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Infection and Immunity, November 2008, p. 5149-5157, Vol. 76, No. 11
0019-9567/08/$08.00+0     doi:10.1128/IAI.01579-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Enhanced Toll-Like Receptor Responsiveness Associated with Mitogen-Activated Protein Kinase Activation in Plasmodium falciparum-Infected Children{triangledown}

Franca C. Hartgers,1* Benedicta B. Obeng,1,3 Astrid Voskamp,1 Irene A. Larbi,3 Abena S. Amoah,3 Adrian J. F. Luty,2 Daniel Boakye,3 and Maria Yazdanbakhsh1

Department of Parasitology, Leiden University Medical Centre, Albinusdreef 2, 2333 ZA Leiden, The Netherlands,1 Department of Medical Microbiology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands,2 Noguchi Memorial Institute for Medical Research, University of Ghana, P.O. Box LG581, Legon, Accra, Ghana3

Received 30 November 2007/ Returned for modification 29 January 2008/ Accepted 7 August 2008


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ABSTRACT
 
Acute Plasmodium falciparum infection is associated with strongly upregulated cytokine responses that are at least partly the result of activation of Toll-like receptors (TLRs). Whether and how TLR expression/responsiveness changes upon malarial infection is, however, currently not well understood. To assess this, we examined expression of TLRs and used the TLR ligand lipopolysaccharide (LPS) and Pam3Cys to stimulate peripheral blood mononuclear cells (PBMCs) from Ghanaian schoolchildren who live in a rural area where P. falciparum is endemic. Expression of TLR2 was higher, and responses to its ligand, Pam3Cys, were enhanced in P. falciparum-infected children compared to their uninfected counterparts. In cells from the same children, stimulation by Pam3Cys resulted in higher p38 mitogen-activated protein kinase activation and higher cytokine production. In vitro experiments confirmed that preincubation of PBMCs with P. falciparum-infected red blood cells enhanced responsiveness to TLR ligands. Taken together, the data indicate that P. falciparum-infected children in areas where malaria is endemic have an altered innate immune system, which might be important for the balance between immunity and pathology when new infections are encountered or when novel vaccines are introduced.


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INTRODUCTION
 
Acute Plasmodium falciparum malaria is associated with strong proinflammatory responses that themselves are thought to play an important role in fighting the infection. The proinflammatory cytokines gamma interferon (IFN-{gamma}) and tumor necrosis factor alpha (TNF-{alpha}) have been shown to control parasitemia by activating macrophages and cytotoxic lymphocytes for increased killing of infected erythrocytes and hepatocytes (17). Although proinflammatory responses are thus thought to be associated with protective immunity to malaria during the early phases of infection, overproduction of IFN-{gamma} or TNF-{alpha} predisposes to severe malarial pathology (7, 16).

Activation of the immune system involves recognition of pathogen-associated molecular patterns (PAMP) by pattern recognition receptors such as Toll-like receptors (TLR). These receptors play an important role in linking the innate immune system to the induction of adaptive responses (21). Plasmodial PAMP identified thus far include the putative P. falciparum toxin glycosylphosphatidylinisitol (13) that is recognized by TLR2 and TLR4, whereas TLR9 appears to respond to parasite DNA complexed with hemozoin (22). Typically, such interactions induce a proinflammatory response. However, it is thought that downregulation of TLR responsiveness might occur in the later stages of infection, analogous to observations during chronic helminth infections (2, 9, 28). In the later stages of Plasmodium yoelii infection in mice, when the infection is resolving, dendritic cells become refractory to TLR stimulation, as reflected by downregulated production of the proinflammatory cytokines, interleukin-12 (IL-12) and TNF-{alpha} (23). Modulation of inflammatory responses in this way is thought to prevent the development of severe pathology. Understanding innate immune responses during an infection is important for control of immunity/pathology not only to the ongoing infection but also to coinfections.

