Plasmodium falciparum infection of the placenta impacts on the T helper type 1 (Th1)/Th2 balance of neonatal T cells through CD4⁺CD25⁺ forkhead box P3⁺ regulatory T cells and interleukin-10

Abstract

Placental malaria infection affects the T helper type 1 (Th1)/Th2 balance in neonatal children. We investigated a potential role of regulatory T cells in this balance by comparing T cell responses of cord blood mononuclear cells (CBMC) from parasitized and non-parasitized placenta of Gambian women. CBMC were depleted of CD4⁺CD25⁺ forkhead box P3 (FoxP3)⁺ regulatory T cells and analysed in vitro for their ability to produce interferon (IFN)-γ, sCD30 and interleukin (IL)-10 in response to phytohaemagglutinin (PHA), live Plasmodium falciparum,schizont extracts and the recombinant P. falciparum blood stage antigen merozoite surface protein 1 (MSP1₁₉). As expected, lower IFN-γ and higher sCD30 responses were observed for the cells from the parasitized group. In addition, higher IL-10 levels were produced by CBMC from the parasitized group. Depletion of regulatory T cells decreased IL-10 production, which resulted in a restoration of IFN-γ expression in response to all stimuli. The Th2 marker sCD30 remained significantly higher in the parasitized group in response to malaria protein antigens while similar levels were recovered between both groups in response to live P. falciparum. Similar effects were observed by adding an antibody that blocks IL-10 function. These results suggest that the impact of P. falciparum infection on Th1 differentiation of neonatal T cells can be ascribed to regulatory T cells through production of IL-10.

Introduction

Malaria parasites cause approximately 300–600 million clinical attacks and 2 million deaths per year [1]. Pregnant women are particularly susceptible. The prevalence of placental malaria, in which parasitized red blood cells are sequestered in the placenta, ranges from 30 to 70% of gravid women in many malaria endemic areas, including The Gambia. This condition is associated with increased risk of abortion, stillbirths, low birth weight and infant mortality [2,3]. Using a birth cohort in 2001 that enrolled pregnant Gambian women, the immunological responses to mitogens and Plasmodium falciparum schizont extracts were compared for cord blood mononuclear cells (CBMC) from parasitized and non-parasitized placentas [4]. We found that placental malaria infection also impacts on the newborn infant's immunological response [4]. Placental infection with P. falciparum inhibited production of the T helper type 1 (Th1) cytokines interferon (IFN)-γ and interleukin (IL)-12 and stimulated expression of the Th2 marker sCD30 when the lymphocytes of neonatal children were examined. These results suggested that the parasite has acquired an ability to manipulate T cell immunity by affecting the Th1/Th2 balance and that an active mechanism of T cell suppression may operate during the course of malaria infection. Regulatory T cells (T_regs) have been shown recently to contribute to the maintenance of P. falciparum infection [5,6]. Moreover, parasite antigen-specific CD4⁺ regulatory cells are generated in utero as a consequence of P. falciparum infection of the placenta [7]. The functional role of T_regs in the balance of Th1/Th2 immune responses during P. falciparum infection in newborns is still poorly understood. We therefore examined the possible role of T_regs in immune dysregulation during active placental P. falciparum infection, using frozen cord blood samples collected from the 2001 Sukuta cohort (pilot study). We compared both the magnitude and the Th1/Th2 balance of cytokines induced in vitro by mitogen, malaria antigens and live parasites, both before and after depletion of T_regs or the addition of anti-IL-10 blocking antibodies to CBMC cultures.

Materials and methods

Population

Details of the study population have already been published [4]. Briefly, in collaboration with the Gambian Ministry of Health, a study was undertaken in the peri-urban village of Sukuta 11 km from the Medical Research Council (MRC) Laboratories in Fajara during the period of September 2001 to January 2002. Primiparous and multiparous pregnant women were recruited into the study. After obtaining written informed consent, up to 50 ml of cord blood was collected upon delivery. Active malaria infection was assessed by microscopic examination of Giemsa-stained placental imprints and cord bloods. All slides were read twice by experienced microscopists and discrepancies resolved by a third reader (limit of detection, approximately 2 parasites/µl) [8]. The study was approved by the Joint Gambia Government/MRC Ethics Committee.

