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Originally published online as doi:10.1189/jlb.0807565 on April 15, 2008

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(Journal of Leukocyte Biology. 2008;84:244-254.)
© 2008 by Society for Leukocyte Biology

Plasmodium falciparum-free merozoites and infected RBCs distinctly affect soluble CD40 ligand-mediated maturation of immature monocyte-derived dendritic cells

Paushali Mukherjee1 and Virander Singh Chauhan

Malaria Research Group, International Centre of Genetic Engineering and Biotechnology, New Delhi, India

1Correspondence: Malaria Group, International Centre for Genetic Engineering and Biotechnology (ICGEB), P.O. Box 10504, Aruna Asaf Ali Marg, New Delhi 110067, India. E-mail: paushali{at}icgeb.res.in


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ABSTRACT
 
Free plasmodium merozoites released from the parasitized hepatocytes and erythrocytes represent a transitory, extracellular stage in its mammalian host. In this study, we compared the effect of Plasmodium falciparum-free merozoites with infected RBCs (iRBCs) on the maturation of human monocyte-derived dendritic cells (DCs) in vitro. Phagocytosed-free merozoites prevented soluble CD40 ligand (sCD40L)-induced, phenotypic maturation of DCs and secretion of IL-12p70 but enhanced IL-10 production and primed, naive CD4+ cells to produce a high level of IL-10 compared with IFN-{gamma}. Free merozoites augmented sCD40L-induced ERK1/2 activation, and inhibition of ERK1/2 with its inhibitor PD98059 markedly abrogated IL-10 production and rescued IL-12 production. Therefore, the molecular mechanisms by which free merozoites antagonized sCD40L-induced DC maturation appeared to involve the activation of the ERK pathway. In contrast, phagocytosed iRBCs by itself induced DCs to semi-maturation, responded to CD40 signaling by maturing and secreting increased levels of TNF-{alpha}, IL-6, and also IL-12p70, and led to a pronounced, proinflammatory response by the allogenic CD4+ T cells. iRBCs regulate CD40-induced p38MAPK. Studies using inhibitors selective for p38MAPK (SB203580) showed that p38MAPK played an essential role in the maturation and function of DCs. Our results reveal the ability of free merozoites and iRBCs to distinctly alter the sCD40L-induced DC functioning by regulating the activation of the MAPK pathway that can inactivate or exacerbate immune responses to promote their survival and the development of parasite-specific pathologies.

Key Words: innate • immunology • human • MAPK signaling


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INTRODUCTION
 
The plasmodium parasite for most of its complex life cycle is located intracellularly. However, at periodic intervals, following pre-erythrocytic and erythrocytic schizogony, parasitized cells rupture to release merozoites that represent a brief extracellular stage in the life cycle of the malarial parasite. Merozoites are extracellular for only a short time; most of them invade fresh erythrocytes immediately, but some are phagocytosed by the fast-acting, innate immune system before they can invade erythrocytes [1 ]. Some studies indicate that merozoites rather than parasitized erythrocytes are the specific target for phagocytic cells in Plasmodium falciparum malaria. Monocytes in the peripheral blood and spleen have been shown to phagocytose P. falciparum merozoites but rarely parasitized or nonparasitized erythrocytes in the absence of immune serum [2 ].

Dendritic cells (DCs) play a central role in the generation of effective adaptive immune responses against most pathogens including plasmodium parasites [3 4 ]. DCs behave as first-line sentinels and respond to microbial signals and transport the antigens they capture from the peripheral tissues to the draining secondary lymphoid organs where they efficiently present antigen to naive T lymphocytes to mount an appropriate immune response [5 ]. Recent studies have looked at the interactions between parasite-infected RBCs (iRBCs) and DCs in the context of their ability to manipulate the host innate immune system. Modulation of DC functions by malaria parasites has been observed ex vivo in acutely infected individuals as well as in vitro using laboratory lines of infected erythrocytes [6 7 8 9 ]. After interaction with intact iRBCs, DC failed to mature and up-regulate the expression of MHC class II, costimulation molecules, and adhesion molecules in response to LPS, CD40 ligand (CD40L), TNF-{alpha}, or monocyte-conditioned medium [6 ]. Although these DCs produced TNF-{alpha}, they failed to activate T cells (with respect to proliferation and IL-2 production) and secreted the immunosuppressive IL-10 cytokine instead of IL-12 [6 ]. When parasitized RBCs rupture to release merozoites, parasite debris like hemozoins and free GPIs are also released into the circulation. Several studies have shown that both hemozoins and free GPIs affect the functioning of DCs and also macrophages (10 11 12 13 14 15) . However, little is known about whether free merozoites can induce, delay, or modify the DC maturation process like the parasite-infected RBCs.

