Background
Malaria is a major public health problem in developing countries, primarily in sub-Saharan Africa and Asia [
1]. There were 229 million clinical cases of malaria resulting in approximately 409,000 deaths reported in 2019, making malaria one of the most prevalent and deadly infectious diseases in the world [
1]. Despite significant efforts to reduce the incidence of malaria in the past decade, progress in controlling and eliminating malaria has stalled in the last 2–3 years, with the number of infections beginning to rise in a number of countries [
1,
2]. Moreover, there is increasing evidence that
Plasmodium falciparum parasites, the causative agent of malaria, are developing resistance to a number of front-line anti-malarial drugs (such as artemisinin), which has the potential to severely impact malaria control in the future [
3,
4]. Consequently, there is still an urgent need to develop new treatments for malaria.
Understanding the critical host pathways that control the susceptibility and resistance of individuals to malaria in endemic countries may afford novel strategies to treat severe malaria. A recent human genome wide association study (GWAS) in Kenya identified single nucleotide polymorphisms (SNPs) in the human plasma membrane calcium ATPase 4 (PMCA4) gene (also called
ATP2B4) to have a very strong association with resistance and severity of malaria infection [
5]. This study confirmed results in two earlier GWAS (one in Ghana and one multi-site across Africa, Asia and Oceania) identifying
ATP2B4 as a significant resistance loci for severe malaria [
6,
7]. The reproducibility in identification of
ATP2B4 across different populations and studies suggests a major role for the gene product of
ATP2B4 in modulating the severity of
Plasmodium infection.
PMCAs are ATP-driven and calmodulin-dependent calcium pumps that eject calcium from the cell cytoplasm to the extra-cellular compartment [
8]. There are four PMCA isoforms that have been identified (PMCA1-4). It is believed that PMCA1 and PMCA4 are ubiquitously expressed whereas PMCA2 and PMCA3 expressions are confined to specific tissues [
9]. Importantly, PMCA4 expression can be detected in red blood cells (RBC), the target cell of
Plasmodium parasites during blood-stage malaria. In RBC, PMCA is the only active calcium extrusion pump required to maintain intracellular calcium in the 20–50 nM range [
10]. Thus, inhibition of PMCA4 activity significantly elevated intracellular calcium in RBCs [
10,
11]. Notably, the growth defect of
Plasmodium parasites in cold stored (4 °C) RBCs has been associated with inhibition of PMCA channel activity and increased intracellular Ca
2+ concentrations [
12]. The increase in intracellular Ca
2+ concentrations can lead to RBC dehydration [
13], and there is evidence that
P. falciparum parasites are unable to invade and grow as efficiently in dehydrated RBCs [
14]. Targeting and manipulating the activity of PMCA4 may, therefore, be a novel strategy for treatment of malaria.
In this study to directly assess the role of PMCA4 in modulating the course of blood stage malaria, mice with a systemic genetic knockout of the Pmca4 gene (PMCA4−/− mice) were infected with different species of murine Plasmodium parasites. Surprisingly, whilst abrogation of PMCA4 influences the morphology and biology of RBCs, it did not substantially affect peripheral parasite levels during Plasmodium yoelii, Plasmodium chabaudi or Plasmodium berghei infections. Inhibition of PMCA4 did, however, slightly increase the resistance of mice to experimental cerebral malaria (ECM). The results suggest that PMCA4 may have an effect on the development of severe malaria disease but raise important questions on the mechanisms involved.
Methods
Ethics statement
All animal work was approved following local ethical review by the University of Manchester (UoM) Animal Procedures and Ethics Committees and was performed in strict accordance with the U.K Home Office Animals (Scientific Procedures) Act 1986 (approved H.O Project Licence P8829D3B4).
