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Zhou et al. Malaria Journal 2012, 11:343http://www.malariajournal.com/content/11/1/343
RESEARCH Open Access
Opsonization of malaria-infected erythrocytesactivates the inflammasome and enhancesinflammatory cytokine secretion by humanmacrophagesJingling Zhou1, Louise E Ludlow4,5, Wina Hasang4,5, Stephen J Rogerson4,5 and Anthony Jaworowski1,2,3*
Background: Antibody opsonization of Plasmodium falciparum-infected erythrocytes (IE) plays a crucial role inanti-malarial immunity by promoting clearance of blood-stage infection by monocytes and macrophages. Theeffects of phagocytosis of opsonized IE on macrophage pro-inflammatory cytokine responses are poorlyunderstood.
Methods: Phagocytic clearance, cytokine response and intracellular signalling were measured using IFN--primedhuman monocyte-derived macrophages (MDM) incubated with opsonized and unopsonized trophozoite-stage CS2IE, a chondroitin sulphate-binding malaria strain. Cytokine secretion was measured by bead array or ELISA, mRNAusing quantitative PCR, and activation of NF-B by Western blot and electrophoretic mobility shift assay. Data wereanalysed using the MannWhitney 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 and IL-6 mRNA by MDM and activated NF-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 or TNF, whereas PPS-opsonized IE induced secretionof IL-1 (18.6 pg/mL [34.2-14.4]) and TNF (113 pg/ml [42117.0]) 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 inMDM showing that Fc receptor-mediated phagocytosis activates the inflammasome. MDM attached to IgG-coatedsurfaces however secreted IL-1 in response to unopsonized IE, suggesting that internalization of IE is notabsolutely required to activate the inflammasome and stimulate IL-1 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 is dependent on Fc receptor-mediated phagocytosis; for IL-1, this occurs byactivation of the inflammasome. The data presented in this paper show that generating antibody responses toblood-stage malaria parasites is potentially beneficial both in reducing parasitaemia via Fc receptor-dependentmacrophage phagocytosis and in generating a robust pro-inflammatory response.
Keywords: Plasmodium falciparum, Human, Monocyte-derived macrophages, Antibody, Fc gamma receptor,Phagocytosis, Pro-inflammatory cytokines
* Correspondence: firstname.lastname@example.orgEqual contributors1Centre for Virology, Burnet Institute, PO Box 2284, Melbourne, Victoria 3001,Australia2Department of Medicine, Monash University, Melbourne, Vic 3004, AustraliaFull list of author information is available at the end of the article
2012 Zhou et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the CreativeCommons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly cited.
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BackgroundThere are an estimated 243 million clinical cases of mal-aria each year, resulting in almost 800,000 deaths (WorldHealth Organization, World Malaria Report 2010). Theburden of disease falls mainly on children under five yearsold and women in their first and second pregnancies (ibid,). The majority of deaths in children, and morbidityassociated with infection in pregnancy, are due to infec-tion by Plasmodium falciparum. The production of anti-bodies to the blood stages of malaria parasites representsan important component of anti-malarial immunity. Thisis most clearly shown by the ability of passively transferredgamma globulin to clear blood-stage infection and allevi-ate clinical illness [2,3]. The mechanism of protectionafforded by gamma globulins purified from hyperimmunedonors is assumed to have occurred by transfer of malariaspecific antibodies, however it cannot be ruled out that al-leviation of disease symptoms may also be contributed bythe immunomodulatory properties of intravenously admi-nistered gamma globulins . The role of antibodies inprotection from malaria is also shown by the protectionafforded to newborn infants from maternal antibodies(although see ) and is suggested by an association ofthe titre of antibodies to malaria antigens with decreasedrisk of disease . Protective antibodies are directedagainst merozoite proteins and variant surface antigensexpressed on infected erythrocytes (IE) [7,8].The mechanism(s) by which antibodies confer protec-
