Abstract
BackgroundAntibody 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.MethodsPhagocytic clearance, cytokine response and intracellular signalling were measured using IFN-γ-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-κB 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.ResultsUnopsonized 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-1β and IL-6 mRNA by MDM and activated NF-κB to a similar extent. Unopsonized IE induced secretion of IL-6 (median= 622 pg/ml [IQR=1,250-240], n=9) but no IL-1β or TNF, whereas PPS-opsonized IE induced secretion of IL-1β (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-1β in response to unopsonized IE, suggesting that internalization of IE is not absolutely required to activate the inflammasome and stimulate IL-1β secretion.ConclusionsIt is concluded that IL-6 secretion from MDM in response to CS2 IE does not require phagocytosis, whereas secretion of TNF and IL-1β is dependent on Fcγ receptor-mediated phagocytosis; for IL-1β, 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 Fcγ receptor-dependent macrophage phagocytosis and in generating a robust pro-inflammatory response.
Highlights
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
Preparation and opsonization of CS2 IE The chondroitin sulphate A (CSA) binding P. falciparum strain CS2 [36] was maintained in unexpired human group O+ erythrocytes (Australian Red Cross Blood Service) in RPMI-HEPES supplemented with 0.5% Albumax II (GIBCO) and 25 mM NaHCO3 and tested for CSA adhesion and Mycoplasma contamination as described [17,35]
IE collected from the 60% layer (92-95% purity) were washed three times and re-suspended in PBS at a density of 1x108 per ml opsonized for 30 min at room temperature with 9% pooled patient immune serum (PPS) from Malawian HIV-uninfected pregnant women with malaria, which demonstrated high levels of antibody to CS2 IE [17], or left unopsonized
Summary
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 majority of deaths in children, and morbidity associated with infection in pregnancy, are due to infection by Plasmodium falciparum. The production of antibodies to the blood stages of malaria parasites represents an important component of anti-malarial immunity. This is most clearly shown by the ability of passively transferred gamma globulin to clear blood-stage infection and alleviate clinical illness [2,3]. Protective antibodies are directed against merozoite proteins and variant surface antigens expressed on infected erythrocytes (IE) [7,8]
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