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Clin Exp Immunol. Sep 2005; 141(3): 475–484.
PMCID: PMC1809463

Toxoplasma gondii regulates recruitment and migration of human dendritic cells via different soluble secreted factors

Abstract

We investigated in vitro the properties of soluble factors produced by Toxoplasma gondii on the recruitment, maturation and migration of human dendritic cells (DC) derived from CD34+ progenitor cells. We used soluble factors including excreted secreted antigens (ESA) produced under various conditions by the virulent type I RH strain (ESA-RH) and the less virulent PRU type II strain (ESA-PRU). Soluble factors of both T. gondii strains appeared to possess a chemokine-like activity that attracted immature DC. This recruitment activity required the presence of functional CCR5 molecules on the cell membrane. Incubation of DC for 24 h with ESA triggered the migration of a large percentage of these cells towards the chemokine MIP-3β; ESA-PRU was more efficient than ESA-RH. ESA produced in absence of exogenous protein and crude extract did not induce DC migration but retained recruitment activity. These data indicate that recruitment activity and migration-inducing activity are not governed by the same factors. Moreover, incubation of DC for 48 h with ESA did not modify the expression of costimulation or maturation markers (CD83, CD40, CD80, CD86 or HLA-DR), but induced a decrease in CCR6 expression associated with an increased expression of CCR7. Taken together, these results suggest that T. gondii controls recruitment and migration of immature DC by different soluble factors and may induce a dysfunction in the host-specific immune response.

Keywords: human dendritic cells, Toxoplasma gondii, chemotaxis

Introduction

Toxoplasma gondii is known to occur abundantly in humans, in whom it can cause life-threatening disease in the fetus and in immunosuppressed individuals [1]. T. gondii infection normally elicits a type 1 cytokine response, in which CD4+ and CD8+ T lymphocytes play an essential role in resistance to infection [2,3]. However, in the absence of regulation, type 1 cytokine production induced by T. gondii can lead to host death [46]. Some recent studies support the fact that T. gondii is a master manipulator of host responses. The parasite simultaneously triggered protective cytokine responses and paradoxically suppressed the same types of immune function [7]. This dual potency of the parasite could allow the establishment of a stable host—parasite interaction.

In the organism, dendritic cells (DC) act as sentinels against pathogens. Their role in the initiation and regulation of the T-lymphocyte response towards T. gondii is fundamental during infection. Firstly, they are able to reach the site of infection (recruitment phase) and secondly, after recognizing the pathogen, they are activated and migrate towards lymph nodes to activate naive T lymphocytes (migration and activation phases) [8]. The movement of DC in the organism is therefore crucial to their immunological functions.

Recent evidence in mice indicates that a molecule released by T. gondii, the cyclophilin C-18, is very similar to MIP-1β, binds CCR5 and recruits immature mouse DC in vitro[9]. This molecule was also involved in the regulation of interleukin (IL)-12 production by DC.

Other studies in mice have demonstrated that T. gondii can activate DC and trigger their migration to the spleen in order to activate T cell proliferation [10]. More generally, after activation with antigen or contact with lipopolysaccharides (LPS), the migration of activated DC to the spleen is controlled by the chemokine MIP-3β which binds on CCR7 [11,12].

We have demonstrated previously [13] that direct infection of human CD34+-derived DC by live T. gondii affected DC migration via control of CCR6/CCR7 expression. This control was strain-dependent and was also observed when DC and parasites were separated by a porous membrane, suggesting that soluble factors produced by the parasite could regulate DC functions.

Soluble proteins are known to be released at precise stages of host cell invasion. Some of these soluble factors, called excreted/secreted antigen (ESA), have been shown as important components in the process of invasion and replication of T. gondii within host cells [1417].

We were interested in studying both aspects of the mobility of human DC (recruitment and migration) which seem to be controlled by soluble factors present in the ESA of T. gondii. We used an in vitro model with human DC derived from CD34+ progenitors and soluble factors, including ESA, produced under various conditions by the highly virulent RH strain or by the less virulent PRU strain of T. gondii. We evaluated the effect of these factors from both strains on: (i) recruitment of DC, (ii) modifications of phenotype and (iii) migration towards MIP-3β.