Mice and infections
PMCA4 global knock out (PMCA4
−/−) mice and wild type (WT) littermates were used in this study. Generation of PMCA4
−/− mice was described in our previous publication [
15]. Uninfected global PMCA4
−/− mice are generally normal, as defined by whole body phenotype analysis [
16]. Mice were bred at the Biological Service Facility (BSF), University of Manchester. All mice were maintained in specific-pathogen free conditions in individually ventilated cages. The
Plasmodium chabaudi used was the non-lethal AS strain obtained from Dr K.N. Brown (National Institute for Medical Research (NIMR), London) [
17]. The
Plasmodium yoelii NL strain was obtained from Dr Brian De Souza (UCL, London) [
18], whereas the
Plasmodium berghei ANKA used was the clone cl15cy1 as described in [
19]. Depending upon the experiment, cryopreserved
P. chabaudi AS,
P. yoelii NL or
P. berghei ANKA parasites were passaged once in C57BL/6 mice before experimental mice were infected via intravenous (i.v.) injection of 10
4 parasitized red blood cells (pRBC) of one of the
Plasmodium species. The course of infection was monitored by assessing peripheral parasitaemia via microscopic examination of Giemsa-stained thin blood smears, by calculating mouse weight on specified days of infection (compared to starting weight at initiation of experiment), and by quantifying erythrocyte numbers in peripheral blood (RBC /ml) by microscopy using a C-Chip disposable haemocytometer (Cambridge Bioscience). ECM was graded during
P. berghei ANKA infection as previously described [
20]: 1 = no signs; 2 = ruffled fur/and or abnormal posture; 3 = lethargy; 4 = reduced responsiveness to stimulation and/or ataxia and/or respiratory distress/hyperventilation; 5 = prostration and/or paralysis and/or convulsions. Stages 2 and 3 were classified as prodromal ECM and stages 4–5 were classified as ECM.
Plasmodium berghei infected mice were euthanized when they reached stage 4/5. To examine the permeability of the blood brain barrier (an important event during ECM), mice were injected i.v. with 200 ml of 1% Evans blue (Sigma Aldrich, UK) on day 6 of
P. berghei infection. After one hour, mice were culled and intra-cardiac whole-body perfusion with 15 ml of PBS was performed, following which brains were removed.
Automated blood analysis
For peripheral blood analysis, mice were anesthetized using 2.5% isoflurane and blood was collected from the jugular vein. Blood samples were analysed within 6 h after collection using an automated blood analyser (Sysmex XT-2000iV) at room temperature. Blood analysis was carried out in 2 batches using a mouse profile.
Western blot
For western blot analysis of erythrocyte PMCA expression, blood was collected from the jugular vein of PMCA4−/− and WT mice under anaesthesia with 2.5% isoflurane, and heparinized. Blood was centrifuged at 1800g for 5 min and the plasma and buffy coat were discarded. Erythrocytes were then washed 4 times in twice their volume of 0.9% saline, with centrifugation for 5 min at 1200g. Packed erythrocytes were then lysed in 10 × their volume of RIPA buffer (containing 1% IGEPAL CA-630, 0.5% sodium deoxycholate, 0.1% SDS, 0.5 mM phenylmethylsulphonyl fluoride, 500 ng/ml Leupeptin, 1 mg/ml Aprotinin and 2.5 mg/ml Pepstatin A), and 10 µl of the resultant lysate was separated by SDS-PAGE and transferred to PVDF membranes (Millipore) using standard protocols.
Membranes were blocked in 3% BSA (for PMCA4 detection) or 3% non-fat milk (for PMCA1 detection) for 1 h at room temperature, before refrigerated incubation overnight with primary antibodies against PMCA4 (clone JA9-Abcam) or PMCA1 (clone F-10-Santa Cruz). Proteins were visualized the following day using enhanced chemiluminescence (GE Healthcare), after incubation with HRP-linked anti-mouse or anti-rabbit secondary antibody (Cell Signalling) on a ChemiDoc XRS Imaging System (Biorad). Membranes were then incubated with β-actin or GAPDH antibody (abcam) as loading control.
Image stream
Heparinized blood obtained from the tail vein was surface stained for 25 min at 4 ΟC with anti-Ter119 (Ter119—BioLegend), anti-CD44 (IM7—Thermofisher) and anti-CD71 (R17217—Thermofisher), before being washed and resuspended in PBS containing DAPI. Cells were analysed with an ImageStream X Mk II (Amnis) and data was analysed with IDEAS (Amnis).
Analysis of RBCs morphology following Plasmodium infection
Images of Giemsa-stained thin blood smears from WT and PMCA4−/− mice on days 7 and 9 of P. chabaudi and P. yoelii infection were uploaded to Image Pro Premier image analysis software. Automated classification of RBCs was performed with a lower size threshold of 3000 pixels (to exclude small cell fragments and circulating material) and a roundness threshold of < 1.2 (to exclude overlapping cell populations). A total of 38 fields of view from 3 mice per group per time point was analysed.
Flow cytometry
Spleens were removed from infected and uninfected PMCA4−/− and WT control mice and homogenized through a 70 µm cell sieve to create single cell suspensions (BD Biosciences). RBCs were lysed in the samples by addition of RBC lysing buffer (BD Biosciences), following which cells were washed and resuspended in HBSS containing 2% FCS (FACS buffer). Brains were isolated from mice after intra-cardial perfusion with PBS, chopped into small pieces using scissors, and passed through a 10 ml syringe, before being incubated on a tube roller at room temperature in FACS buffer with Collagenase (final concentration 1 mg/ml) (Sigma) for 45 min. The resultant brain cell suspensions were filtered through a 70 µm cell sieve, washed in FACS buffer, and layered on a 30% Percoll gradient and centrifuged at 2000g for 10 min. The supernatant was discarded and the cell pellet collected. RBCs were lysed in the samples by addition of RBC lysing buffer (BD Biosciences) and cells were washed and resuspended in FACS buffer. Absolute cell numbers were determined by microscopy using a haemocytometer and live/dead differentiation was performed using the trypan blue exclusion cell viability assay (Sigma).
