Introduction

Plasmodium falciparum (P. falciparum), which causes malaria is a leading root of mortality in the tropical countries. African equatorial regions now account for 94% of cases [1]. P. falciparum species present the most prevalent parasite, which infects humans and causes malaria disease throughout the entirety of Sub-Saharan Africa. It is predominant in the East and Horn of Africa, including Sudan, Ethiopia, Djibouti, Eritrea, South Sudan, and Somalia [2]. One of the key reasons that P. falciparum represents a challenging parasite for humans to deal with and impedes the ability of immune responses against plasmodium, is that protective immunity against infection takes several years to acquire, despite recurrent parasite exposure [3, 4], even in high transmission areas. Besides being parasite-specific, naturally acquired immunity is also stage and antigen-specific, so immunity is developed only against erythrocytic stages of infection [5]. The equilibrium of inflammatory cytokine responses, in addition to the interactions of the regulatory mediators, are fundamental to the clearance of Plasmodium parasites, and for preventing serious malaria disease complications during the infection. So as malaria progresses this balance plays a crucial role in eliminating the infection [6].

The cellular activation phase and the systemic response to inflammation are strongly influenced by cytokines, a type of polypeptide derived from cells. It also plays a significant role in mediating inflammation. Several of these factors are multi-functional and have a local or systemic effect [7], and parasites trigger specific immune responses by encouraging peripheral blood mononuclear cells (PBMCs) to trigger the release of cytokines. It is possible that this may be instrumental in activating phagocytic, natural killers, and other effective immune cells, to fight out the liver and blood stages of the parasite [8, 9]. Over the course of the infection by P. falciparum malaria, inflammatory cytokines, and chemokines are raised peripherally in blood and contribute to parasite eradication, and these factors are also believed to be accountable for a variety of symptoms and pathological changes [10]. However, the approaches and processes by which infected people regulate pro/anti-inflammatory responses in consequence of exposure to malaria infection as a result of P. falciparum species are currently incompletely understood.

Studies have established that inflammatory cytokines and regulating mediators are significant predictors of malaria prognosis and determinants of disease severity. And according to some scholars, the balance of anti-inflammatory cytokines interleukin 4 (IL-4) and IL-10 and pro-inflammatory cytokines IL-6, tumor necrosis factor-α (TNF-α), interferon-gamma (IFN-γ), and IL-8 may increase susceptibility to anaemia induced by malaria parasites [11]. Measurement of these cytokines is suggested to determine the parasitaemia rate, clinical severity, symptoms, and outcome [12]. Furthermore, immunity against malaria stages in the blood requires powerful immune response strategies and mechanisms.

Consistent with these prior findings that stated the changes in immune cellular ratios in peripheral blood [13], here the investigation focused on immunological mediators that regulate immune cellular balance. In this study, various levels of cytokine responses were examined in relation to the parasite density consequences of P. falciparum infection, with an emphasis on malaria severity, which is considered to be more severe in children and their immune mediator’s response levels that differ substantially according to previous investigations [14, 15]. According to our hypothesis, the effective response against malaria infection may reflect in forms of associations between the concentration of the inflammatory cytokines, which might explain the protective roles of the inflammatory mediator cells producer during different infection stages and suggest that, before sufficient cytokine responses are elicited, the parasite density must be attained.

Materials and methods

Results

One hundred and eight participants were recruited in this study. The mean age of participants was 3.25 ± 1.81 years (range 1 to 9 years old), with a sex ratio of 1:4 (male:female), and the variations in cytokine levels based on gender were not observed. To assess the relationship between age and cytokine profile in malaria patients, the IFN-γ was significantly higher in the older children group (more than 6 years) compared to the younger children group (P < 0.05). Though IL-6 and TNF-β seem higher in the older group, there is no significant difference among all groups (P > 0.05) (Table 1).

The relationship between cytokine concentrations among study groups was examined (Table 2). There was a significant relationship between severe malaria and cytokine concentrations among patients in comparison to the healthy control group (P < 0.001). The median concentration of IFN-γ was significant (P < 0.001) in severe malaria (111.7 pg/mL) compared to controls (4.0 pg/mL), as in uncomplicated malaria (P = 0.007). TNF-β levels among participants were higher in the two types of clinical malaria compared to controls. It also showed significant relation for severe with control P < 0.001, severe and uncomplicated P = 0.027, and uncomplicated with control P < 0.001. IL-8 levels in the two types of malaria the median was 21.98 pg/mL for severe cases, 11.29 pg/mL for uncomplicated malaria, and (4.0 pg/mL) for controls, showing significant association (P < 0.001). Severe malaria patients had also the highest IL-6 and IL-10 levels (29.0, 18.0 pg/mL), respectively, however, there was no difference between uncomplicated and severe malaria patients (P > 0.05).

In univariate analysis, the levels of cytokines IFN-γ and IL-8 showed a significant increase in malaria patients compared to controls (P = 0.005; P = 0.003, respectively). Also, multivariate analysis confirmed the considerable alterations in levels of both IFN-γ and IL-8 in malaria patients when compared to control levels (P = 0.008; P = 0.028, respectively) (Table 3).

For exploring whether cytokines correlate with each other in relation to infection densities, statistical approaches revealed a correlation between the cytokines in both severe and uncomplicated malaria. IL-8 was found to be positively correlated with TNF-β (r = 0.760, P < 0.001) for severe and (r = 0.346, P = 0.004) for uncomplicated (Figure 1). Similarly, a positive correlation was noticed too, between IL-6 and anti-inflammatory cytokines IL-10 (r = 0.575, P = 0.010) in severe malaria patients (Figure 2).

