Pro- and anti-inammatory response prole modulates Plasmodium falciparum malaria outcomes among subjects from Baiyeku, Ikorodu, Lagos, Nigeria.

Background Available evidence indicates that the various stages of the malaria parasite life cycle elicit specic immune responses of which the relative levels of pro-inammatory cytokines are key to disease progression, killing the parasite and mediating disease outcomes. This study will inform immunological interventions against malaria and thus malaria vaccine developments programs/efforts. Methods A total of four hundred and sixty-two participants were screened in a community survey for Plasmodium falciparum (P. falciparum) malaria in Baiyeku, Lagos, Nigeria. P. falciparum parasitaemia was determined by Microscopy using thick and thin blood lms stained by Giemsa method using World Health Organization parasitology laboratory protocol whist the serum levels of IL-10, IFNγ and TNFα were determined by Enzyme linked immunosorbent assay [ELISA]. Data analysis was done by One-way Analysis of Variance (ANOVA), Chi square (X²) and Student’s T-test in statistical package for the social sciences (SPSS) version 24 was used to test statistical signicance between the symptomatic groups and asymptomatic in relation to age, gender and BMI of the participants.

the participants with the highest parasite density.
Conclusion The prevalence of P. falciparum obtained in this study area which is endemic for malaria is 15.2% suggesting a signi cant reduction of the disease over time. The awareness of the disease which is now more than before seems to contribute to the lowering of prevalence of the disease in the community.
There was a positive relationship between TNF-alpha levels and body temperature. However, compared with the anti-in ammatory cytokine  in this study, the levels of the pro-in ammatory cytokines (IFNγ and TNF-α) were lower due to the negative action of the anti-in ammatory cytokines. IL-10 value increased as parasitemia increased (p=0.073). These ndings suggest that higher levels of antiin ammatory cytokines, especially IL-10 levels may contribute to pathogenesis of uncomplicated malaria.
Background Page 3/18 Available evidence indicates that the various stages of the malaria parasite life cycle elicit immune responses [1,2]. The pro-in ammatory cytokines tend to play an important role in preventing malaria and killing the parasites. Furthermore, the relative levels of pro-and anti-in ammatory cytokines are essential mediators of malaria anemia production and outcomes [3]. Early production of pro-in ammatory T-helper 1 (Th1) cytokines such as tumor necrosis factor (TNF)-alpha, interleukin (IL)-12 and interferon (IFN)gamma may limit progression to severe complications from uncomplicated malaria. They actually limit the growth of parasites and stimulate monocyte phagocytosis to improve clearance of parasitized erythrocytes while IL-6 is a major mediator of the acute phase response [3,4]. Often involved in the immune response to Plasmodium are other pro-in ammatory cytokines such as IL-17 and IL-22, developed by other cell subtypes including Th17 cells [5]. An increase in peripheral blood IL-17-producing CD4 + T cells has been reported during P. vivax infection, along with the production of the proin ammatory cytokines IFN-γ, IL-10 and the transforming growth factor (TGF)-beta [5]. During murine infection IL-22 activation defends against liver damage [6]. However, if these pro-in ammatory responses during the acute infection are not properly regulated, severe malaria complications may arise [7,8]. Hence, the need for anti-in ammatory responses to regulate the development of pro-in ammatory cytokines and subsequent cytopathic consequences. Regulatory cytokines such as IL-10 play a major role in infection with Plasmodium, neutralizing excessive development of in ammatory Th1 cytokines [9,10]. Anti-in ammatory Th2 cytokines including IL-4 and IL-13 control the humoral immune response, leading to the clearance of parasites and inhibiting the development of Th1 cytokines [11,12].
Given the signi cance of the development of pro-and anti-in ammatory cytokines in the human immune response to infection with P. falciparum malaria, this is not well established in Nigerians.

Description of Study Area
Baiyeku is a rural settlement with favorable ecological features for all year round transmission of malaria. It is an ancient town in the Ikorodu Local Government Area located along the Lagos Lagoon on Latitude

Study Design And Sample Collection
This study was a cross-sectional prospective study involving community survey of Plasmodium falciparum malaria in the study area of Baiyeku town in Ikorudu Local Government Area of Lagos State, Nigeria [ Fig. 1]. The transmission of malaria in this area typically occurs all year round. Sampling was conducted at the peak of transmission (April to August, 2017).
The sample size for the study was derived using an anticipated population proportion of 50%, con dence level of 95% and absolute precision of 5 percent point. The sample size was calculated using the following formula [13]: Where z is the critical value of the standard normal distribution at 5% level (1.96), n is the minimum sample size, P is the anticipated population proportion and d is the absolute precision at 5 percentage point (0.05).
This gave an approximate sample size of 384 participants. A total of 462 were enrolled in the study.

Ethical Approval
Ethical approval of the study was obtained from Nigerian Institute of Medical Research, Institutional Review Board (NIMR-IRB).
Before the commencement of the study, the investigators visited the site and explained the study to the chief of the community who is the traditional ruler known as Baale. The Baale gave his consent and permitted the town announcer to announce our mission to the community. The Baale provided us with the venue (canopy, chairs, and tables). Interested persons in the community that came out for the survey were interviewed and recruited as participants in the study. Informed consent was obtained from the study participants. Socio-demographic data consisting of age, gender, height and weight as well as body temperature were obtained before venous blood samples were collected by standard phlebotomy for malaria parasites testing.

