Abstract
The balance between Th1 cytokines (tumor necrosis factor [TNF]-α, interferon [IFN]-γ) and Th2 cytokines (interleukin [IL]-10, -4) may be critical in the development of severe falciparum malaria. Therefore, plasma concentrations of these cytokines were determined in children with various manifestations of malaria. Plasma levels of IFN-γ and IL-4 were undetectable in most children. However, TNF-α and IL-10 were significantly elevated in children with high-density parasitemia and malaria anemia compared with children in control groups. In children with mild malaria, IL-10, but not TNF-α, was significantly elevated. While the highest concentrations of TNF-α were found in children with malaria anemia, IL-10 levels were highest in children with high-density uncomplicated malaria. The mean ratio of IL-10 to TNF-α was significantly higher in children with mild and high-density parasitemia (4.64, P <.005) than in children with malaria anemia (1.77). Thus, higher levels of IL-10 over TNF-α may prevent development of malaria anemia by controlling the excessive inflammatory activities of TNF-α.
An estimated 1–2 million children die annually in Africa from falciparum malaria [1], usually from complications due to cerebral malaria or severe anemia [2]. Although the pathologic basis for the development of cerebral malaria and malaria anemia is not well understood, it is apparent that cytokines play a significant role. Inflammatory cytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)-1, interferon (IFN)-γ, and IL-6 are highly elevated in acute Plasmodium falciparum infections [3–5]. TNF-α in particular has been associated with cerebral malaria and death in children [4, 5]. Subsequently, it was shown that elevation of TNF-α was not exclusively associated with cerebral malaria but was also associated with anemia and high-density P. falciparum infections [6]. These data notwithstanding, it is important to recognize that not all malaria-infected children with higher levels of TNF-α develop severe malaria. Thus, we reasoned that the cytokine network as a whole, rather than a single cytokine, must be carefully investigated to better understand the interactive role of various cytokines in the severe disease manifestations of malaria.
Experimental studies with rodent malaria models have shown that the up-regulation of Th2 cytokines prevents the development of cerebral malaria and that Th1 cytokines promote disease [7]. However, it is unclear whether Th2 cytokines assist in preventing severe disease in humans. IL-10, a Th2 cytokine, regulates the functional activity and the production of a Th1 cytokine, TNF-α. IL-10 can inhibit P. falciparum—induced TNF-α production [8]. Preliminary studies have shown that IL-10 levels are elevated during malaria infection as are TNF-α, IFN-γ, and other cytokines [9]. Thus, in this study, we investigated the hypothesis that a higher level of Th2 cytokines (IL-4,-10, or both) than Th1 cytokines (TNF-α, IFN-γ, or both) may prevent development of malaria anemia in an area in which malaria is highly endemic by down-regulating the inflammatory activities of Th1 cytokines.
Materials and Methods
Study area
This cross-sectional study was conducted in the population living in Kisumu Town, Nyanza Province, western Kenya. Malaria is transmitted perennially in this area and P. falciparum malaria accounts for ∼97% of malarial infections. Residents experience 100–300 infective mosquito bites per year [10].
Study population
Children aged 3–11 years admitted to the Kisumu District Hospital were enrolled in the study. More than 90% of the population in this area are of the Luo ethnic group. Study participants were categorized into six groups: mild malaria, high-density uncomplicated malaria, malaria anemia, hospital controls, asymptomatic, and aparasitemic. Group characteristics are summarized in table 1. Aparasitemic and asymptomatic children were recruited from the same rural areas in which the hospitalized children lived; they were recruited by cross-sectional surveys conducted in the same season and year. Children were excluded from the study if they were severely immunosuppressed, malnourished, or had other concurrent infections (based on clinical signs determined by a clinical officer) that may cause fever, as were children admitted for malaria who received medication >4 h.
Cytokine response in different groups of children.
Cytokine response in different groups of children.
