Pro- and anti-inammatory immune response proling prevents severe malaria among Nigerians infected with Plasmodium falciparum: the future for malaria vaccines and therapeutics.

Background Available evidence indicates that the various stages of the malaria parasite life cycle have specic immune responses. The pro-inammatory cytokines tend to play an important role in preventing malaria and killing the parasites. Furthermore, the relative levels of pro-and anti-inammatory cytokines are essential mediators of malaria anemia production and outcomes. Natural human immune responses to malaria recognize extracellular sporozoites and merozoites, both of which have surface-exposed antigens, and which are currently being developed for various vaccines. Methods A total of four hundred sixty- two (462) participants were tested for Plasmodium falciparum. The procedure employed were parasite staining using World Health Organization parasitology laboratory protocol [Microscopy] of Giemsa staining and Enzyme linked immunosorbent assay [ELISA]. Results The subjects in this study showed high levels of INF-γ and TNF-α which decreases with increased malaria severity and high parasite density. These results suggest that INF-γ cytokine and TNF-α may contribute to protection against severe malaria anaemia and parasite clearance. Conversely, infected participants showed higher levels of IL-10, which decreases with severe malaria parasite, furthermore IL-10 levels correlated with parasite density. These ndings suggest that higher levels of anti-inammatory cytokines, especially IL-10 levels may contribute to pathogenesis of complicated malaria by inhibiting the INF-γ and TNF-α production. Conclusion Molecular biological and other serological analysis are needed to elucidate the implication of these cytokines and other pro-inammatory cytokines as IL-17, IL-21 and IL-22 in the responses to malaria and consequently their involvement in malaria vaccine construct/development as well as other therapeutics for the treatment and elimination of the malaria parasite in our environment.


Introduction
Available evidence indicates that the various stages of the malaria parasite life cycle have speci c 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 [26,4]. Early production of proin 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 [10,11]. They actually limit the growth of parasites and stimulate monocyte phagocytosis to improve clearance of parasitized erythrocytes. While, cytokineIL-6 is a major mediator of the acute phase response [3,26]. Often involved in the immune response to Plasmodium are other pro-in ammatory cytokines such as IL-17andIL-22, developed by other cell subtypes including Th17 cells [26]. An increase in peripheral blood IL-17-producing CD4+ T cells has been reported during P. vivax infection, along with the production of the pro-in ammatory cytokines IFN-γ, IL-10 and the transforming growth factor (TGF)-beta [12]. During murine infection IL-22 activation defends against liver damage [13]. However, if these pro-in ammatory responses during the acute infection are not properly regulated, severe malaria complications may arise [14,15]. Hence, the need for anti-in ammatory responses to regulate the development of proin 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 [16,17]. Anti-in ammatory Th2 cytokinesincludingIL-4and IL-13 control the humoral immune response, leading to the clearance of parasites and inhibiting the development of Th1 cytokines [18,19].
Given the signi cance of the development of pro-and anti-in ammatory cytokines in the human immune response to infection with Plasmodium falciparum malaria, this is not well established in Nigerians. Accordingly, in this research we examine pro-and anti-in ammatory responses in western Nigerians subjects by evaluating collected plasma cytokines from participants in Baiyeku community according to their malaria status [3,9,26].

Description of Study Area
This was a community surveyed cross-sectional study for Plasmodium falciparum malaria, conducted in Baiyeku Community in the Ikorodu Local Government Area of Lagos State, Nigeria [  a) Participants, adults 18-60 years who have been exposed to malaria and who have lived in endemic areas of malaria for at least six months, each giving informed consent. b) Children 10-17 years of age who have lived in endemic areas of malaria for at least six months, each giving informed consent. c) Children 1-9 years of age who have lived in endemic areas of malaria for at least six months with informed consent from parents / guardians.

Exclusion criteria
Persons who were unwilling or unable to provide informed consent.

Research eligibility evaluation
Persons ful lling inclusion criteria were asked if they wished to participate in the study and the participants were given full disclosure of the necessary information alongside the consent form. They were asked to read or have read to them, the consent form and to sign the form. Individuals not providing informed consent or who chose not to consent their minor were not enrolled.