The ligation of CD40, expressed primarily on APCs by CD40L (CD154), transiently expressed on activated T cells, is one of the most potent physiological stimuli that facilitates the maturation of immature DCs [16 ]. Ligation of CD40 molecules activates a cascade of phenotypic and functional effects on DCs that includes up-regulating the expression of costimulatory molecules and in particular, to be one of the major regulators of IL-12 induction [17 18 19 20 ]. The CD40–CD40L interaction is therefore crucial for bringing DCs to a state of maturation, where they can efficiently trigger immune cascades including T cell priming, memory formation, induction of a Th1 immune response, and the generation of humoral immune responses [16 ]. The CD40 signaling pathway in DCs involves activation of nitrogen-activated protein kinase (MAPKs) or ERKs [21 ]. Recent studies have indicated that p38 and ERK1/2 MAPK pathways mediate cytokine release in DCs and monocytes [22 23 ]. Studies with CD40 and CD154 knockout mice and blocking antibodies to CD40 and CD154 have revealed that the CD40-CD154 interaction is necessary for the development of protective immunity against several parasitic, bacterial, and viral infections [24 25 26 ]. It is not known whether the CD40-mediated signaling plays any role in the APC function of the plasmodium parasite-exposed DC. In this study, we compared the interaction of P. falciparum-free merozoites and iRBCs with monocyte-derived DCs (MDDCs) and its functional consequences. Additionally, we explored the effect free merozoites and iRBCs had on the CD40-mediated activation of DC and whether the alterations in the functional maturation may be correlated with the activation of distinct intracellular signaling pathways. Our data show that as a consequence of the interaction between free merozoites and iRBC with human MDDCs, sCD40L-mediated phenotypic and functional maturation of DC was regulated by activating different MAPK pathways and equipping these cells with distinctive features that enable them to induce pro- and anti-inflammatory responses in vitro.


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MATERIALS AND METHODS
 
Free merozoite and live iRBC
P. falciparum parasites of the 3D7 strains were grown in O+ human erythrocytes in RPMI 1640 supplemented with 25 mM HEPES, 0.225% NaHCO3, 0.1 mM hypoxanthine, 25 g/mL gentamicin, and 0.5% AlbuMax I (all purchased from Invitrogen Life Technologies, Carlsbad, CA, USA) and were maintained under 3% O2/3% CO2/94% N2 according to the method of Trager and Jensen [27 ]. Parasites were synchronized twice by sorbitol treatments and monitored by examination of Giemsa-stained thin blood smears. Briefly, at 10% parasitemia, parasite culture was centrifuged at 1500 rpm for 5 min at room temperature. The cell pellet was suspended in 5 times vol of 5% aqueous D-sorbitol, mixed gently, and incubated at 37°C circulating water bath for 5 min. The cells were washed twice with serum-free RPMI medium to remove sorbitol and resuspended in complete RPMI media mentioned above. Mature schizonts were isolated as described by Schwarzer et al. [28 ]. Briefly, mature schizonts were harvested from synchronized cultures of 10% parasitaemia on a cushion of 80% Percoll gradients (Sigma-Aldrich, St. Louis, MO, USA) to remove extracellular hemozoin and residual trophozoites [28 ]. Parasites were washed and resuspended in a small volume of culture medium. Washed, uninfected erythrocytes [uninfected RBCs (uRBCs)] were used as controls. To obtain free P. falciparum merozoites, mature schizonts were cultured in complete medium without fresh RBCs for 3 h, and free merozoites were collected from culture media by centrifugation (20 min at 2500 g). The parasite pellet was washed thoroughly with PBS before applying to magnetic cell separation LS columns (MACS, Miltenyi Biotec, Bergisch Gladbach, Germany) to remove extracellular hemozoin [29 ]. After the LS column was placed in the MACS separator, the merozoite pellet was suspended in prewarmed PBS (pH 7.4)–0.5% BSA, 37°C, and applied to the column. Free merozoites were collected from the flow-through, as the flow rate was too fast for the retention of merozoites onto the column and contaminating hemozoin having metallic properties adhered to the column.

Human MDDC
Whole blood was drawn from healthy blood donors according to institutional guidelines. PBMCs were isolated from whole blood by Ficoll density gradient centrifugation (Histopaque, Sigma-Aldrich). CD14+ monocytes were isolated by positive selection from PBMCs by magnetic bead separation (Miltenyi Biotec). MDDCs were generated by incubating purified monocytes in RPMI-1640 medium supplemented with 10 ng/mL GM-CSF (Peprotech, Rocky Hill, NJ, USA) and 10 ng/mL IL-4 (PeproTech), and fresh cytokines were added on alternate days. After 7 days of culture, DCs were harvested and washed before use. MDDCs that were generated exhibited forward-scatter and side-scatter characteristics of DCs and had a typical immature phenotype (>87% CD1a+ cells; >95% CD11c+ cells; <1% CD14– cells; >79% CD80low cells; >90% HLA-DR+ cells). MDDCs were gated on CD11c, a specific marker for DCs [30 31 ]. MDDCs were cultured in RPMI 1640 containing 10% FBS, 15 mM HEPES, 200 mM glutamine, 100 µg/mL penicillin and streptomycin, and 50 µg/mL gentamicin. DCs were seeded at 5 x 106 cells/ml and treated with media alone or exposed to P. falciparum-free merozoites (free merozoites; 1:25), iRBCs (1:25), or LPS (1 mg/ml) for 24 h. Postexposure, DCs were washed thoroughly and stimulated with 3 µg/mL soluble CD40L (sCD40L; from Peprotech) for another 24 h. In blocking experiments, MDDCs were preincubated with 10 µg/ml neutralizing anti-10 mAb (R&D Systems, Minneapolis, MN, USA), 10 mM SB203580 or 50 mM PD98059 (Cell Signaling Technology, Beverly, MA, USA) for 1 h before being exposed to culture medium, free merozoites, or iRBCs for 18 h, followed by stimulation with sCD40L for 24 h.

