Circulating Cytokine Profiles of Polyparasitized Individuals in Gabon.

Background Malaria, blood-borne filarial worms and intestinal parasites are all endemic in Gabon. This geographical co-distribution leads to polyparasitism and, consequently, the possibility of immune-mediated interactions between different parasite species. Intestinal protozoa and helminths could modulate anti-malarial immunity, for example, thereby potentially increasing susceptibility to malaria. Methods Blood and stool samples were collected during cross-sectional surveys in five provinces of Gabon. Parasitological diagnosis was performed to detect plasmodial parasites, Loa loa and Mansonella perstans , intestinal helminths (STH) and protozoan parasites. Nested PCR was used to detect submicroscopic plasmodial infection in individuals with negative blood smears. Cytometric Bead Array was used to quantify interleukin (IL)-6, IL-10 and Tumor Necrosis Factor (TNF)-α in plasma of subjects with different parasitological profiles i.e. malaria only, filariasis only, intestinal protozoan only, soil-transmitted helminths (STH) only, malaria/filariasis, malaria/STH, malaria/intestinal protozoa co-infections and in uninfected individuals/control group. Results Median IL-6 (124.5 [36.9–433.9] pg/mL) and IL-10 (224.5 [78.0–657.9] pg/mL) levels and the median IL-10/TNF-α (69.9 [12.5–140-7]) ratio were all significantly higher among individuals with Plasmodium falciparum infection compared to other groups ( p < 0.0001). The median TNF-α level (6.5 [3.5–11.7] pg/mL) and IL-10/IL-6 ratio (3.6 [2.0–11.9]) were higher in subjects with STH (p = 0.09) and P. falciparum -intestinal protozoa co-infection ( p = 0.04), respectively. IL-6 (rho=-0.37; p < 0.01) and IL-10 (rho=-0.37; with helminths and protozoa.


Introduction
In Gabon, malaria and intestinal parasite infections (IPIs) as well as filariasis are frequently diagnosed in the population, with the prevalence varying from 1-75% [1,2]. This geographical co-distribution can lead to polyparasitism with possible interactions between parasite species. Studies on the association between Plasmodium and helminths have shown that helminths could have either a protective, a detrimental or a neutral effect on plasmodial infections. For example in Mali, Schistosoma (S.) haematobium delayed the appearance of clinical malaria in children, while in Gabon and in Senegal it was reported that Ascaris lumbricoides and S. haematobium increased the risk of clinical episodes [3][4][5]. Shapiro and colleagues found no interactions between these STH and malaria parasites [6]. Although the epidemiological patterns and/or the clinical consequences of polyparasitism are being increasingly studied, data concerning the immune responses and the susceptibility to other diseases of individuals exposed to or infected by different helminth or protozoal parasites in endemic areas remain scarce. Relevant information for Gabon does not exist.
Understanding the immune response elicited by each pathogen in the case of co-infection could help with the management or the prevention of the deleterious effects of polyparasitism.
Levels of pro-and anti-inflammatory cytokines that are influenced either by the environment or by other parasites can reflect P. falciparum induced immunity and morbidity of populations living in regions with different malaria endemicity. Indeed, interleukin (IL-) 10 was shown to be a predictor of the occurrence clinical malaria in highly endemic areas of India while IL-6, IL-10 and IL-12 were associated with disease outcome in a non-endemic region [7]. With respect to Plasmodium-helminth co-infections, helminths orient the immune response toward an anti-inflammatory pathway whilst plasmodial infection is known to elicit a strong pro-inflammatory response (Boeuf et al., 2013; Hartgers et al., 2009); filaria-helminth co-infection is associated with a low IL-10 level whereas that of IL-6 was described to be lower in the case of Plasmodium-hookworm co-infection [10].
On the other hand, chronic intestinal helminthic and/or filarial infection are associated with high levels of IL-10 ( Maizels et al. 2004). IL-10 downregulates the functions of immune cells that release proinflammatory cytokines, both preventing the elimination of worms and protecting the host against helminth infection-related symptoms. IL-6 is also described in the literature to increase significantly among populations infected with helminths [12]. However, the level of TNF-α is reported not to vary [12].
In a rural setting, anti-malarial immunity can be modulated by infection with Trichuris trichiura [13], but in urban and suburban areas data on such interactions are lacking. The heterogeneity of Plasmodium spp. prevalence can be due to different susceptibility patterns to P. falciparum infection according to the presence and type of other parasitoses. In Ghana it was shown that the production of inflammatory cytokines was inversely proportional to the level of malaria transmission [14]. The study presented here was conducted in areas of Gabon not yet investigated that have different levels of malaria endemicity. In another hand, in Gabon, intestinal protozoan were found more frequently in coinfection with P. falciparum comparatively to STHs [15,16]. But it is not described if these protozoan, pathogenic or not, interact with P. falciparum-related immunity. Indeed, non-pathogenic protozoan carriage does not constitute a public health problem. But experimental studies on Blastocystis spp., a controversial pathogenic intestinal protozoa, Entamoeba (E.) histolytica and Giardia (G.) intestinalis, have demonstrated that these parasite carriage is associated with the production of pro-inflammatory cytokines [17,18]. In humans, this immunomodulation was equally observed [19]. A study showed that Blastocystis spp. could downregulate immune response to another antigen as helminths [18]. So this survey evaluated the impact of intestinal parasites (helminths or protozoa) and blood filaria on the secretion of cytokines involved in malaria immunity during coinfection with helminths and protozoa.