There is very little information on TLR responsiveness in human malarial infection, and it is unclear whether TLR responsiveness is also down-modulated during the later stages of infection. Older reports indicate that individuals with acute malaria show reduced responsiveness to several vaccines, including tetanus toxoid, meningococcal polysaccharide, Haemophilus influenzae type B conjugate, and whole-cell vaccine of typhoid fever (1, 26, 30). The impact of asymptomatic chronic P. falciparum parasitemia in this context is less clear, and its effects on the innate immune system are unknown. To determine whether such asymptomatic malarial infection modulates TLR responsiveness, we have analyzed TLR responsiveness of schoolchildren living in an area of Ghana, West Africa, where P. falciparum malaria is endemic.


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MATERIALS AND METHODS
 
Study population. The study population consisted of schoolchildren, between 6 and 15 years of age, from the Greater Accra Region of Ghana. All parents were invited to a meeting where information on the study was provided by the researchers. Children whose parents consented by signing or thumb-printing an informed consent form were registered to participate in a large study of immune responses and parasitic infections and allergies. The Institutional Review Board of the Noguchi Memorial Institute for Medical Research, Accra, Ghana, approved the study. In a previous study in this area in Ghana in 2004, we had measured TLR2 mRNA expression in 120 rural children from the Ga district (11) and found that TLR2 expression was higher in malaria-infected than noninfected children. For the present, more detailed study on the influence of malarial infection, 36 children ranging in age from 6 to 12 years were randomly selected from two rural schools in the Dangme East district in Ghana, approximately 75 km from the center of Accra. P. falciparum malaria is endemic in this area, with an overall prevalence of 51%, as determined by standard Giemsa-stained thick blood smear examination. All infected children had low to moderate parasitemia (less than 100 parasites per 100 high-power fields [5]). No child had symptoms of malaria such as fever, and at the time of blood collection none were reported as having taken antimalarial treatments. All samples were used for the whole-blood stimulation assay, while only the first 16 samples were used for peripheral blood mononuclear cell (PBMC) isolation, flow cytometry, and mitogen-activated protein kinase (MAPK) activation experiments.

For the in vitro restimulation experiments, cryopreserved PBMCs were used from four Dutch healthy donors, between 21 and 26 years of age, and from six Gabonese school children, between 6 and 14 years of age and living in the area of Lambaréné. These children were confirmed free of helminth infection and had been enrolled in a study on the role of parasitic infections in atopy, approved by the Ethics Committee of the International Foundation of the Albert Schweizer Hospital (27). The prevalence of malaria infection (mostly P. falciparum) in this area was 46%.

Cytokine measurements. For cytokine analysis, 1.0e6 PBMCs were stimulated with either medium (RPMI 1640 medium supplemented with 10% fetal calf serum; both from Gibco), lipopolysaccharide ([LPS] 10 ng/ml; Sigma Aldrich) or Pam3Cys (200 ng/ml; EMC Microcollections, Germany) in round-bottom 96-well plates (Nunc). Supernatants were collected after 3 days of culture at 37°C in 5% CO2. For the restimulation experiments, cryopreserved PBMCs from Gabonese donors (27) and Dutch donors were seeded at 1.0e5 PBMCs per well in 96-well round-bottom plates in RPMI 1640 medium containing 10% human serum. Prestimulation was performed for 24 h with medium, infected red blood cells (iRBC), uninfected red blood cells (uRBC) prepared according to a standard protocol described in detail elsewhere (18), or 1 ng/ml LPS. After careful removal of the supernatant, cells were restimulated with either LPS (1 ng/ml) or Pam3Cys (1 µg/ml) for six additional hours before the supernatants were harvested. Levels of TNF-{alpha} and IL-10 were determined simultaneously in the supernatants by using a Luminex-100 cytometer (Luminex Corporation, Austin, TX), equipped with StarStation software (Applied Cytometry Systems, Dinnington, United Kingdom). Buffer reagent kits and Luminex cytokine kits (BioSource, Camarillo, CA) were used, and cytokines were measured according to the protocol, with slight modifications (29), and analyzed using the Luminex-100 cytometer. The lower detection limit of the assays was 5 pg/ml for IL-10 and 10 pg/ml for TNF-{alpha}. Samples with concentrations below the detection limit were assigned half the value of this threshold. Background cytokine production (cells cultured with medium only) was subtracted from the values obtained after stimulation with LPS or Pam3Cys.