Blood sample collection
All children had a physical examination. The presence of malarial parasites was determined using a fingerprick Giemsa-stained thick blood smear, and parasitemia was determined by the number of asexual stage parasites per 300 white blood cells. The parasitemia density per microliter of blood was estimated by the formula: (number of parasites/300) × 8000 (the mean number of white blood cells per microliter of blood). Hemoglobin levels were determined by spectrophotometer (Hb; HemoCue, Helsingborg, Sweden). Eligible children were enrolled in the study, and 5 mL of venous blood was collected into sterile heparinized vacutainers from each enrolled child (Becton Dickinson, Rutherford, NJ). Plasma was immediately separated and frozen in liquid nitrogen to avoid denaturation of cytokines in the plasma.
Cytokine ELISA
A two-site sandwich ELISA was used to determine cytokine levels in plasma. TNF-α, IFN-γ, IL-4, and IL-10 levels were estimated using optimal concentrations of antibodies and cytokine standards according to the manufacturer's instructions. Cytokine concentrations in the test samples were determined from a standard curve with various concentrations of cytokines (3–3000 pg/mL; R&D Systems, Minneapolis).
Statistical analysis
Differences between the mean concentration of cytokines in various groups were compared using the non-parametric Kruskal Wallis test. The ratio of IL-10 to TNF-α was calculated for each study participant. The statistical difference in the mean ratios between the mild and severe disease groups was tested using the Wilcoxon test. P <.05 was considered statistically significant.
Results
TNF-α levels
Mean plasma TNF-α levels in children with different clinical symptoms were compared. Results are presented in table 1. The mean TNF-α concentration was lowest in the healthy aparasitemic children and did not vary significantly in the asymptomatic and hospital control children. In children with mild malaria, the TNF-α levels were not different from the control groups. In the high-density uncomplicated malaria group, TNF-α levels were ∼4-fold higher than in the mild malaria group (P <.001). Malaria-anemic children had the highest levels of TNF-α, which was highly significant when compared with any of the control groups (P <.001).
IL-10 levels
The mean plasma concentrations of IL-10 in various patient groups are shown in table 1. Lowest levels of IL-10 were found in the aparasitemic children and asymptomatic children with malaria parasitemia. For children with mild malaria, although TNF-α levels were not elevated when compared with the control groups, IL-10 levels were significantly higher than in control asymptomatic children (P <.004). In contrast to TNF-α levels, mean IL-10 levels were higher in the high-density uncomplicated malaria group than in the malaria anemia group. However, this difference was not statistically significant.
Plasma IL-10-to-TNF-α ratio
Since IL-10 regulates both the production and function of TNF-α, we hypothesized that children with a low IL-10-to-TNF-α ratio may be more likely to have severe disease than children with a higher ratio. Therefore, we determined the individual IL-10-to-TNF-α ratio for each child and compared the means (table 2). In this study, the mild disease group comprised children with mild malaria and high-density uncomplicated malaria; the severe disease group included children with malaria anemia. The mean IL-10—to-TNF-α ratio (4.64) was significantly higher (P <.005) in children with mild disease than in children with severe disease (mean ratio, 1.77). When we compared the IL-10—to—TNF-α ratio between the children with moderate (hemoglobin <8 g/dL to >5 g/dL) and severe malaria anemia (hemoglobin <5 g/dL), the difference was not significant (data not shown).
Mean interleukin (IL)-10-to-tumor necrosis factor (TNF)-α ratios in Kenyan children grouped by mild or severe malaria.
Mean interleukin (IL)-10-to-tumor necrosis factor (TNF)-α ratios in Kenyan children grouped by mild or severe malaria.
IFN-γ and IL-4 levels
Plasma levels of IFN-γ and IL-4 were low in most of the subjects tested, and there was no statistically significant difference between the control groups and any of the symptomatic groups of children (P >.05; table 1).
Discussion
Our hypothesis predicted that higher plasma IL-10 concentrations over TNF-α levels might provide protection against severe malaria anemia by down-regulating the severe pathologic effects of TNF-α. This study showed that children in this holoendemic area with mild disease (mild or high-density uncomplicated malaria) had significantly higher IL-10—to—TNF-α ratios than children with severe disease (malaria anemia). This finding is consistent with our hypothesis that the balance between the Th1 cytokine TNF-α and the Th2 cytokine IL-10 is critical in the pathogenesis of severe disease in P. falciparum—infected persons who live in areas in which malaria is endemic.
