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The Journal of Immunology, 2006, 176: 1840-1847.
Copyright © 2006 by The American Association of Immunologists

Induction of Suppressor of Cytokine Signaling-1 by Toxoplasma gondii Contributes to Immune Evasion in Macrophages by Blocking IFN-{gamma} Signaling1

Stefan Zimmermann*, Peter J. Murray{dagger}, Klaus Heeg{ddagger} and Alexander H. Dalpke2,{ddagger}

* Institute of Medical Microbiology and Hygiene, Philipps University, Marburg, Germany; {dagger} Department of Infectious Diseases, St. Jude Children’s Research Hospital, Memphis, TN 38105; and {ddagger} Department of Hygiene and Medical Microbiology, University of Heidelberg, Heidelberg, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Toxoplasma gondii is an intracellular parasite that survives and multiplies in professional phagocytes such as macrophages. Therefore, T. gondii has to cope with the panel of antimicrobial host immune mechanisms, among which IFN-{gamma} plays a crucial role. We report in this study that in vitro infection of murine macrophages with viable, but not with inactivated, parasites results in inhibition of IFN-{gamma} signaling within the infected cells. Thus, infection of RAW264.7 macrophages with tachyzoites inhibited IFN-{gamma}-induced STAT-1 tyrosine phosphorylation, mRNA expression of target genes, and secretion of NO. These effects were dependent on direct contact of the host cells with living parasites and were not due to secreted intermediates. In parallel, we report the induction of suppressor of cytokine signaling-1 (SOCS-1), which is a known feedback inhibitor of IFN-{gamma} receptor signaling. SOCS-1 was induced directly by viable parasites. SOCS overexpression in macrophages did not affect tachyzoite proliferation per se, yet abolished the inhibitory effects of IFN-{gamma} on parasite replication. The inhibitory effects of T. gondii on IFN-{gamma} were diminished in macrophages from SOCS-1–/– mice. The results suggest that induction of SOCS proteins within phagocytes due to infection with T. gondii contributes to the parasite’s immune evasion strategies.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The protozoan Toxoplasma gondii replicates within any nucleated cell investigated to date. Among the infected cells are macrophages, which, in turn, make use of a wide panel of antiparasitic weapons, including the induction of NO. To establish a productive infection, T. gondii thus has to manipulate macrophage functions (1). The production of immune regulatory cytokines, especially IL-12, Ag presentation, and immediate antiparasitic effector mechanisms are hallmarks of macrophage activation. In the case of T. gondii infection, the former reactions contribute to the induction of strong cellular immunity (2). IFN-{gamma} is of crucial importance for limiting parasitic growth (3), and one of its actions is to fully activate macrophages.

T. gondii has developed the means to inhibit both primary activation of macrophages as well as the antiparasitic actions of IFN-{gamma}. Thus, IL-12 and TNF-{alpha} production are diminished in infected cells (4, 5, 6) concurrent with down-regulation of NF-{kappa}B activation (6, 7, 8). Recently, it has been proposed that T. gondii uses STAT3 activation to mediate this inhibition, thus exploiting a cellular negative regulatory pathway commonly triggered by IL-10 (9). In addition, infection of macrophages leads to an inhibition of IFN-{gamma} signal transduction, as determined by a reduction of IFN-{gamma}-mediated MHC class II up-regulation and inducible NO synthase (iNOS)3 induction (10, 11, 12). However, the precise molecular mechanism involved remained unknown. We, therefore, tested the hypothesis that parasites could use natural host-derived regulatory processes that inhibit IFN-{gamma} signal transduction.

Suppressor of cytokine signaling (SOCS) proteins constitute a family of eight members, including SOCS-1 to -7 and cytokine-inducible Src homology 2 domain-containing protein (CIS). These proteins function as intracellular, inducible feedback inhibitors of cytokine receptor signaling (13, 14, 15, 16). After binding of their respective ligands, type I and II cytokine receptors become phosphorylated by receptor-bound JAKs, generating docking sites for STAT factors, which become tyrosine phosphorylated, dimerize, and translocate to the nucleus. Among the activated genes are SOCS proteins that act as classic feedback inhibitors to limit further signaling (17). In vivo gene deletion studies have established that SOCS-1 is an essential inhibitor of IFN-{gamma} (18, 19) signaling.