Materials and methods

T. gondii strains

PRU [18] and RH [19] strains were routinely maintained in our laboratory by passage in OF1 mice [13].

For preparation of soluble factors, tachyzoites were obtained in vitro after coculture with the human monocyte cell line THP1 (TIB-202, ATCC) in RPMI medium supplemented with 5% heat-inactivated fetal calf serum (FCS, Gibco-BRL Life Technologies, Cergy Pontoise, France) and 1% penicillin-streptomycin. After complete THP1 cell lysis, tachyzoites were recovered and passed through 8-µM and 3-µM filters (Millipore, Saint-Quentin en Yvelines, France). Tachyzoite viability was checked by phase contrast microscopy and parasites that were black, empty or egg-like in shape were considered to be dead [20,21].

Preparation of soluble factors from T. gondii tachyzoites

Excreted secreted antigens (ESA) were prepared using the method of Darcy et al. [22]. Briefly, tachyzoites isolated from THP1 coculture were cultured at 1·5 × 108/ml in RPMI-1640 (Gibco-BRL Laboratories, Grand Island, NY, USA) supplemented with 10% FCS (RPMI-10% FCS) in six-well plates for 3 h at 37°C with periodical agitation. In some experiments, FCS was replaced by 2 g/l bovine serum albumin (BSA, Roche Diagnostics, Paris, France) or 2 g/l human serum albumin (HSA, purified by chromatography from fraction V, Sigma-Aldrich, l’Isle d’Abeau, France). The ProteoPrep™ Blue Albumin Depletion Kit (Sigma-Aldrich) was used according to the recommendations of the manufacturer in order to deplete ESA. The amount of albumin remaining was quantified by analysis of PAGE-SDS electrophoresis and gel integration using the 1D Image Analysis Software from Kodak Digital Science (Rochester, NY, USA).

Supernatant of tachyzoites (S) was prepared according to Aliberti et al. [9]. Briefly, tachyzoites were cultured at 1·5 × 108/ml in Phosphate buffer saline (PBS) in six-well plates for 1 h at 37°C with periodical agitation. For both preparations, a minimum tachyzoite viability of 90% was checked at the end of the incubation period by phase contrast microscopy. Soluble factors were recovered by centrifugation for 10 min at 1000 × g then filtered on a 0·20-µM pore size membrane (Millipore) and stored in liquid nitrogen.

Crude antigens were prepared according to Fatoohi et al. [23], Briefly, 1·5 × 108 tachyzoites were washed in PBS and disrupted in 1 ml PBS by five freeze-thaw cycles. The crude extract was clarified by centrifugation at 2500 × g for 15 min and then filtered and treated in the same manner as the supernatants. Extract of THP1 cells was produced by disruption of 1 × 107 cells in 1 ml by five freeze-thaw cycles. The extract was treated as crude antigens.

Purification and culture of cord blood CD34+ cells

Human cord blood was collected according to institutional guidelines during normal full-term deliveries. Mononuclear cells were isolated by flotation on Ficoll (Lymphoprep, Nycomed Parma AS, Oslo, Norway) and then depleted of adherent cells by overnight culture on a plastic surface. CD34+ cells were purified by immunomagnetic selection with mini-MACS (Miltenyi-Biotec GmbH, Bergisch Gladbach, Germany), which yield a suspension containing more than 95% of CD34+ cells. Isolated progenitors were cultured in RPMI-1640 supplemented with 5% FCS, 5 × 10−5 M 2-mercaptoethanol (Sigma), antibiotics (penicillin-streptomycin-amphotericin B, Sigma), 200 U/ml recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF; specific activity, 2 × 106 U/mg; kindly provided by Schering Plough Laboratories, Dardilly, France) and 50 U/ml recombinant human tumour necrosis factor α (TNF-α; specific activity, 2 × 107 U/mg; Genzyme Corp., Boston, MA, USA).