Subject Registration
Individuals who met and consented to the eligibility criteria were registered as study participants. The study involved voluntary and informed consent (participants 18 years of age) or informed parental consent (children 1-9 years of age) or consent (children 10-17 years of age) prior to blood sample collection. A personal identi cation number (PID #) was assigned to each participant and their blood samples for easy of identi cation and avoidance mix-up.

Blood Collection Techniques
The study recruited four hundred sixty-two (462) participants. 5 ml of venous blood were collected into plain EDTA tube using standard phlebotomy practice and aseptic techniques. The blood samples were stored at-80 ° C for downstream applications as described by Kiechle et al [2].

Ethical Consideration
Ethical approval (Ref no: IRB/17/021) was obtained from the Institutional Review Board of the Nigerian Institute of Medical Research (NIMR), Lagos to conduct the study. Advocacy and necessary permission was obtained from the head of the community as well.

Microscopy
Detection /Examination of blood for parasitemia Procedure A total of four hundred sixty-two (462) participants were tested for infection with Plasmodium falciparum and the procedure for parasite staining using Giemsa staining was adopted from World Health Organization parasitology laboratory protocol [WHO, 2003].
In the center of a labeled frosted slides, small drop of blood were placed and spread, and the smears were allowed to dry. The slides were then stained with a 10% Giemsa solution for 8-10 minitues. The slides were viewed under the microscope at X100 (Olympus CX21, UK). Parasitemia was measured by counting the number of malaria parasites against a number of leucocytes in the thick blood lm, based on a putative mean count of white blood cells density of 8000 white blood cells per μl. The malaria parasite density MPD (Parasite per μl of blood) was calculated [Parasites per μl = number of parasites x 8000/number of leucocytes].

Evaluation of Cytokines by Enzyme Linked Immunosorbent Assay (ELISA)
Levels of Tumor Necrosis Factor Alpha (TNF-α), Interferon Gamma (IFN-γ) and Inerleukin-10 (IL-10) were assessed in sera of the study participants including the controls, using antigen capture ELISA (Enzyme Linked Immunosorbent Assay) kits based on antigen-antibody reaction (Maxisorb; NUNC Denmark) employing the manufacturer's protocol. Samples and standards were added using appropriate diluents into ELISA pre-coated plates. After incubation and washing step (Washer:BioTek Instrumentals, USA), goat anti-human IgG HRP conjugate reagent (Sigma A9544) were added followed by Chromogen solution A. The Optical Density (OD) was read within 15 minutes at 450 nm, on an ELISA reader (BioTek EL 800, BioTek Instrumentals, USA) and the concentration from the standard curve obtained using values for the standards (Voller et al., 1978).

Statistical Analysis of data
Chi-square (χ2) test was used to compare data sets generated, Students-t-test, ANOVA, Pearsons correlations and graphs were also used to analyze the data.

Demographic Pro le of Participants
Four hundred and sixty two (462) participants, consisting of 136 males and 326 females, were screened for Plasmodium falciparum malaria in this study. Of this population, 70 were microscopically positive for Plasmodium falciparum (Table 2), corresponding to a prevalence of 15.2%. The female participants were predominantly positive (70%) while males constituted 30% positives.
Of the 70 participants microscopically con rmed positive for Plasmodium falciparum, 65.7% were participants below 17 years of age while the adult participants aged 18 years and above constituted 34.3%. Overall, the median age of the participants was 21 years.
The seventy (70) age-matched controls on the other hand, were microscopically con rmed negative. The median age for the healthy controls was 21 years [ Table 1].  Figure 2]. Of the 462 participants tested, 70 (15.2%) were microscopically positive for malaria, 21(30%) were male and 49(70%) were female. There was a signi cant difference between male and female participants' (p<0.01) geometric mean parasite density (GMPD). Similarly, the results showed a statistical difference, p<0.01 between the age groups tested, with the highest GMPD in subjects under 5 years of age (P<0. 01) compared with those over the age of 5 [ Table 2].   In this study [ Table 4], the underweight infected participants had a higher parasite load of 68.6% with a parasitemia level of < 100/μl, while those with normal BMI had a parasite density load of 31.4% with parasitemia levels of 1,000<100,000. The overweight and obese had a parasitemia level of < 1,000≥10,000≥100,000 with a parasite load of 2.9% and 8.6% [p>0.01] respectively. Table 5. Evaluation of malaria Parasite density in relation to cytokine levels of participants.
[ Table 6]. Subjects who were grossly infected with Plasmodium falciparum showed an elevated pro and anti-in ammatory immune responses compared to individuals who tested negative to malaria parasite.