Flow cytometry
To determine phagocytosis, free merozoites, iRBCs, or uRBCs were suspended at 5 x 107/ml in PBS and labeled with 2.5 µM CFSE (Molecular Probes, Eugene, OR, USA) for 10 min at room temperature. Labeled parasites were washed four times with complete culture medium and PBS before coculturing with DCs at a 25:1 parasite:DC ratio for 3 h at 37°C. Following 4 h of coculture with parasites, RBCs were lysed, and DCs were washed and analyzed by flow cytometry. The uptake of CFSE-labeled, free merozites, iRBCs, or uRBCs was determined by gating cells on the CD11c+ population.

Following 24 h or 48 h of coculture with parasites, MDDCs were harvested from culture, washed, and resuspended in EDTA-containing medium to dissociate cell clusters. DCs were costained with PE-conjugated anti-human CD11c and FITC-conjugated anti-human mAb, which included anti-CD40, anti-CD80, anti-CD83, anti-CD86, or anti-HLA-DR (BD Biosciences, San Jose, CA, USA) After incubation on ice for 30 min, cells were washed and fixed with 1% paraformaldehyde. To detect phospho-ERK1/2, phospho-p38MAPK, total ERK1/2, and total p38MAPK DCs were fixed in 1% paraformaldehyde and then permeabilized with 0.1% saponin–1% BSA in PBS (saponin buffer) before being incubated with Alexa 488-conjugated phospho-ERK1/2, PE-conjugated phospho-p38MAPK, FITC-conjugated, total ERK1/2, and total p38MAPK (BD Biosciences). Stained cells were analyzed using FACSCalibur (BD Biosciences) and CellQuest software (BD Biosciences), and data were analyzed with FlowJo Version 8.5.2 (Tree Star Inc., Ashland, OR, USA).

CD4+ T cell purification and coculture with DCs
CD4+ T cells were purified from PBMCs by positive selection with the CD4 T cell isolation kit (Miltenyi Biotec). After release of sorted CD4 T cells from the column, the cells were magnetically labeled with CD45RA MicroBeads. Naive CD4+CD45RA+ T cells were isolated by positive selection. The purified CD4+CD45RA+ T cells were frozen at 5 x 106 cells/vial in 10% DMSO/FBS freezing mixture until use. When required, cells were thawed and washed twice with incomplete RPMI 1640 and cultured with DCs as described previously [32 ]. Briefly, MDDCs were cultured under different conditions, washed twice to remove any cytokine, and cocultured with purified, allogeneic, naïve CD4+CD45RA+ T cells at a 1:5 ratio in 24-well culture plates. After 6 days of coculture, DC-primed CD4+ T cells were restimulated for 24 h with plate-bound anti-CD3 (10 µg/ml) and soluble anti-CD28 (2 µg/ml). Cytokine production in the culture supernatant was assessed by ELISA. For intracellular cytokine production, aliquots of 1 x 106 T cells were collected on Day 6 of the culture, washed twice, and restimulated for 6 h with PMA (50 ng/ml) and ionomycin (1 µg/ml) in the presence of brefeldin A (10 µg/ml) added for the last 4 h. To distinguish responder CD4+ T cells from stimulator DCs, harvested cells were washed and stained with anti-CD4-PE-Cy5 before fixing with 2% formaldehyde, permeabilizing with 0.1% saponin, and stained with PE-conjugated mAb to IL-4, IL-10, and IFN-{gamma}. Stained cells were analyzed on a FACSCalibur flow cytometer (BD Biosciences). Data were analyzed using FlowJo software (Tree Star Inc.).

Cytokine analysis
Culture supernatants were collected at 18 h from blood DCs cultured under different conditions, as shown in the figures. To assess the cytokine profile of CD4+ T cells, DC-primed CD4+ T cells were restimulated for 24 h with plate-bound anti-CD3 (10 µg/ml) and soluble anti-CD28 (1 µg/ml). Levels of various cytokines (TNF-{alpha}, IFN-{gamma}, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p40, and IL-12p70) in the culture supernatants were quantitated using commercially available cytokine kits (BD Biosciences). The assays were performed in duplicates.

Statistics
Statistical significance was calculated by a two-sample two-tailed Student’s t-test (InStat, GraphPad Software, San Diego, CA, USA). Comparisons were considered statistically significant at P < 0.05.


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RESULTS
 
P. falciparum-free merozoites and infected erythrocytes induce distinct DC responses
P. falciparum iRBC has been shown to prevent maturation of human MDDCs [6 ], but whether interaction of DCs with P. falciparum-free merozoites could modify its maturation has not yet been investigated. We first determined whether there were any differences in the induction of innate immune responses following phagocytosis of iRBC or free merozoites. Human MDDCs were cocultured with CFSE-labeled, free merozoites and iRBCs at the ratio of 1:25 (DC:parasite) at 37°C. DCs effectively phagocytosed free merozoites as well as live iRBCs and were observed inside the cytoplasmic compartments of DCs (Fig. 1 A ). Flow cytometric analyses revealed that the 65.6%-free merozoite-associated CFSE fluorescence colocalized with CD11c+ DCs compared with 41.8% of iRBC-associated CFSE fluorescence (Fig. 1B) . When the parasite dose was increased to 50:1 or higher, there was a concurrent increase in the internalization of the parasites, but there was also a significant increase in the number of dead DCs (data not shown). Hence, the 25:1 parasite:DC ratio was selected for further experiments.