Phenotypic analysis

Cells were washed with isotonic NaCl/Pi buffer containing 1% BSA, 0·6% acid citrate-dextrose and 0·02% sodium azide (Sigma). Cells (1·5 × 105) were incubated with 10 µl labelled monoclonal antibodies (MoAb) for 30 min at 4°C: CD1a (IgG2a; Dako, Trappes, France), CD80-FITC (IgG2b k; Diaclone, Besancon, France), CD83-FITC (IgG2 k; Immunotech, Marseille, France), CD86-FITC (IgG1), CD40 (IgG1; Diaclone); CCR6 (IgM; BD Bioscience, Pont de Claix, France), CCR7 (IgG2B; R & D Systems, Minneapolis, MN, USA) and HLA-DR (IgG2a; Beckton Dickinson, Mountain View, CA, USA). For unlabelled primary antibodies, a further step with a secondary antibody, GAMIg-FITC (Zymed Laboratories, San Francisco, CA, USA), was performed. After being washed in NaCl/Pi buffer, cells were kept on ice and 40 µl propidium iodide were added (1·7 ng/ml; Sigma) for 10 min before FACS analysis. When FACS analysis had to be delayed, propidium iodide was omitted and cells were fixed with 1% formaldehyde in NaCl/Pi buffer. Analysis was performed on a FACScan Flow Cytometer (Beckton Dickinson) with a gate that excluded dead cells. DC-Lamp (IgG1 clone 104.G4; from Immunotech) expression was detected after intracellular staining of DC. The Dako IntraStain K2311 kit for cell permeabilization was used (DAKO A/S, Glostrup, Denmark).

Recruitment assays

For recruitment assays, DC were immature or activated by incubation for 24 h with detoxified lipopolysaccharides from Escherichia coli (LPS, 25 ng/ml 0127-B8, Sigma). Cell recruitment was assessed using 12-well chemotaxis chambers and inserts with a 8-µM pore polycarbonate filter (Falcon, Beckton Dickinson). Live cells (2 × 105), suspended in 0·4 ml of RPMI-10% FCS, were applied to the upper compartment of the chamber. Three hundred microlitres of MIP-3α at 0·25 µg/ml (R & D Systems), MIP-3β at 0·25 µg/ml (R & D Systems), ESA at a dilution of 1/20 or tachyzoite supernatant at a dilution of 1/20, all diluted in RPMI-10% FCS, were applied to the lower chamber as chemoattractants. For experiments with the competitor, a control with medium used for ESA preparation, MIP-3α, MIP-3β or ESA was added to the upper compartment at the same concentration as that in the lower compartment. Chambers were incubated at 37°C for 24 h. In some experiments, 2 × 105 DC were incubated for 30 min with 0·75 µg MoAb anti-CCR5 (R & D Systems) or anti-CCR6 (BD Bioscience) before the recruitment assay. All cells that had migrated into the lower compartment were recovered and counted by microscopy. Each treatment was performed in triplicate. Results were expressed as the percentage of DC in the lower compartment in relation to the number of DC deposited in the upper compartment.

Migration assays to MIP-3β

For migration assays, DC used were cultured for 24 h with LPS (25 ng/ml), or sequential dilutions of ESA (ESA-RH or ESA-PRU) or tachyzoite supernatant (S-RH or S-PRU) of each T. gondii strain. After 24 h, DC were deposited in the upper part of a migration chamber and MIP-3β (0·25 µg/ml) in the lower part. All reagents were diluted in RPMI-10% FCS, and results were expressed as described in the recruitment assay. In some experiments, DC were incubated with anti-CCR5 or anti-CCR6 before incubation with ESA and before the migration assays.

Statistical analysis

Data were analysed by the nonparametric test of Wilcoxon.

Results

Phenotype of DC used

Suspensions of CD34+ cells cultured in vitro with GM-CSF and TNF-α for 6–8 days regularly contained more than 90% of DC. All cells expressed the phenotype of immature DC with low expression of DC-Lamp, CCR7, CD83 and CD86 (Table 1).