Discussion
Although in many parts of Africa the burden of Plasmodium falciparum malaria is gradually decreasing, it is characterized by spatial and temporal variability, which presents new and evolving challenges for malaria control programmes. Nigeria presents the highest malaria prevalence in sub-Saharan Africa as a result new malaria treatment and therapeutics and possibly vaccines are needed to combat this scourge.
In this study, we assessed the demographic prevalence of malaria parasite, its density and severity among four hundred and sixty-two (462) subjects, and the pro-and anti-in ammatory cytokine pro les of tumor necrosis factor-alpha (TNFα), interferon gamma (IFN-γ) and interlukin-10(IL-10) among western Nigerians for future vaccines and therapeutics arsenals in combating malaria infections. Of these subjects, 134 were males and 328 females. The study participants age groups ranged from 1-9 years of age, 10-17 years of age and 18-60 years of age from the Beiyeku community of Lagos, western Nigeria. A prevalence of 15.2% was observed among the participants in all age groups and gender. One of the immediate presumed reason could be educational level and sample size. Some literatures attributed high malaria prevalence to education levels; for instances a population that is well informed about the use of long lasting treated nets (LLN), insecticide residual spraying (IRS) and intermittent preventive treatment (IPT) of pregnant women could help reduce the prevalence of the disease, it could also be attributed to the sample size involved in various studies, also environmental factors such as rainfall and climatic change plays prominence in biodiversity of the vector. In the present study we employed a larger sample size compared to many other studies conducted in Nigeria and other parts of Africa. Similarly, the malaria parasite geometric density was higher in females compared to males who were positive, the relationship between parasite density and disease incidence is complex and non-linear. A multitude of factors including the sample's age structure, sampling timing in relation to local malaria transmission seasons and the methodology and rigor applied to parasite detection density. Findings such as those reported here were in consonance to those of Irene et al, [7]. The age group less (<5 years) recorded a higher geometric malaria density compared to those over 5 years old. The higher mean parasite density peaks observed in children were as a result of a less developed immune system that cannot effectively clear parasites load compared to adults. Increased in age or adulthood reduced the prevalence of infection and parasite density. The observed decrease in parasite density among adults is most likely due to the development of clinical immunity that is not sterile over time. This immunity controls infection and is usually pronounced in children over 15 years of age and in adults. These people have been exposed to mosquito bites over the years and, consequently, to malaria many times more. Such limited immunity allows people to tolerate serious malaria infection without getting ill even if they can get malaria. Parasite densities in children therefore appear to give a true picture of the intensity of an infection than in adults, which can be a useful indicator for monitoring the intensity of the disease. Similar ndings were reported in the study of Odongo Aginya et al [5] in Uganda, Markell et al [6] and Molineaux [8].
Subject with lower body mass index (BMI) showed a very high prevalence of (46.8%), those with normal (BMI) had a prevalence of (27.3%). The overweight and obese with a Body mass index of [BMI] 25.0 -29.9 [kg/m 2 ] and [BMI] >30) (kg/m 2 ) showed a prevalence of (12.6%) and (13.4%) respectively. This physiological factors had been shown to contribute to low/high level parasitemia in several studies [26]. Similar ndings were observed in the current study. The reason could be attributed to nutritional status, pregnancy and diabetics. Monte et al [10] research ndings however elucidated otherwise, it was observed in the study that high BMI, obesity and diabetes acts as protective factor against malaria. Katja et al [11] proved a total contrast to our nding, the study showed that BMI, obesity and diabetes are factors that increased malaria parasitemia. All of the forgoing studies are linked to physiological sociodemographic indices.