Flow cytometry. For surface staining of monocytes, 2.0e6 PBMCs were seeded in a 24-well plate and stimulated with the same stimuli as for the cytokine assay and harvested by scraping the cells after 24 h of culture. The following antibodies were used: anti-CD14-peridinin chlorophyll protein, anti-CD16-allophycocyanin (both BD Biosciences), and anti-TLR2-phycoerythrin (clone T2.5; eBioscience). Monocytes were gated using CD14 and CD16 double staining in order to be able to remove dead cells from gating. Activation of TLR results in phosphorylation of MAPK p38 (P~p38) and extracellular signal-regulated kinase (P~ERK), which can be measured by intracellular staining with phospho-specific antibodies (12, 14). For this, PBMCs were seeded at a concentration of 0.5e6 to 1.0e6 cells in 24-well plates and rested overnight before stimulation with medium (RPMI 1640 medium with 10% fetal calf serum), 10 ng/ml LPS, or 200 ng/ml Pam3Cys. After stimulation for 30 or 60 min, cells were fixed in the well with 4% formaldehyde, incubated for 10 min at room temperature, and harvested by cell scraping. Cells were washed twice in phosphate-buffered saline containing 0.5% bovine serum albumin before permeabilization in 700 µl of ice-cold 90% methanol. Following incubation for 30 min on ice, cells were frozen at –80°C until further use. Within 5 weeks of permeabilization and freezing, cells were thawed, washed twice in phosphate-buffered saline-0.5% bovine serum albumin, and stained for 2 h at room temperature with antibodies (10 µl per sample). The antibodies used to detect P~p38 and P~ERK were anti-phospho-p38 Alexa Fluor 547 (item 4552) and anti-phospho-p44/42 Alexa Fluor 488 (item 4373), respectively (Cell Signaling Technology). Flow cytometric analyses were performed using a Becton Dickinson FACSCalibur flow cytometer (BD Biosciences) and FlowJo analysis software (Treestar). Since the staining method for P~p38 and P~ERK destroyed the recognition of the monocyte marker CD14, percentages of P~p38- and P~ERK-positive cells were calculated from the total PBMC population. Pilot experiments with European donors have indicated that activity for P~p38 and for P~ERK was detectable only in monocytes. Analysis of the forward-sideward scatter plot of the P~p38- and P~ERK-positive cells of Ghanaian donors confirmed the selective activation of P~p38 and P~ERK in monocytes. The ratio of P~p38 to P~ERK was determined by dividing the mean fluorescence intensity (MFI) of P~p38 by the MFI of P~ERK in MAPK-positive cells.

Quantitative PCR analysis of TLR2 and TLR4 mRNA. Immediately after venipuncture into heparinized tubes, whole blood was mixed 1:5 with Nuclisens lysis buffer (Biomérieux, Boxtel, The Netherlands) to stabilize the RNA; samples were was stored at –80°C. A Nuclisens Isolation kit (Biomérieux) was used for the isolation of total nucleic acid according to the manufacturer's instructions, followed by treatment with RNase-free DNase (Invitrogen, Breda, The Netherlands) to remove genomic DNA. Reverse transcription of RNA was performed using Moloney murine leukemia virus reverse transcriptase (Invitrogen). Gene expression was assessed with real-time quantitative PCR in duplicate in accordance with the Taqman assay instructions using Taqman probes and qPCR Core kit reagents (both Eurogentec, Seraing, Belgium) on the ABI 7500 machine, normalizing for the housekeeping gene 18S rRNA. Sequences of primer and probes were as described previously (15).

Statistical analysis. Analyses were performed using GraphPad Prism software (version 4.0). Differences between P. falciparum-infected and noninfected groups were assessed using a nonparametric Mann-Whitney U test. Assessments of correlations were performed using a nonparametric Spearman rank test. In order to adjust for the effect of age and of helminth infection, associations between cytokine production and malarial infection were assessed by multivariate linear regression analysis using log-transformed cytokine data.


