Pregnancy-associated Plasmodium falciparum malaria (PAM) results, sometimes, in massive intervillous inflammation that contributes to placental insufficiency, impaired intra-uterine growth and consequently to low birth weight in the newborns and a higher risk of dying early in life 1234.

Infants born to women with PAM are more predisposed to P. falciparum infection in their first year of life 567. Immunological mechanisms are generally considered to play an important role in causing this susceptibility. In utero sensitization to transplacentally transferred soluble P. falciparum antigens may constitute the basis for increased susceptibility to malaria episodes in early life. Importantly, it has been demonstrated that cord blood mononuclear cells (CBMC) of neonates born to mother with PAM specifically respond to plasmodial asexual stage antigens, and that cord blood B cells produce anti-malaria specific IgM and IgE antibodies 58910, providing irrefutable evidence of in utero sensitization.

In this context, active infection in the placenta by P. falciparum was associated with hampered T-helper 1 (Th1) responses, as reflected by reduced IFN-γ production upon T-cell stimulation 9. In addition, the anti-inflammatory IL-10 cytokine is more frequently produced by CBMC of those born to mothers with PAM compared with non-infected mothers 11. CD4⁺CD25^high regulatory T-cells (Treg) are a principal source of IL-10 in such cases 12. Treg are found at higher frequency in cord blood (CB) of neonates born to mothers with PAM at delivery as compared to unexposed newborns 12.

Because of their key function in the initiation and regulation of adaptive immune responses, it is reasonable to assume that antigen presenting cells (APC), such as monocytes and dendritic cells (DC), contribute to the modulation of foetal immune responses upon exposure to P. falciparum in utero. Indeed, DC seem to play an important role in both protective and dysfunctional immune responses against malaria in murine models 1314. DC comprise a heterogeneous population of cells; myeloid DC (MDC) that orchestrate T-cell responses through a fine modulation of IL-12 secretion, while plasmacytoid DC (PDC) are an essential component of innate and adaptive immunity through secretion of type I interferons (IFN) in response to pathogens 15. A minor blood MDC population, blood DC antigen (BDCA)-3⁺ cells, has been described sharing the same ontogeny as the more frequent BDCA-1⁺ MDC subset 1617.

The foetal/neonatal immune system exhibits quantitative and functional differences from the adult one and neonatal DC have reduced ability in delivering co-stimulatory signals to T-cells as a consequence of their incomplete maturation 18. They also exhibit a markedly decreased capacity in secreting IL-12 and IFN-α 1920. This probably contributes to the development and relative predominance of Treg in CB 21, although seemingly less marked in Africans vs. Europeans 22.

Whether and how P. falciparum infection in the mother may affect foetal innate immunity is poorly understood. One study conducted in The Gambia reported lower lipopolysaccharide (LPS)-induced IFN-γ and IL-12 activity in CBMC of newborns of mothers with PAM as compared to uninfected mothers 9. A more recent study revealed that CBMC of neonates born of Gabonese mothers with P. falciparum infection exhibit significantly increased IFN-γ responses upon stimulation with toll-like receptor (TLR)3 and TLR4 ligands 22.

Contrasting findings have also been reported on the characterization of DC subsets in CBMC of neonates born to P. falciparum-infected mothers. One study reported a significantly higher frequency of MDC 23, while another reported profoundly reduced numbers of PDC 24 as compared to unexposed newborns.

The mechanistic hypothesis behind the present study is that malaria infection in the mother may cause a dysfunctional activation of foetal APC by parasite-derived products that cross the placenta. An altered activation of foetal APC could be responsible for the impaired T-cell response that is observed in infants born to mothers with PAM.

Using flow cytometry, subpopulations of DC and monocytes were evaluated in CB of neonates from Beninese women with or without malaria infection. In addition, the impact of P. falciparum exposure in utero, on the innate activation of foetal APC was examined by stimulating CBMC with specific TLR ligands; LPS was employed to activate TLR4 on monocytes and BDCA-1⁺MDC, polyinosine-polycytidilic acid (PolyI:C) to selectively stimulate TLR3 expressed in MDC, and CpG-A ODN to specifically activate TLR9 expressed in PDC 161725.

The consensus view is that in utero sensitization to P. falciparum antigens is a common phenomenon during PAM. Parasites do not usually cross the placental barrier and such sensitization is most probably caused by the transplacental passage of soluble P. falciparum antigens 828. Accordingly, no parasite-positive smears were detected in CB samples of malaria-infected women in this study population.

Pregnant women declared to receive either SP or CQ as malaria prevention during pregnancy. In contrast to a previous study performed in the same area 29, no differences were observed for P. falciparum infection rate according to the type of prophylaxis used by the mothers. However, the number of subject included in this study was low and the project was not designed to examine this matter.

In this study, the foetal BDCA-1⁺ and BDCA-2⁺ DC subsets expressed significantly higher levels of MHC class II molecules upon PAM, as indicated by the presence of P. falciparum MP in placenta, which is in agreement with previous data 23. The observation that CD86 expression on foetal DC was unaffected by PAM suggests that P. falciparum stimulation in utero induces only partial activation of these cells. Failure to provide DC with a sufficiently strong costimulatory signals can impair the ability to form stable interactions with T-cells, as recently shown in a murine model of malaria 30. Partial DC maturation can lead to altered T-cell activation and induction of tolerance 31323334, possibly contributing to impaired immune responses that have been observed in the offspring of mothers with PAM 911.

