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The Journal of Immunology, 2006, 177: 2584-2591.
Copyright © 2006 by The American Association of Immunologists

Toxoplasma gondii Genotype Determines MyD88-Dependent Signaling in Infected Macrophages1

Leesun Kim*, Barbara A. Butcher*, Chiang W. Lee*, Satoshi Uematsu{dagger}, Shizuo Akira{dagger} and Eric Y. Denkers2,*

* Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY; and {dagger} Department of Host Defense, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Infection of mouse macrophages with Toxoplasma gondii elicits MAPK activation and IL-12 production, but host cell signaling pathways have not been clearly delineated. Here, we compared macrophage signaling in response to high virulence type I (RH) vs low virulence type II (ME49) strain infection. Tachyzoites of both strains induced p38 MAPK-dependent macrophage IL-12 release, although ME49 elicited 2- to 3-fold more cytokine than RH. IL-12 production was largely restricted to infected cells in each case. RH-induced IL-12 release did not require MyD88, whereas ME49-triggered IL-12 production was substantially dependent on this TLR/IL-1R adaptor molecule. MyD88 was also not required for RH-stimulated p38 MAPK activation, which occurred in the absence of detectable upstream p38 MAPK kinase activity. In contrast, ME49-driven p38 MAPK activation displayed an MyD88-dependent component. This parasite strain also induced MyD88-dependent activation of MKK4, an upstream activator of p38 MAPK. The results suggest that RH triggers MAPK activation and IL-12 production using MyD88-independent signaling, whereas ME49 uses these pathways as well as MyD88-dependent signaling cascades. Differences in host signaling pathways triggered by RH vs ME49 may contribute to the high and low virulence characteristics displayed by these parasite strains.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The intracellular protozoan Toxoplasma gondii is a highly successful food- and waterborne parasite that infects up to 1 billion humans worldwide (1). The high incidence of Toxoplasma in animals also makes this parasite an important zoonotic pathogen (2). Infection in most cases is asymptomatic, a key factor in the success of the parasite. The benign nature of T. gondii infection is likely due to the ability of the parasite to induce strong host protective cell-mediated immunity, characterized by production of Th1 cytokines such as IFN-{gamma} (3). This response is dependent on early production of IL-12 by cells of the innate immune system (4, 5). In this way, T. gondii may limit its own replication, preserving the host and allowing establishment of latent infection. Nevertheless, in situations of immunodeficiency or when the parasite is congenitally acquired, T. gondii emerges as a pathogen that may be lethal if not appropriately treated. Disease most often manifests as encephalitis, retinochoroiditis, or pneumonitis caused by uncontrolled parasite replication in infected individuals (6, 7).

Toxoplasma displays a unique population structure that is dominated by three clonal lineages (types I, II, and III) that are widespread throughout Europe and North America (8, 9, 10, 11). Type I strains are highly virulent in mice with an LD100 as low as a single tachyzoite. Strain types II and III are less virulent (LD100 > 1000) and as such are able to establish chronic infections in mice. The high virulence of type I strains in mice may be linked to overproduction of proinflammatory cytokines as well as increased rates of parasite migration (12, 13, 14). There is evidence that infections with type I parasite strains cause more severe disease manifestations in humans (15, 16, 17, 18).

Because of the central role of IL-12 in resistance to Toxoplasma and other microbial pathogens, considerable interest is focused on signaling pathways involved in induction of this cytokine during infection (19, 20, 21, 22). IL-12 is produced by dendritic cells, macrophages, and neutrophils during T. gondii infection (23, 24, 25). IL-12 production and host resistance is abrogated in MyD88–/– mice during infection with ME49, a type II Toxoplasma strain (26, 27). Because MyD88 is an essential adaptor molecule in TLR-initiated signaling (28, 29), these pattern recognition molecules are strongly implicated in parasite detection. Recently, TLR11 was found to recognize a T. gondii profilin-like protein leading to dendritic cell IL-12 production (30). Insofar as other results implicate TLR2 in parasite-induced responses (31, 32), it seems probable that the parasite expresses more than one type of TLR ligand. In addition to TLR-MyD88 signaling, a T. gondii-derived cyclophilin termed C-18 has been reported to induce dendritic cell IL-12 production through CCR5, a Gi{alpha} protein-coupled chemokine receptor (33).

