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The Journal of Immunology, 2007, 179, 1129-1137
Copyright © 2007 by The American Association of Immunologists, Inc.
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Activation of TLR2 and TLR4 by Glycosylphosphatidylinositols Derived from Toxoplasma gondii1

Françoise Debierre-Grockiego2,3,*, Marco A. Campos3,{dagger}, Nahid Azzouz*, Jörg Schmidt*, Ulrike Bieker*, Marianne Garcia Resende{dagger}, Daniel Santos Mansur{ddagger}, Ralf Weingart4,§, Richard R. Schmidt§, Douglas T. Golenbock, Ricardo T. Gazzinelli5,{dagger},¶,|| and Ralph T. Schwarz5,*,#

* Institut für Virologie, AG Parasitologie, Philipps University, Marburg, Germany; {dagger} Instituto René Rachou, Fundaçao Oswaldo Cruz, Belo Horizonte, Brazil; {ddagger} Department of Microbiology, Biological Sciences Institute, Federal University of Minas Gerais, Belo Horizonte, Brazil; § Fachbereich Chemie, University of Konstanz, Konstanz, Germany; Division of Infectious Disease and Immunology, Department of Medicine, University of Massachusetts Medical School, Worcester, MA; || Department of Biochemistry and Immunology, Federal University of Minas Gerais, Belo Horizonte, Brazil; and # Unité de Glycobiologie Structurale et Fonctionnelle Unité Mixte de Recherche, Centre National de la Recherche Scientifique/Université des Sciences et Technologies de Lille No. 8576–Institut Fédératif de Recherche 118, Villeneuve D’Ascq, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
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-{alpha} production by macrophages exposed to T. gondii GPIs. Importantly, TNF-{alpha} 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-{gamma} production in TLR2-, TLR4-, or CD14-deficient mice. Indeed, splenocytes from T. gondii-infected TLR2–/– and TLR2/4–/– mice produced more IFN-{gamma} 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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Glycosylphosphatidylinositol-anchored proteins dominate the surface of the Toxoplasma gondii tachyzoite (1, 2) and GPI biosynthesis is an essential process for viability in T. gondii (3). Our previous results have shown that GPIs highly purified from T. gondii tachyzoites induced TNF-{alpha} production in macrophages through the activation of the transcription factor NF-{kappa}B (4). The glycan cores and the lipid moieties prepared from the T. gondii GPIs were sufficient to stimulate the synthesis of TNF-{alpha} by macrophages. However, a remaining issue concerns the mechanisms by which the T. gondii GPIs signal the host cells.

TLRs expressed differentially among immune cells (5, 6) recognize microbial components distinct of host self molecules. Stimulus of the TLRs leads to cytokine production and antimicrobial responses through NF-{kappa}B activation (7). Although major advances have been made in the assignment of individual TLRs to bacterial and virus ligands (e.g., LPS, peptidoglycans, lipoteichoic acid, lipoproteins, flagellin, bacterial (CpG) DNA, viral fusion protein, dsRNAs and ssRNAs (8, 9, 10, 11, 12, 13, 14, 15)), this research area is only emerging in the field of protozoan parasites (16). It has been shown that activation of TLR2 is essential for the induction of IL-12, TNF-{alpha}, and NO synthesis by macrophages activated by Trypanosoma cruzi GPIs (17). Similarly, GPIs from Plasmodium falciparum (18) and lipophosphoglycan from Leishmania major (19) activate TLR2, whereas ceramide-containing GPIs from T. cruzi activate TLR4 (20).

The strongest data in vivo for the role of TLRs during infection with protozoan parasites come from MyD88-deficient mice. MyD88, an adaptor molecule used by TLR family members (21, 22), plays an essential role in host resistance and pathogenesis during infection with different protozoan parasites, such as Plasmodium berghei (23), T. cruzi (24), T. gondii (25), and L. major (19). In the case of T. gondii, MyD88-deficient animals succumbed to acute infection, due to a dramatic increase in parasite burden (25, 26). Splenocytes from MyD88-deficient mice exposed to T. gondii soluble tachyzoite Ags failed to produce IL-12 and IFN-{gamma} (25) and developed a Th2 cytokine pattern (CD4-dependent IL-4, IL-5, IL-10, and IL-13) (27). However, upon infection with T. gondii the phenotype of mice deficient in a single TLR is not so obvious (26). More precisely, TLR2–/– as well as TLR4–/– mice show no defect in the early IL-12 and IFN-{gamma} responses to soluble tachyzoite Ags (25) and survived to a regular T. gondii parasite inoculum (26, 28). Although not lethal, T. gondii infection of TLR11-deficient mice resulted in increased cyst numbers associated with impaired IL-12 and IFN-{gamma} production (29). In contrast, TLR9 deficiency appeared to protect mice from intestinal pathology and lethality caused by oral infection with T. gondii (30).