Table 1
Phenotype of DC after 7 days of culture in vitro before contact with T. gondii

T. gondii ESA recruited immature DC

We evaluated in vitro the recruitment of immature DC or LPS-activated DC in vitro using different chemoattractants (MIP-3α, MIP-3β) in comparison with ESA of T. gondii produced by the highly virulent RH strain (ESA-RH) or the less virulent PRU strain (ESA-PRU). These molecules were deposited in the lower compartment of the migration chamber and competitors (RPMI, MIP-3α, MIP-3β, ESA-RH and ESA-PRU) in the upper compartment with the cells (Fig. 1). In the first instance (Fig. 1a), we found that MIP-3α attracted only immature DC, and that addition of MIP-3α as a competitor in the upper compartment blocked the migration, whereas MIP-3β had no significant effect on the migration of immature DC, as expected. Addition of ESA-RH and ESA-PRU to the upper compartment inhibited the migration of immature DC towards MIP-3α.

Fig. 1
ESA-RH and ESA-PRU attract immature DC. DC were incubated for 24 h in RPMI (□) or LPS ([filled square]). After washing, the same number of live DC was deposited in the upper compartment of the migration chamber with either RPMI-10% FCS (control), MIP-3α ...

In Fig. 1b, it is shown that MIP-3β attracted only mature DC (LPS-activated DC). Addition of MIP-3α, ESA-RH or ESA-PRU to the upper compartment did not block the migration. An assay of migration towards ESA-RH and ESA-PRU (Fig. 1c, ,d)d) confirmed that ESA of both strains attracted only immature DC. Moreover, this migration was inhibited when MIP-3α, ESA-RH or ESA-PRU were used as competitors.

As ESA fractions were produced under conditions different from those of the supernatant, we compared the recruitment potency of ESA with supernatants of both strains (S-RH or S-PRU) produced according to Aliberti et al. [9]. We observed that the dose—response were comparable between ESA and supernatants; moreover ESA and supernatant activities were identical in both strains (Table 2). In constrast, no dose-dependent effect was observed with mature DC, and all values were lower than the MIP-3β control. These data indicated clearly that ESA and supernatant of tachyzoites of RH or PRU strains possess chemotactic-like activity towards immature DC.

Table 2
Recruitment of DC towards different T. gondii soluble factors preparations

Recruitment of DC occurred through CCR5

ESA of T. gondii appeared to possess a chemokine-like activity towards immature DC. To determine which receptors were involved in the recruitment of immature DC by ESA, we evaluated the migration of immature DC towards MIP-3α, ESA-RH or ESA-PRU after treatment with CCR5 or CCR6 antibodies (Fig. 2). As expected, after treatment with anti-CCR6, only 18·5% of the DC population migrated towards MIP-3α instead of 47·2% without this treatment. The treatment with anti-CCR5 had no effect on DC migration towards MIP-3α; however, when ESA-RH was used as the chemoattractant, 8·5% of the DC population migrated after anti-CCR5 treatment compared with 41·3% without this treatment. This blockage of migration towards ESA-RH was not found with anti-CCR6 treatment (39·2%). The same results were obtained with ESA-PRU. Thus, the recruitment of immature DC by ESA of both strains involved CCR5 but not CCR6.

Fig. 2
Recruitment of immature DC by ESA of T. gondii occurred via CCR5. DC were incubated with RPMI-10% FCS alone or with CCR5 or CCR6 antibodies diluted in RPMI-10% FCS for 30 min. After washing, immature DC were layered in the upper part of a migration chamber ...

ESA did not trigger maturation of DC

We demonstrated that ESA of T. gondii attracted immature DC. To determine whether incubation of ESA with DC triggered their maturation, we evaluated their phenotype by following expression of maturation markers such as DC-Lamp and CD83, expression of costimulation markers such as CD40, CD80 and CD86 and translocation of HLA-DR molecules after 48 h of contact with ESA.

LPS was used in order to obtain mature DC and LPS-activated DC regularly expressed increased amounts of the different markers (Fig. 3). After 48 h of incubation with ESA-RH or ESA-PRU, expression of CD83, CD40, CD80, CD86 and HLA-DRhigh was not significantly enhanced in comparison with DC incubated in medium alone. Only DC-Lamp expression increased in a part of the DC population after incubation with ESA.