We and others recently found a requirement for p38 MAPK signaling in macrophage IL-12 induction in response to soluble tachyzoite extracts and live parasites of the virulent type I RH strain (34, 35). p38 MAPK activation in response to T. gondii involves parasite-induced autophosphorylation rather than a more conventional pathway of phosphorylation mediated by upstream MAPK kinases (MKK)3 such as MKK3, MKK4, or MKK6 (34). Other recent studies indicate that low virulence Toxoplasma strains such as PTG (a clonal derivative of ME49) induce IL-12 in dependence on MyD88 and that levels of cytokine induced are much higher than those stimulated by RH infection (36).

In this study, we compared the ability of low virulence ME49 and high virulence RH to induce MAPK activation and IL-12 production during macrophage infection. In general agreement with others (36), we found higher IL-12 production in response to ME49 tachyzoites. Interestingly, p38 MAPK activation and IL-12 induction by RH tachyzoites did not require functional MyD88. Nevertheless, during ME49 infection, MyD88 was required for optimal p38 MAPK activation and IL-12 production. We also found a substantial MyD88-independent IL-12 response during in vivo RH infection. Levels of IL-12 were sufficient to allow survival of mice undergoing infection with tachyzoites of an attenuated RH strain variant. These results demonstrate that the Toxoplasma genotype exerts a profound influence on activation of innate immune signaling pathways during intracellular infection. Parasite strain-specific differences in how T. gondii activates cells of innate immunity may explain, in part or whole, the substantially disparate virulence phenotypes displayed by these parasites during in vivo infection.


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

Female C57BL/6 mice (6–8 wk of age) were obtained from Taconic. MyD88–/– animals were engineered as described (28, 29). IL-12p35–/– and CCR5–/– mice were obtained from The Jackson Laboratory. Breeding pairs of MKK3–/– were supplied by R. Flavell (Yale University, New Haven, CT). Mice were housed in the animal facility of the College of Veterinary Medicine at Cornell University (Ithaca, NY). RH, ts-4, and ME49 tachyzoites of T. gondii (The American Type Culture Collection, Manassas, VA) were maintained on human foreskin fibroblast monolayers by biweekly passage in DMEM (Invitrogen Life Technologies) supplemented with 1% heat-inactivated FCS (HyClone), 100 U/ml penicillin (Invitrogen Life Technologies), and 0.1 mg/ml streptomycin (Invitrogen Life Technologies). Parasite cultures were tested every 6–8 wk for Mycoplasma contamination using a commercial PCR-ELISA-based kit (Roche Applied Science).

Reagents and Abs

Abs specific for total and phosphorylated forms of p38, ERK1/2, MKK3/6, and MKK4 were purchased from Cell Signaling Technology. Mouse FITC-conjugated anti-p30 (SAG-1) Ab was obtained from Argene, and rat PE-conjugated anti-IL-12p40/p70 was from BD Pharmingen. SB202190, wortmannin, and pertussis toxin were purchased from Calbiochem. Ultrapure LPS from Salmonella minnesota R595 was purchased from List Biological Laboratories.

Cell culture

Bone marrow-derived macrophages were prepared as described previously (37). A stable transfectant J774A.1 macrophage line expressing a dominant negative form of p38 MAPK was kindly provided by S. Paludan and T. Mogenson (University of Aarhus, Aarhus, Denmark) (38). For in vitro infection, RH and ME49 tachyzoites or LPS were added to macrophages in tissue culture plates (Costar), which were then briefly centrifuged (3 min, 200 x g) to synchronize contact between cells and parasites. In some experiments, macrophages were preincubated for 2 h with wortmannin or pertussis toxin before infection.

Western blotting

Immunoblot analysis was conducted as previously described using ECL-based detection (37). Films were scanned (ScanMaker 8700; Microtek Lab), and results were semiquantitated by measuring band intensity and area followed by normalization to total p38 MAPK. Results were displayed in relative densitometry units.

