Mélanie Dacheux

Véronique Sinou

Christine Payré

Louise Jeammet

Daniel Parzy

Philippe Grellier

Christiane Deregnaucourt
email: christiane.deregnaucourt@mnhn.fr

Gerard Lambeau
email: lambeau@ipmc.cnrs.fr

Gérard Lambeau

Antimalarial activity of human group IIA secreted phospholipase A2 in relation with enzymatic hydrolysis of oxidized lipoproteins

Keywords: Malaria, secreted phospholipase A 2, lipoprotein, oxidation, Plasmodium

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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 [START_REF] Cowman | Malaria: Biology and Disease[END_REF]. In humans, the intraerythrocytic parasite is responsible for the clinical symptoms associated with malaria. The vast majority of clinical cases present as non-specific 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 (http://www.who.int/gho/malaria/en/) 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 entry to fight the disease. The work presented here focuses on the possible antimalarial role of a family of secreted phospholipases A 2 (sPLA 2 ) released by mammalian host cells, with special emphasis on human group IIA secreted PLA 2 (hGIIA sPLA 2 ). sPLA 2 s are structurally-conserved enzymes with a small molecular mass [START_REF] Fenard | Secreted phospholipases A2, a new class of HIV inhibitors that block virus entry into host cells[END_REF][START_REF] Koduri | Bactericidal properties of human and murine groups I, II, V, X, and XII secreted phospholipases A2[END_REF][START_REF] Piris-Gimenez | In vivo protective role of human group IIA phospholipase A2 against experimental anthrax[END_REF][START_REF] Movert | Secreted Group IIA Phospholipase A2 Protects Humans Against the Group B Streptococcus: Experimental and Clinical Evidence[END_REF][START_REF] Pernet | Pseudomonas aeruginosa eradicates Staphylococcus aureus by manipulating the host immunity[END_REF][START_REF] Kim | Lysis of human immunodeficiency virus type 1 by a specific secreted human phospholipase A2[END_REF] which catalyze the hydrolysis of glycerophospholipids at the sn-2 position to release free fatty acids and lysophospholipids [START_REF] Lambeau | Biochemistry and Physiology of Mammalian Secreted Phospholipases A2[END_REF][START_REF] Murakami | Emerging roles of secreted phospholipase A2 enzymes: Lessons from transgenic and knockout mice[END_REF][START_REF] Dennis | Phospholipase A2 enzymes: physical structure, biological function, disease implication, chemical inhibition, and therapeutic intervention[END_REF][START_REF] Murakami | Emerging roles of secreted phospholipase A enzymes: The 3rd edition[END_REF][START_REF] Murakami | A new era of secreted phospholipase A(2)[END_REF]. Mammalian sPLA 2 s exhibit unique tissue and cellular distributions as well as different enzymatic properties [START_REF] Murakami | A new era of secreted phospholipase A(2)[END_REF][START_REF] Valentin | Increasing molecular diversity of secreted phospholipases A2 and their receptors and binding proteins[END_REF][START_REF] Singer | Interfacial kinetic and binding properties of the complete set of human and mouse groups I, II, V, X, and XII secreted phospholipases A2[END_REF][START_REF] Masuda | Diverse cellular localizations of secretory phospholipase A2 enzymes in several human tissues[END_REF], suggesting distinct physiological and pathophysiological roles for each enzyme. Besides their role in the production of lipid mediators such as eicosanoids and lysophospholipids, multiple evidence indicates that sPLA 2 s participate in innate immunity, especially in the first line of host defense against bacteria and other pathogens [START_REF] Vadas | Induction of group II phospholipase A2 expression and pathogenesis of the sepsis syndrome[END_REF][START_REF] Weinrauch | Mobilization of potent plasma bactericidal activity during systemic bacterial challenge. Role of group IIA phospholipase A2[END_REF][START_REF] Koprivnjak | Wall Teichoic Acid Deficiency in Staphylococcus aureus Confers Selective Resistance to Mammalian Group IIA Phospholipase A2 and Human alpha-Defensin 3[END_REF][START_REF] Sibmooh | Increased fluidity and oxidation of malarial lipoproteins: relation with severity and induction of endothelial expression of adhesion molecules[END_REF][START_REF] Fenard | Secreted phospholipases A2, a new class of HIV inhibitors that block virus entry into host cells[END_REF][START_REF] Koduri | Bactericidal properties of human and murine groups I, II, V, X, and XII secreted phospholipases A2[END_REF][START_REF] Piris-Gimenez | In vivo protective role of human group IIA phospholipase A2 against experimental anthrax[END_REF][START_REF] Movert | Secreted Group IIA Phospholipase A2 Protects Humans Against the Group B Streptococcus: Experimental and Clinical Evidence[END_REF][START_REF] Pernet | Pseudomonas aeruginosa eradicates Staphylococcus aureus by manipulating the host immunity[END_REF][START_REF] Kim | Lysis of human immunodeficiency virus type 1 by a specific secreted human phospholipase A2[END_REF][START_REF] Nevalainen | Antibacterial actions of secreted phospholipases A2. Review[END_REF][START_REF] Balestrieri | Group V secretory phospholipase A2 modulates phagosome maturation and regulates the innate immune response against Candida albicans[END_REF][START_REF] Dore | Roles of secreted phospholipase A2 group IIA in inflammation and host defense[END_REF][START_REF] Paganelli | Group IIA-Secreted Phospholipase A2 in Human Serum Kills Commensal but Not Clinical Enterococcus faecium Isolates[END_REF][START_REF] Van Hensbergen | Streptococcal Lancefield polysaccharides are critical cell wall determinants for human Group IIA secreted phospholipase A2 to exert its bactericidal effects[END_REF][START_REF] Edgar | Discovery of glycerol phosphate modification on streptococcal rhamnose polysaccharides[END_REF]. Among sPLA 2 s, human group IIA (hGIIA) sPLA 2 is also known as the inflammatory-type sPLA 2 . It is a strong bactericidal agent present in inflammatory fluids and in the plasma of patients with sepsis [START_REF] Vadas | Induction of group II phospholipase A2 expression and pathogenesis of the sepsis syndrome[END_REF][START_REF] Weinrauch | Mobilization of potent plasma bactericidal activity during systemic bacterial challenge. Role of group IIA phospholipase A2[END_REF][START_REF] Movert | Secreted Group IIA Phospholipase A2 Protects Humans Against the Group B Streptococcus: Experimental and Clinical Evidence[END_REF][START_REF] Nevalainen | Antibacterial actions of secreted phospholipases A2. Review[END_REF][START_REF] Dore | Roles of secreted phospholipase A2 group IIA in inflammation and host defense[END_REF][START_REF] Gronroos | Bactericidal group IIA phospholipase A2 in serum of patients with bacterial infections[END_REF][START_REF] Nevalainen | Timeresolved fluoroimmunoassays of the complete set of secreted phospholipases A2 in human serum[END_REF][START_REF] Tan | The role of group IIA secretory phospholipase A2 (sPLA2-IIA) as a biomarker for the diagnosis of sepsis and bacterial infection in adults-A systematic review[END_REF]. In patients with malaria, hGIIA circulates at abnormally high levels, an observation originally made by Vadas and colleagues in the early 90s' [START_REF] Vadas | Induction of circulating group II phospholipase A2 expression in adults with malaria[END_REF][START_REF] Vadas | Increased serum phospholipase A2 activity in Malawian children with falciparum malaria[END_REF]. However, its role in malaria has remained unknown until nowadays.

