Background
In
Plasmodium spp. infected subjects the ability to control the development of the parasite depends largely on the balance between pro and anti-inflammatory mediators of their immune response [
1,
2]. Acute
Plasmodium falciparum infection is usually associated with an increase of INFγ and TNF, regarded as the markers of the Th1 and pro-inflammatory response [
2,
3]. This pro-inflammatory response is thought to be needed to impede the multiplication of the parasite and favour its clearance, both in human and animal models [
2-
4]. While important for parasite clearance a powerful Th1 and pro-inflammatory response could also be detrimental for the host if uncontrolled, leading to tissue damage and severe disease [
5,
6]. This is supported by the importance of the anti-inflammatory network characterized by an expansion of the regulatory T cells [
7-
10], as well as by the activation of negative regulators like the CTLA4 or PD-1, transmembrane receptors, during malaria infection [
11,
12]. Moreover, as Th1 responses can be counteracted by Th2 cells, the presence of a strong Th2 response might also influence anti-malarial immunity.
In areas where malaria is endemic, it is the norm that
Plasmodium-infected people also suffer from a concurrent helminth infection [
13,
14]. Helminths have repeatedly been shown to modulate the immune system of their host in order to survive [
15]. Chronic helminthiasis is usually characterized by a marked Th2 response [
16,
17] as well as by the induction of a regulatory network [
18,
19] that could consequently impair the host immune response to other antigens [
20]. Whether a concurrent helminth infection of the host can affect his immune response to
Plasmodium spp. co-infection is still debated [
21,
22]. Population-based studies conducted to assess the effect of helminths on malariometric indices and on the immune response of
P. falciparum infected subjects have so far revealed contrasting results. For example, in Senegal, Sokhna
et al. observed that children with
Schistosoma mansoni had an increased incidence of clinical malaria in comparison to their uninfected counterparts [
23], while in Mali, Lyke and colleagues reported a protective effect of
S. haematobium infection against malaria [
24]. A similar divergent picture has also emerged when considering the cellular response of malaria and helminth co-infected subjects. For example in Senegal, Diallo
et al. reported a significant increase of the plasma concentration of TNF and IFNγ measured in
S. haematobium and
P. falciparum co-infected children in comparison to their
P. falciparum single infected counterpart [
25]. In the same studies, they also observed a significant increase of the plasma concentration of TNF, IFNγ, IL-10, TGF-β, sTNF-RI and sTNF-RII rates in co-infected subjects [
25]. Similarly in Ghana, Hartgers
et al. compared the cytokine response of
S. haematobium subjects to uninfected ones when their whole blood were stimulated with
P. falciparum infected red blood cells (iRBCs) and observed that the measured level of IL-10 was significantly higher in the infected group [
26]. Inversely, in Mali Lyke
et al. reported a decreased level of IL-10 in plasma from
S. haematobium and malaria co-infected subjects by comparison to malaria only subjects [
27].
Some reports have suggested that a concurrent helminth infection is associated with elevated cytokines in particular pro inflammatory ones compared to
P. falciparum infected subjects [
25,
26], while in others either no effect or even a decreased in these cytokines [
27-
29]. It is important to note that each of these studies assessed the immune system of infected people from a different angle, either by using different stimuli or by characterizing a different cells type. Moreover none has yet attempted to provide information on how helminths affect both the innate and the adaptive immune response of
P. falciparum-infected subjects within the same cohort.
This study provides information on the cellular immune response of P. falciparum-infected subjects, with or without concurrent S. haematobium infection. Instead of assessing cytokines responses individually, a more global approach was taken to profile the pattern of cytokine responses in the study subjects. The study hypothesis was that a comprehensive and integrative assessment of multiple cytokines involved in the innate or the adaptive immune response of co-infected subjects would provide a better insight into the effect of S. haematobium on the immune response of P. falciparum infected subjects.
Discussion
The main objective of this study was to determine whether chronic
S. haematobium infection was able to affect the cellular immune response of
P. falciparum infected subjects. By measuring the cytokine production after in-vitro stimulation, the innate and adaptive immune responses of the study subjects were profiled. Here, rather than assessing single cytokines, the pattern of cytokine responses of the study subjects using a PCA was evaluated. PCA is a mathematical tool widely used in the field of biology. It has the advantage of summarizing highly correlated variables in new latent and synthetic variables called principal components that can unveil new pattern of responses [
33,
34]. Two PCs (iPCs) that summarize the innate cytokine responses of the study participants were identify as well as 3 PCs (aPCs) for the adaptive cytokine responses. The interpretation of these different PCs shows that cytokines are released with a certain degree of correlation. This is supported by the fact that none of the PCs identified was made of only one cytokine and at least two cytokines were represented in every PC. Moreover, it was noticed that within the same PC cytokines were either negatively or positively correlated. For example in the iPC2, Th1 type cytokines (IFNγ and TNF) were negatively correlated with cytokines released by macrophages (MCP1-MCAF and MIP1β) implying an antagonistic effect that may need further investigations.
In a number of studies it has been shown that
S. haematobium infection can influence the innate immune response of the human host. For instance in population based studies, schistosomiasis has been linked with functional impairment of human myeloid dendritic cells [
35] and their response to TLR ligands [
36-
38].
