Plasmodium falciparum exposure in utero, maternal age and parity influence the innate activation of foetal antigen presenting cells

Abstract

Background: Malaria in pregnancy is associated with immunological abnormalities in the newborns, such as hampered T-helper 1 responses and increased T-regulatory responses, while the effect of maternal Plasmodium falciparum infection on foetal innate immunity is still controversial.

Materials and methods: The immunophenotype and cytokine release by dendritic cells (DC) and monocytes were evaluated in cord blood from 59 Beninese women with or without malaria infection by using flow cytometry.

Results: Accumulation of malaria pigment in placenta was associated with a partial maturation of cord blood myeloid and plasmacytoid DC, as reflected by an up-regulated expression of the major histocompatibility complex class II molecules, but not CD86 molecules. Cells of newborns of mothers with malaria pigment in their placenta also exhibited significantly increased cytokine responses upon TLR9 stimulation. In addition, maternal age and parity influenced the absolute numbers and activation status of cord blood antigen-presenting cells. Lastly, maternal age, but not parity, influenced TLR3, 4 and 9 responses in cord blood cells.

Discussion: Our findings support the view that placental parasitization, as indicated by the presence of malaria pigment in placental leukocytes, is significantly associated with partial maturation of different DC subsets and also to slightly increased responses to TLR9 ligand in cord blood. Additionally, other factors, such as maternal age and parity should be taken into consideration when analysing foetal/neonatal innate immune responses.

Conclusion: These data advocate a possible mechanism by which PAM may modulate foetal/neonatal innate immunity.

Figure 1

(A) Flow cytometric identification of MDC (BDCA-1⁺ or BDCA-3⁺) and PDC (BDCA-2⁺) in CBMC from one subject. R1 gate was set to include only viable mononuclear cells, as determined by forward scatter (FSC) and side-scatter (SSC) characteristics. Furthermore, cells were gated to be both CD14 and CD19 negative (R2) and either BDCA-1, BDCA-3, or BDCA-2 positive (R3). One million events were analysed for DC, 300,000 events for monocytes and 100,000 events for isotype control enumeration. The percentages represent relative levels of the different DC populations from the selected subject. (B) Partial activation of cord blood BDCA-1⁺ and BDCA-2⁺ cells is related to the presence of MP in placenta. The expression levels of HLA-DR and CD86 were measured by flow cytometry on foetal APC subsets from 16 MP-positive (diagonal striped bars) and 39 MP-negative mothers (white bars). Boxplots illustrate the medians and the 25^th and 75^th percentiles. Y-axes report percentage of positive cells and mean fluorescence intensity (MFI) for the activation markers CD86 and HLA-DR in different APC subsets. P-values were calculated by Mann-Whitney U test. The significance limit was P < 0.05.

Figure 2

Association between APC numbers and activation and maternal age or parity. (A) Data were segregated into 2 groups according to the median value for maternal age. Absolute numbers and CD86 expression levels (as percentage of positive cells and mean fluorescence intensity, MFI) on different foetal APC subsets were analysed as a function of maternal age in 31 women ≤ 25 years of age and 24 women > 25 years of age Boxplots illustrate the medians and the 25^th and 75^th percentiles. (B) Women were divided for parity as paucigravidae (1st and 2nd pregnancy) and multigravidae (≥ 3 pregnancies) as we previously observed that women at first and second pregnancy exhibited the same risk of malaria infection (Fievet, unpublished data). Absolute numbers and HLA-DR expression levels (as percentage of positive cells and mean fluorescence intensity, MFI) on different foetal APC subsets were analysed as a function of parity in 37 women undergoing first or second pregnancy and 18 multigravidae. Boxplots illustrate the medians and percentiles. P-values were calculated by Mann-Whitney U test. The significance limit was P < 0.05.

Figure 3

TLR-induced cytokine responses in CBMC obtained from women with or without MP accumulation in placenta. CBMC (2 million/ml) were stimulated or not with synthetic Hz (5 μg/ml), LPS (100 ng/ml), CpG-A (3 μg/ml) or PolyI:C (20 μg/ml). After 18 hours, supernatants were collected and analysed for IL-6, IL-10, IL-12, IFN-α, TNF-α and MIP-1α/CCL3 levels. Data represent median values and percentiles for 59 individuals; 16 MP-positive (diagonal striped bars) and 43 MP-negative mothers (white bars). Cytokine levels in unstimulated cells were subtracted from the values shown. P-values were calculated by Mann-Whitney U test or t test (see Subjects, Materials and Methods). The significance limit was P < 0.05.

Figure 4

Cord blood cytokine levels are affected by maternal age, but not by maternal P. falciparum infection or MP accumulation in placenta. Foetal plasma samples were collected at delivery and subsequently analysed for IL-6, IL-10, IL-12, IFN-γ, TNF-α and MIP-1α/CCL3 levels. (A) Data represent median values and percentiles for 57 individuals divided in 27 P. falciparum negative mothers (Pf-negative, white bars) and 30 P. falciparum positive mothers (Pf-positive, diagonal striped bars). (B) Women were divided in 39 MP-negative mothers (white bars) and 18 MP-positive (diagonal striped bars). (C) Women were divided in 33 mothers ≤ 25 years of age (white bars) and 24 women > 25 years (diagonal striped bars) Values were calculated by Mann-Whitney U test or t test (see Subjects, Materials and Methods). The significance limit was P < 0.05.

Figure 5

TLR-induced cytokine responses in foetal leukocytes are influenced by maternal age. CBMC (2 million/ml) were stimulated with synthetic Hz (5 μg/ml), LPS (100 ng/ml), CpG-A (3 μg/ml) or PolyI:C (20 μg/ml). After 18 hours, supernatants were collected and analysed for IL-6, IL-10, IL-12, IFN-α, TNF-α and MIP-1α/CCL3 levels. Cytokine levels in unstimulated cells were subtracted from the values shown. P-values were calculated by Mann-Whitney U test or t test (see Subjects, Materials and Methods). The significance limit was P < 0.05. Data represent median values and percentiles for 57 individuals; 33 mothers ≤ 25 years of age (white bars) and 24 women > 25 years (diagonal striped bars) (Data on maternal age were missing for 2 subjects, that were therefore not included in this analysis).

Figure 6

TLR-induced cytokine responses in foetal leukocytes are not influenced by parity. CBMC (2 million/ml) were stimulated with synthetic Hz (5 μg/ml), LPS (100 ng/ml), CpG-A (3 μg/ml) or PolyI:C (20 μg/ml). After 18 hours, supernatants were collected and analysed for IL-6, IL-10, IL-12, IFN-α, TNF-α and MIP-1α/CCL3 levels Cytokine level in unstimulated cells was subtracted from the values shown. P-values were calculated by Mann-Whitney U test or t test (see Subjects, Materials and Methods). The significance limit was P < 0.05. Data represent median values and percentiles for Women were segregated on the basis of parity as 41 paucigravidae (1st and 2nd pregnancy; white bars) and 18 multigravidae (≥ 3 pregnancies; striped bars).