When the studies by Vadas et al. were performed, only hGIB (pancreatic-type human group IB sPLA 2 ) and hGIIA were known in humans. Since then, additional genes coding for sPLA 2 s 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) [START_REF] Lambeau | Biochemistry and Physiology of Mammalian Secreted Phospholipases A2[END_REF][START_REF] Murakami | Emerging roles of secreted phospholipase A2 enzymes: Lessons from transgenic and knockout mice[END_REF][START_REF] Valentin | Increasing molecular diversity of secreted phospholipases A2 and their receptors and binding proteins[END_REF]. Very recently, a genome-wide association study of non-severe malaria has suggested the possible role for one or more sPLA 2 s [START_REF] Milet | First genome-wide association study of non-severe malaria in two birth cohorts in Benin: hints towards the involvement of the STAT3 pathway[END_REF] present in a gene cluster containing 6 sPLA 2 genes coding for hGIIA, hGIID, hGIIE, hGIIF, and hGV [START_REF] Valentin | Cloning and recombinant expression of human group IIF secreted phospholipase A2[END_REF], further strengthening our interest for the study of sPLA 2 s in malaria.

In our previous studies, we demonstrated that various venom sPLA 2 s [START_REF] Deregnaucourt | Bee venom phospholipase A2 induces stagespecific growth arrest of the intraerythrocytic Plasmodium falciparum via modifications of human serum components[END_REF][START_REF] Guillaume | Anti-Plasmodium properties of group IA, IB, IIA and III secreted phospholipases A2 are serumdependent[END_REF][START_REF] Guillaume | Interplay between lipoproteins and bee venom phospholipase A2 in relation to their anti-Plasmodium toxicity[END_REF][START_REF] Rouault | Neurotoxicity and Other Pharmacological Activities of the Snake Venom Phospholipase A2 OS2: The N-Terminal Region Is More Important Than Enzymatic Activity[END_REF] as well as several human sPLA 2 s including hGIIF, hGIII, hGV and hGX sPLA 2 s exert potent in vitro antimalarial activity against Plasmodium falciparum, the most virulent species of human parasites [START_REF] Deregnaucourt | Bee venom phospholipase A2 induces stagespecific growth arrest of the intraerythrocytic Plasmodium falciparum via modifications of human serum components[END_REF][START_REF] Guillaume | Anti-Plasmodium properties of group IA, IB, IIA and III secreted phospholipases A2 are serumdependent[END_REF][START_REF] Guillaume | Interplay between lipoproteins and bee venom phospholipase A2 in relation to their anti-Plasmodium toxicity[END_REF][START_REF] Rouault | Neurotoxicity and Other Pharmacological Activities of the Snake Venom Phospholipase A2 OS2: The N-Terminal Region Is More Important Than Enzymatic Activity[END_REF][START_REF] Guillaume | In Vitro Anti-Plasmodium falciparum Properties of the Full Set of Human Secreted Phospholipases A2[END_REF]. However, in these typical in vitro infection assays of red blood cells by P. falciparum where normal human serum is used, hGIIA sPLA 2 was inactive [START_REF] Guillaume | In Vitro Anti-Plasmodium falciparum Properties of the Full Set of Human Secreted Phospholipases A2[END_REF]. We depicted a mechanism by which human sPLA 2 s 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 non-esterified fatty acids (NEFAs) including polyunsaturated fatty acids (PUFAs) which appeared as the key lipid products toxic to the parasite and responsible for the sPLA 2 -dependent parasite death [START_REF] Guillaume | In Vitro Anti-Plasmodium falciparum Properties of the Full Set of Human Secreted Phospholipases A2[END_REF].

Interestingly, it has been shown that hGIIA sPLA 2 hydrolyzes more efficiently oxidized lipoproteins than their native counterparts [START_REF] Pruzanski | Lipoproteins are substrates for human secretory group IIA phospholipase A2: preferential hydrolysis of acute phase HDL[END_REF][START_REF] Pruzanski | Differential hydrolysis of molecular species of lipoprotein phosphatidylcholine by groups IIA, V and X secretory phospholipases A2[END_REF][START_REF] Pruzanski | Hydrolysis of minor glycerophospholipids of plasma lipoproteins by human group IIA, V and X secretory phospholipases A2[END_REF][START_REF] Korotaeva | Opposite effects of native and oxidized lipoproteins on the activity of secretory phospholipase A(2) group IIA. Prostaglandins Other Lipid Mediat[END_REF][START_REF] Korotaeva | Oxidized phosphatidylcholine stimulates activity of secretory phospholipase A2 group IIA and abolishes sphingomyelin-induced inhibition of the enzyme[END_REF]. Oxidation of lipoproteins is observed in malaria [START_REF] Sibmooh | Increased fluidity and oxidation of malarial lipoproteins: relation with severity and induction of endothelial expression of adhesion molecules[END_REF] and in other pathological situations including atherosclerosis, inflammatory syndromes and infectious diseases [START_REF] Khovidhunkit | Effects of infection and inflammation on lipid and lipoprotein metabolism: mechanisms and consequences to the host[END_REF][START_REF] Jairam | Pathophysiology of Lipoprotein Oxidation[END_REF][START_REF] Winklhofer-Roob | Low-density lipoprotein oxidation biomarkers in human health and disease and effects of bioactive compounds[END_REF]. Since our above in vitro experimental conditions using native human lipoproteins were likely not reflecting the in vivo physiopathological conditions of malaria, we sought to re-investigate whether hGIIA and the other human sPLA 2 s would be more effective against Plasmodium in the presence of oxidized lipoproteins.

We found that in vitro oxidation of human lipoproteins converts these latters into a readily hydrolyzable substrate for hGIIA sPLA 2 , revealing its toxic effect towards the parasite.

Oxidation of lipoproteins also enhances the inhibitory effects of hGIIF, hGV and hGX sPLA 2 s.

To provide further in vivo relevance of these results, plasma from healthy and P. falciparuminfected people were analyzed for sPLA 2 content, lipoprotein oxidation and capacity to inhibit P. falciparum in vitro growth. hGIIA sPLA 2 was increased in plasma from infected patients, whereas the other sPLA 2 s were not detected. The level of lipoprotein oxidation was higher in malaria plasma as compared to normal plasma, and only malaria plasma was able to confer in vitro inhibitory activity of exogenously added hGIIA sPLA 2 against P. falciparum.

The in vivo relevance of these observations was challenged by injection of recombinant hGIIA sPLA 2 to Plasmodium-infected mice. A significant decrease of parasitaemia was observed in mice only when the sPLA 2 was injected at the late timepoint in the course of infection, at the time concomitant to increase in plasma peroxidation.

Together, our results suggest that the combined presence of high levels of oxidized lipoproteins and hGIIA sPLA 2 might synergize to help controlling parasite growth in human malaria.

RESULTS

Oxidative modification of lipoproteins enhances hydrolysis by hGIIA and hGIIF but

not other sPLA 2 s -We previously reported the specific activities of different human sPLA 2 s on native lipoproteins (Table 1 and(37)). To analyze the anti-Plasmodium activity of human sPLA 2 s in a context more relevant to malaria where lipoproteins are oxidized [START_REF] Sibmooh | Increased fluidity and oxidation of malarial lipoproteins: relation with severity and induction of endothelial expression of adhesion molecules[END_REF], we examined the capacity of various human sPLA 2 s to hydrolyze LDL and HDL particles after in vitro oxidation.

Hydrolysis of lipoproteins was assessed by measuring the release of non-esterified fatty acids (NEFAs). Seven of the 9 recombinant catalytically-active human sPLA 2 s hydrolyzed oxidized and native LDL and HDL particles with the same specific activities (Table 1). In contrast, hGIIA, and to a lower extent hGIIF, exhibited significantly higher activity on oxidized lipoproteins. Oxidation of both LDL and HDL dramatically increased the activity of hGIIA sPLA 2 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 sPLA 2 s, which are highly active on both native and oxidized lipoproteins.