Because GPIs derived from T. gondii have been shown to promote NF-{kappa}B translocation and activate macrophages to produce proinflammatory cytokines, namely TNF-{alpha} (4), we decided to investigate their ability to activate TLRs. In this report, we evaluate the ability of T. gondii whole GPIs or their fragments to promote NF-{kappa}B translocation in Chinese hamster ovary (CHO)6 cells expressing TLR2 or TLR4, and test whether they activate macrophages from C57BL/6, TLR2-, TLR4-, TLR2/4-, CD14-, or MyD88-deficient mice to produce TNF-{alpha}. Our results show that the glycan cores and phospholipid moieties of T. gondii GPIs activated both TLR2 and TLR4 in CHO cells. In contrast, complete GPIs activated only TLR4. MyD88 deficiency, but not TLR2, TLR4, or CD14 deficiency, resulted in impaired production of TNF-{alpha} by macrophages exposed to either whole GPIs or GPI fragments. Importantly, simultaneous deficiency of both TLR2 and TLR4 led to complete abrogation of TNF-{alpha} production by T. gondii GPIs. Consistently, TLR2/4–/– mice presented increased number of cysts as compared with wild-type (WT) mice. We hypothesize that other MyD88-dependent receptors, like other TLRs or even IL-18R and IL-1R, together with TLR2 and TLR4, contribute to an effective immune response against T. gondii.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Materials

[3H]Glucosamine hydrochloride (25 Ci/mM) was purchased from Hartmann Analytic. GPIa, a GPI molecule of T. gondii, was chemically synthesized according to Pekari et al. (31) and has the following structure: (ethanolamine-PO4)-Man{alpha}1–2Man{alpha}1-6(GalNAcbeta1-4)Man{alpha}1-4GlcN{alpha}- inositol-PO4.

Mice

TLR2–/–, TLR4–/–, and MyD88–/– mice were generated by S. Akira (Osaka University, Osaka, Japan); TLR2/4–/– and CD14–/– mice were generated by D. T. Golenbock and all were backcrossed with C57BL/6 for eight generations. C57BL/6 mice used as WT control were obtained from a colony maintained at the Instituto René Rachou (Fundação Oswaldo Cruz, Belo Horizonte, Brazil). The mouse colonies and the experimental procedures using mice were performed according to the institutional animal care and use guidelines from Fundação Oswaldo Cruz.

Metabolic labeling of tachyzoites

Tachyzoites of T. gondii (strain RH) grown in Vero cells were labeled with [3H]glucosamine for 4 h. After labeling, parasites were released from host cells with the help of glass beads and the Mixer Mill homogenizer (Retsch). Tachyzoites were purified by glass wool filtration (32).

Extraction and purification of GPIs

Tachyzoite glycolipids were extracted according to Menon et al. (33) as described previously. Briefly, labeled and unlabeled glycolipids were extracted with chloroform-methanol-water (10:10:3, by volume), partitioned between water and water-saturated n-butyl alcohol, and separated by thin-layer chromatography (TLC). Chromatograms were scanned for radioactivity, and areas corresponding to individual T. gondii GPIs I–VI (34) were scraped off, re-extracted with chloroform-methanol-water (10:10:3, by volume) by sonication (Branson 3200; 47 MHz), and recovered in the butanol phase after water-saturated n-butyl alcohol-water partition. The absence of endotoxin in purified T. gondii GPIs was checked with the Limulus Amebocyte Lysate kit QCL-100 (BioWhittaker).

Generation of neutral core glycans

TLC-purified GPIs of T. gondii were dephosphorylated, deaminated, and reduced as described (34, 4). Briefly, dephosphorylation was performed by incubation of TLC-purified GPIs with 48% aqueous hydrofluoric acid for 60 h at 0°C. Deamination occurred in freshly prepared 0.1 M sodium acetate (pH 3.5) containing 0.25 M NaNO2 and incubated at room temperature for 3 h. The reaction was terminated by addition of 0.4 M boric acid and 1 M NaOH. The material was reduced overnight at 4°C using 2 M NaBH4 prepared in 0.3 M NaOH and desalted over AG50W-X12 resin.

