (Fig. 4A). Results similar to those obtained for IL-12/23p40 were observed for the other tested cytokines, including TNF (Fig. 4B) and IL-6 (Fig. 4C). Taken together, these findings revealed that UNC93B1 not only interacted with TLR11 but also was essential for initiation of the TLR11-dependent signaling pathway in response to T. gondii profilin in vitro.
TLR11 is a major regulator of IL-12 production in response to T. gondii. TLR11-knock-out mice fail to produce significant amounts of IL-12 in response T. gondii profilin, and as a result, they demonstrate an enhanced susceptibility to parasitic infection (19, 21). Because our in vitro experiments strongly suggest that UNC93B1 is essential for TLR11-dependent induction of IL-12, we next examined whether UNC93B1 is required for TLR11 activation in vivo. First, we injected WT and 3d mice with purified T. gondii profilin and analyzed IL-12/23p40 responses at different time points after injection. We observed that similarly to TLR11- and MyD88-deficient animals, 3d mice failed to produce IL-12/23p40 in response to T. gondii profilin (Fig. 5A). These results revealed that UNC93B1 was essential for TLR11 activation in vivo. To explore further the functions of UNC93B1 in the regulation of IL-12-dependent host resistance to T. gondii, WT and 3d animals were infected with the parasite, and the production of IL-12/23p40 was analyzed during the course of infection. As controls, both TLR11- and MyD88-deficient mice were included in the experiments. We observed that similarly to Tlr11−/− and Myd88−/− animals, mice that lacked functional UNC93B1 protein did not produce IL-12 in response to parasite infection (Fig. 5B). This finding suggests that UNC93B1 is essential for TLR11 activation not only in response to purified profilin but also during the course of T. gondii infection.
A major downstream function of IL-12 is the regulation of Th1 responses to T. gondii (42,–44). Thus, we next analyzed the effects of UNC93B1 deficiency on T cell responses to the parasite. We observed that T. gondii-infected 3d animals had substantially lower numbers of IFN-γ-secreting CD4+ and CD8+ T cells than did WT controls (Fig. 5C). These data strongly argued that the lack of functional UNC93B1 precluded TLR11/MyD88-dependent regulation of Th1 responses to the parasite. To test whether UNC93B1 was also required for host resistance to parasitic infection, the survival of 3d mice was assessed after infection with T. gondii brain cysts. The 3d mice were highly susceptible to T. gondii infection; all of the mice died within 2 weeks after parasite infection (Fig. 5D). To test whether the failure of 3d mice to control T. gondii was due to the defective production of IL-12, we treated the mice with recombinant IL-12 and examined the survival of the treated and control mice during experimental toxoplasmosis. We observed that all 3d mice treated with IL-12 during the 1st week after infection were resistant to the parasite as is evident from 100% survival rate, whereas the nontreated 3d mice or vehicle (PBS)-treated animals succumbed to the parasitic infection within the first 2 weeks after infection (Fig. 5D). These results suggest that the major defect causing acute death in T. gondii-infected 3d mice lies in the failure to secrete IL-12 in response to the parasite.
A surprising finding of our experiments is that TLR11, an innate immune receptor for the parasitic protein profilin, has an intracellular localization and interacts directly with the ER-resident protein UNC93B1. The 3d mice, which express a nonfunctional UNC93B1, not only failed to produce IL-12 in response to the TLR11 ligand T. gondii profilin but also were highly susceptible to T. gondii infection and demonstrated almost complete ablation of Th1 priming in response to parasitic infection. Our experiments revealed that in addition to nucleic acid-sensing TLRs, UNC93B1 was essential for TLR11-dependent innate immune recognition of T. gondii, production of IL-12, and host resistance to parasitic infection.
In addition to TLR11 signaling, antigen presentation and cross-presentation are impaired in the absence of functional UNC93B1 protein (17), and this protein can associate with the parasite in infected cells (45). In has been demonstrated that, within infected macrophages, UNC93B1 is involved in TLR- and IFN-γ-independent mechanisms of parasite clearance and that this function is dependent upon direct association of UNC93B1 with the parasitic vacuole (45). Thus, in addition to the regulation of antigen presentation, UNC93B1 may play two nonredundant roles during infection with T. gondii: coordinating TLR11-dependent IL-12 induction and TLR-independent parasite clearance within already infected cells. This combination of all these factors likely contributes to a very high sensitivity of 3d mice to T. gondii. Nevertheless, because injection of IL-12 rescued 3d mice from susceptibility to the parasitic infection, the principal defect in UNC93B1 mutant T. gondii-infected mice is the lack of a protective IL-12 response required for host resistance to the parasite.
Early recognition of invading pathogens, which is the key function of TLRs, depends on efficient discrimination between host and microbial molecules. Whereas bacterial and fungal pathogens possess unique and essential cell wall components, including LPS, lipoprotein, and zymosan, which provide a biochemical basis for efficient self/nonself discrimination, viruses and parasites share numerous metabolic similarities with the host and thus provide a limited target for innate recognition. Unlike bacteria and fungi, the recognition of viral infections relies predominantly on the detection of nucleic acids. Considering the inherent similarities between host and viral nucleic acids, the compartmentalization of TLR3, TLR7, and TLR9 is essential for selective recognition of foreign molecules, and concomitantly, these receptors must be protected from the induction of proinflammatory reactions in response to self DNA or RNA. Failure to maintain a physical separation of host nucleic acids and endosomal TLRs is frequently associated with autoimmune disorders.
The intracellular localization of TLR11 raised questions regarding the biological importance of this compartmentalization. The TLR11 ligand profilin, an actin-binding cytosolic protein, is not immediately accessible for recognition by DCs. Profilin must be released from the parasite prior to detection by its receptor. Although it is tempting to suggest that the intracellular localization of TLR11 facilitates recognition of the invading parasite, our experiments revealed that infection of DCs by T. gondii was not required for TLR11 activation. In contrast, others and our group have observed that T. gondii-infected DCs fail to up-regulate the necessary co-stimulatory molecules and produce significantly diminished amounts of proinflammatory cytokines compared with noninfected DCs (45,–47). Consequently, the infected DCs demonstrate a decreased capacity to induce efficient parasite-specific T cell responses. Thus, early detection of T. gondii is vital for the initiation of protective immune responses against the parasite. Indeed, TLR11 can be activated by very low levels of T. gondii profilin, and splenic DCs are capable of initiating TLR11-dependent secretion of IL-12 in response to very low doses of the parasite in vivo. This enhanced sensitivity of TLR11 may be achieved by an accumulation of parasitic protein within endosomal compartments, thus increasing the probability of TLR11 activation by T. gondii profilin. Future studies are essential for dissecting mechanisms regulating this enhanced sensitivity of TLR11 toward the parasitic protein. Because our previous experiments have established that only profilin from T. gondii and other closely related parasites can activate TLR11 in DCs (19, 20), we do not favor a model in which the intracellular localization of TLR11 is required for the prevention of DC activation by host profilins. Nevertheless, this possibility cannot be formally excluded because the biochemical mechanism responsible for the activation of TLR11 by profilins remains to be established.
In summary, the present data demonstrate that TLR11, a protein-recognizing receptor, is an intracellular innate immune sensor that requires UNC93B1 for its activation and control of immunity against T. gondii. Future studies are needed to dissect the biochemical mechanism responsible the intracellular localization of TLR11. This knowledge is needed for a complete understanding of the innate immune pathways that regulate host resistance against protozoan parasites.
2The abbreviations used are: