Antimalarial Activity of Human Group IIA Secreted Phospholipase A₂ in Relation to Enzymatic Hydrolysis of Oxidized Lipoproteins

The level of human group IIA secreted phospholipase A₂ (hGIIA sPLA₂) is increased in the plasma of malaria patients, but its role is unknown. In parasite culture with normal plasma, hGIIA is inactive against Plasmodium falciparum, contrasting with hGIIF, hGV, and hGX sPLA₂s, which readily hydrolyze plasma lipoproteins, release nonesterified fatty acids (NEFAs), and inhibit parasite growth.

ABSTRACT

The level of human group IIA secreted phospholipase A₂ (hGIIA sPLA₂) is increased in the plasma of malaria patients, but its role is unknown. In parasite culture with normal plasma, hGIIA is inactive against Plasmodium falciparum, contrasting with hGIIF, hGV, and hGX sPLA₂s, which readily hydrolyze plasma lipoproteins, release nonesterified fatty acids (NEFAs), and inhibit parasite growth. Here, we revisited the anti-Plasmodium activity of hGIIA under conditions closer to those of malaria physiopathology where lipoproteins are oxidized. In parasite culture containing oxidized lipoproteins, hGIIA sPLA₂ was inhibitory, with a 50% inhibitory concentration value of 150.0 ± 40.8 nM, in accordance with its capacity to release NEFAs from oxidized particles. With oxidized lipoproteins, hGIIF, hGV, and hGX sPLA₂s were also more potent, by 4.6-, 2.1-, and 1.9-fold, respectively. Using specific immunoassays, we found that hGIIA sPLA₂ is increased in plasma from 41 patients with malaria over levels for healthy donors (median [interquartile range], 1.6 [0.7 to 3.4] nM versus 0.0 [0.0 to 0.1] nM, respectively; P < 0.0001). Other sPLA₂s were not detected. Malaria plasma, but not normal plasma, contains oxidized lipoproteins and was inhibitory to P. falciparum when spiked with hGIIA sPLA₂. Injection of recombinant hGIIA into mice infected with P. chabaudi reduced the peak of parasitemia, and this was effective only when the level of plasma peroxidation was increased during infection. In conclusion, we propose that malaria-induced oxidation of lipoproteins converts these into a preferential substrate for hGIIA sPLA₂, promoting its parasite-killing effect. This mechanism may contribute to host defense against P. falciparum in malaria where high levels of hGIIA are observed.

INTRODUCTION

Malaria is due to protozoan parasites of the genus Plasmodium that are transmitted to vertebrates by mosquitoes. In mammalian hosts, Plasmodium spends most of its lifetime in red blood cells (1). In humans, the intraerythrocytic parasite is responsible for the clinical symptoms associated with malaria. The vast majority of clinical cases present as nonspecific febrile illnesses that are relatively easily terminated (uncomplicated malaria), but a minority of cases progress to severe, life-threatening disease. According to the WHO World Malaria Report 2015 (2), there were 214 million cases of malaria globally in 2015 and 438,000 malaria deaths attributed to major complications. In this context, a better knowledge of the actors of malaria physiopathology remains a key element to fight the disease. The work presented here focuses on the possible antimalarial role of a family of secreted phospholipase A₂ (sPLA₂) released by mammalian host cells, with special emphasis on human group IIA secreted PLA₂ (hGIIA sPLA₂).

sPLA₂s are structurally conserved enzymes with a low molecular mass (14 to 19 kDa) that catalyze the hydrolysis of glycerophospholipids at the sn-2 position to release free fatty acids and lysophospholipids (3–7). Mammalian sPLA₂s exhibit unique tissue and cellular distributions as well as different enzymatic properties (7–10), suggesting distinct physiological and pathophysiological roles for each enzyme. Besides their role in the production of lipid mediators, such as eicosanoids and lysophospholipids, multiple pieces of evidence indicate that sPLA₂s participate in innate immunity, especially in the first line of host defense against bacteria and other pathogens (11–26).

Among sPLA₂s, hGIIA sPLA₂ is also known as the inflammatory-type sPLA₂. It is a strong bactericidal agent present in inflammatory fluids and in the plasma of patients with sepsis (11, 12, 18, 21, 23, 27–29). In patients with malaria, hGIIA circulates at abnormally high levels, an observation originally made by Vadas and colleagues in the early 1990s (30, 31). However, its role in malaria has remained unknown until recently.

When the studies by Vadas et al. were performed, only hGIB (pancreatic-type human group IB sPLA₂) and hGIIA were known in humans. Since then, additional genes coding for sPLA₂s have been identified in the human genome, and up to 12 genes are now identified (IB, IIA, IID, IIE, IIF, III, V, X, XIIA, XIIB, otoconin-95, and the pseudogene IIC) (3, 4, 8). Very recently, a genome-wide association study of nonsevere malaria suggested a role for one or more sPLA₂s (32) present in a gene cluster containing 6 sPLA₂ genes coding for hGIIA, hGIID, hGIIE, hGIIF, and hGV (33), further strengthening our interest in the study of sPLA₂s in malaria.

In our previous studies, we demonstrated that various venom sPLA₂s (34–37) as well as several human sPLA₂s, including hGIIF, hGIII, hGV, and hGX sPLA₂s, exert potent in vitro antimalarial activity against Plasmodium falciparum, the most virulent species of human parasites (34–38). However, in these typical in vitro infection assays of red blood cells by P. falciparum where normal human serum is used, hGIIA sPLA₂ was inactive (38). We depicted a mechanism by which human sPLA₂s exert their killing effect against P. falciparum indirectly by hydrolyzing phospholipids from human native lipoproteins present in the parasite culture medium and generating lipid products such as nonesterified fatty acids (NEFAs), including polyunsaturated fatty acids (PUFAs), which appeared as the key lipid products toxic to the parasite and responsible for sPLA₂-dependent parasite death (38).

Interestingly, it has been shown that hGIIA sPLA₂ more efficiently hydrolyzes oxidized lipoproteins than their native counterparts (39–43). Oxidation of lipoproteins is observed in malaria (14) and in other pathological situations, including atherosclerosis, inflammatory syndromes, and infectious diseases (44–46). Since our in vitro experimental conditions described above using native human lipoproteins likely were not reflecting the in vivo physiopathological conditions of malaria, we sought to reinvestigate whether hGIIA and the other human sPLA₂s would be more effective against Plasmodium in the presence of oxidized lipoproteins.

We found that in vitro oxidation of human lipoproteins converts these into a readily hydrolyzable substrate for hGIIA sPLA₂, revealing its toxic effect toward the parasite. Oxidation of lipoproteins also enhances the inhibitory effects of hGIIF, hGV, and hGX sPLA₂s. To provide further in vivo relevance for these results, plasma from healthy and P. falciparum-infected people were analyzed for sPLA₂ content, lipoprotein oxidation, and capacity to inhibit P. falciparum in vitro growth. hGIIA sPLA₂ was increased in plasma from infected patients, whereas the other sPLA₂s were not detected. The level of lipoprotein oxidation was higher in malaria plasma than normal plasma, and only malaria plasma was able to confer in vitro inhibitory activity of exogenously added hGIIA sPLA₂ against P. falciparum. The in vivo relevance of these observations was challenged by injection of recombinant hGIIA sPLA₂ into Plasmodium-infected mice. A significant decrease of parasitemia was observed in mice only when the sPLA₂ was injected at the late time point in the course of infection, at the time concomitant to an increase in plasma peroxidation.

Together, our results suggest that the combined presence of high levels of oxidized lipoproteins and hGIIA sPLA₂ synergize to help control parasite growth in human malaria.

RESULTS

Oxidative modification of lipoproteins enhances hydrolysis by hGIIA and hGIIF but not other sPLA₂s.

We previously reported the specific activities of different human sPLA₂s on native lipoproteins (Table 1 and reference 38). To analyze the anti-Plasmodium activity of human sPLA₂s in a context more relevant to malaria where lipoproteins are oxidized (14), we examined the capacity of various human sPLA₂s to hydrolyze low-density lipoprotein (LDL) and high-density lipoprotein (HDL) particles after in vitro oxidation.

TABLE 1.

Specific activities of recombinant human sPLA₂s on lipoproteins

Human sPLA2	n	Specific activity (μmol NEFA/min·mg of sPLA2)				Fold changea (oxidized/native)		P valueb
		Oxidized		Native				Oxidized versus native		LDL versus HDL
		LDL	HDL	LDL	HDL	LDL	HDL	LDL	HDL	Native	Oxidized
hGIB	4	0.06 ± 0.03	0.45 ± 0.39	0.05 ± 0.02	0.25 ± 0.22	1.2	1.8	0.1133	0.1250	0.0625	0.0625
hGIIA	7	0.16 ± 0.09	0.10 ± 0.06	0.02 ± 0.02	0.03 ± 0.03	8.0	3.3	0.0156*	0.0313*	0.5982	0.0156*
hGIID	2	0.04	0.03	0.03	0.04	—	—	—	—	—	—
hGIIE	2	0.03	0.02	0.02	0.02	—	—	—	—	—	—
hGIIF	6	1.04 ± 0.28	1.56 ± 0.64	0.74 ± 0.21	1.58 ± 0.59	1.4	1.0	0.0313*	0.8438	0.0139*	0.1563
hGIII	4	4.29 ± 3.84	0.82 ± 0.51	6.29 ± 5.37	1.06 ± 0.70	0.7	0.7	0.2297	0.0961	0.0447*	0.0931
hGV	6	0.99 ± 0.37	3.27 ± 2.57	1.05 ± 0.44	3.88 ± 2.20	0.9	0.8	0.8438	0.1563	0.0313*	0.0625
hGX	7	2.66 ± 1.27	5.31 ± 2.12	2.58 ± 1.60	7.92 ± 5.08	1.0	0.6	0.6875	0.1094	0.2188	0.0313*
hGXIIA	2	<0.01	<0.01	<0.01	<0.01	—	—	—	—	—	—

^aValues are means ± SD (μmol NEFA/min·mg of sPLA₂). —, not determined. n, number of independent experiments.

^bFor statistical analysis, the nonparametric Wilcoxon matched-pair test was used. *, P < 0.05.

Hydrolysis of lipoproteins was assessed by measuring the release of nonesterified fatty acids (NEFAs). Seven of the 9 recombinant catalytically active human sPLA₂s hydrolyzed oxidized and native LDL and HDL particles with the same specific activities (Table 1). In contrast, hGIIA and, to a lesser extent, hGIIF, exhibited significantly higher activity on oxidized lipoproteins. Oxidation of both LDL and HDL dramatically increased the activity of hGIIA sPLA₂, whereas oxidation of LDL but not HDL slightly increased the activity of hGIIF. A slight fold change was also observed for hGIB on LDL and HDL, but this change did not reach significance. However, the specific activity of hGIIA on oxidized LDL and HDL remained lower than that of hGIIF, hGIII, hGV, and hGX sPLA₂s, which are highly active on both native and oxidized lipoproteins.

Oxidized LDL and HDL pretreated with human sPLA₂s, including hGIIA, are active in vitro against P. falciparum.

We next compared the anti-Plasmodium activity of oxidized lipoproteins before and after hydrolysis by recombinant human sPLA₂s. Assays were carried out with lipoproteins at concentrations close to the physiological ones (0.2 mg/ml of phospholipids in the culture medium). Inhibitory concentrations of sPLA₂-hydrolyzed LDL and HDL against P. falciparum growth are presented in Table 2. Without sPLA₂ pretreatment, oxidized LDLs were barely inhibitory toward Plasmodium, whereas oxidized HDLs were not. Pretreatment with 20 nM hGIIF, hGIII, hGV, and hGX sPLA₂s, but not other sPLA₂s, increased the toxicity of oxidized LDL and rendered oxidized HDL toxic to the parasite. However, oxidized lipoproteins were not more potent than their native counterparts after hydrolysis. Oxidized LDL were less potent than native LDL after hydrolysis by hGIII sPLA₂.

TABLE 2.

Anti-P. falciparum activities of lipoproteins after hydrolysis by human sPLA₂s

sPLA2 pretreatment (20 nM)	Activity ofa:
	Oxidized lipoproteins			Native lipoproteins
	IC50 (μg PC/ml)		P value (oxidized LDL versus oxidized HDL)	IC50 (μg PC/ml)		P value
	LDL	HDL		LDL	HDL	LDL (oxidized versus native)	HDL (oxidized versus native)
None	220.1 ± 61.7	>250.0	—	>250.0	>250.0	—	—
hGIB	207.5 ± 59.5	185.2 ± 56.6	—	>250.0	>250.0	—	—
hGIIA	220.7 ± 59.3	>250.0	—	>250.0	>250.0	—	—
hGIID	222.3 ± 60.6	>250.0	—	226.6 ± 40.7	>250.0	—	—
hGIIE	224.7 ± 54.8	>250.0	—	>250.0	238.7 ± 43.3	—	—
hGIIF	151.4 ± 49.7	147.6 ± 49.7	0.8447	190.0 ± 89.5	157.4 ± 68.9	0.3970	0.4375
hGIII	166.4 ± 63.1	196.8 ± 77.5	0.0209*	115.0 ± 43.9	210.2 ± 99.9	0.0498*	0.6747
hGV	195.6 ± 67.9	141.6 ± 45.5	0.0089**	241.6 ± 133.8	181.2 ± 84.5	0.2604	0.1198
hGX	101.8 ± 16.5	75.6 ± 11.2	0.0355*	111.6 ± 37.3	81.8 ± 13.3	0.5155	0.4294
hGXIIA	228.2 ± 54.1	>250.0	—	>250.0	>250.0	—	—

^aValues are means ± SD from 5 independent experiments. For statistical analysis, the nonparametric Wilcoxon matched-pair test was applied, with P < 0.05 (*) and P < 0.01 (**) considered significant. —, not determined.

Since the circulating levels of hGIIA sPLA₂ can be very high in severe cases of malaria (30, 31) while pretreatment of oxidized lipoproteins with 20 nM hGIIA was ineffective against P. falciparum, we evaluated the anti-Plasmodium potency of oxidized lipoproteins when treated at higher concentrations. As shown in Table 3, pretreatment with 100 nM hGIIA sPLA₂ moderately increased the toxicity of oxidized LDL, whereas 250 nM induced a marked toxicity of both oxidized LDL and HDL and rendered native lipoproteins toxic.

TABLE 3.

Anti-P. falciparum activities of lipoproteins after hydrolysis with high concentrations of hGIIA sPLA₂

hGIIA sPLA2 pretreatment	Anti-Plasmodium activity of LDL and HDLa (IC50 value [μg PC/ml])
	Native LDL	Oxidized LDL	Native HDL	Oxidized HDL
None	>250	220.1 ± 61.7	>250	>250
100 nM	±250	162.3 ± 22.4	>250	>250
250 nM	215.3 ± 26.4	87.0 ± 39.2	216.6 ± 23.5	87.3 ± 9.2

^aValues are means ± SD from 3 independent experiments.

The anti-Plasmodium activity of human sPLA₂s, including hGIIA, is enhanced in culture medium containing oxidized lipoproteins.

To further analyze the effects of human sPLA₂s under conditions closer to physiology where all classes of lipoproteins are present, a total lipoprotein fraction (oxidized or not) instead of purified LDL or HDL was used without enzymatic pretreatment, and incubation was prolonged for 96 h to cover two parasite intraerythrocytic cycles instead of one. As expected, hGIIF, hGV, and hGX sPLA₂s were inhibitory in culture medium containing native lipoproteins (Table 4). hGIIF and hGX sPLA₂s exhibited 50% inhibitory concentration (IC₅₀) values of 19.8 and 1.5 nM, close to the IC₅₀ values previously measured in human plasma (10.7 nM and 2.9 nM, respectively [38]), whereas hGV sPLA₂ was found to be more active (IC₅₀ of 21.9 nM) than in previous assays with plasma (IC₅₀ of 94.2 nM [38]). hGIIA sPLA₂ was not inhibitory in normal plasma (38) or with native lipoproteins (Table 4). Remarkably, all four sPLA₂s had enhanced inhibitory activities in the presence of oxidized versus native lipoproteins. hGV and hGX sPLA₂s were 2-fold more active and hGIIF sPLA₂ was 4.6-fold more active, consistent with its increased capacity to hydrolyze oxidized lipoproteins. hGIIA sPLA₂ was inhibitory in the presence of oxidized lipoproteins, with an IC₅₀ value of 150 nM, while it was completely inactive with native lipoproteins.

TABLE 4.

Anti-P. falciparum activities of human sPLA₂s in the presence of native or oxidized lipoprotein fraction from human plasma

Total lipoprotein	Anti-P. falciparum activity of human sPLA2sa (IC50 value [nM])
	hGIIA	hGIIF	hGV	hGX
None	>250.0	>50.0	>80.0	>3.0
Native	>250.0	19.8 ± 13.3	21.9 ± 3.3	1.52 ± 0.74
Oxidized	150.0 ± 40.8	4.3 ± 4.0	10.2 ± 2.7	0.78 ± 0.26

^aValues are means ± SD from 4 independent parasite growth inhibition assays. The lipoprotein fraction was 0.2 mg PC/ml in culture medium.

Oxidized PUFAs are not responsible for enhanced anti-Plasmodium activity of sPLA₂s.

To understand why sPLA₂s exhibit greater anti-Plasmodium activities in assays with oxidized lipoproteins, we tested whether oxidized PUFAs have greater toxicity than native PUFAs. As far as we know, only one study, by Kumaratilake et al. (47), reported the anti-Plasmodium activity of oxidized PUFAs, namely, oxidized arachidonic acid (AA) and docosahexaenoic acid (DHA). The authors found that these PUFAs were more active after oxidation. Assuming that oxidized phospholipids present in lipoproteins can be hydrolyzed by sPLA₂s such as hGIIA and hGX to release the corresponding oxidized PUFAs (40, 48), we compared the effects of native and self-oxidized AA, eicosapentaenoic acid (EPA), and DHA on parasite growth in dose-response assays. When added to culture medium containing human plasma, these PUFAs were inhibitory to P. falciparum in both native and oxidized states (Table 5). The IC₅₀ values for the three PUFAs in their native states were in good accordance with those reported by Kumaratilake et al. (47). However, once oxidized, they were not more efficient, exhibiting similar (AA) or even higher (EPA and DHA) IC₅₀ values (Table 5). Thus, these results are different from those by Kumaratilake et al., who reported an ∼5-fold (AA) and ∼2.0-fold (DHA) better capacity of these PUFAs to inhibit Plasmodium after oxidation (47). The discrepancy might come from differences in the amount and molecular identity of oxidation products as well as differences between experimental settings for oxidation. This indicates that there is no clear-cut difference between the capacity of oxidized versus native PUFAs to inhibit Plasmodium growth and suggests that the enhancement of the anti-Plasmodium activity of sPLA₂s in the presence of oxidized lipoproteins comes from an increased activity of sPLA₂s at hydrolyzing oxidized particles and from prolonged incubation during the inhibition assay, resulting in a greater production of various toxic lipids beyond the oxidized ones.

TABLE 5.

In vitro anti-Plasmodium effect of native and oxidized PUFAs

PUFA	Anti-P. falciparum activity of native and oxidized PUFAsa (IC50 value [μM])		P valueb (native versus oxidized)
	Native	Oxidized
Arachidonic acid (C20:4, n-6)	10.6 ± 2.1	16.1 ± 2.7	0.1293
Eicosapentaenoic acid (C20:5, n-3)	7.4 ± 1.4	25.9 ± 5.1	0.0030**
Docosahexaenoic acid (C22:6, n-3)	10.5 ± 1.0	65.3 ± 23.2	0.0315*

^aIC₅₀ values are means ± SEM from 3 independent experiments in triplicate.

^bUnpaired t test was applied for statistical analysis, with P < 0.05 (*) and P < 0.01 (**) considered significant.

Circulating levels of hGIIA sPLA₂, but not hGIIF, hGV, and hGX sPLA₂s, are increased in uncomplicated P. falciparum malaria.

To provide a pathophysiological relevance for our in vitro results, we tested the sPLA₂ enzymatic activity and the presence of hGIIA, hGIIF, hGV, and hGX sPLA₂s in plasma from 41 P. falciparum-infected Vietnamese adults versus 28 nonparasitized, healthy donors from the same geographic area. None of the parasitized individuals presented signs of complicated malaria at the time of blood sampling. As shown in Fig. 1A, sPLA₂ enzymatic activity was dramatically increased in the plasma of P. falciparum-infected patients relative to that of healthy donors (median [interquartile range, or IQR], 260.0 [148.5 to 367.1] versus 16.20 [10.46 to 29.06] cpm/min·μl, respectively; P < 0.0001). No correlation was found between the level of enzymatic activity and blood parasitemia (P = 0.8079, Spearman’s r = 0.03969; data not shown).

FIG 1.

sPLA₂ enzymatic activity and hGIIA sPLA₂ protein concentration are increased in malaria plasma. Plasma from 41 P. falciparum-parasitized patients (parasitized plasma) and 28 nonparasitized healthy donors (normal plasma) were analyzed for sPLA₂ activity using the [³H]oleate E. coli membrane assay (A) and hGIIA sPLA₂ content by quantitative TR-FIA using hGIIA-specific antibodies (B). Medians with IQR are shown. The nonparametric Mann-Whitney U test for independent samples was applied. ***, P < 0.0001. (C) Correlation between plasma hGIIA sPLA₂ concentration and plasma sPLA₂ enzymatic activity (Spearman r = 0.9542; ***, P < 0.0001). (D) Correlation between plasma hGIIA sPLA₂ concentration and parasitemia from donors (Spearman r = 0.06894; ns, not significant).

Plasma samples next were analyzed for the presence of hGIIA, hGIIF, hGV, and hGX sPLA₂s by a sandwich enzyme-linked immunosorbent assay (ELISA)-like method (time-resolved fluoroimmunoassay [TR-FIA]) using specific antibodies and assays for each sPLA₂ (28). Protein levels of hGIIA sPLA₂ were significantly increased in the plasma from parasitized patients (median [IQR], 1.6 [0.7 to 3.4] nM, with a maximum value of 9.1 nM) compared to those of the plasma from healthy donors (median [IQR], 0.0 [0.0 to 0.1] nM) (P < 0.0001) (Fig. 1B). In contrast, hGIIF, hGV, and hGX sPLA₂s were not detected in the parasitized plasma (not shown), indicating that these enzymes do not circulate, at least at detectable levels, in uncomplicated malaria. In the parasitized plasma, sPLA₂ enzymatic activity and hGIIA protein concentration were highly correlated (P < 0.0001, Spearman’s r = 0.9542) (Fig. 1C), indicating that hGIIA sPLA₂ is the predominant sPLA₂ responsible for the circulating enzymatic activity. No correlation was found between the plasma concentration of hGIIA and the parasitemia from infected donors (P = 0.6684, Spearman’s r = 0.0689) (Fig. 1D).

Malaria, but not normal, plasma inhibits in vitro parasite growth when spiked with recombinant hGIIA sPLA₂.

The capacity of hGIIA sPLA₂ endogenously present in the plasma from malaria patients to inhibit Plasmodium development was evaluated under conventional in vitro culture conditions. Ten plasma samples containing various levels of endogenous hGIIA sPLA₂, up to 9 nM, were tested (Fig. 2A). It is important to note that in our culture conditions where RPMI was supplemented with 8% human plasma, the final hGIIA concentrations were only in the range of 0.1 to 1 nM. The growth rates of P. falciparum measured in the presence of plasma from the different malaria patients were variable but not significantly lower than those measured in the presence of plasma from different healthy donors (median growth rate [IQR], 6.1 [4.3 to 14.4] in malaria plasma versus 8.1 [6.7 to 9.5] in normal plasma, P = 0.4278) (Fig. 2A). We then evaluated the possible role of hGIIA sPLA₂ present in the diluted plasma by adding to the culture medium LY311727 (3-{[3-(2-amino-2-oxoethyl)-2-ethyl-1-(phenylmethyl)-1H-indol-5-yl]oxy}propyl]-phosphonic acid; 164083-84-5), a specific active-site inhibitor of hGIIA sPLA₂ (9). Addition of LY311727 did not modify the parasite growth rate in the plasma samples (multiplication factor after 96 h of cultivation, median [IQR], 7.08 [2.87 to 17.5] without LY311727 versus 6.65 [4.14 to 10.7] with LY311727, P = 0.1550) (data not shown). The absence of effect of LY311727 (i) indicates that the variable capacity of the different malaria plasma samples to support parasite growth is not due to hGIIA, and (ii) might be explained by too-low concentrations of endogenous hGIIA under our assay conditions to be effective at inhibiting the growth of Plasmodium. To test this possibility, P. falciparum was grown for 96 h in plasma from malaria patients versus healthy donors spiked with 100 nM recombinant hGIIA sPLA₂. This concentration was chosen based on hGIIA plasma concentrations reported in severe cases of malaria (30, 31). As shown in Fig. 2A, hGIIA spiked in malaria but not normal plasma inhibited parasite development, demonstrating that (i) the concentration of endogenous hGIIA under our assay conditions was not sufficient to inhibit parasite growth but that higher concentrations were clearly effective, and (ii) malaria-induced modification of plasma (possibly including oxidation of lipoproteins) is required to reveal the anti-Plasmodium activity of hGIIA. We also tested the spike of malaria plasma with 10 nM hGIIF or 0.5 nM hGX recombinant sPLA₂s for comparison. Both enzymes were inhibitory for P. falciparum in malaria plasma and were found to be more active than hGIIA sPLA₂ (Fig. 2B), as anticipated from the in vitro results with human native plasma.

FIG 2.

(A) Malaria, but not normal, plasma spiked with recombinant hGIIA inhibits the growth of P. falciparum. P. falciparum was grown for two intraerythrocytic cycles (96 h) in RPMI containing 8% malaria (M; n = 10) or normal plasma (N; n = 8) in the absence or presence (+IIA) of 100 nM recombinant hGIIA sPLA₂. Parasitemia at time zero and 96 h (t₀ and t₉₆) was measured from Giemsa-stained smears. Multiplication factor was expressed as parasitemia t₉₆/parasitemia t₀. Paired t test was applied, with a P value of <0.05 (*). (B) hGIIF and hGX sPLA₂s are inhibitory to P. falciparum in malaria plasma. Parasites were grown for 96 h in RPMI containing 8% malaria plasma supplemented with 100 nM hGIIA (IIA), 10 nM hGIIF (IIF), or 0.5 nM hGX (X) or not supplemented with sPLA₂ (w/o). Experiments were performed with 14 (w/o; hGIIA) and 11 (hGIIF and hGX) malaria plasma samples. The nonparametric Wilcoxon signed rank test was applied for statistical analysis. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Lipoproteins from P. falciparum-infected plasma are oxidized.

To add further support to our hypothesis that the anti-Plasmodium activity of hGIIA sPLA₂, and maybe other sPLA₂s, is promoted by the presence of oxidized lipoproteins in the plasma from infected patients, we evaluated the level of lipoproteins and their oxidative status in malaria plasma versus controls. In accordance with previous observations reporting a drop in HDL content in patients’ blood during malaria (49, 50), the level of lipoproteins, as measured by total phosphatidylcholine (PC) concentration, was found to be lower in plasma from malaria patients than from healthy donors (median [IQR], 1.20 [1.00 to 1.46] mg/ml versus 1.36 [1.19 to 1.68] mg/ml, P = 0.0158) (Fig. 3A). Oxidation of lipoproteins in plasma was estimated using the specific Mercodia oxidized LDL immunoassay kit (see Materials and Methods). LDL oxidation was clearly increased in the plasma from malaria patients compared to that from healthy donors (median [IQR], 33.13 [25.65 to 39.79] versus 15.41 [12.13 to 18.69] U/liter, P < 0.001) (Fig. 3B).

FIG 3.

Phospholipid content and LDL oxidation in malaria plasma. Plasma samples from uncomplicated malaria patients (n = 41) and healthy individuals (n = 28 for phospholipid analysis, n = 19 for oxidation analysis) were analyzed for phospholipid content using the phospholipid B-kit from WAKO Chemicals (A) and lipoprotein oxidation using the Ox-LDL kit (Mercodia) (B) by following manufacturers’ instructions. Medians with interquartile ranges (IQR) are shown. The Mann-Whitney test was applied. *, P < 0.05; ***, P < 0.001.

Together, our results demonstrate that malaria-induced modifications of plasma include oxidation of lipoproteins that most likely promotes the anti-Plasmodium activity of hGIIA sPLA₂ and enhances the inhibitory capacity of hGIIF and hGX sPLA₂s when spiked in malaria plasma.

The anti-Plasmodium activity of hGIIA sPLA₂ in malaria plasma requires its catalytic activity.

To demonstrate that the anti-Plasmodium activity of hGIIA sPLA₂ in malaria plasma requires enzymatic hydrolysis of oxidized lipoproteins, we first assessed the ability of the enzyme to hydrolyze phospholipids from malaria plasma. Plasma samples were incubated with hGIIA sPLA₂ as well as hGX sPLA₂, since the latter actively hydrolyzes both native and oxidized lipoproteins. hGIIA sPLA₂ did not release NEFAs from healthy plasma, whereas a small but significant release occurred from malaria plasma (P = 0.0093) (Fig. 4A). As expected, hGX sPLA₂ induced a net increase in NEFAs from both malaria (P = 0.0020) and healthy donor (P = 0.0019) plasma (Fig. 4A).

FIG 4.

(A) Exogenous hGIIA sPLA₂ hydrolyzes phospholipids from malaria but not normal plasma. Plasma from parasitized (n = 11; malaria) and nonparasitized healthy subjects (n = 10; normal) were incubated at 37°C for 18 h with 400 nM hGIIA sPLA₂ (hGIIA) or 8 nM hGX sPLA₂ (hGX) or without sPLA₂ (w/o). NEFA content (μg/μl of plasma) before and after incubation was measured by using the NEFA-C kit. NEFA release was calculated by subtracting NEFAs measured at time zero of incubation from NEFAs after 18 h of incubation. For statistical analysis, the nonparametric Wilcoxon signed rank test was applied. **, P < 0.01. (B) Parasite inhibition by hGIIA sPLA₂ depends on its catalytic activity. P. falciparum was tested for growth in 7 malaria plasma samples supplemented with 100 nM hGIIA sPLA₂, either WT or catalytically inactive (H48Q mutant). The growth control was growth without sPLA₂ (w/o). Multiplication factor after 96 h of incubation under culture conditions was calculated from parasitemia at time zero versus that at 96 h of incubation. For statistical analysis, paired t test was applied. *, P < 0.05; ns, P > 0.05. (C) The rate of parasite inhibition increases in parallel to the NEFA concentration measured in the supernatant of a culture established in malaria plasma supplemented with recombinant hGIIA sPLA₂. P. falciparum was grown for 96 h in RPMI-calcium with 8% malaria plasma (a mixture of 5 malaria plasma samples) supplemented with 20, 100, or 200 nM recombinant hGIIA sPLA₂. Aliquots were taken every 24 h to determine parasitemia and measure NEFAs released by hGIIA in the culture supernatant. Curves indicate parasite multiplication factor (i.e., percent parasitemia at each time point divided by percent parasitemia at time zero of incubation) in the presence of 20 (○), 100 (□), and 200 nM hGIIA (■) or in its absence (◆). Histograms show NEFAs from the supernatant of the parasite culture incubated with 20 (white bar), 100 (light gray bar), or 200 nM (dark gray bar) hGIIA. Levels of NEFAs at each time point were normalized by subtracting the amount of NEFAs in the culture supernatant at time zero of incubation.