Generation of diacylglycerols

TLC-purified GPIs of T. gondii were cleaved using 1 U of Bacillus cereus phosphatidylinositol phospholipase C (Sigma-Aldrich) in 100 µl of 0.1 m of Tris-HCl (pH 7.4), 0.1% sodium deoxycholate overnight at 37°C. The enzyme was inactivated by treatment for 3 min at 100°C. Diacylglycerols were recovered in the butanol phase after partition between water and water-saturated n-butyl alcohol.

CHO cell lines and flow cytometry analysis

The CHO reporter cell lines (CHO/CD14, expressing endogenous functional TLR4; 7.19/CD14/TLR2, expressing TLR2; and the 7.19 clone, expressing neither TLR2 nor functional TLR4) were generated as described (35). These cell lines contain a CD25 gene reporter under the control of E-selectin promoter, which contains an NF-{kappa}B-binding site (35, 36). Cells were plated at 1 x 105/ well in 24-well tissue-culture dishes. The next day, the different molecules from T. gondii were added to the culture. Medium alone, 100 ng/ml LPS (from Escherichia coli serotype 055:B5; Sigma-Aldrich), UV-killed Staphylococcus aureus at a ratio of S. aureus/cell of 500:1 (American Type Culture Collection 12.692), and 10 ng/ml MALP-2 (Alexis Biochemicals) were used as controls. In the experiments done to check absence of endotoxin in GPI samples, polymyxin B (Sigma-Aldrich) was added at 1 µg/ml 15 min before GPIs or LPS. After 18 h stimulation, cells were stained with PE-labeled anti-CD25 (mouse mAb to human CD25, PE conjugate; Caltag Laboratories). The cells were examined by flow cytometry (BD Biosciences) and analyses were performed using CellQuest software (BD Biosciences) (17, 35).

Primary macrophages culture

Thioglycolate-elicited peritoneal macrophages were obtained from C57BL/6, TLR2–/–, TLR4–/–, TLR2/4–/–, CD14–/–, and MyD88–/– mice by peritoneal washing. Macrophages were stimulated with the different molecules of T. gondii. Medium alone (RPMI 1640 (Sigma-Aldrich) containing 5% FCS), LPS, and UV-killed Staphylococcus aureus were used as controls. The amount of TNF-{alpha} in supernatants was determined after 24 h of stimulation using an ELISA kit (Duoset; R&D Systems).

Experimental infections

C57BL/6, TLR2–/–, TLR4–/–, TLR2/4–/–, and MyD88–/– mice were infected i.p. with ~10 cysts (in 0.2 ml of PBS) of the ME-49 strain of T. gondii (37). After 40 days, the surviving mice were sacrificed by cervical dislocation, the brains were removed and homogenized in 1 ml of PBS, and the cysts were counted in duplicate. Spleen cells were also collected, washed with RPMI 1640 medium, and treated for 2 min with lysis buffer (9 vol of 0.16 M NH4Cl and 1 vol of 0.17 M Tris-HCl (pH 7.5)). The erythrocyte-free cells were cultured (5 x 105 cells/100 µl) for 48 h with medium alone (DMEM; Sigma-Aldrich) or in the presence of RH T. gondii extracts, named F3, and prepared as previously described (38). The amounts of IL-12 and IFN-{gamma} in supernatants were determined using ELISA kits (Duoset; R&D Systems).

Statistics

The Kruskal-Wallis nonparametric test and the unpaired Student t test were adopted for statistical evaluation and p < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Induction of NF-{kappa}B-dependent expression of CD25 on TLR2- or TLR4-expressing CHO cells in response to T. gondii GPIs, GPIa, core glycans, and lipid moieties

The six different T. gondii GPIs separated by TLC (Fig. 1, A and B) and a chemically synthesized GPI, lacking the lipid moiety (GPIa, Fig. 1C), are able to induce TNF-{alpha} in macrophages (4). We investigated the ability of these GPIs to activate NF-{kappa}B by using CHO/CD14 reporter cell lines expressing either human TLR2 or endogenous TLR4. These cells express a CD25 reporter gene, which is under control of the NF-{kappa}B-binding site. NF-{kappa}B activation was assessed by measuring the expression of surface CD25 by flow cytometry. In all experiments, UV-killed S. aureus or MALP-2 and LPS were used as controls for TLR2 and TLR4 activation, respectively (data not shown). No induction of CD25 expression was observed on control cells (without functional TLR2 and TLR4) and on TLR2 cells exposed to the six different T. gondii GPIs (Fig. 2A). In contrast, induction of CD25 expression by the GPIs was obtained on the TLR4+ cells. To exclude the possibility that this result was due to a contaminating molecule, we have tested the chemically synthesized GPI of T. gondii, GPIa (Fig. 1C). Like the natural GPIs, the synthetic molecule was able to induce CD25 expression on TLR4+, but not TLR2+, cells (Fig. 2B). Cells were stimulated with GPIs in the presence of polymyxin B to rule out a possible effect of a contaminating endotoxin. Polymyxin B completely abrogated the ability of LPS to induce CD25 expression, but GPI induction of CD25 was not, or was only slightly, depressed (Fig. 3).