Patients And Methods Study sites and populations
This study was nested in cross-sectional surveys on asymptomatic malaria carried out from September 2013 to June 2016 in five out the nine provinces of Gabon with different degrees of urbanization (Fig. 1). Samples taken from individuals participating in villages located around the main towns of the Ogooué-Ivindo and Haut-Ogooué provinces, and those from patients at the Centre Hospitalier Régional (CHR) d'Oyem, (Woleu-Ntem province), the CHR de Koula-Moutou and Dienga (Ogooué-lolo province), the CHR de Melen and the Lalala Public Health Center (Estuaire province) were used for this study. Lalala is a suburban area of the capital city of Libreville, an urban area; the CHR of Melen is located in a suburban area and the CHR d'Oyem, of Koula-Moutou as well as villages in the Ogooué-Ivindo and Haut-Ogooué and Dienga are located in rural areas.
Inclusion criteria for sample selection included for the study participants: absence of fever (axillary temperature ≤ 37.5 °C) or absence of history of fever the day of the screening and during the week preceding the consultation, absence of other clinical symptoms suggestive of malaria, absence of antimalarial drug uptake the last two weeks, absence of any other severe medical condition and sickle cell disease, permanent residence in the study area, agreement to fill the questionnaire and written informed consent.
All the plasma controls were those from individuals with no history of or current P. falciparum or intestinal parasite infection.

Procedures For Sample Selection For This Immunological Substudy
During the main surveys, patient recruitment, demographic, socioeconomic, environmental and parasitological data were collected in standardized case report forms. Among the 843 patients recruited for the main surveys, 400 provided blood and stool samples. After the microscopic diagnosis of blood and intestinal parasites, blood samples of those with negative blood smears were screened

Ethical Consideration
This nested study received ethical clearance from "Comité National d'Ethique pour la Recherche" (CNER) of Gabon. The protocol and the questionnaire were also approved by the Ministry of Health.

Sample Processing
After collection, whole blood was used for microscopic malaria and blood filarial diagnosis. After centrifugation blood pellets were stored at − 20 °C for the molecular detection of P. falciparum; plasmas were directly stored at -20 °C on site, then transported to the laboratory in a cooled container and stored at − 80 °C prior to immunological analysis. The participants were provided with clean, labelled stool collection pots with clear instructions to ensure that stool samples were collected correctly.