Second, we tested whether the enzymatic activity is essential for the anti-Plasmodium effect of hGIIA sPLA₂. Parasite growth inhibition assays were performed using the H48Q catalytically inactive mutant of hGIIA (<0.5% of wild-type [WT] enzymatic activity [51]). The parasite development was not affected in malaria plasma spiked with the H48Q mutant, indicating that hydrolysis of plasma phospholipids is required for parasite inhibition (Fig. 4B).

Lastly, we analyzed the relationship between Plasmodium inhibition in malaria plasma supplemented with hGIIA sPLA₂ and the amount of NEFAs released by the enzyme in the culture supernatant. We found that both parasite inhibition and amount of NEFA in the culture medium increase with the time of incubation and concentration of hGIIA sPLA₂ (Fig. 4C). Interestingly, a small but noticeable inhibition of parasite development was observed in the presence of 20 nM hGIIA beyond 48 h of incubation, suggesting a long-term effect induced by relatively low concentrations of the enzyme.

Together, these results indicate that the anti-Plasmodium activity of hGIIA sPLA₂ observed in the plasma from malaria patients is mediated by hydrolysis of oxidized lipoproteins.

Recombinant hGIIA sPLA₂ inhibits parasite development in Plasmodium-infected mice only when injected at the time of plasma peroxidation.

To challenge our hypothesis that hGIIA sPLA₂ helps to control parasite development in an in vivo situation where lipoprotein oxidation does occur, we injected recombinant hGIIA sPLA₂ into C57BL/6J mice infected with P. chabaudi at different time points postinoculation (p.i.). The recombinant enzyme was injected twice daily for 3 consecutive days, either at the onset of the patent phase (when the first parasites are observed on blood smears) or later, just prior to the parasitemia peak. Interestingly, a significant decrease by about 20% of the parasitemia peak was observed after the late injection of hGIIA (Fig. 5B) but not when hGIIA was injected at early time points (Fig. 5A).

FIG 5.

Injection of recombinant hGIIA sPLA₂ affects Plasmodium development in mice in a time-dependent manner. (A) Eleven C57BL/6J mice were inoculated at day 0 with P. chabaudi chabaudi 864VD. At days 8, 9, and 10 p.i., a group of 6 mice was injected twice daily i.p. with 0.125 mg/kg of recombinant hGIIA sPLA₂ (■), while control mice (n = 5) were injected with vehicle (□). (B) Twelve mice were infected as described for panel A, and then a group of 7 mice were injected with hGIIA at days 12, 13, and 14 p.i., and control mice (n = 5) were injected with vehicle. Parasitemia from tail blood was determined at days 6, 9, 12, 13, 14, 16, 19, and 21 p.i. Two-way ANOVA with Bonferroni posttests was applied for statistical analysis (*, P < 0.05; ***, P < 0.001). Arrowheads point out days of injection. (C) Plasma peroxidation in P. chabaudi-infected mice is increased at the peak of parasitemia. Mice from 3 independent experiments were sacrificed at day 0 or at days 8, 14, and 21 postinoculation of 1 × 10⁶ RBC parasitized with P. chabaudi chabaudi 864VD. Plasma was analyzed for TBAR content (MDA equivalent). Values from the 3 experiments have been pooled. One-way ANOVA (Kruskal-Wallis test) with posttest (Dunn’s multiple-comparison test) was applied for statistical analysis (***, P < 0.001). Means with standard errors of the means are shown. Values from each plasma sample are presented as dots.

To assess the potential involvement of oxidized lipoproteins in the in vivo anti-Plasmodium effect of hGIIA, the plasma from infected mice was analyzed for lipid peroxidation. The levels of thiobarbituric acid-reactive substances (TBARs) were increased at day 14 following parasite inoculation but not before (Fig. 5C), i.e., at the time coincident with the one when injection of hGIIA was found to be effective against Plasmodium infection.

DISCUSSION

Since the discovery in the early 1990s of an increase of hGIIA sPLA₂ in the plasma from malaria patients (30, 31), no comprehensive study on the potential role of this enzyme has been made in the context of malaria physiopathology. Interestingly, an association of plasma PLA₂ enzymatic activity and related phospholipid products with brain swelling in pediatric cerebral malaria was reported in 2015 (52), suggesting a role of hGIIA sPLA₂ in the inflammatory events operating in cerebral malaria. More recently, a possible role for the gene cluster encoding multiple sPLA₂s (genes for hGIIA, hGIID, hGIIE, hGIIF, and hGV sPLA₂s) has been suggested from results of a genome-wide association study performed on noncomplicated malaria susceptibility (32), highlighting the possible role for one or more human sPLA₂s in malaria.

In line with the above-described findings, we provide here the first evidence that hGIIA sPLA₂ and maybe other sPLA₂s contribute as host defense factors to control Plasmodium development. Our results indicate that hGIIA, as well as some other sPLA₂s, operate through hydrolysis of lipoproteins and, more precisely, oxidized lipoproteins that are produced during the course of Plasmodium infection in malaria (14). This was demonstrated by combining a series of in vitro studies on parasite infection, by analysis of plasma from malaria patients, and finally by in vivo analysis of the anti-Plasmodium effect of hGIIA sPLA₂ in a mouse model of chronic malaria.

We previously demonstrated that hGIIF, hGIII, hGV, and hGX, but not hGIIA, sPLA₂s inhibit the in vitro growth of P. falciparum through hydrolysis of native lipoproteins present in human plasma (38). We now show that oxidized lipoproteins are also substrates for these enzymes and participate in the inhibition of parasite growth, at least in vitro. Focusing on hGIIA sPLA₂ because of its presence in the plasma from malaria patients, we also show that (i) hGIIA hydrolyzes much more potently oxidized LDL and HDL than their native counterparts (where no detectable hydrolysis was observed), a finding in accordance with previous results (39, 40, 43), and (ii) hGIIA exhibits anti-Plasmodium activity only in the presence of oxidized lipoproteins.

Under normal culture conditions, hGIIF, hGIII, hGV, and hGX sPLA₂s hydrolyze lipoprotein phospholipids, producing toxic free fatty acids, mainly long-chain PUFAs (38). It is known that hGIIA sPLA₂ has little activity on native lipoproteins but higher activity on oxidized counterparts (39, 40, 43, and this study). Identification and quantification of the lysophospholipids and fatty acids produced upon hydrolysis of native lipoproteins by hGIIA and other sPLA₂s have been reported by us and others (38–40). It is also known that hGIIA sPLA₂ hydrolyzes oxidized lipoproteins to generate nonoxidized lipids and likely oxidized products (39, 40, 42, 43). In vitro and in vivo antimalarial properties of native as well as oxidized lipids, especially PUFAs, have been reported elsewhere (47, 53). Kumaratilake et al. have shown that oxidized PUFAs are more active than native PUFAs against P. falciparum in vitro (47). However, we did not find evidence that oxidized PUFAs are more toxic than native PUFAs, yet PUFAs were indeed toxic. These differences might be due to experimental variations between the two studies, and, obviously, additional investigations will be necessary to solve the question of the role of oxidized lipids in the anti-Plasmodium activities of sPLA₂s. Considering the other sPLA₂s, it is interesting that hGIIF, hGV, and hGX sPLA₂s displayed increased capacities to inhibit Plasmodium when incubated with oxidized lipoproteins from the whole lipoprotein fraction and for a long incubation time (96 h, i.e., two parasite cycles), whereas LDL or HDL first purified and then oxidized and prehydrolyzed with 20 nM of those sPLA₂s did not exhibit greater toxicity than the native ones in the classical 48-h inhibitory assay. This indicates that experimental conditions are crucial to reveal the anti-Plasmodium properties of sPLA₂s and suggests that a specific pathophysiological environment is a key factor in promoting their enzymatic and biological activities. In line with our findings, many studies have now shown that hGIIA sPLA₂ is only active when acting on specific noncellular and cellular phospholipid substrates, including microparticles, exosomes, or free mitochondria released by activated human platelets or other immune cells, or damaged and apoptotic cells after oxidation or scramblase-mediated phosphatidylserine externalization (6, 54–65).

