Opsonization of malaria-infected erythrocytes activates the inflammasome and enhances inflammatory cytokine secretion by human macrophages

Background: Antibody opsonization of Plasmodium falciparum-infected erythrocytes (IE) plays a crucial role in anti-malarial immunity by promoting clearance of blood-stage infection by monocytes and macrophages. The effects of phagocytosis of opsonized IE on macrophage pro-inflammatory cytokine responses are poorly understood. Methods: Phagocytic clearance, cytokine response and intracellular signalling were measured using IFN-gamma-primed human monocyte-derived macrophages (MDM) incubated with opsonized and unopsonized trophozoite-stage CS2 IE, a chondroitin sulphate-binding malaria strain. Cytokine secretion was measured by bead array or ELISA, mRNA using quantitative PCR, and activation of NF-kappa B by Western blot and electrophoretic mobility shift assay. Data were analysed using the Mann-Whitney U test or the Wilcoxon signed rank test as appropriate. Results: Unopsonized CS2 IE were not phagocytosed whereas IE opsonized with pooled patient immune serum (PPS) were (Phagocytic index (PI)=18.4, [SE 0.38] n=3). Unopsonized and opsonized IE induced expression of TNF, IL-1 beta and IL-6 mRNA by MDM and activated NF-kappa B to a similar extent. Unopsonized IE induced secretion of IL-6 (median=622 pg/ml [IQR=1,250-240], n=9) but no IL-1 beta or TNF, whereas PPS-opsonized IE induced secretion of IL-1 beta (18.6 pg/mL [34.2-14.4]) and TNF (113 pg/ml [421-17.0]) and increased IL-6 secretion (2,195 pg/ml [4,658-1,095]). Opsonized, but not unopsonized, CS2 IE activated caspase-1 cleavage and enzymatic activity in MDM showing that Fc receptor-mediated phagocytosis activates the inflammasome. MDM attached to IgG-coated surfaces however secreted IL-1 beta in response to unopsonized IE, suggesting that internalization of IE is not absolutely required to activate the inflammasome and stimulate IL-1 beta secretion. Conclusions: It is concluded that IL-6 secretion from MDM in response to CS2 IE does not require phagocytosis, whereas secretion of TNF and IL-1 beta is dependent on Fc. receptor-mediated phagocytosis; for IL-1 beta, this occurs by activation of the inflammasome. The data presented in this paper show that generating antibody responses to blood-stage malaria parasites is potentially beneficial both in reducing parasitaemia via Fc. receptor-dependent macrophage phagocytosis and in generating a robust pro-inflammatory response.