Figure 1
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  		FIGURE 1. Structure of T. gondii GPIs. A, TLC scan of [3H]glucosamine-labeled glycolipids of T. gondii. Parasite GPIs were purified by sequential extraction and separated by TLC. Glycolipids from metabolically labeled parasites with [3H]glucosamine were used as tracers. Chromatograms were scanned for radioactivity using a Berthold LB 2842 linear analyzer. B, Structures of GPIs of T. gondii (34 ): GPIs I and II: (ethanolamine-PO4)-Man{alpha}1–2Man{alpha}1–6(Glc{alpha}1–4GalNAcbeta1–4)Man{alpha}1–4GlcN{alpha}-inositol-PO4-lipid; GPI III: (ethanolamine-PO4)-Man{alpha}1–2Man{alpha}1–6(GalNAcbeta1–4)Man{alpha}1–4GlcN{alpha}-inositol-PO4-lipid; GPIs IV and V: Man{alpha}1–2Man{alpha}1–6(Glc{alpha}1–4GalNAcbeta1–4)Man{alpha}1–4GlcN{alpha}-inositol-PO4-lipid; and GPI VI: Man{alpha}1–2Man{alpha}1–6(GalNAcbeta1–4)Man{alpha}1–4GlcN{alpha}-inositol-PO4-lipid. GPIs I and II and GPIs IV and V differ in their fatty acid composition with palmitic and stearic acids as predominant lipids. C, Structure of GPIa, a chemically synthesized GPI of T. gondii, with a phosphate group instead of the lipid moiety (Man{alpha}1–2Man{alpha}1–6(GalNAcbeta1–4)Man{alpha}1–4GlcN{alpha}-inositol-PO4).

 

Figure 2
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  		FIGURE 2. GPIs purified from T. gondii tachyzoites and GPIa activate TLR4. A, CHO cells expressing TLR2 (TLR2+), TLR4 (TLR4+), or neither (TLR2–/TLR4–) were either untreated (black) or exposed to the six different GPIs (I–VI, from 109 tachyzoites) (gray line). B, CHO cells were either untreated (black) or exposed to 1–100 µM of the synthetic GPI without lipid moiety, GPIa (gray line). CD25 expression was measured by flow cytometry 18 h after stimulation. The figure is representative of three independent experiments. Percentage = percentage of CD25 expression (M2) on GPI-stimulated cells – percentage of CD25 expression (M2) on nonstimulated cells.

 

Figure 3
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  		FIGURE 3. TLR4 activation is not due to contaminating endotoxins in GPI samples. Polymyxin B (PB, 1 µg/ml) was added 15 min before medium alone (black) or the six different GPIs (I–VI, from 109 tachyzoites) (gray line) on CHO cells expressing TLR4 (TLR4+) or not (TLR2–/TLR4–). CD25 expression was compared with those obtained without PB treatment. Positive control for polymyxin B activity was obtained on LPS-treated cells (LPS 100 ng/ml). CD25 expression was measured by flow cytometry 18 h after stimulation. The figure is representative of two independent experiments. Percentage = percentage of CD25 expression (M2) on GPI- or LPS-stimulated cells – percentage of CD25 expression (M2) on nonstimulated cells.

 
The fragments of T. gondii GPIs induce TNF-{alpha} production (4). To answer the question of whether they also use a TLR, neutral core glycans were prepared by dephosphorylation, nitrous deamination, and reduction of the six T. gondii GPIs (Fig. 4A). Interestingly, the glycans could activate CD25 expression on both TLR2- and TLR4-expressing cells (Fig. 4B). This suggests that the lipid moiety of the whole GPIs or the phosphate inositol of GPIa may disturb their binding to TLR2. Lipid moieties were then prepared by the cleavage of the six T. gondii GPIs with phosphatidylinositol phospholipase C (Fig. 5A). The diacylglycerols were able to activate not only TLR4 like the whole GPIs, but also TLR2 (Fig. 5B). Nevertheless, the TLR2 activation by the lipid moieties (except those from GPI I) was lower than those obtained with the core glycans.


