Autophagy Pathways in the Genesis of Plasmodium-Derived Microvesicles: A Double-Edged Sword?

Malaria, caused by Plasmodium species (spp.), is a deadly parasitic disease that results in approximately 400,000 deaths per year globally. Autophagy pathways play a fundamental role in the developmental stages of the parasite within the mammalian host. They are also involved in the production of Plasmodium-derived extracellular vesicles (EVs), which play an important role in the infection process, either by providing nutrients for parasite growth or by contributing to the immunopathophysiology of the disease. For example, during the hepatic stage, Plasmodium-derived EVs contribute to parasite virulence by modulating the host immune response. EVs help in evading the different autophagy mechanisms deployed by the host for parasite clearance. During cerebral malaria, on the other hand, parasite-derived EVs promote an astrocyte-mediated inflammatory response, through the induction of a non-conventional host autophagy pathway. In this review, we will discuss the cross-talk between Plasmodium-derived microvesicles and autophagy, and how it influences the outcome of infection.


Quantification of Gene Expression
Gene expression quantification was performed using RT-qPCR RT-qPCR on 1 × 10 6 cells. Cell treatment and processing were conducted as described by Leleu et al. [28].

Host Autophagy Pathways in Malaria
The following two types of autophagy pathways are known to be engaged during the development of Plasmodium in the mammalian host: (a) Non-selective autophagy, which is an adaptive response for cellular remodeling upon unfavorable stress conditions. It is characterized by a double-membrane-bound autophagosome, decorated by microtubuleassociated protein 1 light chain 3 beta (LC3-II) molecules. These autophagosomes engulf the parasite, and also support its nourishment during the intrahepatic stage [12,29]. (b) selective autophagy, which is initiated by unc-51-like autophagy-activating kinase-1 (Ulk) and class III phosphatidylinositol 3-kinase (PI3K) complexes. Selective autophagy specifically targets intracellular parasites and promotes their elimination through ubiquitination [8,30,31].
Recently, we have demonstrated the involvement of an unconventional LC3-mediated autophagy pathway (LAP), independent of Ulk1, in the transfer and degradation of P. berghei ANKA (PbA)-MVs inside astrocytes. LAP is an intermediary pathway between autophagy and phagocytosis, involving ATG proteins and the recruitment of LC3-II directly on the single-membrane LAPosome formed around the engulfed microbe, prior to its fusion with lysosomes [32]. The LAP pathway was also found to be an inducer of astrocyte pro-inflammatory responses that play a major role in the pathogenesis of CM [28].

