Glycosylphosphatidylinositol (GPI) anchors and glycoinositolphospholipids (GIPLs) from parasitic protozoa have been shown to exert a wide variety of effects on cells of the host innate immune system. However, the receptor(s) that are triggered by these protozoan glycolipids has not been identified. Here we present evidence that Trypanosoma cruzi-derived GPI anchors and GIPLs trigger CD25 expression on Chinese hamster ovary-K1 cells transfected with CD14 and Toll-like receptor-2 (TLR-2), but not wild-type (TLR-2-deficient) Chinese hamster ovary cells. The protozoan-derived GPI anchors and GIPLs containing alkylacylglycerol and saturated fatty acid chains or ceramide were found to be active in a concentration range of 100 nM to 1 μM. More importantly, the GPI anchors purified from T. cruzi trypomastigotes, which contain a longer glycan core and unsaturated fatty acids in the sn-2 position of the alkylacylglycerolipid component, triggered TLR-2 at subnanomolar concentrations. We performed experiments with macrophages from TLR-2 knockout and TLR-4 knockout mice, and found that TLR-2 expression appears to be essential for induction of IL-12, TNF-α, and NO by GPI anchors derived from T. cruzi trypomastigotes. Thus, highly purified GPI anchors from T. cruzi parasites are potent activators of TLR-2 from both mouse and human origin. The activation of TLR-2 may initiate host innate defense mechanisms and inflammatory response during protozoan infection, and may provide new strategies for immune intervention during protozoan infections.

GPIs isolated from Toxoplasma gondii, as well as a chemically synthesized GPI lacking the lipid moiety, activated a reporter gene in Chinese hamster ovary cells expressing TLR4, while the core glycan and lipid moieties cleaved from the GPIs activated both TLR4- and TLR2-expressing cells. MyD88, but not TLR2, TLR4, or CD14, is absolutely needed to trigger TNF-α production by macrophages exposed to T. gondii GPIs. Importantly, TNF-α response to GPIs was completely abrogated in macrophages from TLR2/4-double-deficient mice. MyD88^−/− mice were more susceptible to death than wild-type (WT), TLR2^−/−, TLR4^−/−, TLR2/4^−/−, and CD14^−/− mice infected with the ME-49 strain of T. gondii. The cyst number was higher in the brain of TLR2/4^−/−, but not TLR2^−/−, TLR4^−/−, and CD14^−/−, mice, as compared with WT mice. Upon infection with the ME-49 strain of T. gondii, we observed no decrease of IL-12 and IFN-γ production in TLR2-, TLR4-, or CD14-deficient mice. Indeed, splenocytes from T. gondii-infected TLR2^−/− and TLR2/4^−/− mice produced more IFN-γ than cells from WT mice in response to in vitro stimulation with parasite extracts enriched in GPI-linked surface proteins. Together, our results suggest that both TLR2 and TLR4 receptors may participate in the host defense against T. gondii infection through their activation by the GPIs and could work together with other MyD88-dependent receptors, like other TLRs or even IL-18R or IL-1R, to obtain an effective host response against T. gondii infection.

TLRs function as pattern recognition receptors in mammals and play an essential role in the recognition of microbial components. We found that the injection of glycoinositolphospholipids (GIPLs) from Trypanosoma cruzi into the peritoneal cavity of mice induced neutrophil recruitment in a TLR4-dependent manner: the injection of GIPL in the TLR4-deficient strain of mice (C57BL/10ScCr) caused no inflammatory response. In contrast, in TLR2 knockout mice, neutrophil chemoattraction did not differ significantly from that seen in wild-type controls. GIPL-induced neutrophil attraction and MIP-2 production were also severely affected in TLR4-mutant C3H/HeJ mice. The role of TLR4 was confirmed in vitro by testing genetically engineered mutants derived from TLR2-deficient Chinese hamster ovary (CHO)-K1 fibroblasts that were transfected with CD14 (CHO/CD14). Wild-type CHO/CD14 cells express the hamster TLR4 molecule and the mutant line, in addition, expresses a nonfunctional form of MD-2. In comparison to wild-type cells, mutant CHO/CD14 cells failed to respond to GIPLs, indicating a necessity for a functional TLR4/MD-2 complex in GIPL-induced NF-κB activation. Finally, we found that TLR4-mutant mice were hypersusceptible to T. cruzi infection, as evidenced by a higher parasitemia and earlier mortality. These results demonstrate that natural resistance to T. cruzi is TLR4 dependent, most likely due to TLR4 recognition of their GIPLs.