Figure 4
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  		FIGURE 4. Core glycans activate TLR2 and TLR4. A, Structures of the two core glycans (32 ). Core glycan A from GPIs III and VI: Man{alpha}1–2Man{alpha}1–6(GalNAcbeta1–4)Man{alpha}1–4-anhydromannitol; core glycan B from GPIs I, II, IV, and V: Man{alpha}1–2Man{alpha}1–6(Glc{alpha}1–4GalNAcbeta1–4)Man{alpha}1–4-anhydromannitol, B, CHO cells expressing TLR2 (TLR2+), TLR4 (TLR4+), or neither (TLR2–/TLR4–) were either untreated (black) or exposed to core glycans (gray line) isolated from the six GPIs (I–VI, from 2.5 x 108 tachyzoites). The figure is representative of three independent experiments.

 

Figure 5
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  		FIGURE 5. Diacylglycerols activate TLR2 and TLR4. A, Structures of diacylglycerols. Fatty acids are mainly composed of palmitic and stearic acids (n = 14/16). B, CHO cells expressing TLR2 (TLR2+), TLR4 (TLR4+), or neither (TLR2–/TLR4–) were either untreated (black) or exposed to diacylglycerols (gray line) isolated from the six GPIs (I–VI, from 5 x 108 tachyzoites). The figure is representative of three independent experiments.

 
Simultaneous engagement of TLR2 and TLR4 for macrophage responses to highly purified GPIs derived from T. gondii tachyzoites

To determine whether TLRs are involved in TNF-{alpha} production by cells in response to T. gondii GPIs, peritoneal macrophages from MyD88–/–, TLR2–/–, TLR4–/–, TLR2/4–/–, CD14–/–, and control (C57BL/6) mice were incubated with individual GPIs, and TNF-{alpha} secretion was measured in supernatants. UV-killed S. aureus and E. coli LPS were used as controls for TLR2- and TLR4-dependent TNF-{alpha} production, respectively, and for CD14- and MyD88-dependent pathways. All of the six T. gondii GPIs significantly induced the production of TNF-{alpha} by macrophages of WT mice. On the contrary, almost no TNF-{alpha} was found in the supernatant of MyD88–/– macrophage cultures (Fig. 6A), indicating the requirement of a member of the TLR/IL-1R family in the response to GPIs. Contrary to GPIs of T. cruzi (17) and P. falciparum (18), no difference in TNF-{alpha} production was observed between macrophages of WT and TLR2–/– or TLR4–/– mice in response to GPIs (Fig. 6, A and B), as well as their core glycans and diacylglycerols (data not shown). As demonstrated for P. falciparum GPIs (18), GPIs of T. gondii might be cleaved by phospholipases expressed at the macrophage cell surface and the resulting glycan and lipid moieties could activate TLR4–/– macrophages through TLR2. When macrophages were deficient for both TLR2 and TLR4, the production of TNF-{alpha} in response to GPIs was completely abrogated (Fig. 6C). This indicates that both TLR2 and TLR4 are involved in the signaling pathway leading to production of TNF-{alpha} by macrophages exposed to T. gondii GPIs.


Figure 6
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  		FIGURE 6. Whole GPIs highly purified from T. gondii require MyD88 and both TLR2 and TLR4 to induce TNF-{alpha} production in macrophages. Peritoneal macrophages from C57/BL6 (A–C), MyD88–/– (A), TLR4–/– (A), TLR2–/– (B), CD14–/– (B), and TLR2/4–/– (C) mice were incubated for 24 h in the presence of the six different GPIs (I–VI) extracted from 5 x 108 T. gondii tachyzoites. LPS and UV-killed S. aureus (S. a) were used as positive controls. Peritoneal macrophages incubated with medium alone (M) were used as negative control. Supernatants were assayed for TNF-{alpha} production using a sandwich ELISA. Values represent the mean ± SD of triplicate samples and are representative of two independent experiments.

 
Simultaneous TLR2 and TLR4 deficiency leads to increased cyst numbers in mice infected with T. gondii

The results obtained during in vitro activation by T. gondii GPIs raise the question of the importance of TLR2 and TLR4 during in vivo infection. Intraperitoneal infection of C57BL/6 (WT), TLR2–/–, TLR4–/–, and CD14–/– mice with 10 cysts from the ME-49 strain resulted in 86% survival 40 days postinfection, whereas the MyD88–/– mice died after 9 days of infection (Fig. 7A). In terms of cysts numbers, no significant difference was observed between WT and TLR2–/–, TLR4–/–, or CD14–/– mice (Fig. 7B). Because of their early death, cysts could not be counted in brain of MyD88–/– mice at day 40. The survival rate of TLR2/4–/– mice reached 100% despite a higher number of brain cysts in TLR2/4–/– mice in comparison with control brains (Fig. 7).