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RESULTS
 
Enhanced levels of TLR2 expression associated with asymptomatic P. falciparum infection. In order to test whether asymptomatic P. falciparum infection in a population residing in an area of endemicity affects TLRs, we assessed the expression of two TLR family members, TLR2 and TLR4, that are expressed at relatively high levels in mononuclear cells, using real-time quantitative PCR on mRNA extracted from whole-blood samples derived from a population of schoolchildren resident in rural Ghana. The levels of TLR2 mRNA were significantly higher in samples from P. falciparum-infected children, whereas mRNA levels of TLR4 were similar in both groups (Fig. 1a and b). To determine whether this difference translated into higher cell surface expression of the TLR2 protein, PBMCs were isolated from 36 rural schoolchildren (see Materials and Methods), half of whom were infected with P. falciparum (Table 1). There were no differences in either age or sex ratio of the P. falciparum-infected (n = 18) and noninfected (n = 18) children. Helminth infections were also prevalent in this area; 19 of 36 were infected with Schistosoma haematobium, and three of these children were also infected with Ascaris lumbricoides (Table 1). TLR2 expression on T and B cells was low: less than 2% of either T or B cells expressed TLR2, and even this was at very low levels (data not shown). TLR2 expression was primarily on monocytes that could be divided into two populations, either cells expressing high levels of CD14 and TLR2 and low levels of CD 16 (CD14hi CD16lo TLR2hi) or CD14lo CD16hi TLR2lo cells (Fig. 1c and d). Monocytes of P. falciparum-infected children expressed significantly higher levels of TLR2 than those of noninfected children (P < 0.05) (Fig. 1e). The increase in MFI was the result of both an increased MFI per cell, which did not reach statistical significance, and a significantly increased percentage of CD14hi CD16lo TLR2hi cells within the monocyte population of infected children compared to noninfected children (Fig. 1F). TLR4 cell surface protein expression was too low to allow accurate detection by flow cytometry.


Figure 1
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  		FIG. 1. TLR2 expression is enhanced by asymptomatic P. falciparum infection. Relative mRNA expression of TLR2 (a) and TLR4 (b) was determined in blood of P. falciparum-infected (mal pos) and noninfected (mal neg) individuals by real-time PCR. Horizontal bars represent median values per group. PBMCs cultured for 24 h with medium were stained with antibodies to CD14, CD16, and TLR2. CD14hi CD16lo (m1) and CD14lo CD16hi (m2) cells were gated (c) and analyzed for the surface expression of TLR2 (d). In panel d, the gray line represents the isotype control antibody for m1 cells; the dashed line represents m2 cells, and the solid line represents m1 cells. (e) The geometric mean of TLR2 fluorescence of both populations was determined per donor and compared between P. falciparum-infected donors and negative donors. (f) The percentage of CD14hi CD16lo cells in the total monocyte population was determined in P. falciparum-infected and uninfected donors. *, P < 0.05. freq, frequency; Max, maximum.


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  		TABLE 1. Characteristics of study population

P. falciparum infection is associated with an increased frequency of monocytes expressing P~p38 and an increased P~p38/P~ERK ratio. We next examined whether higher TLR expression might result in increased TLR signaling by stimulating PBMCs from a subset of 16 children for 30 or 60 min with either the TLR4 ligand LPS or the TLR2 ligand Pam3Cys, followed by measurement of phosphorylation of MAPKs, which are the first signaling steps following TLR activation. The phosphorylation of the MAPK p38 and ERK was determined by flow cytometry. Stimulation with LPS or Pam3Cys increased the number of cells with activated P~p38 at both time points in all individuals and for P~ERK only after 30 min (Fig. 2). Although the staining of CD14 was incompatible with the fixation method for the detection of the MAPK, analysis of the forward-sideward scatter plot indicated that the main population of cells positive for MAPK activation consisted of monocytes (data not shown). P. falciparum-infected children had a higher frequency of cells with activated P~p38 than did noninfected children, and this difference was significant for Pam3Cys after 30 min of stimulation (P < 0.05) and decreased after 60 min of stimulation (Fig. 2c and d). Stimulation with LPS did not result in significant differences between the infected and noninfected children, although there was a trend for higher P~p38 activation in the infected group at both time points (Fig. 2c and d). There was variation in P~ERK activation, but between infected and noninfected children no significant differences were detected (Fig. 2e and f). However, as the ratio of P~p38 to P~ERK is important for the type of cytokine response that is induced (20), we compared the ratio in infected and uninfected subjects. The ratio of activated P~p38/P~ERK was higher after 60 min of stimulation with LPS, whereas in response to Pam3Cys the response was highest at 30 min after stimulation. No P. falciparum infection-related differences were seen in this P~p38/P~ERK ratio in response to LPS, but in response to Pam3Cys the ratio was significantly higher after 30 min in those infected with P. falciparum than in noninfected children (P < 0.01) while this difference disappeared at the later time point (Fig. 3b).