Schistosoma haematobium excretory-secretory products can prime dendritic cells to shape the adaptive response toward a Th2 phenotype [
38,
39]. While this immune profile is thought to limit the damage caused by schistosomes in the human host, it could alter the host immune response to a concurrent
P. falciparum co-infection. What was observed in this study is that
P. falciparum infection was marked by an increase of the iPC1 and aPC1, which represented the innate and adaptive general immune responsiveness. Interestingly, this was not the case for
S. haematobium infection, which was associated with an increased level of chemokines (MCP1-MCAF and MIP1b) and the decrease of pro-inflammatory cytokines, namely INFγ and TNF. This indicates that the immune system responds differently to
P. falciparum and to
S. haematobium infection. In
P. falciparum-infected subjects the increase of the iPC1 and aPC1 component is in line with the immune profile seen in asymptomatic
P. falciparum infected subjects [
40]. This is also in line with the literature indicating that in subjects chronically infected with
S. haematobium there is a down modulation of the pro inflammatory response that is thought to allow the survival of the parasites [
18,
19]. These observations regarding
S. haematobium are in line with results of two independent studies that assessed the innate immune response of schistosome-infected subjects. In the first study, Turner
et al. observed that upon stimulation of whole blood with schistosome excretory/secretory products,
S. haematobium infected subjects had an enhanced production of IL-10, an anti inflammatory cytokine, whereas the level of the pro-inflammatory cytokine TNF was not different from the uninfected subjects [
41]. In the second study, Van der Kleij
et al. observed that
S. haematobium infection was associated with a significant decrease in responsiveness to LPS irrespective of pro or anti inflammatory cytokines [
37]. However, a study by Meurs
et al. reported that PBMC of
S. haematobium infected subjects produced significantly more TNF after stimulation with Pam3 a TLR2/1 ligand in comparison to their
S. haematobium uninfected counterparts [
36]. These differences are difficult to reconcile but the culture methods [whole blood versus PBMC], seasonal fluctuation in immune responses, or other factors such as different environments or co infections [
42,
43], need to be taken into consideration when comparing studies.
This study did not observe an effect of
S. haematobium on the innate and adaptive cytokine profile of
P. falciparum infected subjects. The current body of evidence on helminth and malaria co-infection and its effect on the host immune response has so far given contrasting results. For example a cross sectional study showed no impact of light intensity Ascaris infection on the immune response of malaria infected subjects [
44]. In a study conducted in Mali,
S. haematobium infected and uninfected subjects were followed up until the time to the first malaria episode and serum cytokines were measured at the time of inclusion and at the time when study subjects became infected with
P. falciparum [
27]. At baseline the level of IL-4, IL-6, IL-10 and IFNγ cytokines were all higher in subjects infected with
S. haematobium by comparison to uninfected subjects. However, when these participants developed an acute episode of malaria, IL-6 and IL-10 cytokines increased considerably in all groups, but to a higher extent in subjects who were free of schistosome infection [
27], which would suggest that
S. haematobium impedes the cytokine storm. It has to also be noted that, looking at the results differently, which is that at the time of malaria infection, the baseline differences in IL-6 and IL-10 in the
S. haematobium infected and uninfected subjects, fell short of statistical significance, one might conclude that there is no difference between subjects with single malaria versus those who were coinfected. In contrast, in a study in Senegal, where
P. falciparum infected participants were compared to
S. haematobium and
P. falciparum co-infected subjects; Diallo
et al. reported that the plasma concentration of IL-10, TGFβ, INFγ (but not INFα) was higher in co-infected subjects than in those with single infection. The same authors, when examining
in vitro production of cytokines by mononuclear cells stimulated with
P. falciparum schizont extracts and MSP1-19 antigens reported an increase of IL-10 and INFγ but not TGFβ, IL-12 or IL-13 in subjects with
P. falciparum infection compared with subjects co-infected with
P. falciparum and
S. haematobium [
45]. Finally, a study conducted by Hartgers
et al. in Ghana showed higher response to malaria antigens in terms of IL-10 but not INFγ, IL-6, TNF in helminth infected subjects in comparison to those free of helminth infection [
26]. It is important to emphasize that in the study of Hartgers
et al., the response to malaria antigens was compared between
S. haematobium-infected and uninfected subjects and, therefore, malaria infection was artificially mimicked by the use of antigens from
P. falciparum. Regarding, the Senegal studies, the
P. falciparum singly infected individuals originated from a village where
S. haematobium infection was never reported before, whereas co-infected subjects were from an entirely different village endemic for both
S. haematobium and
P. falciparum. Therefore, it is possible that the differences reported, mirror the exposure to different environmental factors rather than to
S. haematobium. This is supported by the work by Smolen and colleagues who compared the immune response of children across four different continents. Using a standardized procedure they observed considerable heterogeneity in the cytokine responses in the different geographical areas [
42].
One obvious limitation of the present study is that it is cross sectional and one could argue that it does not provide information on history of past helminth infections that are capable of imprinting the host immune system. For example, the innate immune system has been shown to be able to keep a “memory” of early exposure to PAMPs through a process called “trained immunity” which is not addressed in this study [
46]. Additional limitation concerns the sample size of the study that may not be sufficient to detect an effect of helminths on
P. falciparum modulated immune responses. However, this study was carried out in a relatively small community where it was possible to enroll all the school-aged children willing to participate and fulfilling the inclusion criteria. Despite these limitations the present study provides original information on the cellular immune response of
S. haematobium and/or
P. falciparum infected subjects. It showed that
P. falciparum, but not
S. haematobium, infection was associated with an increase of the immune responsiveness of the study subjects but it did not evidenced an effect of
S. haematobium on the immune response that were measured in the
P. falciparum-infected participants.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
UAN, JFZ, RFKK carried on the study on the field. They were responsible of the screening and the enrolment of the study participants. UAN carried on the different immunological assays, performed the statistical analysis and wrote the first draft. AAA, MML and BM advise on the epidemiological aspect of the study. HS advise on the immunological aspect of the study. PGK, MY and AAA designed and coordinated the study. All authors participated in the manuscript preparation, read and approved the final version of the manuscript.