In this study we show that infection of macrophages with T. gondii results in an inhibition of IFN-{gamma}-mediated cellular activation measured by NO generation and induction of target genes. IFN-{gamma} signaling was inhibited proximally at the level of STAT1 tyrosine phosphorylation. We also demonstrate that T. gondii induces endogenous SOCS-1 and CIS, and that this contributes to the parasite’s inhibition of IFN-{gamma}. The results describe a molecular mechanism by which T. gondii is able to sabotage macrophage activation by IFN-{gamma}.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cells and culture conditions

RAW 264.7 cells, a murine macrophage cell line, were a gift from R. Schumann (Institute for Microbiology and Hygiene, Charité, Berlin, Germany). Cells were cultured in Clicks/RPMI 1640 supplemented with 5% FCS, 50 µM 2-ME, and antibiotics (penicillin G and streptomycin). SOCS-1 knockout mice were generated from heterozygous intercrosses (mixed 129 x BL/6 background) and have been described in detail previously (20). Bone marrow-derived macrophages were prepared from newborn wild-type littermates or SOCS-1 knockout mice as previously described (21). Briefly, bone marrow cells were seeded overnight in RPMI 1640 supplemented with 10% FCS, antibiotics, and 20% L929-conditioned medium as a source of M-CSF. Nonadherent cells were further propagated for 7–9 days, with a boost of fresh medium on day 4. Finally, adherent cells were scraped and used for the experiments. In the same way, macrophages were obtained from IL-10-deficient mice (22). RAW macrophages stably overexpressing SOCS proteins were established by cotransfection of SOCS expression plasmids and a neomycin resistance cassette, as described by us previously (23, 24). BV-2 cells and bone marrow-derived dendritic cells were used as previously described (25).

Infection with T. gondii

Tachyzoites from the T. gondii strain BK were prepared by coculture with RAW264.7 macrophages. Freshly hatched tachyzoites were used for infection experiments at different parasite to host ratios (multiplicity of infection (MOI)). Where indicated, tachyzoites were killed by repeated freeze/thaw cycles or were labeled with CSFE. To this, 1 x 108 tachyzoites were incubated in 5 ml of 2 µM CSFE at 37°C for 15 min, followed by PBS for 30 min.

Determination of NO secretion

Cells (1.5 x 105) were infected with tachyzoites in 96-well plates as stated in the respective experiment and were treated with recombinant murine IFN-{gamma} (Tebu). Supernatants were harvested after 26 h of stimulation and analyzed. NO accumulation was measured photometrically (550 nm) by mixing equal parts of supernatant and Griess reagent (1/1 mixture of 1 g% sulfanilamide/5% H3PO4 and 0.1% naphthyl-ethylenediamine dihydrochloride).

Cell viability

Cell viability was assessed by determination of MTT turnover.

Quantitative RT-PCR

Cells (1 x 106) were stimulated either in 24-well plates or in Transwells (0.4 µm). Total RNA was isolated using a HighPure RNA kit (Roche), which included DNase I digestion. Total RNA (1 µg) was reverse transcribed with a cDNA synthesis kit (MBI Fermentas). Then, cDNA was diluted 1/4 and used as a template in the quantitative PCR-mix according to the manufacturer’s standard protocol (Eurogentec; ABI PRISM 7700; Applied Biosystems). The primer sequences have been previously described (26) and are available on request. Quantifications were made using either fluorogenic probes (FAM/TAMRA) or by means of SYBR Green. The specificity of RT-PCR was controlled by no template and no reverse transcriptase controls. PCR efficiencies for all reactions were determined and were similar (0.96–1.0). Threshold values were normalized to the expression of beta-actin. Quantitative PCR results are expressed either as n-fold induction to nonstimulated cells or as relative expression (1/2(Ct target gene – Ct actin)).

