Background
Malaria, caused by
Plasmodium falciparum, continues to cause significant morbidity and mortality particularly in young children from sub-Saharan Africa [
1]. Despite advances in control and elimination efforts, malaria parasites and the mosquitoes that transmit them are increasingly developing resistance to currently available drugs and insecticides, respectively [
2‐
4]. Novel control measures are urgently warranted in the global fight against malaria. The development of alternative clinical interventions is hindered by the limited understanding of malaria pathogenesis, particularly the complex interactions between the parasite and the host's immune system.
After mosquito inoculation, malaria sporozoites infect the liver, where the parasites replicate silently. The subsequent blood-stage infection leads to acute uncomplicated febrile illness, which can progress to severe life-threatening disease, as the parasite load increases [
5]. However, individuals who survive and are repeatedly re-infected acquire clinical immunity, which limits parasite load and thereby reduces the likelihood of developing clinical symptoms [
6]. Since naturally acquired immunity does not completely clear the parasites, individuals remain susceptible to asymptomatic infections [
7]. Typically, the highest parasite loads are seen in individuals with severe malaria, lower in symptomatic uncomplicated malaria, and lowest (often-times only positive by molecular methods) in asymptomatic infections. Yet, there is overlap in the parasite loads amongst these groups, such that some individuals with relatively high parasite load can be asymptomatic and others with relatively low parasite load can develop symptoms or even severe complications [
8]. The mechanisms of disease progression remain poorly understood and it is likely that there is either constitutive or acquired variation in the ability of individuals to tolerate a given parasite load without symptoms.
Blood-stage infection causes all of the pathological consequences of malaria, suggesting that alteration of any blood parameters could directly influence the risk of disease manifestations [
9]. Immune cells like T (CD3 +) cells and natural killer (NK) cells are known to play crucial roles in the immune response generated against pathogens. CD4 + T cells and NK cells produce pro-inflammatory cytokines, such as interferon-γ (IFN-γ), that promote parasite killing and clearance of infected erythrocytes by activating monocytes and macrophages, and enhancing B cell function [
10‐
12]. The function of CD8 + T cells during
P. falciparum blood-stage infection is poorly understood, although recent data suggest their role in severe diseases, such as cerebral malaria [
13]. γδ T cells are known to expand following malaria infection and correlate with protection from infection, but this response is attenuated with repeated exposure [
14]. Activated B-cells produce antibodies that play vital roles in controlling the blood-stage parasites. Effector functions of antibodies include inhibition of merozoites invasion of erythrocytes [
15], opsonization of infected erythrocytes for rapid clearance by macrophages [
16], and antibody-dependent cytotoxicity and cellular killing (ADCC) of parasites by natural killer (NK) cells [
17]; the underlying mechanism for the latter remains unclear. It is now widely accepted that endemic individuals have antibodies against a broad range of parasites antigens [
18], yet these individuals remain susceptible to infection. In addition to contributing to the protection, activation of immune cells, when dysregulated, also promote disease pathogenesis. While neutrophils can mediate parasite killing when activated [
19], they also release molecules such as proteases or neutrophil extracellular traps (NETs), which can also be very destructive to host tissues [
20,
21]. Pro-inflammatory mediators, such as IFN-γ and tumor necrosis factor (TNF), that support parasite killing and clearance can cause uncontrolled inflammation and lead to the sequestration of infected erythrocytes. Thus, the regulation of inflammatory responses, orchestrated by CD4 + T cells and monocytes/macrophages, is key to the successful resolution of malaria blood-stage infection [
22,
23].
Changes in peripheral blood leucocyte counts during
P. falciparum infection have been described. The reduction in the percentages and absolute cell counts of the major lymphocyte populations (total T cells, CD4 + T helper cells, CD8 + cytotoxic T cells, and B cells) and NK cells seems to be a feature of acute malaria infection [
24‐
27]. However, there are also conflicting reports, where no differences in the percentages of T cell subsets were identified during acute mild malaria relative to uninfected control groups [
28,
29]. It has also been suggested that factors such as age, sex, geographical location (linked to transmission intensity) and host genetic factors may alter the distribution of T cell populations during acute infection [
25]. For example, γδ T cells were found to rapidly expand following acute
P. falciparum infection in adults [
24], with no evidence of an increase in the numbers of this cell type in children [
28].
