This study is the first to analyse the influence of single or multiple parasitic infections on the plasma cytokine profile of Gabonese individuals. The main aim was to determine the relationship between intestinal parasitoses, as well as loiasis, and the plasma concentrations of different cytokines implicated in malaria pathophysiology.
Lower parasitaemias were observed among participants carrying co-infections of either P. falciparum-intestinal parasites or P. falciparum-filariasis compared to those with only P. falciparum, suggesting an effect of intestinal parasites and filariae on P. falciparum multiplication. Lower parasite burdens, in the presence of intestinal helminth infections, have been reported elsewhere [24, 25]. According to these authors, one explanation could be the existence of immune cross-reactivity between intestinal parasites and Plasmodium sp. Indeed, both parasites induce Th2 immune response and specific IgG3 produced as a result of intestinal parasitic infection could neutralize plasmodial parasites [26]. In the case of protozoan co-infections, no impact of intestinal protozoa on Plasmodium sp parasitaemia has been described.
P. falciparum-infected participants, who were asymptomatic when included, had higher median levels of IL-6 and IL-10 compared to the control group, but no difference in the level of TNF-α level. In the context of IL-10, this result is consistent with the findings of earlier studies [27, 28]. The immune-regulatory and anti-inflammatory cytokine IL-10 is known to downregulate Th1-type cytokines such as IL-6, TNF, and IL-1 [29]. Here, we found IL-10 to be implicated in the inhibition of the production of the TNF-α but not of IL-6 during plasmodial infection. It is possible that the downregulation of TNF is correlated with the absence of clinical signs. TNF-α did not vary in the control group compared to Plasmodium-infected participants with. TNF-α is the cytokine most implicated in the development of the clinical signs of malaria [30], whilst IL-10 and IL-6 are present at high levels in patients with symptomatic malaria [31–33]. The asymptomatic status of participants at the time of the study presented here could indicate the acquisition of premunition which limits the appearance of clinical symptoms, and/or that participants were sampled at an early stage of the infection, before progression toward symptomatic disease [34]. It is important to note that, in the current Gabonese context, younger children are found to be less frequently infected by malaria parasites than older children [15, 35] and the number of adults presenting with clinical malaria is increasing, suggesting a loss of malaria premunition. This epidemiological picture most likely primarily reflects the switch, since 2003, to the use of artemisinin combination therapy for malaria in under-five year old children.
Previous studies conducted in Gabon showed malaria to be co-endemic with other parasitic infections that are present at a similar or higher prevalence (compared to malaria) and that can alter immunity to malaria (Akue et al., 2011; Bouyou-Akotet et al., 2015; M’Bondoukwé et al., 2016, 2018). Here, intestinal parasite infection (IPIs) and blood filariasis were associated with decreased Th1 (IL-6) and Treg (IL-10) responses. Blastocystis sp., the most prevalent intestinal parasite, have immunomodulatory effects with induction of pro-inflammatory cytokine responses [38]. The prevalence of Blastocystis has increased across the country [15, 16]. Therefore, it was hypothesized that, together with its implication in dysbiosis, Blastocystis could affect cytokine production and influence P. falciparum carriage. A study in Pakistan showed that Blastocystis sp. type 1 was associated with low IL-10 production in the blood, but that in stool samples Blastocystis sp. generates an anti-inflammatory environment [19, 39].
The impact of other pathogenic intestinal protozoa on the cytokine profile was not investigated here because of the low number of infected patients and the absence of single E. histolytica and G. duodenalis mono-infection or co-infection with P. falciparum. However, during intestinal protozoa/P. falciparum infection, a significant reduction of pro-inflammatory cytokines (IL-6, TNF-α) was observed, suggesting that infection with G. duodenalis, E. histolytica, or Blastocystis sp. decreases the P. falciparum-induced Th1 response, thereby contributing to the absence of clinical symptoms. This would also explain the difference in IL-10 levels between participants with intestinal protozoa and those from the control group. The implication of each of these three protozoan parasites as well as the different subtypes of Blastocystis sp. on the global and specific cytokine response merits further exploration.