Anthropometric Measurements
Anthropometric data were obtained in accordance with internationally recommended procedures [14].
Weight measurements were taken using a scale (Tefal, Paris, France) precise to the nearest 100g. Recumbent length measurements were taken for children under 2 years of age, while standing height was measured for older participants using a stadiometer (SECA, Hamburg, Germany). BMI (Kg/m²) was calculated for the participants, those within the range 18.5-24.9 were grouped as normal, those below this range were categorized as underweight, those having BMI range of 25.0 -29.9 were grouped as overweight while those who had BMI above 30 were categorized as obese. The body temperatures of the volunteers were obtained using non-contact infrared thermometer (Proactive Medical, NY, USA) at 5 cm away from the participant's forehead. Participants with body temperatures below 37.5°C were asymptomatic participants whereas body temperatures 37.5°C or above were categorized symptomatic.

Sampling and Analyses
3ml of venous blood was obtained from each participant using standard phlebotomy practice and aseptic techniques for malaria diagnosis and immunoassay for (TNF-α), Interferon Gamma (IFN-γ) and Inerleukin-10 (IL-10) by ELISA method. Thick and thin blood lms for malaria diagnosis were made for each participant and examined under the light microscope for P. falciparum following the World Health Organization [14] procedure. Serum for ELISA assay was obtained by dispensing the blood into plain tube, allowed to clot and the serum separated [2]. Malaria parasite density MPD (Parasite per μl of blood) was calculated using the formula: Parasites per μl = number of parasites x 8000/number of leucocytes.
Levels of TNF-α, IFN-γ and IL-10 were assessed in sera of the study participants using antigen capture ELISA (Enzyme Linked Immunosorbent Assay) kits based on antigen-antibody reaction employing the manufacturer's protocol. The concentrations were extrapolated from the standard curve obtained using values for the standards [15].

Statistical Analysis of Data
One-way Analysis of Variance (ANOVA) was used to analyze the group means. Chi square (X²) statistics was used to test statistical signi cance between the symptomatic groups and asymptomatic in relation to age, gender and BMI of the participants. Where appropriate Chi-square (χ2) test was used to compare data sets generated. P value <0.05 was considered statistically signi cant. Geometric Mean Parasite Density (GMPD) for parasite density was determined using SPSS statistical software version 21. The geometric mean parasite density (GMPD) was estimated for the positive participants.

Demographic Pro le of Participants
A total of 462 participants, consisting of 136 (29.4%) males and 326 (70.6%) females, were screened for Plasmodium falciparum malaria in this study. Seventy of these were microscopically positive for Plasmodium falciparum, corresponding to a prevalence of 15.2% (Table 2). Seventy percent were females and 30% males while 65.7% were participants below 17 years of age and 34.3% were aged 18 years and above. Overall, the median age of the participants was 21 years.

BMI Status of Participants
A total of 46.8% of the participants were underweight (Table 3). However, 27.3% had normal BMI (BMI=18.5 -29.5) and 13.4% were obese. Parasite Intensity in Different BMI Status.
Among the participants included in this study, the category with parasitemia ≥1,000 but 100,000 had more parasitemia than other BMI groups. The intensity of Plasmodium falciparum parasitemia was highest among the participants underweight BMI (Table 4).

Evaluation of Cytokine response of Tested Participants
Serum levels of TNF-Alpha, IFN gamma and IL-10 were determined for all the participants in this study. The levels of IFN gamma was highest among the cytokines tested in the participants ( Table 5). The serum levels of all the cytokines (TNFα, IFNγ and IL-10) were signi cantly (p 0.05) higher in the infected than the uninfected participants ( Table 6). The IL-10 levels were the most elevated amongst the participants with the highest parasite densities relative to TNF-α and IFN-γ, although not statistically signi cant. Table 7.
Serum levels of TNF alpha increased with increasing parasitemia (Table 8).

Cytokine levels in Asymptomatic and Symptomatic Participants
IFN-γ values were signi cantly (P=0.014) higher among the symptomatic participants than the asymptomatic, while there was no signi cant difference (P>0.05) in the levels of TNF-α and IL-10 between the symptomatic and asymptomatic participants (Table 8). Cytokine levels in the asymptomatic and symptomatic infected participants compared with uninfected (controls).