pMVs and Autophagy-Related Responses during Pre-Erythrocytic Development
During the liver stage of development, packed inside a transient vacuole (TV), the sporozoites injure and traverse several hepatocytes, before finally settling in one to initiate infection [2]. The invading sporozoite is contained in the PV, wherein it first differentiates into a trophozoite, before switching on replicative processes to form a schizont, containing thousands of merozoites. These merozoites egress the hepatocyte in a merosome (see below), invade erythrocytes, and start the blood-stage infection [2,33,34]. The two vacuolar organelles, TV and PV, constitute the first Plasmodium-derived vesicles during the parasitic life cycle. They allow the parasite (i) to escape hepatocyte elimination strategies, and (ii) to use host cell nutrients and develop alternative scavenging pathways for its survival [5,6,35,36]. As summarized in Figure 1A, the following two host cell pathways for clearing the parasite are triggered during PbA sporozoite development in the liver: (1) in the early intrahepatic stage, parasites inside a TV, or in a deficient PV, are cleared by the induction of a PI3Passociated sporozoite elimination (PASE) process that differs from autophagy, but induces a lysosomal acidification pathway; (2) in the late intrahepatic stage, the PV membrane (PVM) engulfed parasite is targeted by LAP-like non-conventional autophagy that occurs independently of PI3K, RB1-inducible coiled-coil 1 (Rb1cc1), and Ulk1, and without the formation of reactive oxygen species (ROS) ( Figure 1A) [37][38][39][40]. The absence of PI3K and Becn1 (beclin 1, autophagy related) in this process argues in favor of the involvement of alternative non-canonical autophagy, distinct from LAP, in the elimination of the parasite during this late hepatic stage [37,41]. Interestingly, prolonged expression of LC3-II molecules has been observed on the PVM [41]. The lipidation and incorporation of LC3-II at the PVM is promoted by Atg5, and is necessary for the subsequent binding of ubiquitin, sequestosome 1 (Sqstm11/p62), and neighbor of BRCA1 gene 1 (Nbr1) at the membrane [42]. Boonhok et al. (2016) have described an interferon-gamma (IFN-γ)-mediated non-canonical autophagy machinery for the elimination of P. vivax sporozoites in primary human hepatocyte cultures, called Plasmodium-associated autophagy-related (PAAR) responses ( Figure 1A) [43]. PAAR is characterized by the expression of MAPLC3/LC3 on the PVM, and is dependent upon Becn1, PI3K, and Atg5, but not Ulk1 [43]. Although some intra-hepatocyte parasites are eliminated by LAP-like autophagy, others can escape cellular autodigestive elimination [41]. Indeed, to control and avoid elimination by autophagy, mature schizonts remodel LC3 + PVM by shedding autophagic proteins trapped in the tubovesicular network (TVN), and by redirecting the lysosome to the TVN [36,39]. In addition, the interaction of PbA, upregulated in infective sporozoites gene 3 (UIS3) protein, with host MAP1LC3/LC3 inhibits the autophagy function, and allows parasite replication [38,44]. In parallel, PbA is able to divert the non-selective host autophagy and exploits it as a nutritive source for supporting parasite growth [41,45,46]. Together, these mechanisms favor the egress of hepatic merozoites, which are released from infected hepatocytes as merosomes, another pMV that consists of hundreds of parasites surrounded by a host cell membrane [47][48][49][50]. During this process, Plasmodium seems to use vesicle encapsulation as a mechanism to hide from host immune mechanisms, and to escape elimination. others can escape cellular autodigestive elimination [41]. Indeed, to control and avoid elimination by autophagy, mature schizonts remodel LC3 + PVM by shedding autophagic proteins trapped in the tubovesicular network (TVN), and by redirecting the lysosome to the TVN [36,39]. In addition, the interaction of PbA, upregulated in infective sporozoites gene 3 (UIS3) protein, with host MAP1LC3/LC3 inhibits the autophagy function, and allows parasite replication [38,44]. In parallel, PbA is able to divert the non-selective host autophagy and exploits it as a nutritive source for supporting parasite growth [41,45,46]. Together, these mechanisms favor the egress of hepatic merozoites, which are released from infected hepatocytes as merosomes, another pMV that consists of hundreds of parasites surrounded by a host cell membrane [47][48][49][50]. During this process, Plasmodium seems to use vesicle encapsulation as a mechanism to hide from host immune mechanisms, and to escape elimination. . If parasites are unable to discard their unnecessary organelles, or correctly remodel their vacuole, the PI3K complex is formed at the vacuole membrane, and leads to parasite elimination by cytosolic lysosomes. Although LC3 is observed transiently at the vacuole membrane, it is not essential for efficient Plasmodium degradation. This elimination, called PI3P-associated sporozoite elimination (PASE), occurs during the early intrahepatic stage. During the late intrahepatic stage, when the parasite starts its differentiation and multiplication, the PVM enclosing the parasite can be labelled by LC3 promoted by Atg5, resulting in the host Plasmodium-associated autophagy-related (PAAR) response. This pathway, associated with ubiquitin, sqstm1/p62, and Nbr1, is independent of PI3k, Rb1cc1, and Ulk complexes, and does not necessarily . If parasites are unable to discard their unnecessary organelles, or correctly remodel their vacuole, the PI3K complex is formed at the vacuole membrane, and leads to parasite elimination by cytosolic lysosomes. Although LC3 is observed transiently at the vacuole membrane, it is not essential for efficient Plasmodium degradation. This elimination, called PI3Passociated sporozoite elimination (PASE), occurs during the early intrahepatic stage. During the late intrahepatic stage, when the parasite starts its differentiation and multiplication, the PVM enclosing the parasite can be labelled by LC3 promoted by Atg5, resulting in the host Plasmodium-associated autophagy-related (PAAR) response. This pathway, associated with ubiquitin, sqstm1/p62, and Nbr1, is independent of PI3k, Rb1cc1, and Ulk complexes, and does not necessarily lead to clearance of the parasite, as it can avoid fusion with lysosomes by remodeling its vacuolar membrane. (B) After leaving the liver, merozoites invade RBCs. The parasite is able to produce Plasmodium-derived microvesicles by autophagy, using PbAATG, in order to transport genetic information and promote gametocytogenesis. However, pMVs can also induce a pro-inflammatory response. (C) PbA-MVs are transferred from iRBCs to astrocytes inside a PVM directly targeted by LC3-II to form a LAPosome. This then fuses with lysosomes, resulting in parasite clearance. This LC3-associated phagocytosis (LAP) pathway, an unconventional autophagy pathway, induces a pro-inflammatory response in astrocytes, leading to ECM.

