Background
Infection with
Plasmodium falciparum (
P. falciparum) is associated with a marked increase in systemic inflammation. Whereas this immune activation could contribute to parasite clearance, an enhanced immune response could also contribute to disease progression by promoting tissue damage rather than protection and through induction of immune exhaustion [
1]. The immune response during
P. falciparum infection involves several immune cells as well as their interactions. Thus,
P. falciparum infection is associated with a profound T-cell activation with the Th1-derived cytokine interferon γ as a major pathogenic factor [
2,
3]. Monocytes/macrophages are one of the main sources of cytokines in malaria-infected individuals and whereas some of these may be of importance for parasite clearance (e.g., interleukin 12) [
4], others may be major players in disease progression (e.g., tumor necrosis factor) [
5]. Moreover, whereas the inflammatory M1 macrophages could contribute to a sustained and harmful inflammation during
P. falciparum infection, the anti-inflammatory and pro-resolving M2 macrophages may prevent the development of overt disease. Neutrophils are the most abundant leukocyte population in the bloodstream, the primary compartment of
P. falciparum, and several studies support a role of these cells in
P. falciparum infection [
6‐
8]. The release of proteases and myeloperoxidase (MPO) could contribute to tissue damage and parasite invasion, whereas the formation of neutrophil extracellular traps (NETs) could contribute to parasite clearance.
There are several studies on immune activation and inflammation during
P. falciparum infection, and some of these have also examined T-cells, monocytes/macrophages and neutrophils [
9‐
12], but the role of these cells during falciparum malaria are far from clear. In particular, most studies have focused on one of these cell subsets, and fewer have examined the activation pattern of these cells simultaneously. In the present study we aimed to study the activation of T-cells, monocytes/macrophages and neutrophils during infection with
P. falciparum by using sCD25, sCD14, sCD163 and MPO as markers of T-cell, monocyte/macrophage and neutrophil activation, respectively. Moreover, whereas sCD163 at least to some degree could reflect M2 activity [
13], sCD14 is regarded as a more overall marker of monocytes/macrophage activation. Levels of these markers were related to the degree of parasitemia as assessed by quantitative PCR measurements as well as disease severity. The study was performed in Mozambique that has one of the highest global incidences of co-infection with HIV and falciparum malaria. Co-infection with those two infections poses an increased risk of severe malaria disease [
14]. We therefore also examined how HIV infection influenced plasma levels of sCD25, sCD14, sCD163 and MPO in these patients.
Discussion
Infection with
P. falciparum is characterized by a marked immune activation that may be both beneficial (i.e., anti-parasitic) and harmful (i.e., tissue destruction) for the host. We have previously demonstrated an approximately 30-fold increase in interferon inducing protein (IP-10) levels compared with healthy controls in this population of patients with falciparum malaria [
23] indicating a marked immune response affecting T-cell activation in these patients. Herein we extend these findings by showing enhanced activation of neutrophils, T-cells and monocyte/macrophages as assessed by the soluble activation markers MPO, sCD25, sCD14 and sCD163, with particular high levels of MPO and sCD25 in malaria patients that were co-infected with HIV. MPO, sCD25 and sCD14 were significantly correlated with clinical disease severity and sCD25 and in particular MPO were also strongly associated with degree of parasitemia as assessed by quantitative
P. falciparum PCR in plasma. Finally, patients with falciparum malaria also had elevated plasma levels of granzyme B and CX3CL1 suggesting enhanced activation of CD8
+ T-cells and effector memory T-cell subsets, and the marked increase in TIM-3 suggest some degree of T-cell exhaustion during falciparum malaria particularly in those co-infected with HIV. Our findings further underscore the link between immune activation and immune exhaustion during severe falciparum malaria potentially contributing to disease severity.
MPO and neutrophils have been implicated in the pathogenesis of falciparum malaria. A recent study of
Plasmodium yoelii non-lethal infection in wild-type and MPO deficient mice as a murine malaria model, suggested that MPO could contribute to a protective anti-parasite response [
24]. However, very few studies have analyzed MPO levels in human falciparum malaria. Recently, Rocha et al. showed that circulating neutrophils from malaria patients are highly activated, as indicated by a strong type I interferon transcriptional signature, increased expression of surface activation markers, enhanced release of ROS and MPO, associated with increased liver damage [
25]. Herein we show a strong association between MPO levels and the degree of
P. falciparum parasitemia as assessed by qPCR analyses, suggesting that neutrophils and MPO could promote rather than attenuate parasitemia. Moreover, if the high MPO levels reflect degranulated and exhausted neutrophils, the outcome could also be harmful to the host. MPO release has more recently been related to NET formation that could have anti-parasitic effects, but NETs could also promote vascular pathology during falciparum malaria, illustrating the immune response as a double edge sword during falciparum malaria [
26].