Western blot

Cells (2 x 106) were infected with tachyzoites in medium containing 0.5% FCS before IFN-{gamma} stimulation. Cells were lysed for 30 min on ice in 250 µl of lysis buffer (50 mM Tris-HCl (pH 7.4); 1% Igepal; 0.25% sodium deoxycholate; 150 mM NaCl; 1 mM EDTA; 1 mM PMSF; 1 µg/ml each of aprotinin, leupeptin, and pepstatin 1; 1 mM Na3VO4; and 1 mM sodium fluoride). Lysates were cleared by centrifugation at 4°C for 10 min at 11,000 x g. Equal amounts of lysates were fractionated by 10% SDS-PAGE and electrotransferred to polyvinylidene difluoride membranes. Membranes were stained as indicated, and proteins were detected using an ECL system (Amersham Biosciences). Phosphotyrosine-specific STAT1 Ab was purchased from Cell Signaling Technology; STAT1, STAT3, and actin Abs were obtained from Santa Cruz Biotechnology.

Confocal microscopy

RAW264.7 macrophages (3 x 104) were grown on chamber slides in medium containing 0.5% FCS overnight. Then cells were infected with CFSE-labeled tachyzoites and subsequently stimulated with IFN-{gamma}. Cells were fixed in 4% paraformaldehyde/PBS for 20 min and permeabilized in –20°C methanol for 1 h. Cells were incubated with pY-STAT1 (1/50) at 4°C overnight, stained with tetramethylrhodamine isothiocyanate-secondary Ab (1/60; DakoCytomation) for 1 h, and analyzed on a Zeiss LSM 510 meta confocal microscope.

STAT1 activation

STAT1 activity in nuclear extracts was determined using a commercially available kit (STAT1p91 DuoSet; R&D Systems).

Proliferation

Proliferation of T. gondii was determined using 3H-labeled uracil, which is specifically incorporated into the DNA of parasites (27). Cells were infected in 96-well plates and pulsed with 0.03 MBq [3H]uracil/well. The assay was performed in triplicate.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
To proliferate within macrophages, T. gondii has to evade activated immune cells and especially the macrophage-activating effects of IFN-{gamma}. Among the stimulating effects of IFN-{gamma}, generation of nitric radicals is an important antimicrobial mechanism in mice. We observed that infection of murine RAW264.7 macrophages with freshly prepared tachyzoites of T. gondii led to an inhibition of IFN-{gamma}-mediated NO production (Fig. 1a). These effects were dependent on the ratio of parasites to host cells. Moreover, the inhibitory effects could only be observed with tachyzoites displaying full infectiveness; thus, parasites had to be used within a short period after release from the propagating cells. Therefore, the MOIs for effective inhibition differed slightly between experiments. To control for this fact, we determined inhibition of NO production in each of the following experiments. In contrast, parasites killed by freeze/thaw cycles, and thus unable to infect cells, failed to inhibit and even dose-dependently increased IFN-{gamma}-mediated NO generation (Fig. 1b).


Figure 1
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  		FIGURE 1. T. gondii infection inhibits IFN-{gamma} actions. RAW264.7 macrophages were treated with either viable (a and c) or killed (repeated freeze/thaw cycles; b) T. gondii tachyzoites at the indicated MOI. Cells were activated by parallel addition of rIFN-{gamma}. After 26 h of incubation, cell-free supernatants were analyzed for nitrite production (a and b), and cells were examined for viability by MTT assay (c). Shown is one of at least four independent experiments (mean of triplicate determinations ± SD). *, p < 0.05. d, BV-2 cells and bone marrow-derived dendritic cells from BALB/c mice were left uninfected or were infected overnight with 10:1 parasites. Cells were stimulated with 100 U/ml IFN-{gamma} for 2 h and analyzed by RT-PCR for IIGP1 expression (mean ± SD). e, RAW macrophages were left uninfected (control) or were pretreated for the indicated time period with parasites (MOI, 5:1). Cells were stimulated with 50 U/ml IFN-{gamma} for 2 h and analyzed for the expression of iNOS, MIG, and IIGP1 by quantitative RT-PCR (relative expression; mean ± SD; one of two experiments).