Despite the insights gained from previous studies, it is striking that the majority of these reports have only focused on contrasting symptomatic and uninfected cases. Very few studies have considered asymptomatic individuals. Since asymptomatic parasite carriers are clinically immune, they might have immune responses that differ from symptomatic individuals. Studies that compare symptomatic and asymptomatic children of similar age could provide insight into the nature of immune responses that mediate both resistance and tolerance to parasites. This present work addresses this question by profiling leucocyte subpopulations in Ghanaian children with symptomatic and asymptomatic P. falciparum infection and assessing the relationship between cell numbers and measures of parasite load.
Discussion
Numerous studies have indicated that
Plasmodium infection impacts the frequencies of peripheral blood leucocyte subsets. Yet, the majority of these studies have focused exclusively on symptomatic paired healthy cases. The present study was undertaken to address the limited information on the differences between the peripheral blood leucocyte profiles of children with symptomatic and asymptomatic
P. falciparum infections. Here, both the proportions (Figs.
1,
2,
3) and absolute counts (Additional file
2: Fig. S2, Additional file
3: Fig. S3, Additional file
4: Fig. S4) of different leucocyte populations were reported. Despite the outcomes for both were comparable in the present study, it is important to emphasize that absolute cell counts of leucocyte subpopulations are independent of each other, whereas proportions are not. It is conceivable that if the proportion of one cell type increases, one or more of the others decreases.
The present study showed that symptomatic infections were associated with decreased proportions and absolute counts of total CD3 + T cells, CD4 + and CD8 + T cells, CD19 + B cells, and CD11c + cells compared to asymptomatic infections, corroborating previously reported data [
25,
27]. The numbers of peripheral blood CD14 + and γδ + cell counts were not significantly different amongst the groups. Notably, the levels of CD15 + neutrophils were significantly higher in children with acute symptomatic malaria, and the numbers positively correlated with measures of parasite load, suggesting a causal link. The results also indicate that the cellular compositions of peripheral blood in children with asymptomatic malaria infections are very similar to uninfected children. The results strongly suggest that parasite load may be a key determinant of peripheral cell numbers and proportions during malaria infections.
Two measures of parasite load were used in this study:
PfHRP2 levels and parasite densities. Even though a strong positive correlation between
PfHRP2 levels and parasite densities was seen in the combined data of symptomatic and asymptomatic children, it is noteworthy that results of other studies directly addressing the correlation between parasite density and
PfHRP2 have been inconsistent. Some studies have demonstrated a strong relationship [
32], whereas others found no correlation [
33].
PfHRP2 concentrations are thought to be more representative of the total parasite load in the body because
PfHRP2 can be released from both sequestered and circulating parasites [
34]. When the ratio of sequestered to circulating parasites is relatively constant, then
PfHRP2 and parasite density will correlate well. However, when a greater proportion of parasites are sequestered, as has been described in some forms of severe malaria, then
PfHRP2 may be a better measure of parasite load [
35].
Acute symptomatic malaria infection is characterized by a rapid reduction of peripheral blood lymphocyte subsets: CD4 + , CD8 + , CD3 + T cells, and CD19 + B cells [
25]. These cells play a critical but complex role in parasite clearance and disease progression. Malaria parasites tend to sequester for a large portion of the asexual stage, a mechanism that has been shown to favour parasite growth by evading the filtering activity of the spleen [
36]. Accordingly, to carry out their anti-malarial effector functions, it is obvious that lymphocytes will continuously recirculate from the peripheral blood and patrol other body parts and tissues where they may be required [
37]. Studies in animal models and humans revealed that cytokine production is increased during acute malaria infection with consequent induction of fever and other malaria-related symptoms [
23,
38,
39]. It is noteworthy that high cytokine production correlates with increased expression of adhesion molecules on endothelial cells [
40] and leucocytes [
41]. In turn, activated endothelial cells will interact and entrap circulating leucocytes into the microvasculature of organs and this is a crucial step in inflammation. Indeed, it has been shown that IFN-γ, which is significantly elevated in acute malaria as compared to asymptomatic infections [
42], drastically caused the retrafficking of leucocytes into lymph nodes [
43]. Similarly, mice lacking the α-chain of the IFN-γ receptor were characterized by the absence of leucocyte accumulation in the brain vasculature [
44]. Therefore, the loss of peripheral blood lymphocytes subpopulations observed in the symptomatic participants may be due to an enhanced tissue infiltration and attachment [
25]. Alternatively, abnormal cell death through apoptosis has also been indicated as another mechanism by which lymphocytes are depleted from blood during acute malaria infections [
45]. This explanation is corroborated by the observation of high expression of exhaustion and senescence markers on T cells from children with symptomatic malaria compared to asymptomatic participants [
46,
47]. It is becoming increasingly clear that many chronic human infectious diseases, including
P. falciparum infections [
48,
49] and HIV [
50], are associated with the accumulation of atypical memory B cells that are characterized by the expression of exhausted markers, and a reduction of [
51]. Although the present study did not look for atypical memory B cells, it has been demonstrated that peripheral B cells of both asymptomatic and uninfected children living in malaria endemic settings were dominated by atypical memory B cells [
48]. Although atypical memory B cells are phenotypically exhausted, a recent study has shown increased expression of these cells to be associated with greater protection against malaria [
52]. Thus, further work is needed to ascertain the roles of exhausted immune cells in asymptomatic
versus symptomatic malaria infections.