Oxidized LDL and HDL pretreated with human sPLA 2 s 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 sPLA 2 s. 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 sPLA 2 -hydrolyzed LDL and HDL against P. falciparum growth are presented in Table 2. Without sPLA 2 pretreatment, oxidized LDLs were barely inhibitory towards Plasmodium, whereas oxidized HDLs were not.

Pretreatment with 20 nM hGIIF, hGIII, hGV and hGX sPLA 2 s, but not other sPLA 2 s, 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 sPLA 2 .

Since the circulating levels of hGIIA sPLA 2 can be very high in severe cases of malaria [START_REF] Vadas | Induction of circulating group II phospholipase A2 expression in adults with malaria[END_REF][START_REF] Vadas | Increased serum phospholipase A2 activity in Malawian children with falciparum malaria[END_REF] 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 sPLA 2 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.

The anti-Plasmodium activity of human sPLA 2 s including hGIIA is enhanced in culture medium containing oxidized lipoproteins -To further analyze the effects of human sPLA 2 s in 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 sPLA 2 s were inhibitory in culture medium containing native lipoproteins (Table 4): hGIIF and hGX sPLA 2 s exhibited IC 50 values of 19.8 and 1.5 nM, close to the IC 50 values previously measured in human plasma (10.7 nM and 2.9 nM, respectively (37)), whereas hGV sPLA 2 was found to be more active (IC 50 = 21.9 nM) than in previous assays with plasma (IC 50 = 94.2 nM, (37)). hGIIA sPLA 2 was not inhibitory in normal plasma [START_REF] Guillaume | In Vitro Anti-Plasmodium falciparum Properties of the Full Set of Human Secreted Phospholipases A2[END_REF] nor with native lipoproteins (Table 4). Remarkably, all four sPLA 2 s had enhanced inhibitory activities in the presence of oxidized versus native lipoproteins. hGV and hGX sPLA 2 s were 2-fold more active, and hGIIF sPLA 2 was 4.6-fold more active, consistent with its increased capacity to hydrolyze oxidized lipoproteins. hGIIA sPLA 2 was inhibitory in the presence of oxidized lipoproteins with an IC 50 value of 150 nM while it was completely inactive with native lipoproteins.

Oxidized PUFAs are not responsible for enhanced anti-Plasmodium activity of sPLA 2 s -To understand why sPLA 2 s 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. [START_REF] Kumaratilake | Antimalarial properties of n-3 and n-6 polyunsaturated fatty acids: in vitro effects on Plasmodium falciparum and in vivo effects on P. berghei[END_REF] reported the anti-Plasmodium activity of oxidized PUFAs, namely oxidized AA and DHA. The authors found that these PUFAs were more active after oxidation. Assuming that oxidized phospholipids present in lipoproteins can be hydrolyzed by sPLA 2 s such as hGIIA and hGX to release the corresponding oxidized PUFAs [START_REF] Pruzanski | Differential hydrolysis of molecular species of lipoprotein phosphatidylcholine by groups IIA, V and X secretory phospholipases A2[END_REF][START_REF] Gora | The proinflammatory mediator Platelet Activating Factor is an effective substrate for human group X secreted phospholipase A2[END_REF], we compared the effects of native and self-oxidized AA, 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 IC 50 values for the three PUFAs in their native state were in good accordance with those reported by Kumaratilake et al. [START_REF] Kumaratilake | Antimalarial properties of n-3 and n-6 polyunsaturated fatty acids: in vitro effects on Plasmodium falciparum and in vivo effects on P. berghei[END_REF]. However, once oxidized, they were not more efficient, exhibiting similar (AA) or even higher (EPA, DHA) IC 50 values (Table 5). These results are thus different from those by Kumaratilake et al. who reported a ≈ 1.5-fold (AA) and ≈ 2.0-fold (DHA) better capacity of these PUFAs to inhibit Plasmodium after oxidation [START_REF] Kumaratilake | Antimalarial properties of n-3 and n-6 polyunsaturated fatty acids: in vitro effects on Plasmodium falciparum and in vivo effects on P. berghei[END_REF]. The discrepancy might come from differences in the amount and molecular identity of oxidation products as well as differences between experimental settings for oxidation. Anyway, 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 sPLA 2 s in the presence of oxidized lipoproteins might rather come from an increased activity of sPLA 2 s 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.

Circulating levels of hGIIA sPLA 2 , but not hGIIF, hGV or hGX sPLA 2 s, are increased in uncomplicated P. falciparum malaria -To provide a pathophysiological relevance of our in vitro results, we tested the sPLA 2 enzymatic activity and the presence of hGIIA, hGIIF, hGV and hGX sPLA 2 s 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 figure 1A, sPLA 2 enzymatic activity was dramatically increased in the plasma of P. falciparum-infected patients relative to healthy donors (median (interquartile range: IQR): 260.0 (148.5-367.1) versus 16.20 (10.46-29.06) cpm/min x µL respectively, P value < 0.0001).

No correlation was found between the level of enzymatic activity and blood parasitaemia (P value = 0.8079, Spearman's r = 0.03969, not shown).

Plasma samples were next analyzed for the presence of hGIIA, hGIIF, hGV and hGX sPLA 2 s by a sandwich ELISA-like method (Time-resolved fluoroimmunoassay, TR-FIA) using specific antibodies and assays for each sPLA 2 [START_REF] Nevalainen | Timeresolved fluoroimmunoassays of the complete set of secreted phospholipases A2 in human serum[END_REF]. Protein levels of hGIIA sPLA 2 were significantly increased in the plasma from parasitized patients (median (IQR): 1.6 (0.7-3.4) nM, with a maximum value of 9.1 nM, as compared to the plasma from healthy donors (median (IQR): 0.0 (0.0-0.1) nM) (P value <0.0001) (figure 1B). In contrast, hGIIF, hGV and hGX sPLA 2 s 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, sPLA 2 enzymatic activity and hGIIA protein concentration were highly correlated (Figure 1C; P value <0.0001, Spearman's r = 0.9542), indicating that hGIIA sPLA 2 is the predominant sPLA 2 responsible for the circulating enzymatic activity. No correlation was found between the plasma concentration of hGIIA and the parasitaemia from infected donors (Figure 1D; P value = 0.6684, Spearman r = 0.0689).

Malaria but not normal plasma inhibits in vitro parasite growth when spiked with

recombinant hGIIA sPLA 2 .

The capacity of hGIIA sPLA 2 endogenously present in the plasma from malaria patients at inhibiting Plasmodium development was evaluated in conventional in vitro culture conditions. Ten plasma samples containing various levels of endogenous hGIIA sPLA 2 up to 9 nM were tested (Figure 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 was variable but not significantly lower than that measured in the presence of plasma from different healthy donors (median growth rate (IQR): 6.1 (4.3-14.4) in malaria plasma versus 8.1 (6.7-9.5) in normal plasma, P = 0.4278; Figure 2A). We then evaluated the possible role of hGIIA sPLA 2 present in the diluted plasma by adding to the culture medium LY311727, a specific active site inhibitor of hGIIA sPLA 2 [START_REF] Singer | Interfacial kinetic and binding properties of the complete set of human and mouse groups I, II, V, X, and XII secreted phospholipases A2[END_REF].

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 (2.87-17.5) without LY311727 versus 6.65 (4.14-10.7) with LY311727, P = 0.1550) (not shown). The absence of effect of LY311727 i) indicates that the variable capacity of the different malaria plasma to support parasite growth is not due to hGIIA, and ii) might be explained by too low concentrations of endogenous hGIIA in 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 sPLA 2 .