Autophagy and pMV Crosstalk during Blood-Stage Infection
Merozoites eventually released from merosomes invade RBCs to initiate the blood stage of infection [3,51]. The parasite uses its autophagy machinery (Pf ATG) to recycle unnecessary proteins to support its nourishment and biogenesis ( Figure 1B). Pf ATG also allows for survival in the case of nutrient starvation, especially in the intraerythrocytic stage [52]. However, unlike hepatocytes, RBCs do not generate intracellular autophagic host defense mechanisms for parasite clearance [52][53][54]. Interestingly, merosomes are potent immunoregulators. They induce macrophage activation via Toll-like receptor 4 (TLR4) and myeloid differentiation primary response 88 (MyD88) pathways, resulting in an increase in CD40 expression and tumor necrosis factor alpha (TNF-α) secretion [55]. Blood-stage P. falciparum-derived MVs (Pf MVs) express parasite antigens capable of stimulating human peripheral blood mononuclear cells (PBMCs) and macrophages, upregulating pro-inflammatory cytokine secretion and activating neutrophil migration [20]. In P. vivaxinfected patients, circulating pMVs can adhere to human splenic cells by interacting with intercellular adhesion molecule-1 (ICAM-1) [56]. Recently, Pf MVs have been shown to induce natural killer (NK) cell responses during malaria, after interacting with the melanoma differentiation-associated protein 5 (MDA5), a RIG-I-like receptor (RLR) [57]. PMVs isolated from P. falciparumand P. vivax-infected patients have been shown to transport miRNA and parasite proteins [14,58]. Such vesicles can be internalized by iRBCs, resulting in the transfer of genetic information inside exosome-like vesicles, and these vesicles have been found to promote gametocytogenesis [20,59]. In addition, pMVs derived from human brain endothelial cells stimulate the proliferation/activation of T cells [60,61]. These later observations reinforce the hypothesis of a role for pMVs in the pathogenesis of CM.

Role of Autophagy-Dependent pMVs in CM
CM is a lethal complication of Plasmodium infection. It causes around 0.4 million deaths per year, principally in children <5 years of age and in immunosuppressed individuals [1]. The pathophysiology is a consequence of the sequestration of iRBCs in brain microvessels and the detrimental immune response, characterized by neuroinflammation and the lymphocyte migration that results from it [62][63][64][65]. In C57BL/6 CM-susceptible (CM S ) mice infected with PbA, astrocytes are activated early during infection, and release pro-inflammatory factors that contribute to neuroinflammation and death [66][67][68]. We have previously described the in vitro transfer of PbA-MVs to astrocytes after contact with iRBCs ( Figure 1C) [68]. We propose that iRBCs located in the perivascular space could interact with pseudopodia from activated astrocytes during neuropathogenesis [66,69]. As shown in Figure 2A, the transfer of PbA-MVs occurs at a contact point between the astrocytic foot and PbA-iRBCs in CM S_ derived primary astrocyte cultures stimulated for 6 h. This transfer of PbA-MVs to astrocytes occurs via the unconventional LAP autophagy pathway, which is independent of Ulk1 and calcium binding and coiled-coil domain 2 (Calcoco2/Ndp52) [28] ( Figure 1C). The LAP pathway is activated via Becn1, the RUN domain, and the cysteine-rich domain, containing Beclin 1-interacting protein (Rubcn), Atg16L1, Atg5, and Sqstm1/p62. The parasite material transferred to the astrocyte is contained in PV-expressing LC3-II molecules after activation. LAP mediates the degradation of PbA-MVs in astrocytes ( Figure 2B,C). Eventually, an organelle called LAPosome fuses with lysosomes to degrade PbA-MVs ( Figure 2B,C). The treatment of PbA CM S mice with bafilomycin A 1 , an autophagy inhibitor, has been shown to prevent ECM [28]. Confocal microscopy revealed that, in contrast, in CM S -derived astrocytes in CM R -derived cells, PbA-MVs transferred from iRBCs were not internalized, but, instead, remained at the astrocyte extracellular membrane ( Figure 2B). Indeed, RT-qPCR only detected a small quantity of P. berghei 18S ribosomal (PB18S) gene in these astrocytes ( Figure 2C). Unlike in the case of CM S -derived astrocytes, increased expression of MAP1LC3/LC3, ATG5 and ATG16L1 LAP autophagy genes was not observed in CM R -derived cells ( Figure 2D). with lysosomes to degrade PbA-MVs ( Figure 2B,C). The treatment of PbA CM S mice with bafilomycin A1, an autophagy inhibitor, has been shown to prevent ECM [28]. Confocal microscopy revealed that, in contrast, in CM S -derived astrocytes in CM R -derived cells, PbA-MVs transferred from iRBCs were not internalized, but, instead, remained at the astrocyte extracellular membrane ( Figure 2B). Indeed, RT-qPCR only detected a small quantity of P. berghei 18S ribosomal (PB18S) gene in these astrocytes ( Figure 2C). Unlike in the case of CM S -derived astrocytes, increased expression of MAP1LC3/LC3, ATG5 and ATG16L1 LAP autophagy genes was not observed in CM R -derived cells ( Figure 2D). Astrocytes are key players in the innate immune response in the brain [68,70,71]. During malaria, they secrete pro-inflammatory cytokines/chemokines, and express major histocompatibility complex (MHC) class I and II molecules [66][67][68]. We demonstrated that the secretion of chemokine (C-X-C motif) ligand 10 (CXCL10), chemokine (C-C motif) ligand 2 (CCL2), and TNF-α by astrocytes is dependent on the transfer and degradation of PbA-MVs by the LAP pathway [28]. It is known that the production of CXCL10 in the brain is a prerequisite for the recruitment of effector CD8 + T cells expressing the chemokine (C-X-C motif) receptor 3 (CXCR3) involved in ECM [65,67,72,73]. We also evidenced that astrocytes from CM R mice produced significantly lower levels of proinflammatory cytokines/chemokines than CM S -derived cells after stimulation with PbA-iRBCs ( Figure 3A). This is probably due to the smaller number of PbA-MVs transferred to astrocytes from CM R mice. However, we also observed upregulation of leukaemia inhibitory factor (LIF), transforming growth factor beta (TGF-β), and interleukin 10 (IL10) gene expressions in CM S -derived astrocytes after contact with PbA-iRBCs ( Figure 3A).
In contrast to what was observed in astrocytes derived from CM S mice, in ECM-resistant TLR3 knockout (KO) mice, the expression of PB18S and autophagy genes was significantly decreased following PbA-MVs transfer in astrocytes ( Figure 3C,D). In addition, the production of CXCL10, CCL2, TNF-α, IL-10, and TGF-β was totally abolished in astrocytes from TLR3KO mice, unlike in cells from CM S mice ( Figure 3E). These observations strongly suggest a link between the LAP pathway and TLR3-dependent pathway in the transfer of PbA-MVs and the induction of pro-inflammatory cytokine and chemokine responses in astrocytes from CM S mice. Therefore, a TLR3-TRIF-dependent pathway could also participate in neuroinflammation during ECM, similar to what has been reported for the intrahepatic stage [77].