Parasite cultivation and schizont collection

P. falciparum parasites (Pf164 line) were cultured in vitro, as described previously [9]. Parasites at a parasitaemia of approximately 5% ring stages were synchronized using repeated treatments with 5% sorbitol for 20 min at 37°C [10] and then mature parasite-infected erythrocytes were harvested by flotation on a cushion of Plasmagel (a final concentration of 2% gelatine in incomplete culture medium) [11]. The harvested parasites were mixed with fresh uninfected red blood cells and cultured to a final parasitaemia of 10%. The stages of parasite development were assessed by microscopy, as described previously [12].

Parasite cultures were tested at regular intervals for contamination with Mycoplasma spp. by using a polymerase chain reaction (PCR)-based mycoplasma-detection kit (American Type Culture Collection, Manassas, VA, USA). A crude preparation of P. falciparum antigens was obtained by freezing and thawing of parasite-infected erythrocytes. The extracts were titrated on cells from semi-immune adults to identify conditions for optimal induction of cell activation, which was set as a concentration of 10 µg/ml. Extracts from uninfected erythrocytes were used as negative controls.

Culture of cord blood mononuclear cells

Selected frozen samples of CBMC of the Sukuta cohort were thawed, washed with RPMI-1640 medium (Sigma, St Louis, MO, USA) and resuspended in RPMI-1640 medium supplemented with 20 µg/ml gentamycin (Sigma), 2 mM L-glutamine (Sigma) and 10% human antibody serum (Sigma). CBMC (viability reached 85%) were cultured (2 × 10⁵ cells in 200 µl complete medium) for either 3 days in the presence of phytohaemagglutinin (PHA-L, 2·5 µg/ml; Sigma) or 6 days in the presence of erythrocytes infected with P. falciparum (Pf164) at 10% parasitaemia or schizont extracts at 10 µg/ml. Uninfected erythrocytes or extracts from them were used as negative controls, respectively. Merozoite surface protein 1 (MSP1₁₉) [13] at a final concentration of 5 µg/ml was used to examine the T cell response to a blood stage antigen. All assays were performed in triplicate. Cytokines (IFN-γ, IL-10 and soluble sCD30) in the culture supernatants were measured using enzyme-linked immunosorbent assay (ELISA).

CD4⁺CD25⁺ T cell depletion and analysis

CBMC were depleted of CD4⁺CD25⁺ cells using Miltenyi beads (Miltenyi Biotec, Bergisch Gladbach, Germany), following the manufacturer's instructions. Briefly, the depletion was performed in two steps. First, a cocktail of biotinylated antibodies [with specificity for CD16, CD14, CD8, CD19, CD56 and γδ T cell receptor (TCR)] together with anti-biotin microbeads (microbeads conjugated to monoclonal anti-biotin antibody) was used to separate the non-CD4⁺ T cells from the CD4⁺ T cells. Then anti-CD25 antibody and anti-biotin microbeads were added to the CD4⁺ fraction to deplete the CD4⁺CD25⁺ cells. The resultant CD4⁺CD25^- T cells were then added back to the non-CD4⁺ fraction to reconstitute the cell population for in vitro stimulation. The positively selected CD4⁺CD25⁺ T cells were also eluted from the columns and cultured (10⁶/ml) overnight in vitro with PHA to assess their IL-10 production in comparison with CD4⁺CD25^- fraction.

Anti-CD25-PE and anti-CD4-fluorescein isothiocyanate (FITC) antibodies (Becton-Dickinson, UK) were used for quality control of the separations and also to compare frequency of CD4⁺CD25⁺ population in groups from parasitized and non-parasitized placenta. Briefly, in this later case, selected CBMC samples were thawed and stained for 20 min at 4°C, washed with phosphate-buffered saline–bovine serum albumin (PBS–BSA) 2% and analysed using a fluorescence activated cell sorter (FACS)Calibur (Becton-Dickinson).