Fig. 3
Phenotype of DC 48 h after incubation with ESA of T. gondii. DC were cultured with RPMI-10% FCS (Control), or LPS (LPS), or ESA of RH (ESA-RH) or ESA of PRU (ESA-PRU) strains diluted in RPMI-10% FCS for 48 h. Open profiles correspond to cells labelled ...

Twenty-four hours of preincubation with ESA triggered migration of DC towards MIP-3β

ESA of T. gondii did not appear to trigger maturation of DC. However, similarly to LPS, ESA were able to down-regulate expression of CCR6 and, at the same time, increase CCR7 expression (Fig. 4a). Since CCR7 is involved in the migration of DC towards MIP-3β, we evaluated the migration of DC towards this chemokine after incubation with sequential dilutions of ESA for 24 h (Fig. 4b). Our results showed that ESA from both strains induced migration of DC (Fig. 4b), and that a greater effect was obtained with ESA-PRU than with ESA-RH (50·3% and 23·6% of migrating cells, respectively, at a 1/20 dilution).

Fig. 4
Treatment of DC with ESA of T. gondii triggers an increase in expression of CCR7 and migration of DC towards MIP-3β. (a) Expressions of MIP-3α receptor CCR6 and MIP-3β receptor CCR7 after 48 h of incubation with RPMI-10% FCS (Control) ...

In contrast with recruitment (Fig. 2), treatment of DC with anti-CCR5 or anti-CCR6 before contact with ESA had only a weak effect on migration towards MIP-3β (Fig. 5).

Fig. 5
Migration towards MIP-3β of DC treated by LPS or by ESA-PRU of T. gondii did not occurred via CCR5. DC were incubated with RPMI-10% FCS alone or with CCR5 or CCR6 antibodies diluted in RPMI-10% FCS for 30 min. After washing, DC were incubated ...

As for recruitment, we compared ESA with tachyzoite supernatants of both strains prepared according to Aliberti et al. [9]. In contrast with the recruitment assay, the migration inducing factor present in ESA was not recovered in the tachyzoite supernatants (Table 3). As supernatants were prepared without FCS and with a shorter incubation period than ESA, we prepared a batch of ESA in RPMI without FCS in order to evaluate the role of FCS. ESA in RPMI without FCS had no activity on human DC migration (Table 3). When ESA were prepared with RPMI supplemented with 2 g/l of BSA or HSA, the activity was present at similar levels, indicating that it was independent of the nature of the protein. FCS, BSA, HSA and THP1 cell lysate by themselves did not induce the activity (data not shown). After depletion of albumin from ESA, the activity was maintained despite the fact that the amount of albumin recovered was below 20% of the initial amount (Fig. 6). A final control consisted of crude antigens prepared by parasite lysis to confirm that the observed effect of ESA was not related to eventual parasite lysis during preparation of ESA. This fraction appeared to induce only a weak activity compared with ESA-FCS (Table 3).

Fig. 6
The presence of protein is required for the production of active ESA but not for the assays. ESA-PRU were produced in PBS or RPMI-10% FCS or in RPMI supplemented with 2 g/l of BSA or HSA. Part of the ESA was depleted of albumin. (a) assays of migration ...
Table 3
Migration towards MIP-3β of DC incubated with different preparations of T. gondii soluble factor

Discussion

Our observations in vitro indicate that T. gondii can control the motility of human DC through the secretion of soluble factors. T. gondii recruits immature DC via soluble proteins which bind on CCR5, and then induces migration of these cells towards MIP-3β via induction of CCR7 expression. Migration of DC was induced despite the fact that the cells remained immature as shown by labelling of maturation markers.