Parasites Microscopic Diagnosis
The detection of plasmodial parasites was performed by thin and thick smears using the Lambaréné method [20]. Whole blood direct microscopic examination and leukoconcentration techniques allowed for the identification of blood filariae L. loa and M. perstans [21]. To determine the presence of intestinal parasites, three techniques were performed: direct stool examination, Merthiolate-Iodine-Formaldehyde coloration and parasite culture. These techniques are described in detail elsewhere Dna Extraction And Submicroscopic Plasmodial Infection DNA was extracted from peripheral blood collected in EDTA tubes using the QIAamp® kit (QIAGEN®) according to the manufacturer's instructions. The 18S rRNA malarial genes were amplified by nested PCR according to the protocol described by Snounou and Singh [22]. Briefly, for the first reaction,

Circulating Cytokines Measurement
A Cytometric Bead Array human cytokine kit from Becton Dickinson (CBA kit, BD Biosciences, San Diego, CA, USA) was used to measure plasma levels of IL-6, IL-10 and TNF-α according to the manufacturer's instructions. Samples were centrifuged and one volume of the supernatant obtained was diluted into two of the assay diluent. Then, the technique was realized as described by Böstrom and colleagues [23]. The highest standard concentration was 2500 pg/mL and the lowest was 5 pg/mL. The calibration, the sample acquisition and the standard was performed using a BD FACSCalibur flow cytometer (FACSCalibur, Becton Dickinson, Le pont de Claix Cedex, France), and results were analysed with FCAPArray v1.0.1 software (SoftFlow, Pécs, Hungary). The detection limits of cytokines were 1.6 pg/mL, 0.13 pg/mL and 1.2 pg/mL for, respectively, IL-6, IL-10 and TNF-α. If the cytokine concentration was below the detection limit, a value corresponding to half the detection limit was assigned to the sample. perform multivariate analysis, R software was used to build and evaluate some models. F-tests were performed to statistically test the equality of means into different groups, according to age, location, parasitaemia. A p-value less than 0.05 was considered significant.

Results
Samples of 240 participants were selected for the immunological analysis (Fig. 2). Out of the 208 patients for whom the area was known, 172 (82.7%) were from a rural area (Table 1). Age was recorded for 224 of them and the median age was 22.5 [6.0-48.8] years old. Adults represented more than half of the study population (58.0%; 130/224) and children less than 5 years old, 21.0% (47/224). The sex ratio was 0.8 and did not differ according to age and site. concentrations than P. falciparum/STH co-infected ones. Cytokine level was also lower in those with intestinal parasitism and filariasis (Table 3). Intestinal protozoan infected-subjects had a higher IL-10/IL-6 ratio compared to uninfected individuals (p = 0.04).

Multivariate Analysis
Comparison of the IL-6, IL-10 and TNF-alpha levels, and of the IL-10/IL-6 and IL-10/TNF-alpha ratios in the 8 different parasitic groups adjusting by age, urbanization and P. falciparum parasitaemia showed no statistically significant differences (p > 0.05). Cytokine concentrations and cytokine ratios were similar across the age groups after adjusting for parasitic infections and urbanization.

Discussion
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. falciparumintestinal 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 immuneregulatory 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][32][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] 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]. 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. Plasmodium falciparum in Gabon also seem to alter the susceptibility to P. falciparum infection according to age. Those more than 5 years old and adults seem to be more at risk of infection when co-infected with filariasis associated with a higher IL-10/TNF-α ratio, and when co-infected with intestinal parasites associated with a higher IL-10/IL-6 ratio.

Declarations
Ethics approval and consent to participate This nested study received ethical clearance from "Comité National d'Ethique pour la Recherche" (CNER) of Gabon. Participants were informed before to enrolled in the study and accepted that plasma will used for immunological studies. The protocol and the questionnaire were also approved by the Ministry of Health.

Consent for publication
Not applicable Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.   represents a flow chart ranging to the enrolled population, followed by prevalence of parasites found in the study population to different groups with different parasite profile.

Figure 3
Cytokines profiles stratified by areas according to age in months. IL-10 level decreased when age increased in rural area. IL-10/TNF-alpha ratio increased with age in rural are but decreased in urban area. : Box plot displaying IL-6, TNF-α and IL-10 cytokine production and IL-10/TNF-α, IL-10/IL-6 median ratios according to different parasitic profile. Mann-Whitney test was carried to pairwise comparisons. Values used for the graphical representation were log-transformed.