Role of complement and specific antibodies in macrophage internalization of Echinococcus granulosus Laminated Layer particles

Abstract

Echinococcus granulosus, a parasitic cestode, is the causative agent of cystic echinococcosis (hydatidosis). Its larval stage (hydatid) develops within the host’s organ parenchyma shielded by a thick acellular layer called the laminated layer (LL), mainly composed of highly O-glycosylated mucins and calcium myo-inositol hexakisphosphate (InsP6) deposits. As the hydatid grows, LL particles are released into the extracellular environment. The manner in which the host deals with these materials probably has an impact on the immune responses to the parasite. In humans, LL materials are detected at the lesion site and draining lymph nodes. In experimental mice, either infected or injected with LL particles, LL materials circulate systemically and accumulate in the liver, specifically within the Kupffer cells (KC). This selective uptake is mediated mainly by the C-type lectin receptor Clec4F, which in rodents is exclusively expressed by KC. However, Clec4F independent uptake is also observed, both by hepatic and extrahepatic cell types. Understanding these Clec4F independent mechanisms is important towards understanding human infection, as humans do not express Clec4F. In this study we focused on the role of opsonization by the complement system (CS) and specific antibodies in the uptake of LL materials by immune cells. This encompasses the study of a possible antibody-independent mechanism based on the known binding of the calcium InsP6 component of the LL to complement C1q which initiates limited activation of the classical pathway (CP). It also includes the study of the impact of specific antibodies, known to opsonize the LL, and potentially activating the CP. We performed in vitro assays with mouse peritoneal macrophages from C57BL/6 mice (protocol n° 070151-000038-25). We first assessed the internalization of a soluble/colloidal LL preparation (scLL) containing or lacking the InsP6 calcium deposits by cultured mouse peritoneal cavity macrophages. Experimental conditions included addition of serum from E. granulosus-infected mice as a source of LL-specific antibodies and addition of serum from wild-type or C1q KO mice, with active or inactive CS activity in presence/absence of C1q. The LL-specific antibodies were characterized by ELISAshowing a high titer of both IgM and IgG, with presence of subtypes IgG1, IgG2b, IgG2c and IgG3. Results showed that neither the interaction of C1q with calcium InsP6 nor the opsonization by specific antibodies by themselves promoted LL uptake. However, the combined presence of both C1q and specific antibodies, in the presence of an active CS, significantly increased uptake. Injection of wild type or C1qA deficient mice with scLL containing or not InsP6 calcium deposits showed that the InsP6-C1q interaction does not alter the uptake of the LL materials uptake by KC or by splenic macrophages, in the absence of antibody opsonization, in Clec4F-deficient or in wild-type mice. In conclusion, our in vitro results suggest that internalization of scLL requires CP activation by antibodies bound to the mucins. Ongoing experiments (protocol n° 070151-000024-23) will evaluate whether antibody opsonization affects cellular uptake of the material in vivo, in the presence/absence of C1q and also in the presence/absence of Clec4F.