This concentration was chosen based on hGIIA plasma concentrations reported in severe cases of malaria [START_REF] Vadas | Induction of circulating group II phospholipase A2 expression in adults with malaria[END_REF][START_REF] Vadas | Increased serum phospholipase A2 activity in Malawian children with falciparum malaria[END_REF]. As shown in Figure 2A, hGIIA spiked in malaria but not normal plasma inhibited parasite development, demonstrating that 1) the concentration of endogenous hGIIA in our assay conditions was not sufficient to inhibit parasite growth but that higher concentrations were clearly effective, and 2) malaria-induced modifications 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 sPLA 2 s for comparison. Both enzymes were inhibitory for P. falciparum in malaria plasma and found more active than hGIIA sPLA 2 (Figure 2B), as anticipated from the in vitro results with human native plasma.

Lipoproteins from P. falciparum-infected plasma are oxidized -To add further

support to our hypothesis that the anti-Plasmodium activity of hGIIA sPLA 2 , and may be of other sPLA 2 s, 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 [START_REF] Krishna | Variation in common lipid parameters in malaria infected patients[END_REF][START_REF] Visser | Serum Lipids and Lipoproteins During Uncomplicated Malaria: A Cohort Study in Lambarene, Gabon[END_REF], the level of lipoproteins, as measured by total PC concentration was found to be lower in plasma from malaria patients as compared to healthy donors (median (IQR): 1.20 (1.00-1.46) mg/mL versus 1.36 (1.19-1.68) mg/mL, P = 0.0158) (Figure 3A). Oxidation of lipoproteins in plasma was estimated using the specific Mercodia oxidized LDL immunoassay kit (see methods). LDL oxidation was clearly increased in the plasma from malaria patients as compared to healthy donors (median (IQR): 33.13 (25.65-39.79) vs 15.41 (12.13-18.69) U/L, P < 0.001) (Figure 3B). Together, our results demonstrate that malaria-induced modifications of plasma include oxidation of lipoproteins that most likely promotes the anti-Plasmodium activity of hGIIA sPLA 2 and enhances the inhibitory capacity of hGIIF and hGX sPLA 2 s, when spiked in malaria plasma.

The anti-Plasmodium activity of hGIIA sPLA 2 in malaria plasma requires its catalytic activity -To demonstrate that the anti-Plasmodium activity of hGIIA sPLA 2 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 sPLA 2 as well as hGX sPLA 2 , since this latter actively hydrolyzes both native and oxidized lipoproteins. hGIIA sPLA 2 did not release NEFAs from healthy plasma, whereas small but significant release occurred from malaria plasma (P = 0.0093) (Figure 4A).

As expected, hGX sPLA 2 induced a net increase in NEFAs from both malaria (P = 0.0020) and healthy donor (P = 0.0019) plasma (Figure 4A). Second, we tested whether the enzymatic activity is essential for the anti-Plasmodium effect of hGIIA sPLA 2 . Parasite growth inhibition assays were performed using the H48Q catalytically-inactive mutant of hGIIA (<0.5% of WT enzymatic activity ( 50)). 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 (Figure 4B). Last, we analyzed the relationship between Plasmodium inhibition in malaria plasma supplemented with hGIIA sPLA 2 and the amount of NEFAs released by the enzyme in the culture supernatant. We found that both parasite inhibition and NEFA amount in the culture medium increase with the time of incubation and concentration of hGIIA sPLA 2 (Figure 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 possible long-term effect induced by relatively low concentrations of the enzyme.

Together, these results indicate that the anti-Plasmodium activity of hGIIA sPLA 2 observed in the plasma from malaria patients is mediated by hydrolysis of oxidized lipoproteins.

Recombinant hGIIA sPLA 2 inhibits parasite development in Plasmodium-infected mice only when injected at the time of plasma peroxidation.

To challenge our hypothesis that hGIIA sPLA 2 might help controlling parasite development in an in vivo situation where lipoprotein oxidation does occur, we injected recombinant hGIIA sPLA 2 to C57BL/6J mice infected with P. chabaudi at different time points post-inoculation. 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 parasitaemia peak. Interestingly, a significant decrease by about 20% of the parasitaemia peak was observed after the late injection of hGIIA (Figure 5B), but not when hGIIA was injected at early time points (Figure 5A).

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 TBARS were increased at day-14 following parasite inoculation but not before (Figure 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 90's of an increase of hGIIA sPLA 2 in the plasma from malaria patients [START_REF] Vadas | Induction of circulating group II phospholipase A2 expression in adults with malaria[END_REF][START_REF] Vadas | Increased serum phospholipase A2 activity in Malawian children with falciparum malaria[END_REF], no comprehensive study on the potential role of this enzyme has been made in the context of malaria physiopathology. Interestingly, an association of plasma PLA 2 enzymatic activity and related phospholipid products with brain swelling in pediatric cerebral malaria has been reported in 2015 [START_REF] Pappa | Lipid metabolites of the phospholipase A2 pathway and inflammatory cytokines are associated with brain volume in paediatric cerebral malaria[END_REF], suggesting a role of hGIIA sPLA 2 in the inflammatory events operating in cerebral malaria. More recently, a possible role for the gene cluster encoding multiple sPLA 2 (genes for hGIIA, hGIID, hGIIE, hGIIF and hGV sPLA 2 s) has been suggested from results of a genome-wide association study performed on noncomplicated malaria susceptibility [START_REF] Milet | First genome-wide association study of non-severe malaria in two birth cohorts in Benin: hints towards the involvement of the STAT3 pathway[END_REF], highlighting the possible role for one or more human sPLA 2 s in malaria.

In line with the above findings, we provide here the first evidence that hGIIA sPLA 2 and maybe other sPLA 2 s might contribute as host defense factors to control Plasmodium development. Our results indicate that hGIIA, as well as some other sPLA 2 s, might operate through hydrolysis of lipoproteins, and more precisely oxidized lipoproteins that are produced during the course of Plasmodium infection in malaria [START_REF] Sibmooh | Increased fluidity and oxidation of malarial lipoproteins: relation with severity and induction of endothelial expression of adhesion molecules[END_REF]. This was demonstrated by combining a series of in vitro studies on parasite infection, analysis of plasma from malaria patients and finally by in vivo analysis of the anti-Plasmodium effect of hGIIA sPLA 2 in a mouse model of chronic malaria.

We previously demonstrated that hGIIF, hGIII, hGV and hGX but not hGIIA sPLA 2 s inhibit the in vitro growth of P. falciparum through hydrolysis of native lipoproteins present in human plasma [START_REF] Guillaume | In Vitro Anti-Plasmodium falciparum Properties of the Full Set of Human Secreted Phospholipases A2[END_REF]. We now show that oxidized lipoproteins are also substrates for these enzymes and participate to the inhibition of parasite growth, at least in vitro. Focusing on hGIIA sPLA 2 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 [START_REF] Pruzanski | Lipoproteins are substrates for human secretory group IIA phospholipase A2: preferential hydrolysis of acute phase HDL[END_REF][START_REF] Pruzanski | Differential hydrolysis of molecular species of lipoprotein phosphatidylcholine by groups IIA, V and X secretory phospholipases A2[END_REF][START_REF] Korotaeva | Oxidized phosphatidylcholine stimulates activity of secretory phospholipase A2 group IIA and abolishes sphingomyelin-induced inhibition of the enzyme[END_REF], and ii) hGIIA exhibits anti-Plasmodium activity only in the presence of oxidized lipoproteins.