We measured concentrations of hGIIA sPLA₂ up to 9 nM in the plasma from a Vietnamese cohort of 41 patients with uncomplicated malaria. These concentrations are consistent with a previous study by Vadas et al. in a small cohort of 14 Canadian adult patients with uncomplicated malaria (30), where the mean level of hGIIA sPLA₂ was 12.1 ± 4.8 nM (169.7 ± 67.3 ng/ml). We found that P. falciparum develops normally in such malaria plasma (diluted to 8%, i.e., at a relatively low concentration of hGIIA in the nanomolar range), although it contains oxidized LDL. In a subsequent study by Vadas et al. on Malawian children with severe malaria, the mean level of hGIIA sPLA₂ was estimated to be around 70 nM, with the most severe cases exhibiting levels up to 200 nM (31). Of note, in these studies, including ours, plasma samples were likely collected at different times during the course of infection, and it is not known how much the concentration of hGIIA varies during the infection and according to levels of parasitemia. In line with such high concentrations, we show that addition of 100 nM hGIIA sPLA₂ to uncomplicated malaria plasma induces significant parasite inhibition, whereas the same addition of hGIIA to normal plasma is ineffective. Furthermore, it should be considered that hGIIA sPLA₂ has high affinity for various heparan sulfate proteoglycans lining the vascular endothelium and various cells, which likely act as a reservoir of hGIIA in the circulation, and may lead to an underestimation of the actual concentration of hGIIA in blood (66–69). This is supported by the study by Nakamura et al. where the hGIIA sPLA₂ activity measured in plasma was increased by about 3-fold between heparinized and nonheparinized patients (70). This indicates that conventional collection of plasma or serum without heparin pretreatment leaves behind numbers of hGIIA molecules bound to the heparan sulfates in the vasculature or circulating immune cells. With these data in mind, the 100 nM concentration of hGIIA sPLA₂ used in our in vitro experiments may not be that far from the concentration in the vascular compartment of malaria patients, especially in severe cases of malaria. A last point to consider when comparing our in vitro data with what might occur in vivo is the fact that hGIIA needs an appropriate substrate to be effective, in our case oxidized lipoproteins. Besides the various levels of hGIIA circulating in patients, the quality and quantity of this particular substrate might also vary among individuals, leading to variable anti-Plasmodium host responses.

By investigating the effects of injection of recombinant hGIIA sPLA₂ into parasitized mice, we show that Plasmodium development is reduced in those mice. Interestingly, the enzyme is effective only when injected in the late course of infection (at days 12 to 14 p.i.) and not at earlier time points (at days 8 to 10 p.i.). Consistent with the in vitro results, plasma peroxidation was increased at day 14 but not at day 8, in line with the time window at which the injected hGIIA sPLA₂ is effective against Plasmodium in parasitized mice.

It must be noted that the drop in parasitemia induced by injection of hGIIA is rather modest, i.e., about 20%. This may reflect a transient effect of the injected enzyme due to its rapid in vivo clearance or capture by the vascular wall components and, hence, lower bioavailability of the soluble enzyme at hydrolyzing plasma oxidized lipoproteins. This situation may differ from that in infected patients where the enzyme is continuously produced by sustained inflammation and circulates in plasma at steady-state high levels. The use of transgenic mice continuously overexpressing the human enzyme may be more relevant in this context, as they were previously used to demonstrate the antibacterial effect of hGIIA sPLA₂ in vivo (17–19, 71–73) and have been shown to have altered lipoprotein profiles (74, 75).

One may also consider that the in vivo antimalarial activity of hGIIA sPLA₂ results from multiple mechanisms of action, with the release of PUFAs directly toxic to the parasite being only one among several mechanisms. Indeed, there is considerable evidence that hGIIA sPLA₂ is an important effector of the innate immune response and may activate platelets or neutrophils among different immune cells involved in malaria (23, 63, 64, 76, 77). The production of lipid mediators by sPLA₂s as an integral component of the inflammatory reaction plays a major role in protecting the host against invading pathogens (4, 6, 23). However, lipid products might also induce deleterious effects on the host, as exemplified by recent metabolomics analyses showing a positive association between lipid products of the PLA₂ pathway and brain swelling in pediatric cerebral malaria (52).

Concerning the possible in vivo role of other human sPLA₂s, we showed that exogenous hGIIF and hGX sPLA₂s are active against Plasmodium in plasma from malaria patients as well as healthy donors, in accordance with their ability to hydrolyze lipoproteins under native or oxidized states. However, these sPLA₂s were not detected in the plasma of P. falciparum-infected or healthy subjects. It is tempting to speculate that they act locally at sites relevant to Plasmodium infection. Indeed, while there is now considerable evidence that hGIIA sPLA₂ is the only sPLA₂ highly present in the circulation under pathological conditions associated with inflammation, tissue injury, or infection, there is little evidence for the presence of other sPLA₂ isoforms in serum, except for hGIB sPLA₂ in some disease situations (28, 78). However, several sPLA₂s other than hGIIA are also involved in various types of infection, suggesting that these sPLA₂s exert their effects in diverse pathologies locally, within the microenvironment of the disease, and either in an autocrine or paracrine manner (4, 6, 7). Examples include studies on the role of GIII, GV, and GX sPLA₂s after infection with various pathogens (20, 22, 79–82), GIII and GX sPLA₂s in cancer (7, 83, 84), or GIIF, GIII, GV, and GX sPLA₂s in cardiovascular or skin diseases and allergy or asthma (85–90). It is also interesting that hGX sPLA₂ has been found in atherosclerotic plaques but not in plasma from patients with atherosclerosis (91), suggesting that the enzyme is produced locally but not systemically. Altogether, it is possible that sPLA₂s other than hGIIA also contribute to malaria within the vascular wall or via local secretion from immune cells, even though these enzymes are not detected in plasma.

In summary, we have shown that the concomitant presence of high concentrations of hGIIA sPLA₂ and oxidation of lipoproteins, as found in the serum of malaria patients, might participate in the host defense mechanism against P. falciparum. The mechanism of action would be based on a synergistic effect between the enzyme and its capacity to hydrolyze oxidized lipoproteins serving as one of its preferred substrates to release toxic lipids for Plasmodium, yet other indirect mechanisms may be involved. By extrapolation, this mechanism of action of hGIIA on oxidized lipoproteins might also be involved in other pathological conditions where both hGIIA sPLA₂ and oxidized lipoproteins are present, including atherosclerosis or other infectious diseases. It also remains to be determined if other sPLA₂s, such as hGIIF, hGIII, hGV, and hGX, which are active in vitro, also are active in an in vivo situation, even though these enzymes are not found in plasma from malaria patients.

MATERIALS AND METHODS

Materials.