RNA extraction and reverse transcription

Total RNA was extracted from CD4⁺CD25⁺ T cell fraction and CD4⁺CD25^- cell fraction using a total RNA isolation system (Promega Corporation, Madison, WI, USA). The reaction was incubated at 37°C for 1·5 h, and stopped by incubation at 95°C for 5 min. Semi-quantitative reverse transcription–polymerase chain reaction (RT–PCR) was used to analyse forkhead box P3 (FoxP3) expression using specific primers. Glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) served as a positive control for each cDNA sample. Amplification reactions were performed in a volume of 25 µl containing 1 µl cDNA as template, 0·2 µmol of each primer, 0·5 U of Taq polymerase (Boehringer Mannheim, Mannheim, Germany), 0·1 µmol of each dNTP and 1×Taq polymerase buffer. GAPDH primer sequences were 5′-GCAAATTCCATGGCACCGT (forward) and 5′-TCGCCCCACTTGATTTTGG (reverse). FoxP3 primer sequences were 5′-AGCTGGAGTTCCGCAAGAAAC (forward) and 5′-TGTTCGTCCATCCTCCTTTCC (reverse).

Human cytokine determinations

IFN-γ, IL-10 and sCD30 levels in culture supernatants were determined using ELISA kits from Biosource (IFN-γ, IL-10; BioSource Europe, Fleurus, Belgium), MedSystems (sCD30; MedSystems Diagnostics GmbH, Vienna, Austria), following the manufacturer's instructions.

Statistical analysis

Paired and non-paired t-tests were performed on log-transformed data (which normalized the distribution) to compare the significance of results between the groups with and without detectable parasitaemia.

Results

CD4⁺CD25⁺ Fox-P3⁺ T cell frequency in the CBMC

Detailed epidemiological parameters on the Sukuta pilot study have been reported previously [4]. Briefly, one-third of the women (25 of 73, 34·3%) enrolled into the study had active malaria infection in the placenta at delivery. Fifty per cent of selected cord blood samples from parasitized placenta were also positive for the P. falciparum; however, the parasitaemia was very low (mean = 2500 parasites/µl) and all infants were aparasitaemic at 1 week of age.

No association was found with cord blood parasitaemia and Th differentiation [4]. The parasitized and non-parasitized groups were similar for maternal age, parity, placental weight, birth weight and reported chloroquine use. Cord blood samples from both groups were collected and used for evaluation of the Th1/Th2 T cell ratio [4]. A fraction of each sample had also been kept frozen. We used some of these frozen samples to assess the role of T_regs in the changed T cell ratio.

We excluded specific selection of T_regs due to the freezing process, as frozen samples and fresh CBMC displayed similar responses to PHA, protein antigens or allo-mixed lymphocyte reaction (allo-MLR) (not shown).

We first compared frequencies of CD4⁺CD25⁺ FoxP3⁺ T cells in both groups, then depleted these cells from the population and cultured the residual CBMC in the presence of mitogen, live parasite or malaria protein antigen. CD4⁺CD25⁺FoxP3⁺ and CD4⁺CD25⁺FoxP3^low fractions were stimulated overnight with PHA and the higher IL-10 production by CD4⁺CD25⁺ T cells compared to CD4^- CD25^- T cells suggested strongly that all or part of CD4⁺CD25⁺ FoxP3⁺ cells are T_regsand not effector cells (Fig. 1).

Fig. 1.

(a) Quality control of CD4⁺CD25⁺ T cell depletion and (b) their forkhead box P3 (FoxP3) expression and (c) interleukin (IL)-10 production after stimulation with phytohaemagglutinin (PHA) (18 h). Significantly higher FoxP3 mRNA and IL-10 amounts were produced by CD4⁺CD25⁺ (n = 5) compared to CD4⁺CD25^- T cells (n = 6) (*P = 0·0427).

Frequencies of CD4⁺CD25⁺ FoxP3⁺ T cells were low and similar in samples from both groups (1·2% ± 0·2 compared to 1·4% ± 0·3, respectively; P = 0·11). Approximately 10% of CD4⁺ T cells expressed the α-chain of IL-2 receptor CD25, whereas only 1–2% of the CD4⁺ T cells were CD25⁺ in both groups of donors (Fig. 2).

Fig. 2.