Figure 7
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  		FIGURE 7. Susceptibility of C57BL/6 and knockout mice infected with T. gondii. A, Seven C57BL/6, MyD88–/–, TLR2–/–, TLR4–/–, TLR2/4–/–, or CD14–/–, 8-wk-old male mice were i.p. infected with 10 cysts of the ME-49 strain of T. gondii, and mortality was assessed daily. B, After 40 days of infection, the survivors C57BL/6 (C57), TLR4–/–, TLR2–/–, CD14–/–, and TLR2/4–/– mice were sacrificed, and the brain cysts were counted individually. The figure shows means ± SD and is representative of two independent experiments.

 
We also measured the cytokine production by spleen cells from uninfected controls and infected C57BL/6, TLR2–/–, TLR4–/–, TLR2/4–/–, and CD14–/– mice after stimulation with F3, an antigenic preparation enriched in GPI-anchored surface proteins from T. gondii tachyzoites (35). Stimulation of splenocytes from noninfected mice with F3 resulted in lower but consistent levels of IFN-{gamma} and IL-12, and these levels increased in the case of mice infected with the ME-49 strain of T. gondii. This increase was not affected by the absence of TLR4 or CD14 (Table I). On the contrary, F3-stimulated splenocytes from infected TLR2–/– produced higher levels of IFN-{gamma}. A similar increase was shown in splenocytes of TLR2–/– mice infected with T. cruzi (23). Splenocytes from infected TLR2/4–/– mice also produced higher IFN-{gamma} (Table I), further indicating the in vivo role of TLR2 in modulating IFN-{gamma} production.


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  		Table I. Cytokine levels produced by spleen cells from mice infected or not with T. gondii (pg/ml)a

 
Thus, together, the in vitro and in vivo results indicate that activation of both TLR2 and TLR4 by T. gondii GPIs may be critical for eliciting the TNF-{alpha} response by host innate immune cells, but differently from MyD88 and TLR11, are not directly involved in the development of Th1 lymphocytes of mice infected with T. gondii.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Our previous work has shown for the first time that both native and synthetic T. gondii GPIs are capable of activating macrophages to produce TNF-{alpha} (4). Here, we asked the question of whether the T. gondii GPIs activate TLRs. Our results show that intact T. gondii GPIs triggered surface expression of a reporter gene (CD25) under a NF-{kappa}B responsive promoter in CHO cells stably expressing TLR4, but not those expressing TLR2. Similar results, i.e., activation of TLR4 and not TLR2, were obtained with a synthetic molecule, named GPIa. Interestingly, the native core glycans and the diacylglycerols isolated from purified T. gondii GPIs were able to activate both TLR2 and TLR4. Furthermore, we observed that simultaneous deficiency of TLR2 and TLR4 was required to abrogate the in vitro TNF-{alpha} production by macrophages exposed to T. gondii GPIs. Importantly, we found that double TLR2/4–/– mice, but not single TLR2–/–, TLR4–/–, and CD14–/– mice, were more susceptible to infection and presented a higher number of cysts in brain. Thus, together, the in vitro and in vivo results indicate that activation of both TLR2 and TLR4 by T. gondii GPIs may be critical for eliciting the TNF-{alpha} response by host innate immune cells, but differently from MyD88 and TLR11, are not directly involved in the development of Th1 lymphocytes of mice infected with T. gondii.