The dynamic regulation of the number of monocytes and their activation status is crucial in determining infection outcome [
53]. The effect of the parasite on changes of circulating monocytes remains unclear. While some studies have reported increased monocytes count during asymptomatic infections relative to noninfected control [
54], others did not find any differences in monocytes counts between the two groups [
53]. Consequently, the proportions and count of monocytes were the same amongst the groups in the present study. Nonetheless, a positive correlation was observed between parasite densities and both the absolute numbers and the proportions of CD14 + monocytes in the asymptomatic group. Further work is needed to characterize different subtypes (classical, intermediate and nonclassical) of monocytes, since changes in the numbers of these have been implicated in infection outcomes with
P. falciparum infection [
55].
It is intriguing to see different changes in human immune cells in the different clinical states of malaria. Whereas CD4 + , CD8 + , CD3 + , and CD19 + lymphocytes were markedly decreased in patients with acute malaria infections, the percentages and absolute count of CD15 + neutrophils were significantly expanded compared to asymptomatically infected patients. Interestingly, the proportion of CD15 + neutrophils tend to be higher in the asymptomatic group than the control uninfected group although this difference was not significant. A strong positive association was observed between the percentages and absolute cell counts of neutrophil and parasitaemia detected in the symptomatic participants. Whether increased neutrophil numbers promote parasitaemia [
56] or higher parasite load drives neutrophil expansion is not certain at this point. However, the findings imply a possible link between the two. The elevation of neutrophils in proportion to disease severity also suggests that increased neutrophil numbers may contribute to malaria clinical manifestations. This is in support of a previous study showing that depletion of neutrophils prevented the development of cerebral malaria in mice [
57]. Similarly in humans, neutrophil granule protein genes were increased across all severe malaria cases [
35]. Given that increased neutrophils and high parasitaemia are both associated with developing clinical consequences of malaria [
35,
57,
58], it is likely that the presence of a direct relationship between neutrophil cell count and parasite load in the acute
Plasmodium infections may explain why these individuals showed symptoms. However, the absence of association between neutrophils and parasitaemia in asymptomatic infections suggests that neutrophil expansion and the production of neutrophil related cytokines are tightly regulated in the asymptomatic individuals favouring parasite clearance while at the same time avoiding clinical consequences from extensive neutrophilia. It will be interesting to determine whether, beyond the differences in absolute counts, if neutrophils from the different study groups exhibit different phenotypes.
In this study, no changes was observed in the frequencies and absolute count of γδ T cells in any of the study groups, which agreed with a previous study conducted in children [
28]. In contrast, other studies have shown the frequency of γδ T cells to markedly expand during acute
Plasmodium infections in naïve individuals [
24]. Interestingly, Jagannathan et al
. have shown that γδ T lymphocyte cells lose their ability to proliferate following repeated exposure [
14] and this could explain why the present study found no change in the frequency of this cell type. There is a high possibility that the participants in this study may have had repeated previous parasite exposure, suggesting that the γδ T cells may have lost the ability to proliferate upon parasite stimulation. Of course, the possibility of tissue sequestration playing a critical role in determining the outcome of peripheral frequency of γδ T cells in malaria infections cannot be ruled out [
59].
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