The FcB1 strain of the human Plasmodium, P. falciparum Columbia, and the 864VD strain of the murine Plasmodium, P. chabaudi chabaudi, used in this work were from the Unicellular Eukaryotes Collection from the National Museum of Natural History (MNHN; MNHN-CEU-224-PfFcB1). The 864VD strain of the murine P. chabaudi chabaudi was from the MCAM Research Unit’s Plasmodium collection. C57BL/6JOlaHsd mice for the in vivo assays were from Envigo RMS SARL (Gannat, France). Plasma and red blood cells for the in vitro studies were from the O^– or A⁺ blood groups and were supplied by the French National Agency for Blood (convention reference C CPSL UNT 13/EFS/126; Etablissement Français du Sang [EFS]). RPMI 1640 and Albumax II were from Life Technologies (Cergy Pontoise, France). Diff-Quik staining reagents were from Medion Diagnostics AG.

The NEFA-C and the phospholipid B kits, used for quantitative determination of nonesterified fatty acids (NEFAs) and phospholipids, respectively, were from WAKO Chemicals (Oxoid S.A., Dardilly, France). The oxidized LDL ELISA kit was from Mercodia SAS Sales in France. Purified recombinant human sPLA₂s and the hGIII sPLA₂ domain were prepared as described previously (92). The sPLA₂ inhibitor LY311727, which targets hGIIA sPLA₂, was from Sigma.

The polyunsaturated fatty acids (PUFAs) arachidonic acid (AA; C20:4, n-6), eicosapentaenoic acid (EPA; C20:5, n-3), and docosahexaenoic acid (DHA; C22:6, n-3) were from Cayman Chemicals (Interchim). [³H]hypoxanthine monohydrochloride (370 GBq/mmol to 1.11 TBq/mmol) was from Perkin-Elmer. Other high-quality-grade biochemical reagents were from Sigma.

Methods.

(i) P. falciparum cultivation. Under routine culture conditions, P. falciparum was grown in red blood cells from the A⁺ group at 2% hematocrit and 2 to 4% parasitemia in RPMI supplemented with 11 mM glucose, 27.5 mM NaHCO₃, 100 UI/ml penicillin, 100 μg/ml streptomycin adjusted to pH 7.4 (basic medium) or supplemented with 8% heat-inactivated human A⁺ plasma (complete medium) according to the procedure of Trager and Jensen (93). When specified, the serum substitute Albumax II (0.5%, wt/vol, final concentration) was used in culture medium instead of heat-inactivated human plasma. Culture flasks were gassed with 91% N₂, 6% O₂, and 3% CO₂ before being incubated at 37°C. Under those conditions, the intraerythrocytic cycle of the FcB1 strain was 48 h long. Percent parasitemia was established by optical examination of Diff-Quik-stained smears from the formula (infected erythrocytes number/total erythrocytes number) × 100.

(ii) Purification of lipoproteins and oxidation. Nonfasted human plasma was split into aliquots and frozen at –20°C. One aliquot was thawed to prepare LDL and HDL by differential centrifugation according to Havel et al. (94). Briefly, chylomicrons and very-low-density lipoproteins were removed by a first round of centrifugation at 1.006-g/ml density, and then LDL and HDL were purified separately by successive centrifugations at 1.053-g/ml and 1.210-g/ml densities, respectively. When specified, the total lipoprotein fraction was purified by a single run of plasma at 1.210-g/ml density. Lipoproteins were dialyzed at 4°C against NaCl (9 g/liter) and then against RPMI and sterilized by 0.2-μm filtration. Oxidation was achieved by storing lipoproteins in a transparent flask at room temperature under sterile air exchange for 14 days. Native lipoproteins were prepared from another aliquot of the plasma just prior to assays. Experiments were performed within 1 week of lipoprotein storage at 4°C under N₂ in the dark. Phosphatidylcholine (PC) content of lipoproteins was measured by using the phospholipid B dosage kit according to the manufacturer’s instructions.

(iii) Oxidation of PUFAs. Concentrated solutions of commercial AA (76.5 mM), EPA (60 mM), and DHA (100 mM) in ethanol were stored frozen under N₂. An aliquot of 20 μl of each PUFA was allowed to dry in a glass tube and then exposed to air and light for 1 week under sterile conditions. Dried PUFAs were resuspended into 100 μl CHCl₃ and then diluted 1/20 in RPMI containing 8% heat-inactivated human plasma.

Prior to dose-response assays, nonoxidized PUFAs were prepared from another 20-μl aliquot of each concentrated solution, mixed to 80 μl of CHCl₃, and diluted into culture medium as described above.

(iv) Measurement of lipid oxidation in lipoproteins and plasma. The level of TBARs (thiobarbituric acid-reactive substances) was determined as a marker of lipid peroxidation in purified human lipoproteins (95, 96). Briefly, 150 μl of diluted sample containing 0.2% butylated hydroxytoluene was mixed with an equal volume of 0.25 N HCl containing 15% trichloroacetic acid and 0.4% thiobarbituric acid. The mixture was heated at 85°C for 15 min and then cooled on ice. Butanol (150 μl) was added, the sample was vortexed, and then it was left to stand on ice for phase separation to occur. The butanol phase was taken for TBAR determination at 515-nm excitation and 550-nm emission wavelengths on a spectrofluorometer (AMINCO-Bowman series 2). 1,1,3,3-Tetraethoxypropane was used as an external standard. Three independent measurements with plasma lipoproteins from different donors were performed. Before oxidation, TBARs ranged between 0.30 to 0.50 and 0.05 to 0.15 nmol of malondialdehyde (MDA)/mg of protein in LDL and HDL, respectively. After 2 weeks of air-light oxidation, TBAR concentrations had risen to 1.50 to 2.50 (LDL) and 0.30 to 0.50 (HDL) nmol of MDA/mg of protein. For comparison, TBARs in LDL and HDL purified from the serum of malaria cases were reported to be around 1.0 and 0.2 nmol/mg of protein, respectively (14).

Due to the presence in plasma of substances other than MDA that were susceptible to react with thiobarbituric acid (97), the level of lipoprotein oxidation in crude human plasma was determined by quantifying oxidized LDL using the Ox-LDL ELISA kit from Mercodia, a sandwich ELISA based on the mouse monoclonal antibody 4E6, which is directed against a conformational epitope in oxidized ApoB-100. The ELISA was performed according to the manufacturer’s instructions. Oxidation of mouse plasma was measured by quantifying TBARs, because the Mercodia kit is not appropriate for mouse plasma.

(v) Hydrolysis of lipoproteins and plasma by sPLA₂. Native and oxidized LDL and HDL purified from the same batch of human plasma were adjusted to 1 mg phospholipid/ml in RPMI supplemented with 1 mM CaCl₂. They were incubated with and without recombinant sPLA₂ for various times at 37°C. sPLA₂s were used at different concentrations. hGIB was used at 100 nM (n = 4 independent experiments), hGIIA was used at 50, 100, and 250 nM (n = 7), hGIID was used at 100 and 200 nM (n = 2), hGIIE was used at 200 and 250 nM (n = 2), hGIIF was used at 30 and 40 nM (n = 6), hGIII was used at 50 and 100 nM (n = 4), hGV was used at 10, 40, and 50 nM (n = 6), hGX was used at 5, 10, and 20 nM (n = 7), and hGXIIA was used at 100 and 200 nM (n = 2). NEFAs were measured at different time points using the NEFA-C kit (WAKO) by following the manufacturer’s instructions. Values were normalized by subtracting NEFAs measured from lipoproteins incubated without sPLA₂. Specific activity was deduced from the linear part of the curve, [NEFA] = f(t).

To assess the ability of hGIIA and hGX sPLA₂s to hydrolyze phospholipids in plasma from P. falciparum-infected patients versus healthy donors, a 1 mM final concentration of CaCl₂ was added to each plasma sample, and then enzymes were added at 400 nM (hGIIA) and 8 nM (hGX) final concentrations. Samples were incubated overnight at 37°C. The control for endogenous release of NEFAs was sample without added sPLA₂. NEFA content before and after incubation was determined using the NEFA-C kit according to the manufacturer’s instructions.