Flow cytometry

Cells were either stimulated with LPS (100 ng/ml) for 24 h or infected with parasites (tachyzoite-cell ratio, 0.5) for 36 h. Golgi-Block (BD Biosciences) was added 8 h before collection of cells. For detection of IL-12 and intracellular parasites, macrophages were briefly washed with PBS, and FcRs were blocked in PBS containing 1% BSA (Sigma-Aldrich) with 5 µg/ml rat anti-mouse CD16/CD32 (BD Biosciences) and 10% normal mouse serum (Jackson ImmunoResearch Laboratories) for 15 min on ice. Then cells were fixed and permeabilized with BD Cytofix/Cytoperm solution (BD Biosciences) for 20 min on ice. After a washing with BD Perm/Wash solution (BD Biosciences), cells were incubated with FITC-conjugated anti-p30 Ab and PE-conjugated anti-IL-12 Ab for 30 min on ice in the dark. Cells were then washed twice with BD Perm/Wash solution and once with PBS and subjected to flow cytometry using a FACSCalibur flow cytometer (Becton Dickinson Immunocytometry Systems) and analyzed with Flow Jo software (TreeStar).

IL-12p40 ELISA

IL-12p40 was measured by ELISA as described previously (34).

Statistical analyses

Data were analyzed using an unpaired two-tailed Student t test. The significance of survival curves was determined using Kaplan-Meier analysis. p < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
IL-12 derives from infected cells during RH and ME49 infection

We examined IL-12 production during macrophage infection with RH (type I strain, high virulence) and ME49 (type II strain, low virulence) tachyzoites to determine whether Toxoplasma-infected cells, noninfected cells, or a mixture of the two were sources of IL-12. For LPS stimulation, ~22% of the population responded with robust IL-12 production (Fig. 1A). Both ME49 and RH also induced macrophage IL-12 production. Notably, the cytokine was largely restricted to infected cells in both RH and ME49-infected cultures (Fig. 1A). A similar proportion of cells were positive for IL-12 in both cases (29 and 23% for ME49 and RH, respectively). In Fig. 1B, we compared IL-12 release induced by RH and ME49 infection. We found that ME49 induced approximately twice as much IL-12 as RH parasites did. In Fig. 1C, we show that IL-12 induction during both RH and ME49 infection are blocked by SB202190, an inhibitor of p38 MAPK. Because Toxoplasma expresses its own p38 MAPK homolog that could affect IL-12 induction (39), we used a mouse macrophage line expressing a dominant-negative form of host p38 MAPK. These cells failed to produce IL-12 during infection with either parasite strain, in contrast to the nontransfected parent cell line (Fig. 1D).


Figure 1
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  		FIGURE 1. Parasite-induced IL-12 occurs in infected but not bystander noninfected macrophages and requires p38 MAPK activity. A, Bone marrow-derived macrophages were either cultured in medium (Med) alone, stimulated with LPS (100 ng/ml), or infected with ME49 and RH tachyzoites (parasite-cell ratio, 0.5). Cells were subsequently subjected to intracellular staining for IL-12 and tachyzoite surface protein p30 (SAG-1). B, Macrophages were infected with RH or ME49 tachyzoites (parasite-cell ratio, 2:1); then supernatants were collected at the indicated times for IL-12 ELISA. C, Macrophages were infected with tachyzoites (parasite-cell ratio, 1:1) in the presence of p38 MAPK inhibitor (SB202190, 10 µM) or solvent (DMSO). After 36 h, supernatants were collected for cytokine ELISA. D, J774A.1 or dominant negative p38-J774A.1 (DNp38) cells were infected (parasite-cell ratio, 3:1); 36 h later, supernatants were collected for ELISA. Experiments were repeated twice with the same results.

 
Parasite IL-12 induction in bone marrow-derived macrophages does not involve Gi protein-coupled receptors

It was previously reported that signaling through chemokine receptor CCR5 mediated IL-12 production in dendritic cells (40). We examined whether this pathway was involved in IL-12 production by macrophages. As shown in Fig. 2A, production of IL-12 was unaffected by absence of CCR5 during both RH and ME49 infection. Signaling through chemokine receptors involves coupling to Gi proteins, a response cascade that can be blocked with pertussis toxin. However, as shown in Fig. 2B, Pertussis toxin failed to affect IL-12 induction during infection with either parasite strain. We confirmed the effectiveness of the toxin by examining activation of protein kinase B (PKB) that depends on Gi protein signaling during RH infection (Fig. 2C) (41).