tion needs to be defined in order to inform vaccine devel-opment. Mechanisms that have been proposed includeinhibition of parasite growth [9-11], neutralization of sur-face proteins involved in merozoite entry into red bloodcells [12-14], inhibition of IE sequestration , and pro-motion of parasite killing  or phagocytosis via Fcreceptor-dependent mechanisms [16,17](reviewed in ).Antibody responses associated with protection in bothchildren and pregnant women have been shown to bemainly comprised of cytophilic IgG antibodies of the sub-classes IgG1 and IgG3 [19-26], which suggests a role forFc receptors in protection. The role of Fc receptors inmalaria immunity is supported by observations of a linkbetween Fc receptor polymorphisms and outcomes of in-fection [27-30].Erythrocytes infected with trophozoite-stage parasites
are cleared in the spleen, liver and placenta by mono-cytes and monocyte-derived tissue macrophages(MDM).Antibodies to trophozoite-stage IE surface pro-teins opsonize IE and promote their removal byerythrophagocytosis. Engagement of Fc receptors onmyeloid cells increases the efficiency of ingestion byphagocytosis and stimulates cytokine production, butmay also alter the programme of inflammatory gene ex-pression by macrophages in comparison to that inducedby stimulation of innate immune receptors [31,32]. Pro-
inflammatory cytokines are thought to have a role inlimiting malaria parasitaemia in part via activation ofthe innate immune mechanisms of monocytes andmacrophages. They may also play a role in immuno-pathogenesis, as originally postulated by Clark and co-workers from their studies on mouse models of malaria. This dual potential is illustrated by mouse modelsof malaria infection in which the effect of loss of pro-inflammatory cytokine production in IRAK4/ mice,whose monocytes have defective cytokine productiondue to loss of signalling from multiple toll-like recep-tors, has either a beneficial or deleterious effect on out-comes depending upon the susceptibility of the mousestrain . It is, therefore, crucially important to under-stand how opsonization by immune serum affects pro-inflammatory cytokine production in response tomalaria antigens.As part of studies to investigate how opsonization of
IE affects cytokine production by MDM, and how HIVinfection of MDM may alter these responses , the in-fluence of opsonization with immune serum on the pro-duction of the pro-inflammatory cytokines IL-1, TNFand IL-6, by human MDM exposed to trophozoite-stageP. falciparum IE was investigated. To avoid complica-tions of non-opsonic phagocytosis via CD36, the CS2parasite strain , a model for chondroitin sulphate A(CSA) binding maternal malaria parasites which doesnot bind to CD36, was studied. It is shown that mRNAencoding IL-1, TNF and IL-6 is induced by unopso-nized IE in the absence of phagocytosis, and thatopsonization with IgG enhances phagocytosis and IL-6protein secretion, and enhances IL-1 secretion via ac-tivation of the inflammasome. These data show thatpro-inflammatory cytokine gene expression is acti-vated via surface-expressed innate immune receptorsand requires additional signals, which may be derivedfrom Fc receptor signalling pathways or internal pat-tern recognition receptors, to promote robust pro-inflammatory cytokine secretion.
MethodsIsolation of monocytes and culture of monocyte-derivedtissue macrophagesHuman peripheral blood mononuclear cells (PBMC) wereobtained from Buffy Coats separated from volunteer blooddonations (Australian Red Cross Blood Service, South-bank, Victoria, Australia) using Ficoll-Paque density gra-dient centrifugation. Monocytes were isolated from PBMCby countercurrent elutriation using a Beckman J-6M/Ecentrifuge equipped with a JE 5.0 rotor and tested forpurity as described previously . MDM were pre-pared by culturing freshly isolated monocytes adheredto plastic in Iscoves modified Dulbeccos medium(Invitrogen) containing 10% heat-inactivated human
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serum (Red Cross Blood Service, Sydney, Australia)supplemented with 2 mM glutamine, 100 U/mL peni-cillin G and 100 g/mL streptomycin sulphate (IH10medium). MDM were cultured as described , andwhere indicated they were activated for 48 hr with 100ng/ml human IFN- (R&D Systems).
Preparation and opsonization of CS2 IEThe CSA binding P. falciparum strain CS2  wasmaintained in unexpired human group O+ erythrocytes(Australian Red Cross Blood Service) in RPMI-HEPESsupplemented with 0.5% Albumax II (GIBCO) and 25mM NaHCO3 and tested for CSA adhesion and Myco-plasma contamination as described [17,35]. Maturetrophozoite-stage IE were purified from discontinuousPercol