(vi) Anti-Plasmodium activity assays with sPLA₂-hydrolyzed lipoproteins. The ability of human sPLA₂s to promote the toxicity of lipoproteins was tested in dose-response assays as described previously (38). Briefly, LDL and HDL (0.6 mg phospholipid/ml in RPMI) were incubated overnight at 37°C with 20 nM sPLA₂ or alone and then tested for parasite inhibition at an LDL and HDL final concentrations of 0.2 mg/ml (i.e., close to physiological concentrations) in RPMI 0.5% Albumax II for 48 h under normal culture conditions. Albumax II instead of human plasma was used throughout the test to avoid any contribution of lipoproteins from human plasma. Incubations with each sPLA₂ added alone were also performed to check for lipoprotein-independent toxicity of sPLA₂s under these conditions. Assays analyzing the anti-Plasmodium effect of lipoproteins pretreated with higher concentrations of hGIIA sPLA₂ (100 nM and 250 nM) were performed similarly. Assays analyzing the anti-Plasmodium effect of sPLA₂s coincubated with total lipoprotein fraction but without pretreatment were carried out on two parasite cycles (i.e., 96 h).

(vii) Anti-Plasmodium activity assays with oxidized PUFAs. Dose-response assays with oxidized and nonoxidized AA, EPA, and DHA were performed as described previously (38), with some modifications. Decreasing concentrations of each PUFA were established by 3-fold dilution steps in culture medium, distributed in a 96-well plate (50 μl/well), and mixed with a culture of P. falciparum (0.25% parasitemia, 4% hematocrit; 50 μl/well). Parasites were allowed to grow for 48 h under normal culture conditions, and then 9.25 kBq [³H]hypoxanthine was added per well. After an additional 48 h of incubation, plates were frozen and the experiment proceeded as described in reference 38.

(viii) Human plasma collection in Vietnam. Venous whole-blood samples were collected in acid citrate dextrose (ACD) (BD, India) vacutainers from patients attending commune health centers in the Binh Phuoc province, Vietnam. Malaria positivity was evaluated by using the rapid diagnostic test OptiMAL-IT (DiaMed AG, Switzerland). All infected patients had P. falciparum malaria. The final study population included 41 individuals with malaria and 28 healthy donor controls from the same area. From the malaria-infected cohort, there were 33 men and 8 women, with an average age of 27.1 years (range, 16 to 58 years).

Blood samples were centrifuged at 2,400 × g for 5 min at room temperature, and the plasma was immediately stored at –80°C. Plasma samples were then shipped to the National Museum of Natural History (Paris, France), where they were thawed on ice, aliquoted, and stored at –80°C until use.

The study was approved by the Vietnam People’s Army Department of Military Medicine. The purpose of the study was explained to participants in their own language, and oral consent was obtained. Malaria-positive patients were treated with a dihydroartemisinin-piperaquine combination in accordance with the national drug policy of Vietnam.

(ix) TR-FIA. TR-FIAs for hGIIA, hGIIF, hGV, and hGX sPLA₂s in plasma were performed as described earlier (28). The assays used rabbit polyclonal anti-sPLA₂ antibodies to set up ELISA-like sandwiches, which were shown to be highly specific for each sPLA₂ isoform (98). hGIII sPLA₂ could not be measured due to the lack of sensitivity of the corresponding immune serum.

(x) Enzymatic assays on Escherichia coli membranes. sPLA₂ activity in plasma was measured by hydrolysis of Escherichia coli membranes radiolabeled with [³H]oleic acid and autoclaved (99). Briefly, 40 μl of radiolabeled E. coli membranes (100,000 dpm in activity buffer consisting of 0.1 M Tris-HCl, pH 8.0, 10 mM CaCl₂, 0.1% bovine serum albumin [BSA]) was incubated with plasma (20 μl, diluted 1:150 in activity buffer) at 37°C for 30 min. Enzymatic assays were stopped by adding 80 μl of 0.1 M EDTA–0.2% fatty acid-free BSA. Reactions were spun down for 5 min at 10,000 × g, and the supernatant was collected and counted in a 1450 Microbeta counter (Wallac, Perkin Elmer).

(xi) P. falciparum cultivation in malaria plasma and related assays. To analyze the anti-Plasmodium effect of endogenous sPLA₂ in plasma from infected people, the FcB1 strain was grown in red blood cells of the O⁻ group, brought to 0.1 to 0.2% parasitemia, washed, and then transferred at 2% hematocrit into basic RPMI supplemented with 2.5 μM hypoxanthine and 1 mM CaCl₂ (RPMI-calcium). The cell suspension was distributed into wells of a 96-well microplate (100 μl/well), and then 8 μl of plasma from an infected donor or control normal plasma was added per well. To avoid thermal denaturation of the endogenous sPLA₂ (24, 100), plasma samples were not heat inactivated prior to parasite cultivation. The microplate was incubated in a candle jar for 96 h, and then parasitemia in each well was determined from Diff-Quik-stained smears.

To assess parasite inhibition by exogenously added hGIIA sPLA₂, the experiment was carried out as described above, with recombinant hGIIA added at the specified concentration at the start of incubation. To evaluate the contribution of hGIIA catalytic activity, recombinant hGIIA sPLA₂ in its native (WT) or catalytically inactive form (H48Q mutant) (51), and/or the specific hGIIA inhibitor LY311727, were added to the culture at 100 nM (hGIIA WT and H48Q) and 10 μM (LY311727) at the start of incubation.

In vivo assays with Plasmodium chabaudi-infected mice.

Mice were housed in the MNHN animal facilities, accredited by the French Ministry of Agriculture for performing experiments on live rodents. Work on animals was performed in compliance with French and European regulations on care and protection of laboratory animals (EC Directive 2010/63/UE, French Law 2013-118, 1 February 2013). All experiments were approved by the MNHN’s Ethics Committee and registered under deposit no. APAFIS 201802281454598.

C57BL/6J mice (males, 8 to 12 weeks old) were inoculated intraperitoneally (i.p.) with 1 × 10⁶ P. c. chabaudi 864VD-infected mouse red blood cells (RBC) in Alsever’s solution. Tail blood was taken every 2 to 3 days following inoculation. Parasitemia was established by optical examination of Diff-Quik-stained blood smears and counting of 2,000 RBCs.

Parasitized mice were i.p. injected with 100 μl of recombinant hGIIA (0.125 mg/kg of body weight) in phosphate-buffered saline containing 1% mouse serum. Injections were performed twice daily for 3 days, either at the beginning of the patent phase (early injection) or later, i.e., prior to the parasitemia peak (late injection). Blood parasitemia was determined every 2 to 3 days within 21 days following P. chabaudi inoculation. Three independent experiments were performed: one early injection experiment with 5 control mice and 6 hGIIA-injected mice and two late injection experiments, one with 5 (control) and 7 (injected) mice and one with 5 (control) and 6 (injected) mice.

To measure plasma peroxidation, mice were sacrificed by exsanguination at day 0 before parasite inoculation and at days 8, 14, and 21 postinoculation (p.i.). Blood was recovered into EDTA-coated tubes and centrifuged at 800 × g for 15 min at room temperature. Plasma was frozen at –80°C before TBAR measurement.

Statistical analysis.

Data were analyzed using GraphPad Prism software (San Diego, CA). Normality of groups with numbers superior to 6 was tested using the Shapiro-Wilk test. When sampling distribution was normal, the parametric unpaired or paired t test with a two-tailed P value was applied. When sampling distribution was not normal and/or the number was too small (≤6), the nonparametric Mann-Whitney U test for independent samples or the Wilcoxon matched-pair test for dependent samples was used. The effect of hGIIA sPLA₂ injection into P. chabaudi-infected C57BL/6 mice was analyzed by using two-way analysis of variance (ANOVA) with Bonferroni posttest. Statistical analysis of plasma peroxidation level in the course of infection was performed using one-way ANOVA with Dunn’s multiple-comparison test. Differences with P values of <0.05 (*), <0.01 (**), and <0.001 (***) were considered statistically significant.