Spleen and brain samples were surface stained for 20 min with anti-mouse CD4 (GK1.5—Thermofisher), anti-mouse CD8a (53–6.7—Thermofisher), anti-mouse CD11a (M17/4—Thermofisher), anti-mouse CD11b (M1/70—BioLegend), anti-mouse CD31 (390—BioLegend), anti-mouse CD45 (30-F11—Thermofisher), anti-mouse CD49d (R1-2—BioLegend), anti-mouse ICAM-1 (YN1/1.7.4—BioLegend), anti-mouse Ly6C (HK1.4—BioLegend) and anti-mouse Ly6G (1A8—BioLegend). Intracellular staining for granzyme B (GB11—BioLegend) was performed for 45 min, after treatment with Foxp3 fixation/permeabilization buffer (Thermofisher). Dead cells were identified and exluded from analysis using LIVE/DEAD® Fixable Blue Dead Cell Stain Kit (Life Technologies). Fluoresce minus one controls were used to set gates. Cells were analysed with a BD LSR II (Becton Dickinson) using BD FACSDiva software (Becton Dickinson). Data was analysed with FlowJo (Tree Star Inc.).
Statistical analyses
Data was tested for normality using the Shapiro–Wilk test. Unpaired two tailed t test (for parametric data) or the Mann–Whitney U test (for non-parametric data) was used for comparison between two groups. One-way ANOVA, with Tukey post hoc analysis, or the Kruskal–Wallis test, with Dunn post hoc analysis, were used for comparisons between three or more groups, for parametric and non-parametric data, respectively. Survival data was analysed using the Mantel-Cox test. Results were considered as significantly different when p < 0.05.
Discussion
The key finding of our study is that genetic ablation of PMCA4 expression in mice slightly improved the survival against P. berghei infection, which is a common murine model of cerebral malaria. However, PMCA4 deficiency did not alter the course of non-lethal malaria during P. chabaudi and P. yoelii infections. Moreover, the deletion of PMCA4 did not impact peripheral parasite levels during any of the murine malaria models examined.
The PMCA4 SNPs that have been associated with risk to severe malarial disease are believed to control the level of PMCA4 expressed on RBCs [
34]. Specifically, PMCA4 SNPs that correspond with reduced PMCA4 expression (and consequent increased intracellular Ca
2+ levels) appear to be associated with protection against severe malaria [
34]. PMCA4-deficiency in red blood cells has previously been shown to lead to increased mean corpuscular haemoglobin concentration (indicating reduced RBC hydration), likely due to Ca-induced activation of potassium channel resulting in potassium efflux and volume loss (the Gardos effect) [
13]. The hydration state of RBCs has been demonstrated to strongly influence the invasion and growth of
Plasmodium parasites, with
P. falciparum parasites being unable to invade dehydrated cells, potentially due to membrane-cytoskeletal alterations precluding correct orientation of merozoites [
14,
35]. Thus, it was very surprising that there did not appear to be any obvious differences in the status of intra-erythrocytic parasites, or the morphology of parasitized RBCs, in PMCA4
−/− mice compared with WT mice, and that peripheral parasitaemia levels were not altered in PMCA4
−/− mice during any of the investigated murine
Plasmodium spp. infections within this study.
Whilst the above analyses did show differences in the mean corpuscular haemoglobin levels in RBCs from PMCA4
−/− mice, the majority of other analysed RBC properties (size, morphology, maturation) were only minorly affected or were unaffected by abrogation of PMCA4 expression. Indeed, whilst the results from the automated blood analyser suggested that mean corpuscular volume of RBCs was lower in PMCA4
−/− mice, this was not recapitulated within the image stream analysis. There were also no obvious signs of increased proportions of crenated and dehydrated RBCs, or difference in RBC diameter, perimeter and circulaty in infected PMCA4
−/− mice when studying Giemsa-stained thin blood smears. Thus, the data suggests that abrogation of PMCA4 in murine RBCs may not perturb RBC properties to a sufficient level to affect
Plasmodium parasite invasion, growth or survival. Indeed, the effect of RBC dehydration on parasite invasion and growth appears to be a graded phenomenon that only becomes significant above a threshold level of dehydration [
14]. The finding of this study, therefore, appears to question the dominant impact of PMCA4 in controlling
Plasmodium invasion, growth or survival within RBCs (normocytes or reticulocytes) during murine malaria.