Figure 1
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  		Figure 1. P. falciparum-free merozoites and infected erythrocytes are phagocytosed by MDDCs. (A, B) MDDCs were incubated with CFSE-labeled, free merozoites, iRBCs, or uRBCs at a ratio of 1:25 for 3 h at 37°C. Following coculture, nonphagocytosed RBCs were lysed, DCs were stained with PE-labeled, anti-CD11c mAb, and CFSE fluorescence was determined by flow cytometry. CFSE-staining profiles of gated CD11c+ DCs are shown. All data represent two independent experiments.

To investigate the effect of phagocytosed malaria parasites on MDDC maturation, we cultured MDDCs with medium (Ctr-DC), free merozoites (merozoite-DC), and iRBCs (iRBC-DC) and compared them with LPS-induced DC maturation (LPS-DC). Immature DCs stimulated with LPS matured, and there was induction of the maturation marker CD83 and up-regulation of HLA-DR, CD40, CD80, and CD86. Phagocytosed free merozoites were significantly less effective than the iRBCs in inducing up-regulation of maturation markers on the surface of DCs (Fig. 2 ). Merozoite-DCs showed no noticeable changes in the expression of the HLA-DR, CD40, CD80, and CD86 molecules and failed to induce the expression of CD83 when compared with Ctr-DC (Fig. 2) . iRBC-DCs displayed a "semimature" phenotype and expressed increased levels of HLA-DR, CD40, CD80, and CD83 when compared with Ctr-DCs. Although the expressions of stimulatory and costimulatory molecules were increased compared with Ctr-DC, the induction of these molecules was less compared with LPS-DCs (Fig. 2 and Table 1 ). We observed a reduction in the percentage of cells expressing mature DC markers in merozoite-DCs in comparison with iRBC-DCs. The differences in the percentages of CD80+ cells in iRBC-DCs and merozoite-DCs (48.0% vs. 26.0%; P<0.05), CD86+ cells (65.2.0% vs. 45.2%; P<0.05), and CD83+ cells (18% vs. 6.1%; P<0 0.01) were statistically significant (Table 1) . Although the total percentage of HLA-DR+ DC was 89–94%, there was a moderate increase in the expression of HLA-DR in iRBC-DCs compared with merozoite-DCs (331.6 vs. 186.7 MFI; P<0 0.05; Fig. 2 and Table 1 ).


Figure 2
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  		Figure 2. Effects of P. falciparum-free merozoites and iRBCs on maturation of MDDCs. Immature MDDCs were exposed to free merozoites or iRBCs at a ratio of 1:25 with LPS (1 µg/ml) used as internal control for 24 h, and the expression of stimulatory and costimulatory molecules was analyzed by flow cytometry. Isotype controls are indicated by a thin line and medium-stimulated cells as filled, gray histograms, and thick black lines represent specific staining of the indicated cell surface markers. Results are representative of four independent experiments.


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  		Table 1. P. falciparum-Free Merozoites and Parasite iRBCs Induce Distinct Expression of Maturation Markers on Human MDDCsa

To further analyze the effect of free merozoite and iRBCs on DC functional maturation, we studied the kinetics of a parasite-induced cytokine response. Exposure to free merozoites or to iRBCs led to the secretion of TNF-{alpha}, IL-6, and IL-10 but no IL-12p70 by MDDCs (Table 2 ). TNF-{alpha} and IL-6 were significantly produced within 3 h and reached a peak at 12 h in iRBC-DC (ten- to 20-fold up-regulation compared with medium; P<0.05; Fig. 3 ). However, merozoite-DC produced less TNF-{alpha} and IL-6 and reached a peak response late at 18 h. Compared with iRBC-DC, merozoite-DCs produced significantly more IL-10 (P<0.02), and maximal response was observed at 24 h (Fig. 3) . Taken together, these data indicate that free, merozoite- and iRBC-exposed DCs induced distinct phenotype and cytokine profiles.


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  		Table 2. Cytokine Released by P. falciparum-Free Merozoite or Parasite iRBC-Exposed Human MDDCs


Figure 3
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  		Figure 3. Differential effects of P. falciparum-free merozoites and iRBCs on cytokine production by MDDCs. Immature MDDCs were exposed to free merozoites and iRBCs at a ratio of 1:25 with LPS (1 µg/ml) used as internal control, and cell-free culture supernatants were collected at different times and assayed for (A) TNF-{alpha}, (B) IL-6, (C) IL-12, and (D) IL-10 by ELISA. Data are expressed as means ± SD of triplicate cultures in a representative experiment of four independent experiments.

DCs exposed to free merozoites or iRBCs respond distinctly to CD40 maturation stimulus
As ligation of CD40 is a potent signal to induce terminal differentiation of DCs into mature, professional APCs [16 ], we examined the effect of free merozoites and iRBCs on the phenotypic changes induced by CD40 ligation on MDDCs (Fig. 4 ). Stimulation with sCD40L, which triggers DCs specifically through CD40 receptor, induced a strong up-regulation of the costimulatory molecules CD80, CD86, and CD40 of HLA-DR molecules and of the DC maturation marker CD83. Prior exposure to free merozoites or iRBCs strikingly modulated sCD40L-induced maturation of MDDCs. In merozoite-DC, the sCD40L-induced phenotypic changes were noticeably impaired: There was reduction in the frequency of CD40-, CD80-, CD83-, and HLA-DR-positive cells and in the MFI of these molecules (Fig. 4A and 4B) , except for noticeable increase in the CD86 expression in the percentage of positive cells and the MFI when compared with DCs treated with sCD40L alone (Fig. 4A and 4B) . These data suggest that prior exposure to free merozoites disrupted the CD40-dependent maturation of DCs by effectively impeding expression of stimulatory and costimulatory molecule.