Figure 2
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  		FIG. 2. P. falciparum infection is associated with an increased percentage of P~p38-positive cells. PBMCs were stimulated with medium, LPS, or Pam3Cys for 30 or 60 min; cells were fixed, permeabilized, and stained with antibodies specific for P~p38 and P~ERK (pp38 and pERK, respectively). Examples are shown of staining at 30 min for a P. falciparum-negative donor (a) and a P. falciparum-positive donor (b), indicating percentages of P~p38- and P~ERK-positive cells and frequency of P~p38-positive (c and d) and P~ERK-positive cells (e and f) 30 min (c and e) or 60 min (d and f) after stimulation with medium, LPS, or Pam3Cys in the total PBMC live gate for P. falciparum-negative (n = 6) and -positive (n = 10) individuals. In panels c to f, box-whisker plots show median values, with 25th and 75th percentiles in boxes and 10th and 90th percentiles as whiskers. Thin-lined boxes represent P. falciparum-negative donors (n = 6); thick-lined boxes represent P. falciparum-infected donors (n = 10). *, P < 0.05. freq, frequency; pos, positive.


Figure 3
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  		FIG. 3. P. falciparum infection is associated with an enhanced ratio of P~p38 to P~ERK (pp38/pERK). Ratio of MFI of P~p38 to MFI of P~ERK in double-positive cells after stimulation for 30 and 60 min with LPS (a) or Pam3Cys (b). Box-whisker plots show median values, with 25th and 75th percentiles in boxes and 10th and 90th percentiles as whiskers. Thin-lined boxes represent P. falciparum-negative (mal neg) donors (n = 6); thick-lined boxes represent P. falciparum-infected (mal pos) donors (n = 10). The differences between P. falciparum-positive and -negative children were compared using a nonparametric Mann-Whitney test. **, P < 0.01.

Increased production of IL-10 and TNF-{alpha} in response to TLR2 activation. To determine whether increased MAPK activation was associated with increased cytokine production, production of TNF-{alpha} and IL-10 by PBMCs stimulated with LPS or Pam3Cys was assessed. There was a trend toward higher cytokine production by cells of P. falciparum-infected children after stimulation with LPS, while significantly higher levels of both IL-10 and TNF-{alpha} were produced by PBMCs of infected children than PBMCs of noninfected children following TLR2 stimulation with Pam3Cys (Fig. 4a and b, respectively) (P < 0.05 and P < 0.01). Adjustment for the presence of helminth infections or for age did not affect the relationship between malarial infection and cytokine production (linear regression analysis) (data not shown).


Figure 4
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  		FIG. 4. Increased production of IL-10 and TNF-{alpha} in response to TLR2 activation in P. falciparum-infected children. Cytokine responses of PBMCs from P. falciparum-infected (mal pos; n = 18) and -noninfected (mal neg; n = 18) children after stimulation with LPS or Pam3Cys. Values obtained after stimulation with medium were subtracted. TNF-{alpha} (a) and IL-10 (b) were measured in the supernatant after 72 h of stimulation. P. falciparum-infected individuals are represented with closed symbols; noninfected individuals are represented with open symbols. Horizontal lines indicate median values per group. The cytokine responses between the groups were compared for each stimulus using a nonparametric Mann-Whitney test. *, P < 0.05; **, P < 0.01. (c) Correlation plot of TNF-{alpha} levels and ratio of P~p38/P~ERK (pp38/pERK) 30 min after stimulation with Pam3Cys (both infected and noninfected children). A Spearman rank test was used to test for correlation.