Figure 2
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  		FIGURE 2. CCR5 and Gi protein-coupled receptor signaling is not involved in macrophage IL-12 production. A, Bone marrow-derived macrophages from WT or CCR5 KO mice were infected with RH and ME49 tachyzoites (parasite-cell ratio, 1:1); supernatants were collected 36 h later for cytokine analysis. B, Macrophages were preincubated for 2 h with pertussis toxin (PTx); then cells were infected at a ratio of 1:1. Supernatants were collected for ELISA after 36 h. C, Macrophages were pretreated with pertussis toxin and then infected with RH parasites. At the indicated times postinfection, cells were lysed, and activated PKB was assessed by phospho (p)-specific immunoblotting. Experiments were performed three times with similar results. Med, Medium; p, phosphorylated.

 
Toxoplasma strain-specific dependence on MyD88 for MAPK activation and IL-12 production

Because p38 MAPK is involved in IL-12 induced by both T. gondii and LPS (Fig. 1; Refs. 34 , 42 , and 43), we examined activation of MAPK during macrophage infection as well as LPS stimulation. In wild-type (WT) macrophages, both parasite strains and endotoxin induced phosphorylation of p38 and ERK1/2 MAPK within 10 min (Fig. 3A). However, in contrast to activation kinetics, the strengths of MAPK activation differed. The degree of p38 and ERK1/2 MAPK activation was strongest for LPS. Although ME49-induced MAPK phosphorylation was weaker than that triggered by LPS, it was higher and longer lasting relative to RH infection. Infection rates were 93 and 89% for RH and ME49, respectively. Therefore, differences in RH vs ME49-induced MAPK activation cannot be explained by dissimilar rates of infection.


Figure 3
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  		FIGURE 3. MyD88 requirement in Toxoplasma-induced p38 and ERK1/2 MAPK activation depends on parasite strain. A, Bone marrow-derived macrophages from WT or MyD88 KO mice were infected with RH and ME49 tachyzoites (6:1 MOI, >90% infection in both cases) or stimulated with LPS (100 ng/ml). At the indicated time points, cells were collected, and lysates were subjected to Western blot analysis with phosphorylated (p) MAPK Ab and total p38 MAPK Ab as a loading control. B, Densitometric analysis of results shown in A. R, RH; M, ME49; L, LPS. Experiments were repeated three times with similar results.

 
One major signaling pathway leading to MAPK activation occurs through the TLR adaptor molecule MyD88. Accordingly, we examined whether absence of MyD88 influenced activation of p38 and ERK1/2 MAPK. As shown in Fig. 3, absence of MyD88 had no effect on RH-induced MAPK activation. In contrast, the level of p38 and ERK1/2 phosphorylation was decreased in MyD88–/– relative to WT macrophages infected with ME49. Nevertheless, the kinetics of MAPK activation was unaffected by the absence of functional MyD88, in contrast to LPS-induced macrophage responses, which displayed an MyD88-dependent delay in maximal p38 and ERK1/2 activation, as well as a decrease in total levels of phosphorylation, relative to WT cells (Fig. 3). Delayed MAPK activation is a characteristic of MyD88-independent signaling through TLR4 that instead involves the alternate adaptor molecule TRIF (29, 44).

Next, we examined the MyD88 requirement for macrophage IL-12 production in response to RH, ME49, and LPS. As expected, LPS-induced IL-12 was totally dependent on MyD88 (Fig. 4). Consistent with the lack of an MyD88 requirement for p38 MAPK activation, RH-induced IL-12 production was unaffected by the absence of this TLR/IL-1R adaptor molecule. In contrast, levels of IL-12 triggered by ME49 infection were decreased in the absence of MyD88 (Fig. 4). Nevertheless, there was also clearly an MyD88-independent IL-12 response. Fig. 4 also shows that at lower multiplicities of infection (MOI) (0.3–0.7) induction of IL-12 in WT macrophages is approximately equivalent for both parasite strains. This is consistent with data in Fig. 1A that also shows no major difference in intracellular IL-12 at lower MOI.