Selected cord blood mononuclear cells (CBMC) samples from parasitized (n = 8) and non-parasitized groups (n = 8) were stained with anti-CD25-phycoerythrin and anti-CD4-fluorescein isothiocyanate and analysed using a fluorescence activated cell sorter (FACS)Calibur. One of eight samples is shown.

Effect of T_regs on Th1/Th2 cytokines produced in response to PHA

As expected from the previous study [4], placental infection decreased IFN-γ production significantly (p2 = 0·03) from cord blood cells in vitro in response to PHA without affecting expression of sCD30 (Fig. 3). Interestingly, we found higher levels of IL-10 in cultures of CBMC from parasitized placenta (p5 = 0·025). Depletion of T_regs resulted in a significant decrease in IL-10 production of both groups (p3 = 0·028; p4 = 0·035) and a significant increase in some of the other cytokines measured. In particular, depletion of T_regs resulted in recovery of IFN-γ production in cultures of cells from the parasitized group (p1 = 0·02). A lower but non-significant decrease in IL-13 production by cells from the parasitized group (not shown) and slightly higher levels of sCD30 in both groups were observed subsequent to depletion of T_regs (Fig. 3).

Fig. 3.

Cytokine production by cord blood mononuclear cells (CBMC) in response to phytohaemagglutinin (PHA). CBMC from newborns whose mothers had malaria infection of the placenta at delivery (black bars, n = 21) or were free of infection (grey bars, n = 34) were cultured for 3 days with PHA and assessed for their cytokine production and sCD30 release. T helper type 1 (Th1) [interferon (IFN)-γ] and Th2 (sCD30) markers, as well as the anti-inflammatory cytokine interleukin (IL)-10 were measured using enzyme-linked immunosorbent assay (ELISA). Measurements were made with (+) or without (−) CD4⁺CD25⁺ regulatory T cell depletion. As already reported [4], significantly less IFN-γ was produced by CBMC obtain from the group with parasitized placenta (P2 = 0·003), while the levels of the Th2 markers were similar between groups. Higher IL-10 levels were produced by cells from the parasitized placenta group (P5 = 0·025). Depletion of regulatory T cells decreased IL-10 production in cells from both groups (p3 = 0·028, p4 = 0·035) and restored IFN-γ production (p1 = 0·02). Results are shown as mean ± standard error of the mean.

Effects of T_regs on the Th1/Th2 CBMC cytokines measured in response to live parasites and malaria antigens

CBMC were also tested for their responses to live P. falciparum-infected red blood cells, Pf164 schizont extracts, and purified MSP1₁₉ protein after 6 days of culture. CBMC cultured in the presence of uninfected red blood cells, in the presence of extracts of red blood cells and in medium alone were used as negative controls, respectively (Fig. 4).

Fig. 4.

Cytokine production by cord blood mononuclear cells (CBMC) in response to live Plasmodium falciparumand P. falciparumantigens. CBMC from non parasitized (−) (n = 21) and parasitized (+) (n = 25) placenta were assessed for cytokine production and sCD30 release in response to treatment with the merozoite surface protein 1 (MSP1₁₉) protein antigen and an extract from P. falciparumPf164 schizonts, or upon stimulation with live parasitized red blood cells. Unstimulated CBMC or CBMC stimulated with either uninfected red blood cell extracts or uninfected intact erythrocytes were used as negative controls, respectively. Measurements were made with (grey bars) or without (black bars) CD4⁺CD25⁺ regulatory T cell depletion. Lower interferon (IFN)-γ production (Pf164schizonts p1 = 0·0485; MSP1₁₉ p2 = 0·0497, live Pfp6 = 0·015), higher sCD30 (Pf164schizonts p7 = 0·0245; MSP1₁₉ p8 = 0·021; livepf p9 = 0·028) and higher interleukin (IL)-10 production (Pf164schizonts p12 = 0·048; MSP1₁₉ p13 = 0·0478; live Pfp15 = 0·038) in response to malaria protein antigens Pf164schizonts and MSP119 and live parasite. CD4⁺CD25⁺ T cell depletion decreased IL-10 in both groups and significantly in response to live Pf(p14 = 0·0315), maintained the higher sCD30 production in response to malaria protein antigens in the parasitized group (Pf164schizonts (p10 = 0·032; MSP1₁₉ p11 = 0·041) and restored IFN-γ production in response to live parasite (p3 = 0·0209). Results are shown as mean ± standard error of the mean (minus backgrounds).