In addition, 30 min after stimulation with Pam3Cys, but not with LPS, the ratio of P~p38/P~ERK correlated positively with the levels of TNF-{alpha} produced after 3 days of stimulation (r = 0.65; P < 0.01) (Fig. 4c), indicating that TLR2 activation is associated with a stronger early signal in P. falciparum-infected children, which has consequences for cytokine production profiles.

In vitro prestimulation with P. falciparum antigens. Since the results obtained from individuals living in an area in Ghana in which malaria is endemic indicated that TLR responsiveness was increased in P. falciparum-infected subjects, we wished to determine whether prestimulation of PBMCs with malarial antigens in vitro would also result in increased TLR responsiveness. To establish that it is exposure to P. falciparum parasites and not other factors associated with P. falciparum infection that mediates this effect, PBMCs from healthy European donors were prestimulated with P. falciparum-infected RBC, followed by stimulation with either LPS or Pam3Cys and quantification of cytokine (TNF-{alpha} and IL-10) production. Preexposure of cells to LPS resulted in TLR unresponsiveness, whereas preexposure to iRBC led to a greatly enhanced TNF-{alpha} response to both LPS and Pam3Cys compared with preexposure to uRBC, as shown in Fig. 5a. IL-10 levels were too low to be detected. Since most European donors would not have been exposed to malaria, we also performed these prestimulation experiments with PBMCs of the Gabonese donors who had had frequent exposure to P. falciparum infection (Fig. 5b). The responsiveness of these cells to both LPS and Pam3Cys was also increased following prestimulation with iRBC, indicating that the exposure to iRBC enhances TLR2 responsiveness in both naïve and malaria-exposed subjects, which confirms the results with samples obtained after in vivo exposure to P. falciparum.


Figure 5
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  		FIG. 5. In vitro prestimulation with P. falciparum antigens leads to enhanced TLR responsiveness. Per well, 1.0e5 PBMCs were stimulated for 24 h with 1.0e6 iRBC, 1.0e6 uRBC, or LPS; cells were washed and restimulated for 6 h with LPS or Pam3Cys. TNF-{alpha} was measured in the supernatants. Mean values with standard errors of the means are given per group of donors. (a) European donors (n = 4). (b) Gabonese donors (n = 6). Differences between samples prestimulated with uRBC and samples prestimulated with iRBC were tested using a paired t test. *, P < 0.05; **, P < 0.01.


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DISCUSSION
 
Our data show that TLR expression and responses are enhanced in individuals carrying asymptomatic P. falciparum infection. The expression levels of the TLR2 gene, the number of TLR2-expressing monocytes, and TLR2 signaling per cell induced by the TLR2 ligand Pam3Cys were all increased in P. falciparum-infected subjects. The latter was reflected by increased MAPK activation that resulted in higher production of TNF-{alpha} and IL-10. A recent report on TLR responsiveness during acute experimental P. falciparum infection of malaria-naive volunteers showed that both TLR4 and TLR2 responses were enhanced 8 days after infection and that these responses returned to baseline after treatment (18). Thus, in naive as well as malaria-experienced populations, the presence of iRBC is associated with increased TLR2 responsiveness of the innate immune system.

All the children in our study population were more than 5 years old with relatively low levels of P. falciparum parasitemia and no clinical symptoms of malaria at the time of inclusion, a condition generally referred to as asymptomatic malaria. The response to the TLR2 ligand Pam3Cys was clearly enhanced in infected subjects, whereas this was less pronounced when TLR4 was targeted. This is consistent with results showing that TLR2 but not TLR4 mRNA expression was significantly higher in infected subjects. By flow cytometry we found an increased number of monocytes expressing high surface levels of TLR2 in samples from P. falciparum-infected individuals. Recently, data have been published showing high expression levels of TLR2 mRNA in whole blood of patients with tuberculosis (4), but not all infections lead to enhanced TLR expression. Helminth infections, for example, appear to lead to decreased TLR2 expression (2) (11).