In normal culture conditions, hGIIF, hGIII, hGV and hGX sPLA 2 s hydrolyze lipoprotein phospholipids, producing toxic free fatty acids, mainly long chain PUFAs [START_REF] Guillaume | In Vitro Anti-Plasmodium falciparum Properties of the Full Set of Human Secreted Phospholipases A2[END_REF]. It is known that hGIIA sPLA 2 has little activity on native lipoproteins but higher activity on oxidized counterparts ( [START_REF] Pruzanski | Lipoproteins are substrates for human secretory group IIA phospholipase A2: preferential hydrolysis of acute phase HDL[END_REF][START_REF] Pruzanski | Differential hydrolysis of molecular species of lipoprotein phosphatidylcholine by groups IIA, V and X secretory phospholipases A2[END_REF][START_REF] Korotaeva | Oxidized phosphatidylcholine stimulates activity of secretory phospholipase A2 group IIA and abolishes sphingomyelin-induced inhibition of the enzyme[END_REF] and this study). Identification and quantification of the lysophospholipids and fatty acids produced upon hydrolysis of native lipoproteins by hGIIA and other sPLA 2 s have been reported by us and others [START_REF] Guillaume | In Vitro Anti-Plasmodium falciparum Properties of the Full Set of Human Secreted Phospholipases A2[END_REF][START_REF] Pruzanski | Lipoproteins are substrates for human secretory group IIA phospholipase A2: preferential hydrolysis of acute phase HDL[END_REF][START_REF] Pruzanski | Differential hydrolysis of molecular species of lipoprotein phosphatidylcholine by groups IIA, V and X secretory phospholipases A2[END_REF]. It is known also that hGIIA sPLA 2 hydrolyzes oxidized lipoproteins to generate non-oxidized lipids and likely oxidized products [START_REF] Pruzanski | Lipoproteins are substrates for human secretory group IIA phospholipase A2: preferential hydrolysis of acute phase HDL[END_REF][START_REF] Pruzanski | Differential hydrolysis of molecular species of lipoprotein phosphatidylcholine by groups IIA, V and X secretory phospholipases A2[END_REF][START_REF] Korotaeva | Opposite effects of native and oxidized lipoproteins on the activity of secretory phospholipase A(2) group IIA. Prostaglandins Other Lipid Mediat[END_REF][START_REF] Korotaeva | Oxidized phosphatidylcholine stimulates activity of secretory phospholipase A2 group IIA and abolishes sphingomyelin-induced inhibition of the enzyme[END_REF]. In vitro and in vivo antimalarial properties of native as well as oxidized lipids, especially PUFAs, has been reported elsewhere [START_REF] Kumaratilake | Antimalarial properties of n-3 and n-6 polyunsaturated fatty acids: in vitro effects on Plasmodium falciparum and in vivo effects on P. berghei[END_REF][START_REF] Krugliak | Antimalarial effects of C18 fatty acids on Plasmodium falciparum in culture and on Plasmodium vinckei petteri and Plasmodium yoelii nigeriensis in vivo[END_REF]. Kumaratilake et al.

have shown that oxidized PUFAs are more active than native PUFAs against P. falciparum in vitro [START_REF] Kumaratilake | Antimalarial properties of n-3 and n-6 polyunsaturated fatty acids: in vitro effects on Plasmodium falciparum and in vivo effects on P. berghei[END_REF]. 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 sPLA 2 s. Considering the other sPLA 2 s, it is interesting to note that hGIIF, hGV and hGX sPLA 2 s 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 sPLA 2 s 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 sPLA 2 s, 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 sPLA 2 is only active when acting on specific "non-cellular" 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 [START_REF] Murakami | Emerging roles of secreted phospholipase A enzymes: The 3rd edition[END_REF][START_REF] Fourcade | Secretory phospholipase A2 generates the novel lipid mediator lysophosphatidic acid in membrane microvesicles shed from activated cells[END_REF][START_REF] Murakami | Different functional aspects of the group II subfamily (Types IIA and V) and type X secretory phospholipase A2s in regulating arachidonic acid release and prostaglandin generation. Implications of cyclooxygenase-2 induction and phospholipid scramblase-mediated cellular membrane perturbation[END_REF][START_REF] Boilard | Interaction of low molecular weight group IIA phospholipase A2 with apoptotic human T cells: role of heparan sulfate proteoglycans[END_REF][START_REF] Birts | Catalytic and non-catalytic functions of human IIA phospholipase A2[END_REF][START_REF] Boilard | A novel anti-inflammatory role for secretory phospholipase A2 in immune complex-mediated arthritis[END_REF][START_REF] Subra | Exosomes account for vesicle-mediated transcellular transport of activatable phospholipases and prostaglandins[END_REF][START_REF] Nelson | Investigation into the role of phosphatidylserine in modifying the susceptibility of human lymphocytes to secretory phospholipase A(2) using cells deficient in the expression of scramblase[END_REF][START_REF] Gibbons | Role of membrane oxidation in controlling the activity of human group IIa secretory phospholipase A(2) toward apoptotic lymphoma cells[END_REF][START_REF] Olson | Kinetic evaluation of cell membrane hydrolysis during apoptosis by human isoforms of secretory phospholipase A2[END_REF][START_REF] Boudreau | Platelets release mitochondria serving as substrate for bactericidal group IIA-secreted phospholipase A2 to promote inflammation[END_REF][START_REF] Duchez | Platelet microparticles are internalized in neutrophils via the concerted activity of 12lipoxygenase and secreted phospholipase A2-IIA[END_REF][START_REF] Murakami | Lipoquality control by phospholipase A2 enzymes[END_REF].

We measured concentrations of hGIIA sPLA 2 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 [START_REF] Vadas | Induction of circulating group II phospholipase A2 expression in adults with malaria[END_REF], where the mean level of hGIIA sPLA 2 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 nM 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 sPLA 2 was estimated to be around 70 nM, with the most severe cases exhibiting levels up to 200 nM [START_REF] Vadas | Increased serum phospholipase A2 activity in Malawian children with falciparum malaria[END_REF]. Of note, in these studies including ours, plasma were likely collected at different times during the course of infection, and it is not known how varies the concentration of hGIIA during the infection and according to levels of parasitaemia. In line with such high concentrations, we show that addition of 100 nM hGIIA sPLA 2 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 sPLA 2 has high affinity for various heparan sulfate proteoglycans lining the vascular endothelium and various cells, which likely acts as a reservoir of hGIIA in the circulation, and may lead to an underestimation of the actual concentration of hGIIA in blood [START_REF] Sartipy | Molecular basis for the association of group IIA phospholipase A2 and decorin in human atherosclerotic lesions[END_REF][START_REF] Murakami | Molecular nature of phospholipases A2 involved in prostaglandin I2 synthesis in human umbilical vein endothelial cells. Possible participation of cytosolic and extracellular type II phospholipases A2[END_REF][START_REF] Murakami | Functional association of type IIA secretory phospholipase A2 with the glycosylphosphatidylinositol-anchored heparan sulfate proteoglycan in the cyclooxygenase-2-mediated delayed prostanoid-biosynthetic pathway[END_REF][START_REF] Murakami | Distinct arachidonatereleasing functions of mammalian secreted phospholipases A2 in fibroblastic and mastocytoma cells through heparan sulfate shuttling and external plasma membrane mechanisms[END_REF]. This is supported by the study by Nakamura et al. where the hGIIA sPLA 2 activity measured in plasma was increased by about 3-fold between heparinized and nonheparinized patients [START_REF] Nakamura | Heparin-enhanced plasma phospholipase A2 activity and prostacyclin synthesis in patients undergoing cardiac surgery[END_REF]. 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 sPLA 2 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 response.