The results indicating that only the core glycans and the diacylglycerols isolated from purified T. gondii GPIs activate TLR2 suggests that the whole molecules may have a conformation, which does not permit the activation of TLR2. Indeed, we have previously found that the core glycan from GPIa was more efficient to induce TNF-{alpha} than GPIa itself. This could be explained by the activation of TLR2 and TLR4 by the core glycan, but only of TLR4 by the phosphate-inositol-containing whole molecule. In addition, differences in the TLR2 activation were observed by the diacylglycerols isolated from the six T. gondii GPIs. This could be due to distinct fatty acid associations. The GPIs differ in their fatty acid composition with palmitic and stearic acids as predominant lipids but the exact distributions are unknown. The lipid moiety could determine the specificity of GPIs in terms of TLR they activate, which would be consistent with studies performed with bacterial glycolipids (39, 40, 41, 42). In the same way, L. major GPI-anchored lipophosphoglycan, but not delipidated lipophosphoglycan, activates TLR2, showing that the lipid part is important (19). We and others suggest that a minimal length of fatty acid is required for cell activation. Indeed, we have previously shown that a diacylglycerol containing two 16:0 chains did not induce TNF-{alpha} in macrophages, whereas one with two 18:0 chains did (4). Similarly, van der Kleij et al. (43) showed that cells failed to respond to a commercially available lyso-PS 16:0. The lipid moieties of the GPIs II–VI, which lower activate TLR2, could contain a 16:0 chain or could be lyso-forms with only one C18:0 chain. Two independent signaling pathways were reported in response to the two moieties of P. falciparum GPI. The evolutionary conserved core glycan, Man{alpha}1–2Man{alpha}1–6Man{alpha}1–4GlcN-myoinositol, activates protein tyrosine kinases, while the GPI-associated diacylglycerols are required for mobilization of the calcium-independent {epsilon} isoform of protein kinase C (44). With regard to our results with the core glycans and the lipid moieties of T. gondii GPIs, it could be possible that these independent activated pathways are due to the binding to different TLRs. Additional analysis of phosphorylation of protein kinases that are specifically induced after activation of TLR2 and/or TLR4 may help elucidate this question. Thus, we postulate the hypothesis that the specificity of GPIs in terms of the TLR they activate is determined by an interplay between both the glycan and lipid moieties.

The next step was to define which TLR was responsible for the induction of TNF-{alpha} production in macrophages exposed to T. gondii GPIs. Results obtained with macrophages from knockout mice indicate that as for T. cruzi and P. falciparum GPIs (17, 18), TLR2 is also a critical receptor for T. gondii GPI activity. TLR4 was also shown to be critical. Thus, abrogation of TNF-{alpha} production elicited by T. gondii GPIs was observed in macrophages from double TLR2/4–/– mice, but not from single TLR2–/– or TLR4–/– mice. We assume that cleavage of GPIs by macrophage-specific enzymes into glycan and lipid moieties may explain the TLR2-dependent production of TNF-{alpha} in murine macrophages. Consistently, GPIs of P. falciparum are also recognized by TLR2 and TLR4. Indeed, the levels of TNF-{alpha} produced by TLR2–/– and TLR4–/– macrophages in response to P. falciparum GPIs correspond to 20 and 72% of the amount produced by the WT macrophages, respectively (18). Further, in a recent study, a particular subset of free GPIs containing ceramide, purified from T. cruzi parasites, was shown to trigger the synthesis of proinflammatory cytokines via TLR4 (20). Although different studies in vitro have addressed the issue of whether TLR2 and TLR4 are involved in the host innate immune cell responses to T. gondii (25, 45, 46), no previous attempt was made to define a specific TLR2/TLR4 agonist in T. gondii parasites, as shown here likely to be the tachyzoite-derived GPI.

An important question raised by this study is related to the relevance of TLR2 and TLR4 activation by T. gondii GPIs during in vivo infection with this parasite. Would both TLR4 and TLR2 be simultaneously activated during in vivo infection with T. gondii by intact and fragments (i.e., core glycan and diacylglycerol moieties) of tachyzoite GPIs? As already suggested by Mun et al. (26), the role of TLRs during T. gondii infection seems to depend on the genetic background of the mice, infective inoculums, and on the parasite strain used. Indeed, the C57BL/6 mice were resistant to infection with the Fukaya strain, and the absence of MyD88, but not TLR2 or TLR4, led to the death of the animals infected with 100 cysts (26, 28). Our data confirm that MyD88–/– mice are highly susceptible to T. gondii infection, because all MyD88–/– mice died 9 days after i.p. infection. Although the mice did not die, an increase of parasite burden was observed by Chen et al. (28) in the lungs of TLR2- and TLR4-deficient mice infected with the Fukaya strain as compared with the C57BL/6 WT mice. Parasites were also detected in kidneys of TLR2-deficient mice, and to a lesser extent, of TLR4-deficient mice infected with the Fukaya strain, associated with functional and histological changes of the kidneys (47). A recent study also shows that death of TLR4–/– mice ~35 days postinfection is accompanied with a higher number of brain cysts and less secretion of IL-12 and IFN-{gamma} by splenocytes than WT mice (48). Furthermore, the presence of TLR4 antagonists ameliorated T. gondii-induced ileitis and cells from ileum of TLR4–/– mice produced less IFN-{gamma} (49). In contrast, we failed to observe impaired production of IL-12 and IFN-{gamma}, enhanced susceptibility, and increased cyst numbers in the brain from TLR2–/– and TLR4–/– mice. Consistent with our in vitro studies indicating the engagement of both TLR2 and TLR4 by T. gondii GPIs, we observed increased cyst numbers in double TLR2/4–/– mice.