Figure 4
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  		Figure 4. Effects of P. falciparum-free merozoites and iRBCs on sCD40L-induced DC maturation. Immature MDDCs were exposed to free merozoites and iRBCs at a ratio of 1:25 for 24 h and were then stimulated with sCD40L (3 µg/ml). After 24 h of stimulation, cells were harvested, and expression of maturation markers was measured by flow cytometry. (A) Percentage of cells staining positive (% Positive) and (B) MFI for each marker were determined from histogram analysis. Results are expressed as means ± SD of four independent experiments. MFI values for each marker were calculated after subtraction of the MFI obtained with the matched isotype control mAb. *, P < 0.05; **, P < 0.01, indicate statistically significant differences when compared with stimulation with sCD40L alone.

However in iRBC-DC, sCD40L-induced maturation of DCs was not inhibited (Fig. 4) . As shown in Figure 4 , iRBC-DCs displayed a more mature phenotype after stimulation with sCD40L with higher expression of the activation and maturation markers CD83, CD40, CD80, CD86, and HLA-DR in terms of percentage of positive cells (Fig. 4A) and fluorescence intensity (Fig. 4B) . There was a moderate increase in frequency of CD40-positive (56.1%), CD80-positive (80.6%), and CD83-positive DCs (59.1%) relative to DCs treated with sCD40L alone (48.7% for CD40, 73.2% for CD80, 39.2% for CD83), indicating the maturation of iRBC-DCs after stimulation with sCD40L. Although the percentage of CD86 and HLA-DR expressing positive cells did not show significant changes, there was approximately a twofold increase in MFI of HLA-DR, whereas fluorescence intensity of CD86 was reduced on iRBC-DCs in response to sCD40L stimulation (Fig. 4B) .

Free merozoites and iRBCs distinctly modulate a CD40-induced cytokine profile of MDDCs
IL-12p70 secretion by human DC could only be amplified by CD40L in the presence of innate signals initiated by microbial stimuli [33 ]. We therefore investigated the effect of CD40 ligation on the cytokine response in free merozite- and iRBC-exposed DCs (Fig. 5 ). Stimulation with sCD40L resulted in higher levels of IL-12p70 (401±65 pg/ml) and IL-10 (419±37 pg/ml) production by MDDCs compared with DC exposed to medium. sCD40L-stimulated merozoite-DC secreted lower levels of IL-12p70 compared with sCD40L-stimulated DC (136±19 vs. 401±65 pg/ml; P<0.02; Fig. 5 ). However, no major effect was observed in the production of TNF-{alpha} and IL-6. In contrast, IL-10 production was enhanced (up to fourfold) in merozoite-DCs after sCD40L stimulation (1484±83 pg/ml vs. 419±37pg/ml; P<0.01; Fig. 5 ).


Figure 5
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  		Figure 5. P. falciparum-free merozoites and iRBCs affect sCD40L-induced cytokine profile of MDDCs. Immature MDDCs which were cultured for 48 h with medium, free merozoites, or iRBCs at a ratio of 1:25 with sCD40L (3 µg/ml) were added during the last 24 h. Cell-free culture supernatants were harvested 48 h later, and TNF-{alpha}, IL-6, IL-12p70, and IL-10 secretion was analyzed by ELISA. Mean ± SEM from three independent experiments is shown. *, P < 0.05; **, P < 0.01, indicate statistically significant differences when compared with stimulation with sCD40L alone.

In iRBC-DCs, after CD40 ligation, there was an increase in IL-12p70 production compared with DCs treated with sCD40L alone (623±94 pg/ml vs. 401±65 pg/ml; P<0.05; Fig. 5 ). TNF-{alpha} and IL-6 but not IL-10 were also increased in iRBC-DCs after CD40 ligation (Fig. 5) . Together, these results suggest that prior exposure of DCs to free merozoites skewed the response toward a pronounced, anti-inflammatory response, whereas iRBCs induced a proinflammatory response on CD40 signaling.

Free merozoites but not iRBCs modify CD40-induced MAPK activity in DCs
To understand the potential intracellular signaling mechanisms that may influence the free merozoites or iRBC-exposed DC responses, we looked at the MAPK signaling pathway. In response to free merozoites alone, MDDCs were found to exhibit phosphorylation of ERK1/2 but little phosphorylation of p38MAPK (Fig. 6 A ). sCD40L induced significant phosphorylation of p38MAPK and ERK1/2 in DCs treated with either medium or LPS (Fig. 6) . In merozoite-DC, there was a significant increase in the sCD40L-induced phosphorylation of ERK1/2 but a decrease in p38MAPK phosphorylation (Fig. 6B) . To confirm this, we next studied the effect of PD98059, which specifically blocks the ERK1/2 pathway. As expected, ERK1/2 phosphorylation induced by sCD40L in merozoite-DC was sensitive to pretreatment with PD98059 (Fig. 6C) . In addition, we observed that when the ERK1/2 pathway was blocked with PD98059, there was an increase in the sCD40L-induced phosphorylation of p38MAPK in merozoite-DC (Fig. 6C) . These results indicate that free merozoites effectively activate the ERK pathway.