Lower IFN-γ production (Pf164schizonts p1 = 0·0485; MSP1₁₉ p2 = 0·00497) and higher IL-10 levels (Pf164schizonts p12 = 0·048; MSP1₁₉ p13 = 0·0478) were observed in response to malaria antigens Pf164 schizonts and MSP1₁₉ in the parasitized group compared to the non-parasitized group. Moreover, higher levels of sCD30 were observed in response to malaria antigens (Pf164schizonts p7 = 0·0245; MSP1₁₉ p8 = 0·021) in the parasitized group. CD4⁺ CD25⁺ T cell depletion restored levels of IFN-γ production, decreased IL-10 and increased sCD30 production in both groups. In this later case the difference between both groups remained significant (Pf164schizonts p10 = 0·032; MSP1₁₉ p11 = 0·041).

Live parasites induced higher concentrations of all tested cytokines compared to protein antigen. It induced significantly lower levels of IFN-γ (p6 = 0·015), higher levels of IL-10 (p15 = 0·038) and higher amounts of sCD30 (p9 = 0·028) in the parasitized group. CD4⁺ CD25⁺ T cell depletion decreased IL-10 production (p14 = 0·049), restored IFN-γ production (p3 = 0·0209) and increased sCD30 in both groups leading to a similar production between both groups.

Effect of IL-10 production on the Th1/Th2 CBMC cytokines

As shown in Figs 3 and 4, higher levels of IL-10 were detected in CBMC cultures derived from the parasitized group and depletion of CD4 T cells resulted in lower IL-10 production and restoration of IFN-γ expression in CBMC from the parasitized group. To examine the direct role of IL-10, we compared cultures of CBMC from both groups in the presence of live parasites, either after depletion of T_regs or in the presence of neutralizing anti-IL-10 antibodies. As shown in Fig. 5, both depletion of T_regs and anti-IL-10 treatment restored IFN-γ production in cells from the parasitized group. In contrast, anti-IL-10 did not have a significant impact on sCD30 (data not shown).

Fig. 5.

Treatment of cell cultures with anti-interleukin (IL)-10 antibody restored interferon (IFN)-γ production in the cultures of cells from the group with parasitized placenta. Cord blood mononuclear cells (CBMC) from parasitized (upper figure, black bars; n = 10) and non-parasitized (lower figure, grey bars; n = 12) placenta were either depleted of regulatory T cells (T_regs^-) or left untreated and cultured in the presence of anti-IL-10 antibody or an isotype control antibody. Following stimulation with live Plasmodium falciparum Pf164-infected red blood cells, IFN-γ release was measured. Significant IFN-γ recovery was observed after regulatory T cell depletion (P1 = 0·014) and anti-IL-10 blocking antibody (P2 = 0·021). Results are shown as the mean ± standard error of the mean (minus backgrounds).

Discussion

P. falciparum placental infection impacts on newborn's immune responses, as both Th1 and Th2 differentiation of newborn's T cells have been reported [4,14]. Mice models versus human malaria, maternal immunity and placental damage may explain these discrepancies. In our hands, previous results from a Sukuta cohort of Gambian primi- and multiparous pregnant women indicated that active placental infection impacts on IFN-γ, IL-12 and sCD30 production of neonatal T cells (lower IFN-γ and IL-12 and higher sCD30) [4]. This study, together with the reported data on impaired IL-12 function in neonates [15], and the inhibitory effect of P. falciparum on dendritic cell (DC) maturation [16], suggested that the reduced production of IFN-γ could be attributed to an inhibition of antigen-presenting cells (APC) in the cultures from the parasitized group. Recent reports indicated that T_regs might be also incriminated in the maintenance of malaria infection through production of high levels of IL-10 and transforming growth factor (TGF)-β. These cytokines, common to APC and T_regs, are known immunomodulatory molecules and may be involved in the inhibition of Th1 cytokine production by the CBMCs from the parasitized placenta group.