Our data demonstrate that both highly virulent RH and less virulent PRU strains of T. gondii secrete soluble molecules that attract immature human DC and that this recruitment is not strain-dependent. The production by tachyzoites of molecules with recruitment activity for DC has not been yet described in humans. However, such activity was reported in a mouse model with the RH strain [9]. Moreover, we show that comparable levels of activity are observed in ESA and in supernatants produced under the same conditions as those used by Aliberti et al. [9]. These authors showed that T. gondii produces cyclophilin C-18, a homologue of MIP-1β, which binds CCR5 with high affinity and can attract mouse splenic DC. This molecule was reported to be present in tachyzoite supernatants but also in supernatants derived from infected fibroblasts after parasite egress and in tachyzoite lysate, indicating that it was produced constitutively by the parasite. Our data indicated the suppression of recruitment activity by anti-CCR5 treatment which suggests that cyclophilin C-18 might be the active factor in our experiments. This direct mechanism of recruitment does not exclude the existence of another indirect mechanism of recruitment of immature DC by T. gondii, as described in vitro in a recent study [24].

Upon contact with ESA, immature DC modified their expression of CCR6 and CCR7 and became attracted by MIP-3β. This modification of phenotype is not associated with DC maturation as shown by measurements of CD40, CD80, CD83, CD86 and HLA-DR.

In mice, only a few studies have demonstrated in vivo that live T. gondii tachyzoites or a soluble cytoplasmic extract of the virulent strain can activate DC, triggering their migration to the spleen and the redistribution of interdigitating DC precursors into T-cell areas of the spleen [10,25]. In vitro, mouse macrophages were activated upon infection with live tachyzoïtes, but not after incubation with ESA [26]. T. gondii was recently reported to invade immature mouse DC preferentially but failed to activate them [27]. In a previous study [13], we described that direct infection with tachyzoïtes of the RH strain induced DC maturation whereas infection of the PRU strain did not. However, ESA from both strains contained a soluble factor that blocked the DC maturation induced by allergens. The ability of direct infection to activate cells has not been reported regularly and may reflect equilibrium between the virulence of the strain and the amount of soluble inhibitory molecules released in the extra-cellular environment.

The migration-inducing factor is present in the ESA of both strains; however, the ESA of the less virulent PRU strain induced the migration of a significantly larger number of DC than that of the RH strain or LPS. This result confirms our previous data on direct infection with tachyzoites [13].

In contrast to recruitment factors, parameters of ESA production appeared to be important in order to obtain active switching factors. Conditions for the preparation of ESA have been established previously by Darcy et al. [22]. The presence of proteins allows optimal survival of the tachyzoites and active excretion/secretion. Without serum, the reduction in the protein level in the incubation medium was estimated to be about 10-fold, which indicated that excretion was impaired. Under identical conditions, Assossou et al. [21] demonstrated that, after 3 h of incubation, SAG-1, a major membrane antigen of the tachyzoites, was not released whereas proteins such as GRA-1, contained in tachyzoite dense granules, were detected. Our results confirm that proteins need to be present during the incubation of tachyzoites for the preparation of ESA. The nature of the proteins does not appear to be crucial since FCS, BSA and HSA gave the same result; in addition the activity is not contained in the protein because it is not impaired significantly by depletion of albumin. Moreover, lysis of tachyzoites was not responsible for DC migration activity, by consequence, DC migration-inducing factor was not present as a pool of intracytoplasmic molecules in the tachyzoites but was actively secreted by the parasite after stimulation by foreign proteins.

Pretreatment of DC with anti-CCR5 impaired recruitment activity, whereas it allowed the CCR6/CCR7 switch and migration of DC towards MIP-3β. Together, these results suggest that recruitment activity and the migration-inducing factor are governed by different molecules.

In conclusion, our data suggest that, T. gondii may be able to recruit immature DC in vivo in order to infect preferentially this cell type. In a second step, the parasite could use the migratory properties of DC to reach deep tissues of the host such as the central nervous system. Moreover, the ability of T. gondii to evade recognition temporarily could promote dissemination and establishment of the parasite before initiation of the immune response. These mechanisms may encourage the long-term persistence of the parasite in the host during chronic toxoplasmosis [7]. T. gondii possesses sophisticated mechanisms to instruct and subvert host-cell responses by secreting ESA molecules. The identification of such molecules and determination of how they impact host-cell signalling cascades is a high priority research area.

Acknowledgments

We express our appreciation to Jane Mitchell for revision of the English text.

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