Figure 6
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  		Figure 6. P. falciparum-free merozoites and iRBCs influence sCD40L-induced MAPK signaling pathways. (A) MDDCs were exposed to medium, free merozoites, iRBCs at a ratio of 1:25, or LPS as internal control for 30 min. (B) MDDCs were exposed to medium, free merozoites, or iRBCs at a ratio of 1:25 for 15 min and stimulated for 20 min with sCD40L (3 µg/ml). (C) MDDCs were pretreated with or without 10 µM SB203580 or 50 µM PD98059 for 1 h and then exposed to medium, free merozoites, and iRBCs at a ratio of 1:25 for 15 min and stimulated for 20 min with sCD40L (3 µg/ml) in the presence or absence of SB203580/PD98059. After stimulation, MDDCs were washed, fixed, permeabilized, and intracellularly stained with fluorochrome-conjugated, phosphor-specific antibodies for p38MAPK and ERK1/2 and analyzed by flow cytometry. All histograms are gated on live CD11c+ cells. Results are representative of three independent experiments.

In contrast, when DCs were exposed to iRBCs alone, there was low-level phosphorylation of p38MAPK as well as ERK1/2 (Fig. 6A) . sCD40L led to a significant increase in phosphorylation of p38MAPK, whereas ERK1/2 phosphorylation was unaltered in iRBC-DC (Fig. 6B) . A significant decrease was observed in sCD40L-induced p38MAPK phosphorylation when the p38MAPK pathway was blocked with SB203580 in iRBC-DC. However, CD40 ligation failed to augment the ERK1/2 signal in iRBC-DCs pretreated with SB203580 (Fig. 6C) . Thus, phosphorylation of the MAPKs following exposure of DCs to free merozoites was quite different from that of iRBCs in response to CD40 ligation. As a control, we had stimulated MDDCs with uRBCs to determine if uRBCs led to phosphorylation of p38 and ERK MAPK. Stimulation of MDDCs with uRBCs did not result in phosphorylation of p38MAPK or ERK1/2 (Supplementary Fig. 1). Therefore, RBC by itself did not have any stimulatory or inhibitory effect on MDDCs.

ERK1/2 and p38MAPK pathways regulate CD40-induced cytokine production in DCs exposed to free merozoites and iRBCs
We further examined the involvement of p38MAPK and ERK1/2 in modulating sCD40L-induced production of IL-12 and IL-10 in free merozoite or iRBC-exposed MDDCs. Immature DCs were preincubated with the p38MAPK inhibitors (SB203580) or the ERK1/2 inhibitors (PD98059) before exposing to free merozoites or iRBCs and subsequently stimulating with sCD40L. In the presence of PD98059, sCD40L-induced IL-10 production in merozoite-DC was abrogated, signifying that ERK1/2 activation was responsible for increased IL-10 production (Fig. 7 ). Similarly, suppression of sCD40L-induced IL-12 production in merozoite-DC could be also correlated with increased ERK1/2 activity. Blocking ERK1/2 phosphorylation with PD98059 resulted in a slight increase in IL-12p70 production in merozoite-DC plus sCD40L (Fig. 7) . Taken together, the results show that merozoites modulate sCD40L-induced IL-10 and IL-12 production in DCs through the ERK1/2 signaling pathway.


Figure 7
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  		Figure 7. Role of MAPK in sCD40L-induced IL-12 and IL-10 production in MDDCs exposed to P. falciparum-free merozoites and iRBCs. MDDCs were pretreated with or without 10 µM SB203580 or 50 µM PD98059 for 1 h and then exposed to medium, free merozoites, or iRBCs at a ratio of 1:25 for 48 h with sCD40L (3 µg/ml) stimulation during the last 24 h with sCD40L (3 µg/ml) in the presence or absence of SB203580/PD98059. Cell-free supernatants were harvested after 48 h and assessed for IL-12p70 and IL-10 secretions in the culture supernatants by ELISA. The data represent the mean ± SEM of five independent experiments.

We next examined MAPK activation requirements for sCD40L-stimulated IL-12p70 production in iRBC-DC. Blocking sCD40L-induced p38MAPK phosphorylation in iRBC-DCs with SB203580 markedly reduced IL-12p70 secretion (Fig. 7) . In contrast, PD98059 did not significantly affect IL-12p70 induction (Fig. 7) . These results suggest that p38MAPK activation may be involved in sCD40L-induced IL-12p70 production in DCs exposed with iRBCs. Thus, free merozoites and iRBCs differentially regulate the sCD40L-triggered cytokine production in DCs through the activation of the MAPK signaling pathway.

Differential expression of cytokines by allogeneic CD4+ T cells stimulated with merozoite-DC plus sCD40L or iRBC-DC plus sCD40L
We next evaluated the nature and quality of the adaptive immune response induced by MDDCs loaded with free merozoites or iRBCs. Naïve, human CD4+CD45RA+ T cells purified from adult peripheral blood and cocultured with DCs at a 1:5 ratio for 6 days; they were then washed and restimulated for 24 h with anti-CD3 and anti-CD28, and then cytokine production was measured in the culture supernatant by ELISA (Fig. 8 A ). CD4+CD45RA+ T cells primed with iRBC-DCs plus sCD40L produced large amounts of IFN-{gamma} and low amounts of IL-5 and IL-10 (Fig. 8A) . In comparison, CD4+CD45RA+ T cells primed by merozoite-DC plus sCD40L produced large amounts of IL-10 (up to 350 pg/ml) and moderate amounts of IFN-{gamma} but did not produce detectable amounts of IL-5 (Fig. 8A) . By intracellular cytokine staining, striking differences were seen in the percentage of CD4+ T cells that produce IFN-{gamma} or IL-10 in response to stimulation with merozoite-DC plus sCD40L or with iRBC-DC plus sCD40L (Fig. 8B) . iRBC-DC plus sCD40L-primed CD4+ T cells expressed primarily IFN-{gamma}, and only a minor percentage of the CD4+ T cells was found to secrete IL-10 upon PMA plus ionomycin restimulation. In contrast, in merozoite-DC plus sCD40L-primed CD4+ T cells, the frequencies of IL-10-producing CD4+ T cells were higher compared to IFN-{gamma}-producing CD4+ T cells upon PMA and ionomycin activation. Therefore, this short-term mitogen-restimulation assay gave a distinct and quantitative indication of the cytokine-producing potentials for CD4+ T cells primed with iRBC-DC plus sCD40L or with merozoite-DC plus sCD40L. Compared with CD4+ T cells primed with iRBC-DC plus sCD40L, the ratio of pro- to anti-inflammatory cytokines in CD4+ cells primed with merozoite-DC plus sCD40L was skewed toward the anti-inflammatory phenotype (Fig. 8A) .