The higher number of monocytes exhibiting high expression levels of TLR2 was associated with an increased percentage of cells with the activated, phosphorylated form of p38 MAPK following TLR2 activation in infected individuals. Although the number of P~p38-positive cells also increased after stimulation with LPS, this difference was not significant. Since the staining procedure for the MAPK destroyed the binding site for the TLR, it was not possible to directly link TLR2 expression with MAPK phosphorylation. The ratio of activated p38 to activated ERK, which has been linked to the induction of an inflammatory response (20), was higher in infected donors and correlated with the levels of TNF-{alpha} produced after stimulation with Pam3Cys. The enhanced responsiveness of TLR2 and, less strongly, TLR4 in infected individuals might be the result of direct interactions of specific parasite-derived PAMP more strongly with TLR2 than with TLR4. Candidate P. falciparum PAMP include glycosylphosphatidylinisitol membrane structures that preferentially activate TLR2 (13).

The in vitro data we present provide support for the idea that P. falciparum infection enhances TLR responsiveness, which is rather unexpected given that prestimulation of PBMCs with LPS induced (cross) tolerance and almost completely abolished responsiveness to LPS and Pam3Cys in PBMCs from both Dutch and Gabonese donors. Indeed, this is in contrast to the finding that prestimulation experiments with P. falciparum-infected RBCs showed enhanced responsiveness of PBMCs, not only of malaria-naive European subjects, consistent with recently published data (18), but importantly also in those of individuals with chronic exposure to malaria. The absence of LPS-like tolerance and cross-tolerance of monocytes seen following preincubation of PBMCs with P. falciparum-infected RBCs and restimulation with LPS is thus an intriguing aspect of the biology of the host-parasite interaction. It is possible that continuous activation of the innate immune system is required to control parasitemia. Studies in an experimental murine model of malaria have shown that dendritic cells isolated from mice that are resolving the infection produced lower levels of TNF-{alpha} upon TLR stimulation, whereas the levels of IL-10 were increased (23). Our results show increased production of not only IL-10 but also TNF-{alpha} by total PBMCs in infected humans. Concurrent production of IL-10 may serve to reduce the inflammatory action of TNF-{alpha}, helping to prevent an excessive inflammatory response that could lead to clinical symptoms while nevertheless allowing a low but persistent level of parasitemia necessary for maintenance of effective acquired immunity (for a review, see reference 6).

The increased TLR responsiveness in individuals with asymptomatic malaria might also have implications for responses to third-party antigens. The impact of asymptomatic parasitemia on vaccination strategies, for example, has not been clarified. Some studies have indicated decreased responsiveness in asymptomatic subjects (10, 25), whereas others showed no differences in vaccination efficacy (19, 24). In the context of a malaria vaccine trial in small numbers of 5- to 9-year-old children, asymptomatic parasitemia did not appear to affect vaccine immunogenicity (8); however, in another study IFN-{gamma} responses to a malaria vaccine were suppressed in asymptomatic children (1 to 6 years old) (3). Asymptomatic parasitemia is frequent in older children and adults living in areas where malaria is endemic but much less so in infants and young children, who represent the ultimate target groups for vaccination. Any impact is therefore likely to be of particular relevance when candidate asexual-stage malaria vaccines enter age-deescalating phase Ib/IIb trials that will include assessments of immunogenicity. It will be important to study immune responses of different TLR family members and responses to different pathogen extracts in infected individuals in order to anticipate possibly modified responses in areas where malaria is endemic. Essential at the molecular level will be an understanding of why the tolerance generally induced by repeated stimulation of TLRs by pathogens appears not to be the case with P. falciparum.


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ACKNOWLEDGMENTS
 
We are indebted to the children for their participation in this study. We thank Marga van de Vegte-Bolmer for technical expertise in parasite culture; Annemiek Dijkhuis for her contribution to the MAPK experiments; and Zahildul Islam, Willemijn Scholten, and Cell Signaling Technologies for their help in optimizing the MAPK staining protocol.

We have no conflicting financial interests.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Parasitology, Leiden University Medical Centre, Albinusdreef 2, 2333 ZA Leiden, The Netherlands. Phone: 31 71 5265066. Fax: 31 71 5266907. E-mail: F.C.Hartgers{at}lumc.nl Back

{triangledown} Published ahead of print on 18 August 2008. Back

Editor: J. F. Urban, Jr.


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Infection and Immunity, November 2008, p. 5149-5157, Vol. 76, No. 11
0019-9567/08/$08.00+0     doi:10.1128/IAI.01579-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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