Consistent with our findings, recent studies from rodent models have highlighted the significant role IL-10 plays in down-regulating severe malaria. Linke et al. [11] showed that IL-10 gene knockout mice with an intrinsic deficiency for IL-10 production when infected with Plasmodium chabaudi chabaudi succumb to severe disease and higher mortality than their heterozygote counterparts or normal mice [11]. An enhanced Th1 response persisted throughout the course of infection in the IL-10-deficient mice, while in control mice a Th2 response was predominant. In another recent study, the exogenous administration of IL-10 to susceptible CBA mice protected them from Plasmodium berghei—induced cerebral malaria while an in vivo neutralization of IL-10 in resistant BALB/c mice induced a neurologic syndrome [12]. Collectively, these findings support the hypothesis that IL-10 is a critical factor in down-regulating the pathogenesis of severe malaria.
In previous studies [3–6, 9], the interrelationship between IL-10 and TNF-α in severe malaria was not investigated. We addressed this issue using defined clinical groups and showed that children with mild disease have higher IL-10—to—TNF-α ratios than children with malaria anemia. However, it remains to be determined whether the IL-10—to—TNF-α ratio is altered in children with cerebral malaria compared with malaria anemia. In the holoendemic area of Kisumu, cerebral malaria is rare. Therefore, we could not study the IL-10—to—TNF-α ratio in cerebral malaria patients. However, similar studies in areas with seasonal transmission of malaria, where cerebral malaria is common, will be necessary to address this issue.
The in vivo sequence of TNF-α and IL-10 production in humans after a malaria infection is not clear. Since monocytes and macrophages produce both cytokines, we speculate that IL-10 may be produced soon after the production of TNF-α to regulate the inflammatory activities of TNF-α. TNF-α induces fever, and elevated body temperatures can suppress parasitemia. while such TNF-α-induced mechanisms can serve the host to control infection, prolonged exposure to TNF-α may adversely affect the individual by inducing or promoting severe disease. Indeed, a previous study showed that TNF-α suppresses erythropoiesis, thus contributing to anemia development [13]. Therefore, P. falciparum—infected persons who produce balanced levels of IL-10 to regulate excessive TNF-α activity may escape from developing severe or moderate malaria anemia. Recently, it was shown that some of the polymorphism in the IL-10 gene is associated with decreased IL-10 production [14]. Further studies are clearly needed to investigate if polymorphism in the IL-10 gene can contribute to pathogenesis of malaria anemia.
Murine studies have shown that in addition to TNF-α, IFN-γ also plays a critical role in the pathogenesis of cerebral malaria [15]. Previous human studies have shown that in acute P. falciparum infections, especially in persons with cerebral malaria, IFN-γ production is elevated [3, 5] in addition to TNF-α. However, in this study, there was no evidence for the production of IFN-γ in any of the clinical malaria groups except in hospital control children. This finding suggests that this cytokine may not be a critical factor in the development of malaria anemia in holoendemic areas. IL-4 is a regulator of IFN-γ and TNF-α activities, and earlier studies showed that IL-4 negatively regulates the induction of cerebral malaria in a rodent model [7]. However, the lack of IL-4 production in malaria patients, as found in this study, suggests that IL-4 may not be a critical factor in the pathogenesis of malaria anemia.
In conclusion, this study showed that a lower IL-10—to—TNF-α ratio is associated with malaria anemia in children residing in a holoendemic area. A higher IL-10—to—TNF-α ratio was associated with mild disease. Our findings suggest that an imbalance in the IL-10 cytokine regulatory network may contribute to the pathogenesis of malaria anemia.
Acknowledgments
We thank Charles Obonyo for assisting with sample collection; David Anyona, Simon Kariuki, John Ongecha, and other staff members of the Kenya Medical Research Institute (KEMRI)-CDC field station for technical support; study volunteers for participation; and the KEMRI director for approving the publication of this work.