By investigating the effects of injection of recombinant hGIIA sPLA 2 to 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 12-14 p.i.) and not at earlier time points (at days 8-10 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 sPLA 2 is effective against Plasmodium in parasitized mice.

It must be noticed that the drop in parasitaemia 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 the 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 sPLA 2 in vivo [START_REF] Piris-Gimenez | In vivo protective role of human group IIA phospholipase A2 against experimental anthrax[END_REF][START_REF] Movert | Secreted Group IIA Phospholipase A2 Protects Humans Against the Group B Streptococcus: Experimental and Clinical Evidence[END_REF][START_REF] Pernet | Pseudomonas aeruginosa eradicates Staphylococcus aureus by manipulating the host immunity[END_REF][START_REF] Grass | Expression of human group II PLA2 in transgenic mice results in epidermal hyperplasia in the absence of inflammatory infiltrate[END_REF][START_REF] Laine | Protection by group II phospholipase A2 against Staphylococcus aureus[END_REF][START_REF] Laine | Resistance of transgenic mice expressing human group II phospholipase A2 to Escherichia coli infection[END_REF] and have been shown to have altered lipoprotein profiles [START_REF] Ivandic | Role of group II secretory phospholipase A2 in atherosclerosis: 1. Increased atherogenesis and altered lipoproteins in transgenic mice expressing group IIa phospholipase A2[END_REF][START_REF] Tietge | Overexpression of secretory phospholipase A(2) causes rapid catabolism and altered tissue uptake of high density lipoprotein cholesteryl ester and apolipoprotein A-I[END_REF].

One may also consider that the in vivo antimalarial activity of hGIIA sPLA 2 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 sPLA 2 is an important effector of the innate immune response and may activate platelets or neutrophils among different immune cells involved in malaria [START_REF] Dore | Roles of secreted phospholipase A2 group IIA in inflammation and host defense[END_REF][START_REF] Boudreau | Platelets release mitochondria serving as substrate for bactericidal group IIA-secreted phospholipase A2 to promote inflammation[END_REF][START_REF] Duchez | Platelet microparticles are internalized in neutrophils via the concerted activity of 12lipoxygenase and secreted phospholipase A2-IIA[END_REF][START_REF] Menschikowski | Secretory phospholipase A2 of group IIA: is it an offensive or a defensive player during atherosclerosis and other inflammatory diseases?[END_REF][START_REF] Ibeas | Secreted phospholipase A2 type IIA as a mediator connecting innate and adaptive immunity: new role in atherosclerosis[END_REF]. The production of lipid mediators by sPLA 2 s, as an integral component of the inflammatory reaction, plays a major role in protecting the host against invading pathogens [START_REF] Murakami | Emerging roles of secreted phospholipase A2 enzymes: Lessons from transgenic and knockout mice[END_REF][START_REF] Murakami | Emerging roles of secreted phospholipase A enzymes: The 3rd edition[END_REF][START_REF] Dore | Roles of secreted phospholipase A2 group IIA in inflammation and host defense[END_REF]. However, lipid products might also induce deleterious effects to the host, as exemplified by recent metabolomics analyses showing a positive association between lipid products of the PLA 2 pathway and brain swelling in pediatric cerebral malaria [START_REF] Pappa | Lipid metabolites of the phospholipase A2 pathway and inflammatory cytokines are associated with brain volume in paediatric cerebral malaria[END_REF].

Concerning the possible in vivo role of other human sPLA 2 s, we showed that exogenous hGIIF and hGX sPLA 2 s 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. These sPLA 2 s were however not detected in the plasma of P. falciparum-infected or healthy subjects. It is tempting to speculate that they may act locally at sites relevant to Plasmodium infection. Indeed, while there is now considerable evidence that hGIIA sPLA 2 is the only sPLA 2 highly present in the circulation under pathological conditions associated with inflammation, tissue injury or infection, there is little evidence for the presence of other sPLA 2 isoforms in serum, except for hGIB sPLA 2 in some disease situations [START_REF] Nevalainen | Timeresolved fluoroimmunoassays of the complete set of secreted phospholipases A2 in human serum[END_REF][START_REF] Aufenanger | Pancreatic phospholipase A2 activity in acute pancreatitis: a prognostic marker for early identification of patients at risk[END_REF]. However, several sPLA 2 s other than hGIIA are also involved in various types of infection, suggesting that these sPLA 2 s exert their effects in diverse pathologies locally, within the microenvironment of the disease, and either in an autocrine or paracrine manner [START_REF] Murakami | Emerging roles of secreted phospholipase A2 enzymes: Lessons from transgenic and knockout mice[END_REF][START_REF] Murakami | Emerging roles of secreted phospholipase A enzymes: The 3rd edition[END_REF][START_REF] Murakami | A new era of secreted phospholipase A(2)[END_REF]. Examples include studies on the role of GIII, GV and GX sPLA 2 s after infection with various pathogens [START_REF] Kim | Lysis of human immunodeficiency virus type 1 by a specific secreted human phospholipase A2[END_REF][START_REF] Balestrieri | Group V secretory phospholipase A2 modulates phagosome maturation and regulates the innate immune response against Candida albicans[END_REF][START_REF] Mitsuishi | Group V and X secretory phospholipase A2 prevents adenoviral infection in mammalian cells[END_REF][START_REF] Mitsuishi | Human group III phospholipase A2 suppresses adenovirus infection into host cells. Evidence that group III, V and X phospholipase A2s act on distinct cellular phospholipid molecular species[END_REF][START_REF] Degousee | Group V phospholipase A2 in bone marrow-derived myeloid cells and bronchial epithelial cells promotes bacterial clearance after Escherichia coli pneumonia[END_REF][START_REF] Kelvin | Lack of group X secreted phospholipase A(2) increases survival following pandemic H1N1 influenza infection[END_REF], GIII and GX sPLA 2 s in cancer [START_REF] Murakami | A new era of secreted phospholipase A(2)[END_REF][START_REF] Brglez | Secreted phospholipases A2 in cancer: Diverse mechanisms of action[END_REF][START_REF] Schewe | Secreted Phospholipases A2 Are Intestinal Stem Cell Niche Factors with Distinct Roles in Homeostasis, Inflammation, and Cancer[END_REF], or GIIF, GIII, GV and GX sPLA 2 s in cardiovascular or skin diseases, and allergy or asthma [START_REF] Henderson | Importance of group Xsecreted phospholipase A2 in allergen-induced airway inflammation and remodeling in a mouse asthma model[END_REF][START_REF] Bostrom | Group V secretory phospholipase A2 promotes atherosclerosis: evidence from genetically altered mice[END_REF][START_REF] Ait-Oufella | Group X secreted phospholipase A2 limits the development of atherosclerosis in LDL receptor-null mice[END_REF][START_REF] Taketomi | Mast cell maturation is driven via a group III phospholipase A2-prostaglandin D2-DP1 receptor paracrine axis[END_REF][START_REF] Yamamoto | The role of group IIF-secreted phospholipase A2 in epidermal homeostasis and hyperplasia[END_REF][START_REF] Samuchiwal | Harmful and protective roles of group V phospholipase A2: Current perspectives and future directions[END_REF].