Individuals with a low Th2-Treg/Th1 ratio are less susceptible to malaria but have a greater likelihood of clinical disease when infected [40, 41]. Here we observed that IL-10 and IL-6 levels were 1.3 to 18.0-fold lower in those with malaria-intestinal parasite co-infections (both helminths and protozoa) (data not shown) compared to those with plasmodial mono-infections, although still higher compared to uninfected participants. A trend towards a down regulation of TNF-α was observed in co-infections with intestinal parasites. This regulatory effect on pro-inflammatory cytokine production during helminth and P. falciparum co-infections is well-described (Bustinduy et al., 2015, Sinha et al., 2010). Elevated IL-10 levels have been observed in patients with Plasmodium-schistosomiasis and malaria-hookworm co-infection (Bustinduy et al., 2015, Courtin et al., 2011, Diallo et al., 2004; al., 2003). The higher IL-10/TNF-α ratio, in the group of participants with Plasmodium mono-infection compared to Plasmodium/helminth co-infection suggests a lower risk of developing clinical signs in case of STH/P. falciparum co-infection as demonstrated by Frosch & John (2013). Additionally, the IL-10/IL-6 ratios suggest that with Plasmodium and intestinal protozoa co-infected participants seem also to be less at risk to have symptoms. Thus, exposure to both IPI and malaria via the increasing prevalence of intestinal parasites in the country would partly explain the increasing frequency of asymptomatic P. falciparum carriage observed in the country [35, 42, 43]. But a meta-analysis of young African children with helminth-P. falciparum co-infections concluded that they are more susceptible to P. falciparum infection [44]
IL-10/TNF-α ratios show that filariae-infected volunteers more than 5 years old have a higher risk of P. falciparum infection than participants infected with other parasites. But when the IL-10/TNF-α (except for STH infection) and IL-10/IL-6 ratios were analyzed, children with intestinal parasites were found to be more susceptible to P. falciparum infection presenting higher values. Intestinal helminths and filarial infections are associated with higher production of IL-10 and consequently with the inhibition of Th1-type cytokine production [12, 45, 46]. The high IL-10/TNF-α and IL-10/IL-6 ratios observed here in those with helminth co-infections are consistent with such observations.
The development of the anti-infectious immune response is promoted by repeated exposures and the chronicity or persistence of infections. The immune response was studied according to age in uninfected, mono-infected or polyparasitized participants. It differed according to age and type of parasitism. The negative correlation between cytokine levels and age is in favor with a greater stimulation in young children. They are actually more at risk of the studied parasites (with the exception of filariasis) and polyparasitism [15]. Although, the cross-sectional design of this study, does not allow to confirm that the measured cytokine levels were baseline. Nevertheless, the fact that participants lived in the study area since several months without significant interventions related to malaria or IPI, allow a comparison between the different groups determined according to the presence and the type of infection. The low level of IL-10 among children with STH, may be due simply to the reduce time of exposition to induce a typical T-regulatory-related IL-10-dominated response as they are younger, as observed in Kenyan children [10]. The decrease in the IL-6 level in case of intestinal protozoan can be related to an immunoregulatory effect induced by these parasitosis; this is confirmed by the high IL-10/TNF-α ratio. The analysis of the IL-10/IL-6 ratio which shows a different profile than that of each cytokine would highlight the predominance of the anti-inflammatory cytokine response in young children infected with STH and intestinal protozoa. In contrast, in adults, the comparable level between infected and uninfected ones, is probably linked to a better control of these infections through a protective and mature immune response.
The predominance of a pro-inflammatory cytokine profile in the absence of infection may be linked to the greater susceptibility of young children to severe malaria which is determined by high IL-6 and TNF response. This could partly explain the predominance of severe forms of malaria in young children, although they remain less frequently infected [35, 47]. Indeed, the lower basal cytokine Th1 level in healthy children could indicate their greater susceptibility to plasmodial infection due to a less efficient control of Plasmodium multiplication that would quickly reach the pyrogenic threshold. In contrast, the downregulation of IL-10 observed in older children would be in favour of a less control of the parasite multiplication at the beginning of infection and therefore of a higher frequency of parasitaemia in this group compared to the young people.
Limits The cross-sectional design is one of the main limits of this study. Also, plasma cytokines cannot be considered as being specifically induced by any given parasitic infection. Specific and more sensitive techniques such as stimulation of PBMC in vitro to assess responses to P. falciparum, intestinal parasites and filariae would be useful in this context. Equally, next generation sequencing would give a global picture of the immune response by measuring the transcript levels of Th1-, Th2- and Treg-related cytokines. The influence of helminths and intestinal protozoa on innate and adaptive responses to P. falciparum could thus be revealed. The small sample size in groups with different parasitic infections could also explain the lack of association between the cytokine levels and infection profiles in multivariate analysis. However, the present results already give a baseline estimation of the overall in vivo cytokine levels implicated in anti-parasite immunity in exposed individuals according to their age.