 
T. gondii tachyzoites proliferate within macrophages and will destroy the cells ~72 h after infection. The observed inhibitory effects of T. gondii infection on IFN-{gamma} were analyzed after 1 day of infection. Cell destruction was not responsible for diminished NO production, because cell viability, as analyzed by MTT assay, was not affected within the period of observation (Fig. 1c). Of note, IFN-{gamma} by itself decreased mitochondrial activity to some extent.

Inhibition of IFN-{gamma} by T. gondii infection was also observed when the expression of IFN-{gamma}-inducible genes was analyzed at the mRNA level. To this, iNOS, the chemokine monokine induced by IFN-{gamma} (MIG), and interferon-inducible GTPase 1 (IIGP1), a member of the p47 GTPase family recently shown to play an important role in defense against T. gondii (28), were examined. Induction of mRNA expression of all three genes by IFN-{gamma} was severely inhibited when cells were infected with the parasite (Fig. 1e). Furthermore, these down-regulatory effects on IFN-{gamma} signaling showed a clear dependence on the duration of infection. At least a 4- to 8-h period of infection was needed to effectively inhibit IFN-{gamma} signals (Fig. 1e). RT-PCR results were confirmed by ELISA for secretion of MIG (data not shown).

Similar results were observed with primary bone marrow-derived macrophages and peritoneal macrophages (data not shown). Moreover, we analyzed the microglia cell line BV-2 and bone marrow-derived dendritic cells for inhibition of IFN-{gamma} signaling by T. gondii (Fig. 1d). In both cell types, induction of IIGP-1 (as well as that of iNOS and MIG; data not shown) was impaired when cells were infected. Also, in the human colon epithelial cell HT29, induction of CXCL10 by IFN-{gamma} was inhibited after T. gondii infection (data not shown). The data indicate that infection with T. gondii results in a broad inhibition of IFN-{gamma} signaling in multiple cell types.

To further analyze the mode of parasite inhibition, we examined the signaling cascade of IFN-{gamma}. Infection with viable T. gondii tachyzoites resulted in an inhibition of early signal transduction of IFN-{gamma}, as measured by loss of tyrosine phosphorylation of STAT1 (Fig. 2a). Moreover, confocal microscopy with labeled parasites indicated that the inhibitory effects were only observed in infected cells, not in bystander cells, excluding a possible role of paracrine factors (data not shown). This was verified in Transwell experiments (Fig. 2c). Only cells that were in direct contact with the parasites and thus could be infected became refractory to IFN-{gamma}, as measured by inhibition of MIG. In contrast, cells on the opposite chamber of the Transwell were not affected in IFN-{gamma} responsiveness by parasite infection.


Figure 2
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  		FIGURE 2. T. gondii infection inhibits IFN-{gamma}-mediated STAT1 signaling. a, RAW 264.7 macrophages were infected with parasites at the indicated MOI for 24 h and subsequently stimulated with 10 U/ml IFN-{gamma} for 30 min. Equal amounts of cellular lysates were blotted and probed with the indicated Abs. b, Macrophages were infected with T. gondii tachyzoites overnight and subsequently treated with IFN-{gamma} as indicated. Nuclear extracts were prepared and analyzed for STAT1 activity. In parallel, cells were coincubated with parasites and IFN-{gamma} and analyzed for NO production after 26 h. One of two experiments is shown; values are the mean of triplicate determinations ± SD. c, Macrophages were seeded in Transwells (0.4 µm), and the lower compartment was infected with 20:1 parasites overnight. Subsequently, cells were stimulated with 30 U/ml IFN-{gamma} for 2 h, and the expression of the IFN-{gamma}-inducible gene MIG was measured by quantitative RT-PCR in both compartments. One of three experiments is displayed; values are the mean of triplicate determinations ± SD. nd, not detected. d, Bone marrow-derived macrophages from either IL-10–/– mice or wild-type littermates were infected with T. gondii as indicated and treated with IFN-{gamma}. NO was analyzed in the supernatant after 26 h (mean of duplicate determinations ± SD; one of two experiments).

