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

Abstract

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.

Keywords: Plasmodium; astrocytes; autophagy; cerebral malaria; microvesicles; neuroinflammation; pathophysiology.

Figure 1

Host autophagy pathways and Plasmodium-derived microvesicles. (A) Plasmodium sporozoite infects a hepatocyte by invagination of the host cell membrane, thus forming a transient vacuole (TV) or parasitophorous vacuole (PV). 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 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.

Figure 2

PbA-MVs transfer to astrocytes from ECM-sensitive (CM^S) and -resistant (CM^R) mice upon 6-h contact with iRBCs. Primary astrocyte cultures derived from neonatal CM^S or CM^R mice were stimulated for 6 h with GFP-PbA-iRBCs, washed, and followed for 48 h post iRBC contact, as previously described [28]. (A) Transmission electron microscopy revealed that PbA-MVs were transferred to CM^S-derived astrocytes at the contact point with iRBCs (top micrograph, white arrows) and were observed intracellularly (bottom micrograph, white arrows) at the 6-h time point. The red arrow shows the cell membrane of the astrocyte. (B) GFP-PbA-MV (green) and parasite DNA (blue) are enclosed inside a PV labelled by UIS4 (pink; left panel) or LC3-II (red; right panel) to form a LAPosome inside CM^S-derived astrocytes. By contrast, PbA-MVs remained at the cell membrane of CM^R-derived astrocytes. (C) The quantity of PB18S gene detected in CM^R-derived astrocytes was significantly lower than that found in CM^S-derived cells, confirming reduced transfer of PbA-MVs in these cells. (D) LAP-related gene expression did not increase in CM^R-derived astrocytes, as compared to CM^S-derived cells, 6 h after PbA-iRBC contact. Student’s t-test was used to compare median fold change in gene expression in panels C and D (n = 5). * p < 0.05; ** p < 0.01; **** p < 0.0001.

Figure 3

Immune response of astrocytes after PbA-MV transfer and TLR3 engagement. Primary astrocyte cultures, derived from CM^S, CM^R, or TLR3KO neonatal mice, were stimulated for 6 h with GFP-PbA-iRBCs. The cells were then washed and followed up for 48 h post iRBC contact. (A) Pro-inflammatory CXCL-10, CCL-2, and TNF-α genes and anti-inflammatory cytokine LIF, IL10, and TGF-β genes were highly expressed in CM^S-derived astrocytes, as compared to CM^R-derived cells. (B) TLR3 pathway genes were significantly upregulated in CM^S-derived astrocytes after 24 h of contact with PbA-iRBCs. (C) TLR3 gene expression was totally abolished in TLR3KO-derived astrocytes. (D) Decreased PB18S gene expression and downregulation of autophagy-related genes (LC3, RUBCN, and ULK1) were observed in TLR3KO-derived, but not CM^S-derived, astrocytes at 48 h after iRBC stimulation. White bars indicate 0-h stimulation and grey bars indicate 48-h stimulation. (E) The absence of CXCL-10, CCL-2, TNF-α, IL-10, and TGF-β gene expression in TLR3KO-derived astrocytes suggests involvement of the TLR3 pathway in the astrocyte immune response. Student’s t-test (except for (B), where one-way ANOVA was used) was used to compare median fold change (n = 5). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Figure 4

Schematic hypothesis of detrimental/protective pathways of astrocytes involved in the pathogenesis of ECM.