It is also interesting to note that hGX sPLA 2 has been found in atherosclerotic plaques but not in plasma from patients with atherosclerosis (90), suggesting that the enzyme is produced locally but not systemically. All together, it is possible that sPLA 2 s 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 sPLA 2 and oxidation of lipoproteins, as found in the serum of malaria patients, might participate to 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 occur. By extrapolation, this mechanism of action of hGIIA on oxidized lipoproteins might also be involved in other pathological conditions where both hGIIA sPLA 2 and oxidized lipoproteins are present, including atherosclerosis or other infectious diseases. It also remains to determine if other sPLA 2 s such as hGIIF, hGIII, hGV and hGX which are active in vitro, may also be active in an in vivo situation, even though these enzymes are not found in plasma from malaria patients. 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). 3 H-hypoxanthine monohydrochloride (370 GBq-1.11 TBq/mmol) was from Perkin-Elmer. Other high quality grade biochemical reagents were from Sigma.

MATERIALS AND METHODS

Materials

Methods

P. falciparum cultivation -

In routine culture conditions, P. falciparum was grown in red blood cells from the A+ group at 2% haematocrit and 2-4% parasitaemia, in RPMI supplemented with 11 mM glucose, 27.5 mM NaHCO 3 , 100 UI/mL penicillin, 100 µg/mL streptomycin, adjusted to pH 7.4 (basic medium), supplemented with 8% heat-inactivated human A+ plasma (complete medium), according to the procedure of Trager and Jensen [START_REF] Trager | Human malaria parasites in continuous culture[END_REF].

When specified, the serum substitute Albumax II® (0.5% w/v final) was used in culture medium instead of heat-inactivated human plasma. Culture flasks were gassed with 91% N 2 , 6% O 2 and 3% CO 2 before being incubated at 37°C. In those conditions, the intra-erythrocytic cycle of the FcB1 strain was 48-h long. Parasitaemia (%) was established by optical examination of Diff-Quik-stained smears from the formula = [infected erythrocytes number / total erythrocytes number] x 100.

Purification of lipoproteins and oxidation -Non-fasted 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. [START_REF] Havel | The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum[END_REF]. Briefly, chylomicrons and VLDL were removed by a first round of centrifugation at 1.006 g/mL density, then LDL and HDL were purified separately by successive centrifugations at 1.053 g/mL and 1.210 g/mL densities, respectively. When specified, 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/L, 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 N 2 in the dark. Phosphatidylcholine (PC) content of lipoproteins was measured by using the Phospholipid B dosage kit, according to the manufacturer's instructions.

Oxidation of PUFAs -Concentrated solutions of commercial AA (76.5 mM), EPA (60 mM) and DHA (100 mM) in ethanol were stored frozen under N 2 . 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 re-suspended into 100 µL CHCl 3 , and then diluted 1/20 in RPMI containing 8% heat-inactivated human plasma.

Prior to dose-response assays, non-oxidized PUFAs were prepared from another 20 µL aliquot of each concentrated solution, mixed to 80 µL of CHCl 3 and diluted into culture medium as above.

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 [START_REF] Sattler | Methodological approaches for assessing lipid and protein oxidation and modification in plasma and isolated lipoproteins[END_REF][START_REF] Asakawa | Coloring condition of thiobarbituric acid test for detecting lipid hydroperoxide[END_REF]. 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 minutes and then cooled on ice. 150 µL of butanol was added, the sample was vortexed, and then let stand on ice for phase separation to occur. The butanol phase was taken for TBARs 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 0.30-0.50 and 0.05-0.15 nmoles of malondialdehyde (MDA)/mg of protein in LDL and HDL, respectively.

After two weeks of air-light oxidation, TBARS concentration had raised to 1.50-2.50 (LDL) and 0.30-0.50 (HDL) nmoles 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 [START_REF] Sibmooh | Increased fluidity and oxidation of malarial lipoproteins: relation with severity and induction of endothelial expression of adhesion molecules[END_REF].

Due to the presence in plasma of substances other than MDA and susceptible to react with thiobarbituric acid [START_REF] Devasagayam | Methods for estimating lipid peroxidation: an analysis of merits and demerits[END_REF], 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 assay 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.

Hydrolysis of lipoproteins and plasma by sPLA 2 -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 CaCl 2 . They were incubated with and without recombinant sPLA 2 for various times at 37°C. sPLA 2 s were used at different concentrations. hGIB was used at 100 nM (number of independent experiments, n = 4), 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), hGXIIA was used at 100 and 200 nM (n = 2). NEFAs were measured at different time points using the NEFA-C kit (WAKO) following manufacturer's instructions. Values were normalized by subtracting NEFAs measured from lipoproteins incubated without sPLA 2 . Specific activity was deduced from the linear part of the curve [NEFA] = f (t).

To assess the ability of hGIIA and hGX sPLA 2 s to hydrolyze phospholipids in plasma from P. falciparum-infected patients versus healthy donors, 1 mM final CaCl 2 was added to each plasma sample and then enzymes were added at 400 nM (hGIIA) and 8 nM (hGX) final concentration. Samples were incubated overnight at 37°C. Control for endogenous release of NEFAs was without added sPLA 2 . NEFA content before and after incubation was determined using the NEFA-C kit, according to manufacturer's instructions.

Anti-Plasmodium activity assays with sPLA 2 -hydrolyzed lipoproteins -The ability of human sPLA 2 s to promote the toxicity of lipoproteins was tested in dose-response assays as described [START_REF] Guillaume | In Vitro Anti-Plasmodium falciparum Properties of the Full Set of Human Secreted Phospholipases A2[END_REF]. Briefly, LDL and HDL (0.6 mg phospholipid/mL in RPMI) were incubated overnight at 37°C with 20 nM sPLA 2 or alone, then tested for parasite inhibition at a LDL and HDL final concentration of 0.2 mg/mL (ie close to physiological concentrations) in RPMI 0.5% Albumax II® for 48h in 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 sPLA 2 added alone were also performed to check for lipoproteinindependent toxicity of sPLA 2 s in these conditions. Assays analyzing the anti-Plasmodium effect of lipoproteins pre-treated with higher concentrations of hGIIA sPLA 2 (100 nM and 250 nM) were performed similarly. Assays analyzing the anti-Plasmodium effect of sPLA 2 s coincubated with total lipoprotein fraction but without pretreatment were carried out on two parasite cycles (i.e. 96 h).

Anti-Plasmodium activity assays with oxidized PUFAs -Dose-response assays with oxidized and non-oxidized AA, EPA and DHA were performed as described in [START_REF] Guillaume | In Vitro Anti-Plasmodium falciparum Properties of the Full Set of Human Secreted Phospholipases A2[END_REF] 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% parasitaemia, 4% haematocrit, 50 µL/well). Parasites were allowed to grow for 48 h in normal culture conditions, then 9.25 kBq 3 H-hypoxanthine were added per well. After an additional 48 h incubation period, plates were frozen and the experiment proceeded as in [START_REF] Guillaume | In Vitro Anti-Plasmodium falciparum Properties of the Full Set of Human Secreted Phospholipases A2[END_REF].

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 16-58 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 Museum (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. Positive patients were treated with dihydroartemisininpiperaquine combination in accordance with the national drug policy of VietNam. Time-resolved fluoroimmunoassays (TR-FIA) -TR-FIA assays for hGIIA, hGIIF, hGV and hGX sPLA 2 s in plasma were performed as described earlier [START_REF] Nevalainen | Timeresolved fluoroimmunoassays of the complete set of secreted phospholipases A2 in human serum[END_REF]. The assays use rabbit polyclonal anti-sPLA 2 antibodies to set up ELISA-like sandwiches, which were shown to be highly specific for each sPLA 2 isoform [START_REF] Degousee | Groups IV, V and X phospholipases A2 in human neutrophils. Role in eicosanoid production and gram negative bacterial phospholipid hydrolysis[END_REF]. hGIII sPLA 2 could not be measured, due to the lack of sensitivity of the corresponding immune serum.