Figure 4
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  		FIGURE 4. Disparate effects of MyD88 deficiency on RH- and ME49-induced IL-12 production. Bone marrow-derived macrophages from WT or MyD88–/– mice were infected with RH or ME49 Toxoplasma, and with LPS. Supernatants were collected at 36 h (T. gondii-infected cells) and 24 h (LPS stimulation) for cytokine ELISA. Experiments were performed three times with the same results.

 
RH and ME49 tachyzoites differ in activation of MKK4, an upstream p38 MAPK-activating kinase

Since p38 MAPK activation was required for IL-12 production, we next examined the activity of upstream MAPK kinases in the presence and absence of MyD88. Three MAPK kinases are known to activate p38 MAPK, namely, MKK3, MKK4, and MKK6 (45). Fig. 5A shows absence of MKK4 activation after RH infection of both WT and MyD88–/– macrophages. However, ME49 triggered MKK4 phosphorylation, and this response was ablated in MyD88 gene-deleted macrophages (Fig. 5A). Stimulation of cells with LPS also induced MKK4 activation. In this case, the absence of MyD88 resulted in a delay in MKK4 phosphorylation, consistent with MyD88-independent signaling through TLR4 (29).


Figure 5
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  		FIGURE 5. Activation of MAPK kinases involved in p38 MAPK phosphorylation (p) during infection with RH and ME49 tachyzoites. A, Bone marrow-derived macrophages from WT and MyD88 KO mice were infected with RH or ME49 tachyzoites (parasite-cell ratio, 6:1) or LPS (100 ng/ml); then samples were collected at the indicated time points for Western blot analysis. B, Macrophages were set up in a manner identical with that of A, using cells derived from WT and MKK3 KO bone marrow. These experiments were performed twice with essentially identical results.

 
Previously, we showed that infection of macrophages with RH parasites induces activation of only MKK3 (34). Nevertheless, the kinetics of T. gondii-driven MKK3 activation failed to correlate with p38 MAPK phosphorylation, and more importantly activation of p38 MAPK was maintained in the absence of MKK3 (Fig. 5B) (34). During ME49 infection, we found a similar pattern of MKK3 activation and a lack of MKK6 activation. The LPS response was qualitatively similar, but here we were able to detect weak MKK6 phosphorylation (Fig. 5B).

ME49-induced ERK1/2 activation involves PI3K and MyD88 signaling, but RH-triggered ERK1/2 phosphorylation depends solely on PI3K

We report elsewhere Gi protein-coupled receptor-dependent PI3K activity mediates ERK1/2, but not p38, MAPK activation upon RH infection (41). The involvement of PI3K in ERK1/2 activation during ME49 relative to RH infection in the presence and absence of MyD88 was examined using the potent and specific chemical inhibitor wortmannin.

As shown in Fig. 6A, RH-induced ERK1/2 phosphorylation in WT macrophages was nearly completely abrogated by wortmannin inclusion. In MyD88 KO macrophages, RH-triggered ERK1/2 activation was essentially normal, and the response was also sensitive to PI3K inhibition. This suggests that RH activation of ERK1/2 proceeds through a PI3K pathway that does not involve MyD88.


Figure 6
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  		FIGURE 6. Both RH and ME49 trigger ERK1/2 activation in dependence on PI3K. Macrophages from WT (A) and MyD88 KO (B) mice were incubated with wortmannin (WM, 50 ng/ml) for 2 h or solvent alone (DMSO) and then infected with RH or ME49 (MOI 6:1, >90% infection). At each time point, whole cells were lysed and samples were subjected to Western blot analysis with anti-phospho (p)-ERK1/2 and total ERK1/2 Ab as a protein loading control. This experiment was repeated three times with similar results.