The findings presented in this study diverge from those of studies on peripheral blood DC from children with acute malaria, where expression levels of MHC class II on the BDCA-1⁺ DC are reduced compared to healthy controls 3536. Also, no increase in foetal BDCA-3⁺ DC was detected upon maternal malaria infection in this study like others have shown in children with severe malaria 35. Circulating APC are continuously exposed to P. falciparum-infected erythrocytes during malaria episodes in children, which may exert a contact-mediated inhibitory effect on DC functionality, as demonstrated by in vitro studies 437. Conversely, infected erythrocytes are rarely detected in CB of those born to mothers with PAM 2338. Thus, foetal APC would rarely if ever encounter parasitized red-blood cells, but would be primarily exposed to and influenced by parasite-derived soluble compounds.

Interestingly, TLR9 stimulation led to increased pro- and anti-inflammatory responses of CBMC of neonates whose mothers had MP accumulating in placentas, and there was a tendency towards increased IFN-γ response upon TLR3 stimulation in the same group. Responses via other TLR ligands, such as LPS were amplified in CBMC but did not change appreciably as a function of maternal malaria infection. Thus, foetal different TLR responses are independently modulated by in utero exposure to P. falciparum, consistent with a recent study 22.

In humans only PDC and B cells express TLR9 39. In this study, CpG-A, a TLR9 ligand that specifically stimulates PDC 2540, was employed. In concordance with the findings presented in this study, MP or alternatively plasmodial DNA bound to MP activate the TLR9 pathway in human and murine PDC 274142. This would suggest a role for MP derived from maternal parasitic infection in inducing foetal BDCA-2⁺ DC partial maturation and increased sensitization to TLR9 ligands. Nevertheless, only low levels of IL-10 and TNF-α were detected in CBMC cultures upon TLR9 stimulation. This was not unexpected given the low frequency of cells able to specifically respond to such stimulus. The biological significance of a slightly increased release of IL-10 and TNF-α by CBMC upon TLR9 stimulation after in utero exposure to P. falciparum is uncertain.

Notably, MP was the only indicator of maternal malaria infection that was significantly associated with partial activation of foetal DC and to amplified innate response to TLR9 ligation, while other markers of maternal parasitization at delivery such as the presence of parasites in peripheral and/or placental blood, were unrelated to DC activation in the exposed newborns. It has been recently postulated that accumulation of MP in leukocytes is a good indicator of total parasite burden, including parasite sequestration 43, and therefore we can consider accumulation of MP in placenta as a marker of high intensity of maternal malaria infection and/or of prolonged parasite exposure. In addition, accumulation of MP in placental leukocytes has been associated with increased monocyte activation and inflammation 44. As a hypothesis, accumulation of MP may represent a specific activation stimulus and inflammation at the placental level and this may cause partial and inadequate activation of APC in the foetal compartment.

Additionally, maternal age and parity should be taken into consideration when analysing foetal/neonatal innate immunity. Women of higher parity and increased age delivered babies in whom significantly fewer blood APC were found, but these cells exhibited an enhanced activation status. Maternal age but not parity also influenced the APC cytokine responses upon TLR stimulation, such that CBMC of offspring of younger mothers exhibited an increased ability to respond to TLR3, 4 and 9 ligands. These data are in agreement with published data on African 23 and Caucasian 45 women and suggest that maternal age and obstetric history may influence foetal/neonatal immune parameters.

Consequences of increased maternal age and/or multiple parities in terms of neonatal responses to pathogens are poorly understood. Two recent studies indicate that the frequency of malaria episodes is higher among infants of malaria-infected multigravidae as compared to primigravidae 67. The intrinsic effect of multiple pregnancies on malaria susceptibility in the offspring may be at least partially explained by our finding of a significantly reduced number of myeloid APC in foetal blood from multigravidae. How maternal age or alternatively parity can affect the number, activation status and cytokine secretion capacity of cord blood APC is presently unknown.

In conclusion, placental parasitization, as indicated by the presence of MP in placental leukocytes, is significantly associated with partial maturation of different DC subsets and to slightly increased responses to a TLR9 ligand in cord blood. As semi-maturation of DC leads to tolerance 46, such partial foetal APC activation may contribute to the altered T-cell responses often observed in newborns of mothers with PAM 567.

These observations advocate a possible mechanism by which PAM may modulate foetal/neonatal innate immunity. Further evaluation of APC activation and downstream T-cell responses is ongoing in a large cohort of newborns and infants from mothers with PAM to assess the impact of altered DC activation on the neonatal cell-mediated immunity.

As it is known that neonatal immune responses are largely dependent on the innate branch of immunity and can be improved through selective TLR stimulation 4748, our results should be considered in the development of effective vaccine strategies for infants living in areas where malaria is endemic.

The authors declare that they have no competing interests.

NF: conceived the study, participated in its design and coordination, acquired the data and contributed to data analysis and interpretation and drafted the manuscript. SV: participated in the design and coordination of the study, contributed in data analysis and interpretation and drafted the manuscript. IS: acquired the data and contributed to their analysis and interpretation. VB: contributed in interpretation and statistical analysis of data and in critically revising the manuscript. SL: contributed in performing the immunoassays, and contributed in their interpretation, participated in critically revising the manuscript. RP: participated in the design of the study, in the analysis of the data and in critically revising the manuscript. AM: participated in the design and coordination of the study and in critically revising the manuscript. AH: participated in the design of the study, in the analysis of the data and in critically revising the manuscript. MT: participated in the design of the study, in the analysis and interpretation of data and in critically revising the manuscript. PD: participated in the design and coordination of the study and contributed to draft the manuscript. All authors read and approved the final manuscript.