 
Beside the inhibition of STAT1 phosphorylation, levels of STAT1 were decreased especially at higher parasite to host cell ratios (Fig. 2a). This, again, could not be accounted for by cell loss, because the loading control actin as well as STAT3 were unaffected. STAT1 activation was also examined using an ELISA-based assay that quantitatively measured nuclear STAT1 accumulation (Fig. 2b). It was observed that T. gondii infection inhibited activation and subsequent nuclear translocation of STAT1, confirming the above findings. However, we also noted that inhibition of IFN-{gamma}-mediated NO production over time was more effectively inhibited than nuclear translocation of STAT1 when comparing two different MOIs (Fig. 2b).

The Transwell experiment (Fig. 2c) already excluded a role of secreted factors for the inhibitory effects of T. gondii infection; however, we furthermore analyzed whether the inhibitory cytokine IL-10 might play a role. We observed that the inhibition of IFN-{gamma} induced NO by T. gondii was equal in IL-10–/– and wild-type macrophages (Fig. 2d).

SOCS proteins have been reported to be natural inhibitors of cytokine receptor signaling acting proximal in the signaling cascade. Therefore, we analyzed the expression of various SOCS family members upon T. gondii infection. We observed that viable, but not killed, parasites were able to increase mRNA expression of SOCS-1 and CIS. Only weak induction of SOCS-3 was observed (Fig. 3, a–c). Expression of SOCS-1 and CIS increased with higher parasite to host ratios. The characteristics were the same as those observed for the IFN-{gamma} inhibitory effects. Thus, only freshly prepared and highly infectious parasites were able to induce SOCS, and these effects were dependent on the applied MOI. Furthermore, the expression of SOCS-1, which plays a crucial role for inhibition of IFN-{gamma}, started to increase 4 h after infection (Fig. 3d), thus paralleling the kinetics of IFN-{gamma} inhibition (Fig. 1e). Transwell experiments demonstrated that SOCS-1 expression was a direct effect of T. gondii infection and was not mediated via paracrine factors (Fig. 3e) as was the infection-associated inhibition of IFN-{gamma}-mediated effects (Fig. 2c).


Figure 3
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  		FIGURE 3. T. gondii tachyzoites directly induce SOCS expression. RAW264.7 macrophages were infected with either viable or freeze/thaw-inactivated parasites at the indicated MOI for 4 h. Subsequently, cells were analyzed for the expression of SOCS-1 (a), SOCS-3 (b), and CIS (c) by quantitative RT-PCR. IFN-{gamma} (10 U/ml) served as a positive control. Shown is the n-fold induction of mRNA compared with untreated cells. One typical experiment of five is shown. d, SOCS-1 expression was analyzed by RT-PCR after infection with 10:1 tachyzoites for different time periods. Displayed is the relative expression of SOCS-1 normalized to beta-actin (mean of duplicate determinations ± SD). e, RAW264.7 macrophages were incubated in 0.4-µm Transwells. Cells were infected 10:1 with T. gondii in the lower compartment and subsequently analyzed for SOCS-1 expression after 5 and 20 h (one of two experiments; mean of triplicate determinations ± SD).