 
In ME49-infected WT macrophages (Fig. 6B), wortmannin partially blocked ERK/2 activation. This effect was most apparent between 10 and 30 min of infection. Absence of functional MyD88 also resulted in decreased ME49-induced ERK1/2 activation. Interestingly, the effect was most striking at 60 and 120 min of infection, unlike inhibition mediated by wortmannin. Finally, inclusion of wortmannin in cultures of ME49-infected MyD88–/– macrophages decreased ERK1/2 activation to near background levels (Fig. 6B). This experiment argues that ME49 triggers ERK1/2 activation using two distinct pathways. One involves signaling through MyD88, and the other involves PI3K dependent signal transduction.

We asked whether the parasite-induced PI3K pathway or ERK1/2 were involved in parasite-induced IL-12 induction. However, blocking PI3K activity with wortmannin failed to affect RH-induced IL-12 production (Fig. 7A). We confirmed the effectiveness of the wortmannin by evaluating parasite-induced PKB phosphorylation that depends on PI3K signaling (Fig. 7B). A similar lack of effect occurred during ME49 infection (data not shown). Similarly, experiments using pharmacological ERK1/2 inhibitors also did not affect macrophage IL-12 production in response to either strain (data not shown) (37).


Figure 7
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  		FIGURE 7. Induction of macrophage IL-12 during RH infection does not require PI3K activity. A, Bone marrow-derived macrophages were reincubated for 2 h with wortmannin (WM); then cells were infected with RH tachyzoites (parasite-cell ratio, 1:1). Supernatants were collected for ELISA after 36 h. B, Macrophages were pretreated with WM and infected; then at the indicated times postinfection cell lysates were prepared for immunoblot analysis of phosphorylated (p) PKB. Experiments were repeated three times with the same results. Med, Medium.

 
MyD88-independent IL-12 production during in vivo Toxoplasma infection

Finally, we asked whether MyD88-independent IL-12 production occurred during in vivo RH infection. Previously, it was found that ME49 infection in MyD88–/– mice resulted in severely reduced IL-12 levels and increased susceptibility (27). Here, we infected MyD88 WT and KO mice i.p. with RH strain tachyzoites and collected peritoneal exudate cells 4 days later. As shown in Fig. 8A, cells from KO animals displayed a substantial IL-12 response, although levels were clearly lower than those of WT cells. Similarly, levels of IL-12 were raised in the serum but remained lower than levels in WT animals (Fig. 8B). Therefore, the in vivo IL-12 response during RH infection involves both MyD88-dependent and MyD88-independent components. The extreme lethality of RH infection precludes an assessment of MyD88-independent IL-12-mediated resistance mechanisms. However, ts-4 is an attenuated RH mutant that is avirulent in normal mice but lethal in the absence of IL-12. As shown in Fig. 8C, MyD88–/– mice survived ts-4 infection, whereas ts-4-infected IL-12 KO animals succumbed within <10 days. In contrast, and as previously reported (27), ME49 infection was lethal in the absence of MyD88. Therefore, mice lacking MyD88 are able to survive infection with ts-4, a strain that is lethal in the absence of IL-12. The result indicates that levels of MyD88-independent IL-12 are sufficient to enable survival during infection with this RH-derived parasite strain.


Figure 8
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  		FIGURE 8. MyD88-independent resistance during infection in vivo. A, WT or MyD88 KO mice were infected by i.p. injection of 1000 RH tachyzoites. After 4 days, peritoneal exudate cells were collected and cultured in vitro without further stimulation. IL-12 levels were assessed in supernatants collected after 24 h. Contr, Cells from noninfected WT animals. B, In the same mice, serum IL-12 levels were determined 4 days postinfection. Each closed circle represents a single mouse. Contr, IL-12 in sera from noninfected WT animals. C, The indicated mouse strains (5 per group) were inoculated i.p. with ts-4 or ME49 Toxoplasma, and survival was monitored. Experiments were repeated twice with the same results.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Production of IL-12 during Toxoplasma infection is a key event for induction of protective immunity that enables establishment of long term infection. Our results reveal that high and low virulence parasite strains trigger distinct proinflammatory signaling pathways, each leading to induction of IL-12. For infection with RH tachyzoites, representative of virulent type I parasite strains, IL-12 production did not require TLR/IL-1R adaptor MyD88. Activation of p38 MAPK, necessary for parasite-induced IL-12 (34, 35), was also normal in MyD88–/– macrophages infected with RH tachyzoites. p38 MAPK phosphorylation occurred in the absence of detectable MKK3, MKK4, and MKK6 activation in MKK3–/– macrophages. Activation of p38 MAPK in this situation appears to depend on autophosphorylative properties of this molecule rather than activation of upstream p38 MAPK kinases (34).