Figure 8
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  		Figure 8. Polarization pattern of CD4+ T cells stimulated with DCs primed with free merozoites plus sCD40L or iRBCs plus sCD40L. (A) Immature MDDCs were exposed to medium, free merozoites, and iRBCs at a ratio of 1:25 for 48 h and stimulated with sCD40L (3 µg/ml) for the last 24 h. The DCs were washed and cocultured with naïve, human CD4+CD45RA+ T cells, purified from adult peripheral blood at a 1:5 ratio for 6 days. The primed CD4+CD45RA+ T cells were harvested and restimulated with immobilized anti-CD3 mAb and soluble anti-CD28 mAb for 24 h at 37°C. The secretion of pro- and anti-inflammatory cytokines in cell-free culture supernatant was quantified by ELISA, and the data are expressed as mean ± SEM and replicate cultures of a representative experiment. *, P < 0.05; **, P < 0.01, indicates statistically significant differences when compared with stimulation with sCD40L alone. The ratio of pro- to anti-inflammatory cytokines obtained in similar experiments was also calculated for each of the stimuli. (B) Allogeneic CD4+CD45RA+ T cells were cultured for 6 days with Ctr-DC, sCD40L-DC, merozoite-DC plus sCD40L, or iRBC-DC plus sCD40L. After washing, the primed CD4+ T cells were restimulated with PMA and ionomycin for 5 h. Brefeldin A was added to the cultures for the last 4 h, and then, intracellular IFN-{gamma} or IL-10 in the CD4+ T cells was analyzed by flow cytometry. The percentages of the respective cytokine-producing CD4+ T cells are indicated in each dot-blot profile. This figure represents the results from one of three independent experiments.


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DISCUSSION
 
P. falciparum iRBCs have been shown to interfere with DC function and play a role in hindering antimalarial immune response during acute malaria infection [6 ]. Rupture of the schizonts to release components of blood-stage parasites, including merozoites, parasite-derived GPI, and pigments such as hemozoin, is generally assumed to be the key factor that triggers innate immune responses [34 35 36 ]. Although hemozoins have been shown to impair differentiation and maturation of monocytes into fully competent DC [10 ], it is not known whether the presence of large numbers of free merozoites specifically alter the DC functions. In the present study, we have compared the effect of free merozoites with iRBCs on the maturation of human MDDCs, which were able to phagocytose both free merozoites and iRBCs. Although we had found a dose-dependent increase in the internalization of parasites, increasing the dose resulted in the increased proportion of apoptotic DCs in cultures. These observations supported those of recent studies reporting that the majority of DCs cultured in the presence of high-dose parasites was apoptotic [37 38 ]. It has been reported that iRBCs inhibit DC maturation in a dose-dependent manner [38 ]. We found that while inhibition of DC maturation was observed at ratios of 100 iRBCs per DC, lower ratios of iRBCs to DCs (10 iRBCs per DC) did not affect LPS-induced maturation of DCs.

In the present study, we have demonstrated that free merozoites as well as iRBCs have the potential to disrupt the host’s innate immune response. Although free merozoites compared to iRBCs were more effectively internalized by MDDCs, free merozoites failed to induce phenotypic maturation of DCs. While preventing maturation-induced increase in the surface expression of stimulatory and costimulatory molecules (HLA-DR, CD40, CD80, and CD83) of DCs, free merozoites induced low levels of TNF-{alpha} and IL-6 but significantly increased level of immunosuppressive cytokine, IL-10 in DCs. Hence, exposure to free merozoites resulted in DCs to differentiate into maturation-resistant, IL-10-secreting APCs, a characteristic of a tolerogenic type of DC [39 ]. In contrast to free merozoites, internalization of iRBCs resulted in DCs becoming semimature phenotypically and produced high levels of TNF-{alpha} and IL-6 but a moderate level of IL-10. It was apparent from the present study that free merozoites and iRBCs did not directly induce DCs to produce IL-12. IL-12p70 was detected in low amounts in supernatants of DCs exposed to free merozoites or iRBCs when compared with LPS-treated DCs, a feature that is consistent with earlier reports [6 40 ].

