Impact of Plasmodium falciparum small-sized extracellular vesicles on host peripheral blood mononuclear cells

<ns4:p><ns4:bold>Background: </ns4:bold>Exagerated immune activation has a key role in the pathogenesis of malaria<ns4:bold>. </ns4:bold>During blood-stage infection, <ns4:italic>Plasmodium falciparum</ns4:italic> can interact directly with host immune cells through infected red blood cells (<ns4:italic>Pf</ns4:italic>iRBCs), or indirectly by the release of extracellular vesicles (EVs). Here, we compared the impact of <ns4:italic>Pf</ns4:italic>iRBCs and <ns4:italic>P. falciparum</ns4:italic> small-sized EVs (<ns4:italic>Pf</ns4:italic>sEVs, also known as exosomes) from a Kenyan clinical isolate (<ns4:italic>Pf</ns4:italic>KE12) adapted to short-term laboratory culture conditions on host peripheral blood mononuclear cells (PBMC).</ns4:p><ns4:p> <ns4:bold>Methods:</ns4:bold><ns4:italic> Pf</ns4:italic>sEVs were isolated from cell-free culture-conditioned media by ultracentrifugation while mature trophozoite <ns4:italic>Pf</ns4:italic>iRBCs were purified by magnetic column separation. The <ns4:italic>Pf</ns4:italic>sEVs and the <ns4:italic>Pf</ns4:italic>iRBCs were co-cultured for 18 hours with PBMC. Cellular responses were quantified by cell surface expression of activation markers (CD25, CD69) and cytokine/chemokine levels in the supernatant.</ns4:p><ns4:p> <ns4:bold>Results:</ns4:bold> Relative to negative control conditions,<ns4:italic> Pf</ns4:italic>sEVs induced CD25 expression on CD4<ns4:sup>+</ns4:sup>, CD19<ns4:sup>+</ns4:sup> and CD14<ns4:sup>+ </ns4:sup>cells, while <ns4:italic>Pf</ns4:italic>iRBCs induced on CD19<ns4:sup>+</ns4:sup> and CD14<ns4:sup>+</ns4:sup> cells. Both <ns4:italic>Pf</ns4:italic>sEVs and <ns4:italic>Pf</ns4:italic>iRBCs induced CD69 on CD4<ns4:sup>+</ns4:sup>, CD8<ns4:sup>+</ns4:sup> and CD19<ns4:sup>+</ns4:sup> cells. In addition, <ns4:italic>Pf</ns4:italic>iRBCs induced higher expression of CD69 on CD14<ns4:sup>+</ns4:sup> cells. CD69 induced by <ns4:italic>Pf</ns4:italic>iRBCs on CD4<ns4:sup>+ </ns4:sup>and CD19<ns4:sup>+</ns4:sup> cells was significantly higher than that induced by <ns4:italic>Pf</ns4:italic>sEVs. Secretion of MIP1α, MIP1β, GM-CSF, IL-6, IL-8, and TNFα were significantly induced by both <ns4:italic>Pf</ns4:italic>sEVs and <ns4:italic>Pf</ns4:italic>iRBCs whereas MCP-1, IL-10, IL-17α  were preferentially induced by <ns4:italic>Pf</ns4:italic>sEVs and IP-10 and IFN-γ by <ns4:italic>Pf</ns4:italic>iRBCs. Prior exposure to malaria (judged by antibodies to schizont extract) was associated with lower monocyte responses to <ns4:italic>Pf</ns4:italic>sEVs.</ns4:p><ns4:p> <ns4:bold>Conclusions: </ns4:bold><ns4:italic>Pf</ns4:italic>sEVs and <ns4:italic>Pf</ns4:italic>iRBCs showed differential abilities to induce secretion of IL-17α  and IFN-γ, suggesting that the former are better at inducing Th17, whilst  the latter induce Th1 immune responses respectively. Prior exposure to malaria significantly reduces the ability of <ns4:italic>Pf</ns4:italic>sEVs to activate monocytes, suggesting immune tolerance to <ns4:italic>Pf</ns4:italic>sEVs may play a role in naturally acquired  anti-disease immunity.</ns4:p>