In the attempt to define the mechanism by which TLR2/4–/– mice become more susceptible to infection, we evaluated the levels of IL-12 and IFN-{gamma} produced by splenocytes from TLR-deficient, as well as WT, mice infected with T. gondii. These two cytokines were evaluated because they have been shown critical for development of host resistance to T. gondii during the initial stages of infection (50, 51). However, we observed that splenocytes from mice lacking TLR2, including TLR2/4–/– mice, produced even higher levels of IFN-{gamma} after stimulation with T. gondii extracts enriched in GPI-linked surface proteins. A similar increase was shown in splenocytes of TLR2–/– mice infected with T. cruzi (24). It has been reported that TLR2 could be involved in host tolerance and its absence could lead to higher host response illustrated by higher IFN-{gamma} production (52, 53, 54). Thus, our results show that, differently from TLR9 (30) and TLR11 (29), which are involved in induction of IL-12 and IFN-{gamma} by T. gondii, TLR2 and TLR4 may be critical receptors for induction of TNF-{alpha}, a cytokine playing a crucial role in the control of host resistance to later stages of T. gondii infection (55).

A recent study has shown that TLR9-deficient mice have less intense intestinal pathology mediated by IFN-{gamma}-producing CD4+ T cells (30). Furthermore, TLR11 was shown to be important for production of IL-12 by dendritic cells in response to T. gondii profilin and in host resistance to infection, as indicated by the higher number of cysts in the brain of TLR11–/– mice infected with T. gondii (29). Although this is clearly an important study, the infection of TLR11-deficient mice gave only a partial susceptibility phenotype, if compared with MyD88–/– mice, indicating the involvement of additional receptors that use MyD88 in innate host resistance to T. gondii.

Although when the TLRs were discovered it was thought that each TLR would be responsible for the defense against a specific pathogen, now the tendency is to think that several TLRs could work together to resolve the infection (56). For example, it has been recently reported that TLR2, TLR7, TLR8, and TLR9 from dendritic cells are stimulated by the very efficient Yellow Fever vaccine leading to immunity against Yellow Fever disease (57). Furthermore, a combinatory role for TLR2 and TLR9 has been shown in infection of mice with either Mycobacterium tuberculosis or T. cruzi (58, 59). Our results point to the direction that TLR2 and TLR4 are involved in the recognition of molecules of T. gondii tachyzoites and may contribute in collaboration with other MyD88-dependent receptors to innate resistance and development of acquired immunity specific to infection with this protozoan parasite. In addition, TLR11 is not functional in humans, which suggests that the role of other MyD88-dependent receptors, such as TLR2, TLR4, and TLR9 could be even more important for parasite recognition in human toxoplasmosis.


   Acknowledgment
 
We are grateful to Dr. Shizuo Akira for the TLR2–/–, TLR4–/–, and MyD88–/– mice.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the Deutsche Forschungsgemeinschaft, the Fonds der Chemischen Industrie, the Stiftung P.E. Kempkes, the Hessisches Ministerium für Wissenschaft und Kunst, the Deutscher Akademischer Austauschdienst, the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Brazil), Fundação de Amparo à Pesquisa de Minas Gerais (EDT 2400/03 and EDT 24000), National Institutes of Health (1RO1AI071319-01), the Ludwig Institute For Cancer Research, the Millennium Institute for Technology and Vaccine Development–Conselho Nacional de Desenvolvimento Científico e Tecnológico (Conselho Nacional de Pesquisas (CNPq)). M.A.C. and R.T.G. are Research Fellows from CNPq. Back

2 Address correspondence and reprint requests to Dr. Françoise Debierre-Grockiego, Institut für Virologie, AG Parasitologie, Philipps University, Hans-Meerwein-Strasse 2, D-35043 Marburg, Germany. E-mail address: debierre{at}staff.uni-marburg.de Back

3 F.D.-G. and M.A.C. contributed equally to this work. Back

4 Current address: Altana Pharma Deutschland GmbH, Moltkestrasse 4, D-78467 Konstanz, Germany. Back

5 R.T.G. and R.T.S. contributed equally to this work. Back

6 Abbreviations used in this paper: CHO, Chinese hamster ovary; WT, wild type; TLC, thin-layer chromatography. Back

Received for publication August 22, 2005. Accepted for publication May 1, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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