 
Next, we analyzed RAW264.7 macrophages stably overexpressing various SOCS family members. Cells were infected at a low MOI, and under these conditions, IFN-{gamma} was able to increase NO in infected cells (Fig. 4a; control and mock). This went along with a complete inhibition of T. gondii growth (measured specifically by [3H]uracil incorporation), establishing that the antiparasitic effects of IFN-{gamma} were functional in our assay (Fig. 4b; control and mock). Overexpression of SOCS-1, however, ablated IFN-{gamma}-mediated NO secretion and led to an undisturbed proliferation of parasites in infected cells (Fig. 4). Intermediate effects were observed for CIS overexpression, whereas SOCS-2 overexpression, which has no reported role in IFN-{gamma} signaling, did not show any alterations.


Figure 4
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  		FIGURE 4. SOCS-1 inhibits the antiparasitic effects of IFN-{gamma}. RAW264.7 macrophages, which either were not transfected (control) or stably overexpressed SOCS proteins, were infected with a low dose of T. gondii (MOI, 1:1) and treated with 100 U/ml IFN-{gamma} for 20 h. Then nitrite production was determined (a), and proliferation of parasites was measured by pulsing with [3H]uracil for 6 h (b; mean ± SD; one of four independent experiments). *, p < 0.05.

 
Using macrophages generated from bone-marrow of newborn SOCS-1–/– mice, we could also confirm a role of SOCS-1 in T. gondii-mediated IFN-{gamma} inhibition (Fig. 5). As a readout, we used the IFN-{gamma}-induced expression of MIG. In SOCS-1–/– cells, we noted that IFN-{gamma} increased the expression of MIG to higher levels compared with wild-type cells, consistent with a role of SOCS-1 as an intrinsic negative feedback inhibitor. However, although infection of wild-type macrophages with T. gondii decreased IFN-{gamma}-induced expression of MIG to 17% of uninfected control values, infected macrophages from SOCS-1–/– mice showed only an inhibition to 44% (Fig. 5). Similar results were obtained for iNOS induction. In wild-type macrophages, an inhibition to 76% was observed upon infection. Under the chosen conditions, inhibition was weaker than that in RAW264.7 macrophages. However, macrophages from SOCS-1–/– mice did not show any inhibition upon infection (data not shown), confirming the results with MIG.


Figure 5
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  		FIGURE 5. IFN-{gamma} inhibition by Toxoplasma parasites requires SOCS-1. Bone marrow-derived macrophages from either wild-type or newborn SOCS-1–/– mice were infected 3:1 with tachyzoites overnight. Subsequently, cells were challenged with 50 U/ml IFN-{gamma} for 4 h. Expression of the IFN-{gamma}-inducible gene MIG was analyzed by quantitative RT-PCR. Shown are the results from one of two experiments (expression of MIG normalized to beta-actin; mean of duplicate determinations ± SD).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
We report in this study that T. gondii is able to use the host SOCS system for its own purposes, namely, to inhibit IFN-{gamma} signaling. Thereby parasites can evade the antiparasitic effects of IFN-{gamma} in macrophages. We found that viable and infectious parasites from the BK strain, but not killed parasites, are able to inhibit IFN-{gamma} signaling and IFN-{gamma}-induced NO, which are critical mediators of host defense in murine macrophages (2, 3, 29). Although the importance of NO during in vivo infection seems to be dependent on the stage of infection, the mouse strain, and the route of infection (30, 31), it is evident that NO is essential for restricting the growth of parasites. Thus, interfering with the NO-inducing IFN-{gamma} system is a promising point for parasites to evade or retard immune defenses. We found that IFN-{gamma}-inducible genes iNOS, MIG, and IIGP1 were inhibited, confirming and extending earlier findings of different groups (8, 11, 12, 32), which showed modulation of iNOS activity by various T. gondii strains. We consistently observed that the inhibitory potential varied between different experiments and was dependent on the capacity to infect host cells. Thus, the parasite to host ratio to obtain similar inhibition might be different for various strains, as also proposed by others (11).