Enzymatic assays on E. coli membranes -sPLA 2 activity in plasma was measured by hydrolysis of E. coli membranes radiolabeled with [ 3 H]-oleic acid and autoclaved [START_REF] Rouault | Recombinant production and properties of binding of the full set of mouse secreted phospholipases A2 to the mouse M-type receptor[END_REF]. 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 CaCl 2 , 0.1% BSA) were incubated with plasma (20 µL, diluted 1:150 in activity buffer) at 37°C for 30 minutes. 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).

P. falciparum cultivation in malaria plasma and related assays -To analyze the anti-

Plasmodium effect of endogenous sPLA 2 in plasma from infected people, the FcB1 strain was grown in red blood cells of the O-group, brought to 0.1-0.2% parasitaemia, washed and then transferred at 2% haematocrit into basic RPMI supplemented with 2.5 µM hypoxanthine and 1 mM CaCl 2 (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 sPLA 2 [START_REF] Paganelli | Group IIA-Secreted Phospholipase A2 in Human Serum Kills Commensal but Not Clinical Enterococcus faecium Isolates[END_REF][START_REF] Gronroos | Roles of group IIA phospholipase A2 and complement in killing of bacteria by acute phase serum[END_REF], plasma samples were not heat-inactivated prior to parasite cultivation. The microplate was incubated in a candle jar for 96 h, then parasitaemia in each well was determined from Diff-Quik-stained smears.

To assess for parasite inhibition by exogenously added hGIIA sPLA 2 , experiment was carried out as above, with recombinant hGIIA added at the specified concentration at the start of incubation. To evaluate the contribution of hGIIA catalytic activity, recombinant hGIIA sPLA 2 in its native (WT) or catalytically inactive form (H48Q mutant) [START_REF] Edwards | The crystal structure of the H48Q active site mutant of human group IIA secreted phospholipase A2 at 1.5 A resolution provides an insight into the catalytic mechanism[END_REF], 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. Parasitized mice were IP-injected with 100 µL of recombinant hGIIA (0.125 mg/kg) in PBS 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 parasitaemia peak (late injection). Blood parasitaemia was determined every 2-3 days within 22 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, with, respectively, 5 (control) and 7 (injected) mice, and 5 (control) and 6 (injected) mice.

In vivo assays with

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

Statistical analysis -Data were analyzed using the GraphPad Prism software (San Diego, CA). Normality of groups with n 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 n number was too small (≤ 6), the non-parametric Mann-Whitney U test for independent samples or the Wilcoxon matched-pair test for dependent samples were used. The effect of hGIIA sPLA 2 injection to P. chabaudi-infected C57BL/6 mice was analyzed by using the two-

-

  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 was from the Unicellular Eukaryotes Collection from the National Museum of Natural History (MNHN-CEU-224-PfFcB1). The 864VD strain of the murine Plasmodium 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, 03800 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 (Etablissement Français du Sang (EFS)), convention reference: C CPSL UNT 13/EFS/126. RPMI 1640 and Albumax II® were from Life Technologies (Cergy Pontoise, France). Diff-Quick staining reagents were from Medion Diagnostics AG. The NEFA-C and the Phospholipid B kits, respectively used for quantitative determination of non-esterified fatty acids (NEFAs) and phospholipids, were from WAKO Chemicals (Oxoid S.A., Dardilly, France). The oxidized LDL ELISA kit was from Mercodia SAS sales in France. Purified recombinant human sPLA 2 s and the hGIII sPLA 2 domain were prepared as described (91). The sPLA 2 inhibitor LY311727 (3-{[3-(2-Amino-2-oxoethyl)-2ethyl-1-(phenylmethyl)-1H-indol-5-yl]oxy}propyl]-phosphonic acid (164083-84-5)) that targets hGIIA sPLA 2 was from Sigma.

  Plasmodium chabaudi-infected mice Mice were housed in the National Museum of Natural History (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, February 1 st , 2013). All experiments were approved by the MNHN's Ethics Committee and registered under the deposit n° APAFIS 201802281454598. C57BL/6J mice (males, 8 to 12-week-old) were inoculated intraperitoneally (IP) with 1x10 6 P. c. chabaudi 864VD-infected mouse red blood cells (RBC) in Alsever's solution. Tail blood was taken every 2-3 days within 21 days following inoculation. Parasitaemia was established by optical examination of Diff-Quik-stained blood smears and counting of 2,000 RBCs.

Figure 1 .

 1 Figure 1. sPLA 2 enzymatic activity and hGIIA sPLA 2 protein concentration are increased in

Figure 2 :

 2 Figure 2: (A) Malaria but not normal plasma spiked with recombinant hGIIA inhibits the

Figure 3 .

 3 Figure 3. Phospholipid content and LDL oxidation in malaria plasma. Plasma samples from

Figure 4 .

 4 Figure 4. (A) Exogenous hGIIA sPLA2 hydrolyzes phospholipids from malaria but not

Figure 5 .

 5 Figure 5. Injection of recombinant hGIIA sPLA2 affects Plasmodium development in mice in

  FIGURE 1

TABLE 2 . A ti-P. falciparum a ti ities of lipoprotei s after h drol sis hu a sPLA 2 s.

 2 Values are ea ± SD of i depe de t e peri e ts. For statistical a al sis, the o para etric Wilco o atched pairs test as applied, ith:  P < . a d  P < .co sidered sig ifica t. -: ot deter i ed.

		O idized lipoprotei s		Nati e lipoprotei s
	Pretreat e t ith 20 M sPLA 2 o e hGIB hGIIA hGIID hGIIE hGIIF hGIII hGV hGX hGXIIA	IC 50 µg PC/ L LDL HDL . > . ± . . ± . . ± . . > . ± . . ± . > . . ± . > . . ± . . ± . . ± . . ± . . ± . . ± . . ± . . ± . . ± . > .	P alue O idized LDL vs o idized HDL -----. . * . ** . * -	IC 50 µg PC/ L LDL HDL > . > . > . > . > . > . . ± > . . > . . ± . . ± . . ± . . ± . . ± . . ± . . ± . . ± . . ± . > . > .	P alue o idized LDL vs ati e o idized HDL vs ati e ----------. . . * . . . . . --

TABLE . A ti-P. falciparum acti ities of lipoprotei s after h drol sis ith high co ce tratio s of hGIIA sPLA A ti-Plasmodium acti ities of LDL a d HDL

 .

		IC values µg PC/ L
	Pretreat e t ith hGIIA sPLA M	Nati e LDL O idized LDL Nati e HDL O idized HDL
	No e	>	. ± .	>	>
		±	. ± .	>	>
	5	. ± .	. ± .	. ± .	. ± .

Values are ea ± SD of i depe de t experi e ts Ta le 4. A ti-P.

falciparum a ti ities of hu a sPLA 2 s i the prese e of ati e or oxidized lipoprotei fra tio fro hu a plas a. A ti-P. falciparum a ti ities of hu a sPLA 2 s

			IC alues M
	Total lipoprotei s	hGIIA	hGIIF	hGV	hGX
	/o	>	.	> .	> .	> .
	Nati e	>	.	. ± .	. ± .	. ± .
	Oxidized		. ± .	. ± .	. ± .	. ± .

Values are ea ± SD of i depe de t parasite gro th i hi itio assays. The lipoprotei fra tio as .

g PC / L i ulture ediu .

ACKNOWLEDGMENTS

Funding. This work was supported by the ATM blanche 2016-2017 to C. Deregnaucourt from the National Museum of Natural History (Paris, France) and by grants to GL from CNRS, the National Research Agency (MNaims ANR-17-CE17-0012-01) and the Fondation de la Recherche Médicale (DEQ20180339193).