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Figure 1. 

Correlation analysis of IL-8 serum levels with TNF-β among uncomplicated and severe malaria patients

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Figure 2. 

Correlation analysis of IL-6 serum levels with IL-10 among uncomplicated and severe malaria patients

However, during this observation using principal component analysis, it has been found that cytokine profiles were unable to distinguish between children, with either severe or uncomplicated malaria from controls (Figure 3).

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Figure 3. 

Principal component analysis of cytokine responses in children with severe, uncomplicated malaria patients, and controls. PC1: maximum variation in data; PC2: the second most variation in data

Discussion

Researchers have spent considerable time and effort studying the essential characteristics and understanding of host immune responses to P. falciparum infections. Despite the complex interactions of the Plasmodium parasite with immune system cells, it is still poorly understood. Cytokine responses significantly influence the emergence and outcome of malaria as separate immunological tactics for infection control. According to the present study, we confirm previous findings of a different pattern of cytokine balance in patients with symptomatic P. falciparum infection [16, 17]. The association of these patterns was also explored to gain a better understanding of immune response strategies and collaboration during malaria infection in an endemic setting.

In this study, individuals infected with P. falciparum malaria expressed significantly higher levels of IFN-γ. A significant relationship was stated between the malaria-infected groups and proinflammatory IFN-γ levels in severe malaria (111.7 pg/mL) and uncomplicated malaria (1,014.0 pg/mL) compared with healthy controls (4.0 pg/mL). And this is consistent with previous research [18]. Furthermore, IFN-γ levels were significantly higher in the older children group (more than 6 years) compared to the younger children group (P < 0.05) [10], implying a protective role for IFN-γ and its association with the rapid killing of P. falciparum, from liver-stage infection through to blood-stage parasite [19].

Additionally, the findings revealed that TNF-β and IL-8, substantially pro-inflammatory cytokines, significantly responded to clinical P. falciparum infection. Compared to non-infected controls, the levels of TNF-β and IL-8 were higher and differed depending on the severity of the infection, during the comparison of the levels as severe and uncomplicated [20, 21]. And these findings illustrate the essential role of TNF-β in immunological development and promoting immune regulation through the innate immune response against P. falciparum infection [22]. In contrast, the pro-inflammatory cytokine IL-8 stimulates neutrophil cells and is an important predictor of malaria parasitaemia, clinical presentation, and mortality [23]. As this study showed, IL-8 levels were significantly associated with malaria intensity.

By using trustworthy biomarkers, it has been discovered that infected people had considerably greater levels of IL-6 and IL-10, confirming the protective effects of the inflammatory cytokines, but neither uncomplicated nor severe malaria patients showed any variations to support the hypothesis that disease tolerance generated by persistent exposure is represented by plasma cytokine concentration in malaria-endemic regions [24].

In studying the correlation between certain cytokines, IL-8/C-X-C motif chemokine ligand 8 (CXCL8), the major mediator involved in the recruitment of neutrophils degranulation and recruitment of inflammatory cells, and TNF-β, a critical regulator that mediates a wide range of immunostimulatory, which shares an effective function in neutrophil activation. In both severe and uncomplicated malaria cases, IL-8 was shown to be positively correlated with TNF-β. This shows an immunological functional link between macrophages and lymphocyte cells during P. falciparum infection, which aids in the elimination of infected red blood cells (RBCs) from circulation [12, 25, 26].

Furthermore, by targeting affected patients with remarkably high plasma concentrations of the regulatory cytokines IL-10, due to parasite density. According to the results, IL-10, a regulatory cytokine, correlated positively with IL-6 an inflammatory cytokine, in malaria cases with high transmission rates [27]. IL-6 has been demonstrated to have double inflammatory properties. A prior study reported an association between IL6, IL10, and parasite density, which supports this finding [28]. It is also evidence that infection tolerance may represent distinct resistance mechanisms for infection clearance depending on disease severity.

The fundamental weakness of our study is its inability to distinguish the influence of previous malaria exposure on the stated age. The variation in cytokine levels production might be explained by the recurrent exposure in older children to malaria infection, which is indicated by the high amount of IFN-γ, IL-6, and TNF-β, and may be because of age-related intrinsic differences in immune system activity [29, 30]. However, by applying principal component analysis, the cytokine patterns were found not to distinguish between children with severe or uncomplicated malaria and the control group.

And the cytokine assessments at a single time point are insufficient to give reliable insights, into the sequence of immune defence tactics, against P. falciparum infection via anti/pro-inflammatory mediator interaction and clinical illness development.

In conclusion, the prevalent results from this study indicated the effective functions of IFN-γ within malaria parasite intensity during the various phases of P. falciparum infection, revealing the levels of contribution of IFN-γ as the key cytokines, which may efficiently impact the clinical outcome of malaria sickness. TNF-β and IL-8, on the other hand, demonstrated a comparable relationship with the severity of infection. Together, IL-6 and IL-10 have preventive functions in severe instances, and they are linked favourably during the infection. Furthermore, these findings provide convenient evidence that IL-8, an important inflammatory cellular recruiter, is correlated with the regulator and neutrophil activator cytokine TNF-β, implying the correlations and the efficient interactions of these cytokines toward parasite intensity. All these data pointed to a double-edged variable protective immune response strategy in eradicating P. falciparum blood stages, influencing illness outcomes, and sharing evidence of the effective involvement of cell mediator makers, namely natural killer (NK) cells, T-cells, and macrophages, which appear to be efficient contributors in host immune response tactics. More research is needed to have a better knowledge of the correlations, interactions, and cytokine patterns associations with P. falciparum infection in an endemic context.