Surface antigenic changes in P.falciparum infected erythrocytes following treatment with Syk inhibitors and Artemisinin

The human Plasmodium falciparum (P.falciparum) parasite, currently infects more than 200 million people annually, causing about 500 000 deaths a year and imposes considerable morbidity on the surviving population. Since 2001, the WHO has recommended Artemisinin based combination therapies (ACTs) as treatment of choice for falciparum malaria. However the WHO has observed foci of suspected artemisinin resistance in South- east Asia. Because strains of P. falciparum are rapidly emerging that are resistant to all known antimalarial drugs, including artemisinin, quinine, chloroquine, piperaquine, and mefloquine and their derivatives, emphasis is currently laid on comprehension of new therapies with novel mechanisms of action that includes also the patient’s immune response. Delayed parasite clearance (DPC) has been identified as an useful indicator of artemisin resistance but it has been shown that parasite clearance suffers interindividual variability and reactivity to antimalarials may depend on host immunity. Recently, studies have demonstrated Syk Inhibitors (R406) as potentially useful new class of antimalarial drugs reducing parasitemia by two ways i) delaying P.falciparum growth and ii) suppressing merozoite egress. The latter is caused by interfering of Syk inhibitors with the membrane of the parasite harboring host RBC. Aim of this study is to understand whether the efficacy of new antimalarial combinations of Syk inhibitors and artemisinins (ARTs) is paralleled by enhanced immune responses of the host. I tried to identify a role of antimalarial drug treatment in the parasites clearance by host’s innate immunity. To reach the goal, I studied the activating effect of Syk inhibitor R406, dihydroartemisinin (DHA) and the combination of both on cellular immune functions in in vitro experiments with human monocytes. First line defense mechanism against the malaria parasite, such as phagocytosis and oxidative burst were assessed in cultured primary phagocytes using ring-stage parasitized RBC as phagocytosis target without and with previous DHA and R406 treatment. The molecular basis for observed functional changes was investigated studying DHA- and R406-dependent opsonin-binding to ring-stage pRBCs. by flow cytometry, Western blotting and immune-precipitation. Monocytes show an increased phagocytosis level after treatment of parasitized Ring-PRBC with DHA and R406 and highest phagocytosis values when DHA and R406 were supplemented together at concentrations of 0.1uM and 0.5.uM, respectively. Membrane-bound autologous IgG and C3c complement factor were remarkably increased on Ring-PRBC surface after treatment with DHA and R406 as judged by flow cytometry. Immunoprecipitation confirmed Band 3 as main protein that is labelled by IgG in Syk-inhibitor treated pRBC and the decreased IgG/band 3 ratio in treated cells vs. untreated ones supports the band 3 aggregation model as signal for IgG flagging. Enhanced phagocytosis of PRBCs may represent the common mechanism for innate malaria protection in nonimmune individuals. Modifications on band 3 of host cell membranes accumulate by the oxidative challenge of the growing parasite accompanied by binding of haemichromes to the cytoplasmic tail of band 3. At the moment when a threshold of modifications is exceeded mainly at trophozoite stage PRBC are recognized by phagocytes and ingested. We hypothesize Syk kinase inhibitors to anticipate the moment of recognition by an early accumulation of modified band 3 and bound haemichromes already at ring stage. Syk inhibitors are described to specifically inhib phosphorylation used by PRBC to shed off band 3 – rich microparticles from their membrane. Consequently, DHA as radical producing molecule enhances the oxidative challenge in PRBC. In conclusion, my data support the hypothesis that Syk inhibitors are a promising class of antimalarial drugs that can suppress parasitemia by increasing also the antiparasitic immune defense. Particularly, R406 should not lead to the selection of resistant strains, as it targets host cell molecules and will likely avoid immunosuppressive effects of hemozoin due to the anticipated phagocytosis of Ring stage-PRBC. Therefore, Syk inhibitors may represent a strategic partner drug for artemisinin therapies for counteracting artemisinin resistance.