It is important to note that it has yet to be definitively shown in any study that PMCA4 directly affects
Plasmodium parasite growth in RBCs. To directly test this question it will be necessary to isolate RBCs from individuals with different PMCA4 SNPs and culture them in vitro with
P. falciparum parasites. As intracellular Ca
2+ levels dictate many different biophysical properties of RBCs [
36], the possibility that alterations in PMCA4 expression level affect other pRBC properties cannot be discounted. This includes several pRBCs properties, such as transport and membrane expression of
Plasmodium proteins and cellular deformability, that could influence the development of severe malaria, and which may feature more prominently in human blood-stage malaria than murine malaria. Moreover, in extreme cases, alterations in the morphology of RBCs may lead to increased phagocytosis within the spleen by red pulp macrophages, which clear damaged and old RBCs from circulation.
Several studies have shown that some genetic conditions involving haemolytic anaemia (including thalassemia and sickle cell disease) are often associated with “leakiness” of the RBC membrane, which lead to the increase in intracellular Ca
2+ level [
36]. However, other reported observations have shown that the mechanisms underlying protection against malaria in these conditions were reduction in haemoglobin level [
37] and reduction in parasite cytoadherence [
38], but not due to the increase in RBCs’ Ca
2+ level. This is in line with the finding in PMCA4
−/− mice that the levels of parasitaemia following
P. chabaudi and
P. yoelii infection were not different between WT and PMCA4
−/− mice.
Although the focus of the present study was on the potential role of PMCA4 on RBCs in influencing the course of blood stage malaria, the PMCA isoforms 1 and 4 are ubiquitously expressed in most organs and cell types [
9]. Interestingly, PMCAs may have different roles and functions in different cell types. For example, in vascular smooth muscle cells and in cardiac myocytes PMCA4 appears to modulate cellular signalling, including regulating nitric oxide (NO) production by neuronal nitric oxide synthase (nNOS)[
39‐
41]. In endothelial cells, PMCA4 modulates vascular endothelial growth factor (VEGF)-induced angiogenesis via regulation of calcineurin [
42]. In fibroblast cell line L929, PMCA4 is involved in regulating signalling pathways controlling responsiveness to tumour necrosis factor (TNF)[
43], whilst in T-cells, PMCA4 is involved in the production of inflammatory cytokines, such as interleukin-2 (IL-2) [
44]. Endothelial cells and various leucocyte populations including macrophages, neutrophils and T cells, play important roles in the pathogenesis of severe malarial disease syndromes, including cerebral malaria. Consequently, it is foreseeable that the association of PMCA4 SNPs with risk of severe malaria is not solely RBC-dependent, but is multifactorial, involving modulation of host pro-inflammatory immune responses and control of vascular homeostasis. In support of this, we observed qualitatively lower damage to the blood brain barrier in PMCA4
−/− mice compared with control mice during
P. berghei infection, corresponding with the slightly increased resistance of PMCA4
−/− mice to ECM. In addition, there was a trend towards lower numbers of T cells in the brains of PMCA4
−/− mice during
Plasmodium berghei infection, and intracerebral T cells that did accumulate also expressed lower levels of Granzyme B in infected PMCA4
−/− mice. Although the IFN-g production by brain accumulating CD8
+ T cells was not assessed, it can be anticipated that the level of this cytokine may also have been lower within infected PMCA4
−/− mice than in infected control WT mice, given that Granzyme B and IFN-g are sensitive measures of CD8
+ T cell pathogenic activity during
P. berghei infection [
30]. Notably, however, the numbers of monocytes, neutrophils and resident microglial cells within the brain were not significantly altered in PMCA4
−/− mice compared with control WT mice during
P. berghei infection. This suggests that global PMCA4 deletion did not lead to a generalized amelioration of neuroinflammation during
P. berghei infection. Thus, further work will be required to assess whether the specific alterations in T cell activity in the brain of infected PMCA4
−/− mice, and the associated slight increase in resistance to ECM, was due to cell autonomous effects of PMCA4 deficiency or was caused by other discrete indirect effects within the brain. Indirect effects that may influence CD8
+ T cell responses in the brain during
P. berghei ANKA infection could potentially include reduced pRBC accumulation and cross presentation of parasite antigen by brain endothelial cells, or alterations in CXCL9 and CXCL10 production, which are critical events in the pathogenesis of ECM [
45‐
48].
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