We have previously shown that co-infection with HIV during falciparum malaria is associated with enhanced inflammation, increased malaria severity and death [
14,
23]. Several other studies also demonstrate that falciparum malaria is more severe in HIV co-infected patients, in particular in those with decreased CD4 T-cell counts [
27,
28]. Here we extend these findings by showing that HIV-infected patients with falciparum malaria have markedly raised levels of MPO and sCD25 as compared with both malaria patients without HIV infection and HIV-infected patients without malaria. Moreover, sCD25 levels were also significantly correlated with disease severity in particular in those co-infected with HIV. These findings suggest that despite T-cell deficiency, co-infected patients have signs of sustained T-cell activation. In fact, sustained T-cell activation with increased spontaneous release of inflammatory markers could itself contribute to immunodeficiency. Thus, high circulating levels of sCD25 are mostly accompanied by decreased membrane expression of CD25 on T-cells [
29] resulting in attenuated proliferation and IL-2 release upon further challenge by additional stimuli in the microenvironment in vivo such as during falciparum malaria. Also, we found markedly increased TIM-3 levels during falciparum malaria as compared with HIV-infected patients without malaria, with the highest levels in those co-infected with HIV, suggesting some degree of T-cell exhaustion during falciparum malaria in particular in those co-infected with HIV. CD8
+ T-cell-mediated responses seem to be of major importance in the anti-parasitic T-cell responses and our findings of enhanced granzyme B levels could reflect an anti-malaria effect in these patients. Indeed, very recently granzyme B was found to contribute to the cytotoxic CD8
+ T-cell-mediated killing of
P. vivax-infected reticulocytes [
30]. However, granzyme B has also been implicated in the development of cerebral malaria [
31] and the increased granzyme B levels could also reflect degranulated and exhausted CD8
+ T-cells, further illustrating the dual effects of the immune response during malaria infection.
Recent studies have demonstrated that monocytes/macrophages are involved in both protection and immunopathology during malaria infection. Data on sCD14 and sCD163 in human falciparum malaria are, however, relatively scarce. As for sCD163 most data are in children and in pregnant women showing some association with disease severity and birth weight in offspring of pregnant women [
32]. Herein, all three groups (HIV infection only, falciparum malaria only, combined HIV/malaria infection) had elevated sCD14 and sCD163 levels with only minor differences between the groups. As for sCD14, however, we found that patients with falciparum malaria and HIV had higher levels than those with malaria alone, and sCD14 was also associated with clinical disease severity in the malaria group as a whole. Moreover, we found a significant association between sCD163 and the degree of parasitemia. The interpretation of these later data is difficult and could be by chance, but it could also be speculated that an increased anti-inflammatory M2 response could enhance parasitemia. Our data suggest the involvement of monocyte/macrophage activation during human
P. falciparum malaria, but the modest changes and the lack of differences in sCD14 between febrile HIV-infected patients and malaria patients suggests that these data should be interpreted with caution.
The present study has some limitations such as potential selection bias of not including the poorest and the wealthiest patients, lack of CD4 cell count on several HIV-infected patients and even if most patients were severely ill, relatively few died, and the sample sizes are too low to probe associations with mortality. Moreover, correlation analyses do not necessarily mean any causal relationship. Also, soluble markers are only surrogate markers of leukocyte action and may not fully reflect their activation status. In relation to T-cell exhaustion, we should ideally have isolated T-cells from fresh patient samples and investigated responses like loss of IL-2 production, loss of proliferative capacity along with co-expression of inhibitory receptors like PD-1 and CTLA-4 [
33] Also, stronger diagnostic tools than measurements of sCD163, such as flow cytometry and functional studies in addition to mRNA analyses of isolated monocytes/macrophages are needed to discriminate between M1 and M2 macrophages.
Acknowledgements
The authors are indebted to all the patients and their next of kin participating in this study, to the healthy controls, to the medical doctors, to the nurses and the nurses’ aides in the medical wards, to the Intensive Care Unit, to the laboratory coordinators and laboratory personnel in the General Laboratory, the Microbiological Laboratory, and the Anatomic Pathology Laboratory in the Central Hospital of Maputo, Mozambique. Special thanks are extended to Einar Sverre Berg at the Department of Virology, The Norwegian Institute of Public Health, Oslo, Norway, for performing the RNA/DNA-nucleic extraction and HIV- PCR. We acknowledge the kind donation of the reference material of P. falciparum (US 03 F Benin I), from the World Health Organization (WHO) Malaria Specimen Bank, hosted by the Center for Disease Control and Prevention (CDC, Atlanta, USA) with support from the Foundation for Innovative New Diagnostics (FIND).