Discussion
This review examines the role of autophagy in the different developmental stages of the Plasmodium parasite. Autophagy is involved in a multitude of parasite biological processes, such as the regulation of intracellular cytoplasmic protein turnover, organelle differentiation, parasite growth, gametogenesis, and infection dissemination. By virtue of its involvement in the biogenesis and function of Plasmodium-derived EVs, autophagy also influences the stimulation of pro-inflammatory responses of innate immune cells and, hence, the severity of the disease.
Our recent description of the involvement of LAP in the transfer of PbA-derived MVs to astrocytes, and the resulting induction of pro-inflammatory factors, exemplifies the importance of MVs in host-parasite interactions and infection outcomes. We also found that a TLR3-mediated anti-inflammatory response is induced in astrocytes after contact with parasite MVs. These observations suggest a dual, but contrasting, role for autophagy in parasite-astrocyte interactions. Autophagy can favor either (i) detrimental neuroinflammation, through the production of CXCL-10 that exacerbates the inflammatory process, or (ii) a protective outcome, resulting from the production of neuroimmunomodulators, such as LIF, which downregulate the exacerbated inflammation and prevent T cells from infiltrating the brain [78]. Through the genesis of parasite-derived EVs, LAP may also influence antigen presentation by astrocytes to pathogenic CD8 + T cells that have migrated to the brain. It is important to note that the astrocytes' production of pro-/anti-inflammatory factors is regulated by crucial molecular events that are responsible for neuropathology during malaria. These events need to be explored further ( Figure 4).
In summary, Plasmodium-derived MVs interact with autophagy pathways to contribute to protection/pathology during malaria. On the one hand, they promote parasite clearance, stimulate a pro-inflammatory innate immune response, and contribute to the downregulation of brain inflammation during CM. On the other hand, they participate in the dissemination of the parasite within the host, as well as in its differentiation to sexual forms. They also contribute to the activation of pro-inflammatory innate responses. Besides this, they can precipitate severe disease by promoting antigen presentation to pathological CD8 + T cells by astrocytes that infiltrate the brain during CM. In summary, Plasmodium-derived MVs interact with autophagy pathways to contribute to protection/pathology during malaria. On the one hand, they promote parasite clearance, stimulate a pro-inflammatory innate immune response, and contribute to the downregulation of brain inflammation during CM. On the other hand, they participate in the dissemination of the parasite within the host, as well as in its differentiation to sexual forms. They also contribute to the activation of pro-inflammatory innate responses. Besides this, they can precipitate severe disease by promoting antigen presentation to pathological CD8 + T cells by astrocytes that infiltrate the brain during CM.