Stimulation with free merzoite or iRBC alone resulted neither in activation nor maturation of DCs, suggesting that additional stimulus is necessary to facilitate activation of antiparasite immune responses. DCs are known to undergo maturation upon CD40–CDl54 interactions that play a decisive step in the initiation of an adaptive immune response. Signaling through CD40 leads to up-regulation of major stimulatory MHC I and II molecules, costimulatory molecules such as CD80/CD86, and cytokines such as IL-12 and IL-1{alpha}/β, which all participate in activation and differentiation of naive CD4+ T cells [17 20 33 41 42 43 ]. In this study, we observed that free merozoites interfered with sCD40L-induced phenptypic maturation of DCs and selectively suppressed sCD40L-induced IL-12 production but not TNF-{alpha} and IL-6 and instead, led to an increased IL-10 production. DCs treated with free merozoites and sCD40L were able to prime naïve CD4+ T cells into effectors producing large amounts of anti-inflammatory cytokine IL-10 and moderate amounts of proinflammatory IFN-{gamma}. These observations suggest free merozoites differentially regulate sCD40L-induced pro- and anti-inflammatory cytokine production and possibly disrupt the protective antiparasite immunity and help in parasite survival. In contrast to the response provoked by free merozoites, iRBCs augmented sCD40L-induced phenotype maturation and production of IL-12, IL-6, and TNF-{alpha} in human DCs. These cells were also able to prime naïve CD4+ T cells to differentiate into effectors producing proinflammatory IFN-{gamma}. Thus, free merozoites and iRBCs acted as counter-regulators of DC activation induced by CD40–CDl54 interactions.

We focused on the underlying molecular mechanisms by which free merozoites and iRBCs influence sCD40L-induced IL-10 and IL-12 production in DCs. Others have suggested that the strength of CD40 signaling may influence the activation of the MAPK pathway and consequently, affect production of pro- and anti-inflammatory cytokines by the APC population [44 45 ]. Our study demonstrated that free merozoites and iRBCs exerted differential effects on the activation of ERK and p38MAPK when DCs were restimulated with sCD40L. Free merozoite alone resulted in increased ERK1/2 phosphorylation but reduced p38MAPK phosphorylation. We further show that CD40 signaling in merozoite-treated DCs resulted in significant phosphorylation and activation of ERK1/2, whereas phosphorylation of p38 was not increased. Several studies had shown that ERK and p38MAPK differentially regulate IL-10 and IL-12 secretion by DCs [46 47 48 ]. As IL-12 versus IL-10 secretion by DCs has important, functional implications in skewing a pro- or anti-inflammatory response, the regulation of ERK and p38MAPK balance in DCs is one potential way by which P. falciparum-free merozoites possibly hinder CD40-triggered cytokine synthesis. Our results demonstrate that phosphorylation of ERK1/2 is responsible for differences in the CD40-mediated IL-10 and IL-12 production in free merozoite-exposed DCs, as inhibition of ERK1/2 pathways using specific inhibitor PD98059 completely abrogated IL-10 production, whereas IL-12 production was enhanced.

In contrast to the effect of free merozoites, iRBCs alone induced low level p38MAPK and ERK1/2 phosphorylation. In response to sCD40L stimulation, there was significant increase in the phosphorylation and activation of p38MAPK but a concomitant decrease in the ERK1/2 phosphorylation in iRBC-exposed DCs. Blocking the p38MAPK pathway with SB203580 significantly inhibited sCD40L-mediated IL-12 production in iRBC-exposed DCs. These data demonstrate an essential role of the p38MAPK pathway in CD40L-induced expression of IL-12 in these DCs, This is supported by data from the literature pointing to a major role that p38MAPK plays in DC–IL-12 expression after CD40 receptor ligation [49 50 ]. Consistent with these reports, sCD40L-induced activation of the p38MAPK pathway in iRBC-exposed DCs may be, in part, responsible for the proinflammatory-type immune responses. The ability of iRBCs to trigger IL-12p70 production in a CD40L-dependent manner is in agreement with earlier in vitro studies indicating that two signals are essential for production of IL-12p70 by human DCs [51 52 ]. We speculate from our results that the two-signal required for the production of IL-12 possibly represents a safety mechanism for optimal control of potentially harmful proinflammatory responses.

Our study suggests that free merozoites and iRBCs have intrinsic differences in their ability to modulate DC functioning, which plays a crucial role in the innate response. The difference in the efficiency of phagocytosis may be a result of the difference in the size of free merozoites and iRBCs as well as involvement of different sets of receptor–ligand in the adhesion and internalization of free merozoites and iRBCs by DCs. Free merozoites and iRBCs may differ with respect to the antigens that engage a different set of pattern recognition receptors (PRRs) through which DC activation signals are known to occur. Human DCs have been shown to phagocytosis iRBCs through interaction between P. falciparum erythrocyte membrane protein-1 expressed on iRBCs and PRRs such as the scavenger receptor, CD36 on DCs [53 54 ]. Merozoites express numbers of proteins on their surface that may interact with different PRRs expressed by DCs; the specificity of such interacting partners is not known. Further studies are necessary to identify the merozoite-associated antigens that are responsible for the variable effects observed between free merozoites and iRBCs.

In summary, these data indicate that events that occur during the cognate interaction between DCs exposed to different developmental forms of the P. falciparum parasite may manipulate host responses to the parasite. This underlies the inflammatory and immunological responses that can be optimally generated or blunted, as in the case of immune evasion seen during malaria infection. These findings may help in understanding the mechanisms that the malaria parasite uses to modulate the innate as well as adaptive immunity to escape host immunosurveillance.


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ACKNOWLEDGEMENTS
 
This work was supported by grants from Indian Council of Medical Research (63/176/2002-BMS) to P. M. and by the institutional core grant.

Received August 24, 2007; revised February 22, 2008; accepted March 3, 2008.


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