In contrast, during low virulence ME49 infection, IL-12 production was partially dependent on MyD88. Unlike RH, ME49 induced MyD88-dependent MKK4 phosphorylation with kinetics similar to p38 MAPK activation. It seems likely that MKK4 contributes to p38 activation in this situation, although this conclusion awaits formal proof. Consistent with this, p38 MAPK activation was partially MyD88 dependent during ME49 infection.

Similar results were obtained when ERK1/2 activation was examined. Here, RH infection triggered phosphorylation that was dependent upon PI3K signaling. This pathway did not require MyD88. In the case of ME49, optimal ERK1/2 activation required MyD88-independent PI3K signaling, as with RH infection. In addition, ME49-driven ERK1/2 activation displayed an MyD88-dependent component. The latter pathway did not involve PI3K signaling.

In sum (Fig. 9), whereas RH infection appears to trigger MyD88-independent pathways leading to p38 activation and IL-12 production in macrophages, ME49 possesses the additional capacity to trigger MyD88-dependent activation of ERK1/2 and p38 MAPK and production of IL-12. The latter likely accounts for the ability of ME49 to induce higher levels of IL-12 relative to RH parasite infection. In addition, a PI3K cascade, initiated through Gi protein-coupled receptor signaling (41), leads to ERK1/2 activation by both parasite strains.


Figure 9
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  		FIGURE 9. Summary of signaling in Toxoplasma-infected macrophages. During RH (type I strain) infection, the parasite triggers ERK1/2 activation through a Gi protein-coupled receptor (GiPCR)-PI3K pathway. RH also triggers MyD88-independent p38 MAPK activation leading to IL-12 induction. In addition to these two distinct signaling cascades, ME49 (type II strain) triggers an MyD88-dependent cascade resulting in ERK1/2 and p38 MAPK activation. MyD88-dependent p38 MAPK activation contributes to IL-12 production in ME49-infected macrophages.

 
Despite RH induction of IL-12, this parasite strain possesses the ability to suppress TLR4-triggered IL-12, TNF-{alpha}, and NO (46, 47, 48). This is a consequence, at least in part, of the ability of the parasites to directly activate STAT-3 (49). Because Toxoplasma itself induces macrophage IL-12, but not TNF-{alpha} or inducible NO synthase, we hypothesize that the latter two mediators may be the true targets of suppression during infection.

During in vivo infection, RH clearly elicited both MyD88-dependent and MyD88-independent components. Dendritic cells, neutrophils (23), or possibly Gr-1+ monocyte/macrophage lineage cells (50) are strong candidates for an MyD88-dependent component that would likely be initiated through TLR ligation, either by parasite molecules or possibly by endogenous host ligands (30, 31, 51, 52, 53). Although in vivo IL-12 levels were lower in the absence of MyD88 during RH infection, they were nevertheless sufficiently high to allow animals to survive infection with a slow growing Toxoplasma strain derived from RH.

With the exception of p38 MAPK, components in the pathway leading to MyD88-independent IL-12 are largely obscure. Our results show that in macrophages the pathway does not involve CCR5 that has been implicated in dendritic cell IL-12 (40), or other pertussis toxin-sensitive Gi protein-coupled receptors. In addition, insensitivity to wortmannin argues against a role for PI3K signaling.

A recent study by Sibley and colleagues examined IL-12 production by macrophages infected with type I RH and type II PTG Toxoplasma strains (36). The PTG strain is a clonal derivative of ME49. As in our work, low virulence infection triggered MyD88-dependent IL-12, and levels produced were higher than those induced by high virulence RH infection. Nevertheless, whereas their study indicated a >100-fold difference in levels of IL-12 produced, in our studies the differences were consistently on the order of 2- to 3-fold. We do not understand the reason for this difference at present. Nevertheless, it is important to emphasize that our studies are consistent overall and together provide strong evidence that the Toxoplasma genotype exerts a critical influence on triggering of host cell signaling cascades.