Cytokine(s) pro ling were conducted in the various participants, TNF-α Showed (1.98 ± .015) lower preponderance compared to IFN-γ (2.46 ± .05) and IL-10 (2.21 ± .054). Subjects who were grossly infected with Plasmodium falciparum showed an elevated pro and anti-in ammatory immune responses compared to individuals who tested negative to malaria parasite. Several studies showed a markedly similar results. Pro-in ammatory cytokines are elevated during malaria infection. IFN-γ is a key molecule in the defense of human antimalarial host as demonstrated by Wikler et al [12] in uncomplicated P. falciparum malaria patients [14]. Interferon gamma is an active macrophage that contributes to the innate immune response to malaria. It is primarily produced by CD8 and CD4 T lymphocytes in a speci c immune response and by NK cells in an unspeci c response [16]. Interferon gamma is essential for the resolution of primary infection by limiting the initial phase of parasite replication, it also contributes to acute malaria symptoms such as fever, nausea and headache by inducing TNF alpha [17].Th1 cytokines, namely IFN-α and IL-2, have a signi cant effect on the outcome of the disease and on the development of protection against Plasmodium sp infection. IL-10 modulates this pro-in ammatory response [18]. TFN-α have been shown to correlate with malaria severity in several studies [16,20,11], low levels of TNF-α levels were observed in our study. This could be explained by the negative action of IL-10 on proin ammatory responses. IL-10 completely abolishes TNF-α production in response to malarial antigens IL-10 levels increased signi cantly with the degree of parasitemia/malaria density in this study, it was however, lower in asymptomatic individuals compared to infected subjects. IL-10 is a critical proin ammatory cytokine [37,38]. There was also an increased IL-10 plasma levels in infected patients in this study compared to non-infected patients, particularly those who tested positive for malaria. IL-10 levels increased as parasitemia increases. These results are consistent with other studies in which the severity of malaria and increased parasitemia have been associated with increased IL-10 levels [19]. This suggests that P. falciparum invigorates IL-10 generation in dose-dependent manner. IL-10 is utilized by regulatory T cells to control immune responses. P. falciparum-infected erythrocytes actuate regulatory T cell expansion, followed by increased IL-10 and IL-6 production [19,13]. In addition to a direct correlation between T regulatory cell numbers and IL-10 plasma levels in infected patients [15,18], it has been shown in various studies that CD4+CD25+Foxp3+ T cells induced during malaria upregulate the IL-10 expression [22,23]. Other authors also report that regulatory T cells are associated with higher rates of parasite development, suggesting that P. falciparum-mediated induction of regulatory T cells may well be a destructive factor [25]. In summary, this study revealed that the severity of malaria infection due to P. falciparum could be regulated and cleared in the blood systems by pro and anti-in ammatory arsenals and also be future valuable immune tools for vaccines constructs and therapeutics.

Conclusion
The subjects in this study showed high levels of INF-γ and TNF-α among positive malaria cases, which increases with increased malaria severity and high parasite density. These results suggest that INF-γ cytokine and TNF-α may contribute to protection against severe malaria anemia and parasite clearance.
Conversely, infected participants were shown to have higher levels of IL-10, which decreases with severe malaria parasite, furthermore IL-10 levels correlated with parasite density. These ndings suggest that higher levels of anti-in ammatory cytokines, especially IL-10 levels may contribute to pathogenesis of complicated malaria by inhibiting the INF-γ and TNF-α production. Further biological analysis and studies are needed to elucidate the implication of these cytokines and other cytokines such as IL-17, IL-21 and IL- In vivo and in vitro relationship with co-regulatory cytokines IL-12 and IL-10. Clin Immunol.   Tables   Table 1 Demographic profile of study population.     Cytokine levels of participants infected with Plasmodium falciparum in comparison with the healthy controls