Studies performed in vitro suggest that activation of Toll-like receptors (TLRs) by parasite-derived molecules may initiate inflammatory responses and host innate defense mechanisms against Trypanosoma cruzi. Here, we evaluated the impact of TLR2 and myeloid differentiation factor 88 (MyD88) deficiencies in host resistance to infection with T. cruzi. Our results show that macrophages derived from TLR2 ^−/− and MyD88^−/− mice are less responsive to GPI-mucin derived from T. cruzi trypomastigotes and parasites. In contrast, the same cells from TLR2^−/− still produce TNF-α, IL-12, and reactive nitrogen intermediates (RNI) upon exposure to live T. cruzi trypomastigotes. Consistently, we show that TLR2^−/− mice mount a robust proinflammatory cytokine response as well as RNI production during the acute phase of infection with T. cruzi parasites. Further, deletion of the functional TLR2 gene had no major impact on parasitemia nor on mortality. In contrast, the MyD88^−/− mice had a diminished cytokine response and RNI production upon acute infection with T. cruzi. More importantly, we show that MyD88^−/− mice are more susceptible to infection with T. cruzi as indicated by the higher parasitemia and accelerated mortality, as compared with the wild-type mice. Together, our results indicate that T. cruzi parasites elicit an alternative inflammatory pathway independent of TLR2. This pathway is partially dependent on MyD88 and necessary for mounting optimal inflammatory and RNI responses that control T. cruzi replication during the early stages of infection.

Trypomastigote forms of Trypanosoma cruzi, the causative agent of Chagas Disease, shed extracellular vesicles (EVs) enriched with glycoproteins of the gp85/trans‐sialidase (TS) superfamily and other α‐galactosyl (α‐Gal)‐containing glycoconjugates, such as mucins. Here, purified vesicles from T. cruzi strains (Y, Colombiana, CL‐14 and YuYu) were quantified according to size, intensity and concentration. Qualitative analysis revealed differences in their protein and α‐galactosyl contents. Later, those polymorphisms were evaluated in the modulation of immune responses (innate and in the chronic phase) in C57BL/6 mice. EVs isolated from YuYu and CL‐14 strains induced in macrophages higher levels of proinflammatory cytokines (TNF‐α and IL‐6) and nitric oxide via TLR2. In general, no differences were observed in MAPKs activation (p38, JNK and ERK 1/2) after EVs stimulation. In splenic cells derived from chronically infected mice, a different modulation pattern was observed, where Colombiana (followed by Y strain) EVs were more proinflammatory. This modulation was independent of the T. cruzi strain used in the mice infection. To test the functional importance of this modulation, the expression of intracellular cytokines after in vitro exposure was evaluated using EVs from YuYu and Colombiana strains. Both EVs induced cytokine production with the appearance of IL‐10 in the chronically infected mice. A high frequency of IL‐10 in CD4+ and CD8+ T lymphocytes was observed. A mixed profile of cytokine induction was observed in B cells with the production of TNF‐α and IL‐10. Finally, dendritic cells produced TNF‐α after stimulation with EVs. Polymorphisms in the vesicles surface may be determinant in the immunopathologic events not only in the early steps of infection but also in the chronic phase.

Initial host defense to bacterial infection is executed by innate immunity, and therefore the main goal of this study was to examine the contribution of Toll-like receptors (TLRs) duringBrucella abortusinfection. CHO reporter cell lines transfected with CD14 and TLRs showed thatB. abortustriggers both TLR2 and TLR4. In contrast, lipopolysaccharide (LPS) and lipid A derived fromBrucellarough (R) and smooth (S) strains activate CHO cells only through TLR4. Consistently, macrophages from C3H/HePas mice exposed to R and S strains and their LPS produced higher levels of tumor necrosis factor alpha (TNF-α) and interleukin-12 compared to C3H/HeJ, a TLR4 mutant mouse. The essential role of TLR4 for induction of proinflammatory cytokines was confirmed with diphosphoryl lipid A fromRhodobacter sphaeroides. Furthermore, to determine the contribution of TLR2 and TLR4 in bacterial clearance, numbers ofBrucellawere monitored in the spleen of C3H/HeJ, C3H/HePas, TLR2 knockout, and wild-type mice at 1, 3, and 6 weeks followingB. abortusinfection. Interestingly, murine brucellosis was markedly exacerbated at weeks 3 and 6 after infection in animals that lacked functional TLR4 (C3H/HeJ) compared to C3H/HePas that paralleled the reduced gamma interferon production by this mouse strain. Finally, by mass spectrometry analysis we found dramatic differences on the lipid A profiles of R and S strains. In fact, S lipid A was shown to be more active to trigger TLR4 than R lipid A in CHO cells and more effective in inducing dendritic cell maturation. In conclusion, these results indicate that TLR4 plays a role in resistance toB. abortusinfection and that S lipid A has potent adjuvant activity.