Our data and those of others suggest that infection with type II strains such as ME49 or PTG induces a TLR signaling cascade (36). Data supporting this conclusion include dependence on MyD88, particularly with respect to p38 MAPK activation. Evidence that type II strains induce NF{kappa}B nuclear translocation would also be consistent with TLR activation (36). In addition, mice with targeted inactivation of MyD88 display an extreme susceptibility phenotype during ME49 infection that is similar to that of IL-12–/– animals (27, 54).

The recent identification of Toxoplasma PFTG as a profilin molecule that binds mouse TLR11 and induces dendritic cell IL-12 production is clearly of interest in the context of the present findings (30). Whether ME49 triggers MyD88-dependent p38 MAPK activation and IL-12 production independence on PFTG remains to be determined. Regardless, it is paradoxical that the PFTG molecule was isolated from extracts prepared from RH parasites that do not appear to trigger MyD88 pathways during live infections in macrophages (36). It is possible that during intracellular infection, RH lacks the means to efficiently deliver parasite profilin or other molecules to the host cell to trigger efficient MyD88-dependent signaling or that bone marrow-derived macrophages themselves fail to express TLR11. The relative resistance of TLR11–/– mice compared with MyD88–/– animals provides evidence for additional parasite ligands that act through MyD88 pathways (30).

TLR2 has also been reported to be involved during in vitro and in vivo responses to T. gondii (31, 32). This receptor is required for recognition of glycosylphosphatidylinositol-mucin molecules expressed by Trypanosoma cruzi (55, 56, 57). Purified Toxoplasma glycosylphosphatidylinositol molecules have also been reported to stimulate macrophage TNF-{alpha} production (58), and it is possible that TLR2 could be involved in recognition of these molecules. Nevertheless, TLR2–/– animals display increased susceptibility to T. gondii only under high dose conditions, arguing that this may not be a major pathway of immune activation (32).

The finding that ME49 parasites are more effective at inducing proinflammatory signaling in vitro compared with RH tachyzoites is paradoxical. This is because in vivo infections in mice suggest that the high lethality of RH tachyzoites is due at least in part to parasite-induced overproduction of proinflammatory cytokines including IL-12, TNF-{alpha}, and IFN-{gamma} (13, 14). It is possible that lack of early control of RH through suboptimal IL-12 induction allows greater replication of the parasite relative to ME49 (36). At later stages of acute infection, increased Ag load resulting from lack of control may induce high level production of proinflammatory cytokines. Alternatively, increased numbers of RH tachyzoites would be expected to cause greater tissue damage relative to parasite strains with replication that was better controlled. In turn, through expression of endogenous tissue TLR ligands (51, 52, 53), this might activate MyD88-dependent inflammatory signaling leading to immunopathology.

Although we examined only single representative strains from type I and II lineages, the close genetic identity of isolates within strain types suggests that differences in how they trigger immunity will prove generally applicable. Strain types I–III are now believed to have arisen from a single genetic cross that occurred ~10,000 years ago (59). It is striking that from this cross appears to have emerged parasite lineages that differ profoundly in their immune activation properties and that might in turn control the virulence properties of the parasite.


   Acknowledgments
 
We gratefully acknowledge Dr. C. Leifer for enlightening discussion and critical reading of the manuscript. We thank E. Pamer and E. Pearlman, for supplying us with MyD88–/– animals, and R. Flavell, for supplying MKK3–/– 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 Department of Health and Human Services Grant AI50617. Back

2 Address correspondence and reprint requests to Dr. Eric Y. Denkers, Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853. E-mail address: eyd1{at}cornell.edu Back

3 Abbreviations used in this paper: MKK, MAPK kinase; PKB, protein kinase B, WT, wild type; MOI, multiplicity of infection; KO, knockout. Back

Received for publication April 11, 2006. Accepted for publication June 5, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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