Extending the findings on macrophages, we observed that infection with viable T. gondii also led to an impairment of IFN-{gamma} signaling in dendritic cells. Because dendritic cells are a major source for IL-12, which has been reported to be crucial for IFN-{gamma} synthesis and resistance to acute toxoplasmosis (33), it will be interesting to determine whether dendritic cells either activated by toxoplasma lysate or infected by viable parasites behave differentially in terms of induction of primary T cell responses.

Concerning the mode of IFN-{gamma} inhibition, we observed that the signaling pathway was disrupted far proximally at the stage of tyrosine phosphorylation of STAT1. This contrasts with the findings of Luder et al. (10), who did not observe inhibition of phosphorylation. However, they used much higher doses of IFN-{gamma} and a lower parasite to host ratio. Indeed, we also observed that the inhibition can be overcome partially by increasing the IFN-{gamma} dose (data not shown). Our results parallel data obtained in a model of African trypanosomiasis in which trypanosome infection led to an inhibition of IFN-{gamma} signaling via a decrease in STAT1 phosphorylation (34). Moreover, our data indicate that IFN-{gamma} inhibition is not merely due to a decrease in phosphorylated STAT1, but that STAT1 itself becomes degraded (Fig. 2). Effects of T. gondii infection on overall cell viability within the observation period were excluded carefully. Both findings are nevertheless consistent with the proposed mode of action of SOCS proteins. Indeed, SOCS-1 has been reported to inhibit JAKs, and this is achieved via the Src homology 2 and KIR domains (17). Yet, SOCS proteins have another functional domain, the C-terminal SOCS box (35). This domain interacts with elongin B/C, which is part of an E3 ligase, thus mediating ubiquitination and degradation of bound target molecules (36). Accordingly, SOCS-1 would prevent phosphorylation of STAT1 and also initiate degradation. In vivo deletion of the SOCS box alone resulted in a phenotype resembling that of the complete SOCS-1 knockout with milder characteristics, substantiating a role of the SOCS box in gene function (37).

To establish a productive infection in macrophages, T. gondii has to manipulate two branches of macrophage activation. First, the direct recognition of parasites that results in macrophage activation has to be avoided. TLRs are crucial for the initiation of this task. Indeed, molecules prepared from T. gondii can be recognized in an MyD88-dependent manner (38, 39), which is important for downstream TLR signaling. Recently, a T. gondii profilin-like protein has been defined as a new ligand for TLR-11 (40). Thus, it is evident that macrophages are able to sense infectious danger by parasites, and this, in general, results in activation of the NF-{kappa}B pathway and subsequent IL-12 and TNF-{alpha} secretion. However, it has been observed that this pathway is inhibited by infection with viable T. gondii (7, 8). MAPKs, which are another signaling system of TLRs, were also inhibited by T. gondii infection (5). As was the case for our observations, these effects were dependent on a direct contact of parasite with host cells, thus excluding paracrine factors. We also show that the inhibitory potential is directly related to the process of infection (Transwell experiments, live vs killed parasites) and is not mediated by secreted factors, including IL-10. Recently, it has been shown that T. gondii inhibits the NF-{kappa}B pathway in a STAT3-dependent manner (9). STAT3 is the crucial transmitter of the inhibitory cytokine IL-10, yet the inhibitory effects were IL-10 independent. Thus, T. gondii seems to highjack the STAT3-dependent endogenous anti-inflammatory pathway to inhibit an initial branch of macrophage activation. It is unlikely that the SOCS proteins play a role in the inhibition of NF-{kappa}B. Although initial reports argued for an inhibitory role of SOCS in TLR signaling (41, 42), our own work strongly argues against this interpretation (21, 23). Thus, the regulatory actions of SOCS proteins remain restricted to cytokine receptor signaling.