Discussion
In many parts of Africa, the burden of Plasmodium falciparum malaria is gradually decreasing. It is however characterized by the spatial and temporal viability presenting with new and evolving challenges for malaria control programmes. Nigeria currently contributes 25% of the global malaria burden, with the highest malaria prevalence in sub-Saharan Africa [16]. Protective immune responses in malaria are mediated by both T-cell and humoral responses, which are important in malaria control strategies. Tumor necrosis factor-alpha (TNFα) and Interferon gamma (IFNγ) are T helper 1 (Th1) anti-parasitic proin ammatory cytokines with established protective roles in malaria, while interleukin-10 is a T helper 2 (Th2) cytokine with demonstrated immune-regulatory anti-in ammatory roles protective in malaria [17].
This study describes prevalence of Plasmodium falciparum malaria and its relationship with pro-and anti-in ammatory innate immune responses (TNFα, IFNγ and interleukin-10 (IL-10)) among asymptomatic and uncomplicated malaria subjects in Baiyeku community of Ikorodu, Lagos State, Nigeria.
A prevalence of 15.2% was observed among participants in all age groups and gender in tandem with the results obtained by Aina [18] which reported prevalence of 14.7% in Ibeshe coastal community, Ikorodu, Lagos state. However, this result contrasts with that of Olukosi [19] who reported 5.7% malaria prevalence in Ijede in Lagos State during the dry season and that of Sam-Wobo [20] in Abeokuta, Ogun Sate with a prevalence of 71.1%. This difference can be attributed to the seasonal variability in the design of the studies. Olukosi's work, although community-based, was conducted during the dry season when the vectoral population is naturally to be low [21]. The study conducted by Sam-Wobo was notably hospitalbased, of which subjects were symptomatic. Some authors have reported strong correlation between high malaria prevalence and low levels of education; for instance a population that is well informed about the use of long lasting Insecticidal Nets (LLIN), indoor residual spraying (IRS) and intermittent preventive treatment of malaria in pregnancy (IPTp) could help reduce the prevalence of the disease [20,22]. South-West Nigerian communities have the highest malaria prevalence and transmission because rainfall is all year round with appropriate climatic conditions for vector breeding [23,24].
Participants in the age group less than ve years (<5 years) showed higher geometric mean parasite density (GMPD) compared to those above 5 years. The higher GMPD peaks observed in children are the result of less developed immune system that could not effectively clear parasites as observed in adults.
Parasite density reduced with increasing age group which could be as result of developed immunity and premonition against malaria.
In Uganda, a negative but signi cant correlation (r = -0.09271; p < 0.0214) was found [25] between parasite density and age, suggesting that mature individuals' clear parasites more effectively than children. This antimalarial immunity modulates infection outcomes and is more pronounced in children over 15 years of age and adults who have been previously exposed [25].
Body mass index (BMI) is a measure of nutritional status of which malaria have a negative effect on the nutritional status of children less than 5 years [26]. However, there has been only a few studies addressing the relationship between malnutrition and malaria in the face of relevant pro and antiin ammatory cytokines. This study revealed higher infection intensities (GMPD) among participants with low BMI (underweight) than those having normal BMI which constituted 27.3%. This inverse relationship between malaria parasitaemia and BMI corroborates the assumption that malaria thrives among the poor while nutrition plays an important role in the development of immunity against malaria [27].
The serum levels of selected innate immune responses, TNF-α, IFN-γ and IL-10, were assessed amongst participants in this study. Serum levels of IFN-γ were signi cantly (P=0.014) higher in the symptomatic than the asymptomatic participants. This infection-induced increase could be directly linked to the effectiveness of IFN-γ as a pro-in ammatory Th-1 cytokine. This result is consistent with the study by Perara [28] in which both IL-10 and IFN-γ levels were reported elevated in P. falciparum-infected patients.
In this study, serum levels of TNF-α was signi cantly (P= 0.03) high among participants within the highest parasitemia group. This is an agreement with the knowledge that TNF Alpha, and other proin ammatory cytokines protect the host against asexual blood stages of malaria parasite [29,30]. T cell studies have shown that mounting a rapid TNF-α and IL-2 response may protect against severe disease and reinfection [31]. TNF-α is a pro-in ammatory cytokine which mediates in ammation that is crucial in malaria immunity [32].
Conversely, however, low levels of TNF-α were observed in this study, relative to IL-10. This could be explained by the negative action of IL-10 on pro-in ammatory responses. IL-10 completely abolishes TNF-α production in response to malarial antigens. IL-10 is a critical anti-in ammatory cytokine [33,34].
There was an increased IL-10 serum levels in infected patients in this study compared to non-infected patients, IL-10 levels increased as parasitemia increased. These results are consistent with other studies in which the severity of malaria and increased parasitemia have been associated with increased IL-10 levels [3]. Perara [29] found that a high circulating TNF-alpha levels and an inadequate IL-10 response in severe malaria (SM) patients carrying TNF2 allele could have contributed to the development of the severe falciparum malarial disease. Findings by Perara [28] had suggested that IL-10 down-regulates the pro-in ammatory response to P. falciparum.
There was no statistically signi cant difference in the serum levels of TNF-α among symptomatic and asymptomatic participants although Perara [28] reported a strong positive correlation between TNF-α levels and body temperature as shown in their study. TNF-α is a critical mediator of malarial fever [30].
These ndings suggest that higher levels of anti-in ammatory cytokines, especially IL-10 levels may contribute to pathogenesis of uncomplicated malaria by inhibiting the production of IFN-γ and TNF-α.   Serum levels of TNF, IFN and IL-10 among the study participants compared to uninfected participants.