We therefore focused on the possible role of this T cell population in the observed Th1/Th2 imbalance. Using the same cohort, we compared CD4⁺CD25⁺ FoxP3⁺ T cell frequencies and cultured selected samples of CBMC before and after depletion of CD4⁺CD25⁺ FoxP3^low cells in the presence of PHA, Pf164 schizont extracts, MSP1₁₉ and live parasites and tested for their ability to elicit IFN-γ, sCD30 and IL-10 secretion. The results confirm our previous observation that active infection of the placenta by P. falciparum exacerbates the Th2 bias of neonatal lymphocytes, as evidenced by the lower IFN-γ in response to all antigens, and higher sCD30 production in response to protein antigens and live Pf by cells from the parasitized group. We have found a higher IL-10 production in response to all antigens as well as higher IL-10 production by the CD4⁺CD25⁺ population in response to PHA, which supports their regulatory phenotype. As stated above, IL-10 production may be attributed to APC but CD4⁺ CD25⁺ FoxP3⁺ T_regs may also be important [5,6]. To discriminate between the different cell populations we depleted CD4⁺CD25⁺ T cells from the CBMC and reassessed the Th1/Th2 cytokines in vitro. The depletion resulted in lower IL-10 production and restoration of IFN-γ production in response to all tested stimuli. No significant post-depletion effect was observed on IL-13 (not shown). Interestingly, an antigen-dependent response was observed for sCD30, e.g. depletion did not affect the response to protein antigens as the higher sCD30 level observed in the parasitized group remained significant. In contrast, it re-established similar levels between both groups in response to live Pf.

The higher levels of cytokines induced in response to live Pf compared to protein antigens as well as the antigen-associated Th2 differentiation suggest a different and probably a complex mechanism of interaction of APC with these different stimuli.

Despite the similar frequencies of CD4⁺CD25⁺ T cells between both groups, which is in contrast to previous reported data [7], depletion of this population impacted on Th1/Th2 cytokines in vitro. The frequency differences may be ascribed to different placental parasitaemia levels, active and/or chronic infection between both cohorts [7,9], and the post-depletion effects observed in our cohort may be attributed to a different CD4⁺CD25⁺ activation state, a synergistic effect with APC or the interaction with other T cell populations. Furthermore [17], using a mouse model, Mold et al. have shown that T_regs of maternal origin cross the placenta. However, in our hands depletion of T_regs (CD25⁺ T cells) did not affect allo-MLR against maternal samples, as reported by Mold's group, suggesting that P. falciparum antigens and placental inflammatory cytokines primed mainly T_regs from CBMC.

To assess the role of IL-10 on Th1 and Th2 cytokines or markers, live Pf stimulation was privileged to protein antigens, as it induced higher concentrations of all tested cytokines and showed a significant difference before and after CD4⁺CD25⁺ FoxP3⁺ T cell depletion. Blockade of IL-10 function with a monoclonal antibody produced a similar profile of cytokine production to depletion of the T_regs, e.g. restoration of IFN-γ production. However, anti-IL-10 antibody had no significant effect on sCD30, e.g. higher levels were still observed in the parasitized group after this treatment (data not shown). This result suggests that the CD4⁺CD25⁺ FoxP3⁺ T cells in the parasitized group, at least through high levels of IL-10, may have reduced the IL-12 [18] production of APC which, in turn, decreased IFN-γ and increased sCD30 production by T cells. IL-10 antibodies were able to attribute the impact of CD4⁺CD25⁺ FoxP3⁺ population on Th1 cytokines to IL-10. These findings on T_reg effects on Th1 and Th2 differentiation highlight a complex strategy used by P. falciparum and require further investigation.

In summary, Th1/Th2 neonatal T cell imbalance to active P. falciparum infection of the placenta, a possible strategy used by P. falciparum to escape immune responses, implicates T_regs through IL-10 production, as both depletion of T_regs and blockade of IL-10 function restored IFN-γ production. Previous studies [4], as well as the current results, incriminate both APC through lower IL12 production and CD4⁺CD25⁺ FoxP3⁺ T_regs through IL-10 in IFN-γ decrease, and therefore exacerbation of the Th2 bias.

Disclosure

None.