Beside the direct activation by contact with parasites, macrophages are efficiently boosted by the effects of IFN-{gamma}. Moreover, IFN-{gamma} and STAT1 are indispensable for defense of T. gondii infections (3, 43). Targeting IFN-{gamma} is thus a promising strategy for parasites. A similar mechanism has been proposed for murine leishmaniasis. In this study it was found that L. donovani induces SOCS-3, thereby suppressing activation of human macrophages (44). Similar to our results, the induction of SOCS was a result of direct host-pathogen contact. Moreover, it was reported that SOCS-1 deficiency results in decreased numbers of infected macrophages in an in vitro infection with Leishmania major and LPS/IFN-{gamma} stimulation (18). Also, infection with Listeria monocytogenes modulated IFN-{gamma} signaling via induction of SOCS-3 (45).

At present, it is only possible to speculate by which means T. gondii and apparently other parasites induce SOCS expression. We and others (24, 26, 46) have shown that TLR stimulation results in the induction of SOCS proteins. However, we could not find SOCS mRNA upon addition of killed T. gondii parasites, although T. gondii proteins can be recognized via TLRs (40). Furthermore, it would be difficult to induce SOCS in a TLR-dependent manner while simultaneously inhibiting the main NF-{kappa}B pathway.

Regarding the induction of SOCS, an interesting link might develop that involves lipoxin (LXA4), an anti-inflammatory eicosanoid mediator. Lack of LXA4 in 5-lipoxygenase-deficient mice resulted in enhanced mortality with increased IL-12 levels during T. gondii infection (47). Moreover, evidence was given that T. gondii might hijack the endogenous LXA4 system, because parasitic extracts showed 15-lipoxygenase activity (48). Moreover, it has been found that LXs induce SOCS proteins, which contribute to their anti-inflammatory actions (49). Although LXs are soluble mediators, and we excluded paracrine factors being responsible for the observed effects, it might be speculated that sufficient concentrations are only achieved in infected cells, or the mediators might act directly within infected cells.

Lack of immune activation will result in detrimental effects due to unlimited parasite replication, although, in contrast, overshooting immune responses will result in immunopathology. Indeed, it has been shown that T. gondii infection in susceptible mice can lead to tissue destruction in small intestine and liver (50, 51), and this was mediated by IFN-{gamma} and NO. To establish a chronic infection, which is a hallmark of toxoplasmosis, dampening acute inflammation to avoid excessive host damage might be a more promising strategy. In this respect, SOCS could also function in avoiding acute toxicity. Indeed, it was found that when SOCS-1+/– mice were challenged with L. major, they developed larger lesions despite having a similar parasitic load as wild-type mice (52). It will be interesting to analyze whether the different susceptibilities of mouse strains toward model parasites might also have a correlate in the ability to induce SOCS proteins.

In summary, T. gondii has developed means to inhibit both direct activation of macrophages by microbial recognition and indirect activation via immune-active cytokines, especially IFN-{gamma}. For the latter, in this study we have identified a possible mechanism that involves the manipulation of the host SOCS system. Inducing endogenous SOCS by the parasite results in inhibition of IFN-{gamma} signaling at the level of signal transduction and rescues T. gondii from the otherwise detrimental antiparasitic effects of this cytokine.


   Acknowledgments
 
We thank Helene Bykow, Christine Barett, and Claudia Trier for excellent technical support.


   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 (Da 592/1 and He 1452/4), the National Institutes of Health (Grant AI062921), the Sandler Program for Asthma Research, Cancer Center CORE (P30 CA 21765), and American Lebanese Syrian Associated Charities. Back

2 Address correspondence and reprint requests to Dr. Alexander Dalpke, Department of Hygiene and Medical Microbiology, University of Heidelberg, Im Neuenheimer Feld 324, D-69120 Heidelberg, Germany. E-mail address: alexander.dalpke{at}med.uni-heidelberg.de Back

3 Abbreviations used in this paper: iNOS, inducible NO synthase; CIS, cytokine-inducible Src homology 2 domain-containing protein; LXA4, lipoxin A4; MIG, monokine induced by IFN-{gamma}; IIGP1, interferon-inducible GTPase 1; MOI, multiplicity of infection; SOCS, suppressor of cytokine signaling. Back

Received for publication June 13, 2005. Accepted for publication November 15, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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