Characterization of the immunosuppressive environment induced by larval Echinococcus granulosus during chronic experimental infection.

The larval stage of Echinococcus granulosus causes the chronic infection known as cystic echinococcosis, deploying strong inhibitory mechanisms on host immune responses. Using experimental intraperitoneal infection in C57BL/6 mice, we carried out an in-depth analysis of the local changes in macrophage populations associated with chronic infection. In addition, we analyzed T cells and relevant soluble mediators. Infected animals showed an increase in local cell numbers, mostly accounted for by eosinophils, T cells, and macrophages. Within macrophage populations, the largest increases in cell numbers corresponded to resident large peritoneal macrophages (LPM). Monocyte recruitment appeared to be active, as judged by the increased number of monocytes and cells in the process of differentiation towards LPM, including small (SPM) and converting peritoneal macrophages (CPM). In contrast, we found no evidence of macrophage proliferation. Infection induced the expression of M2 markers in SPM, CPM, and LPM. It also enhanced the expression of the co-inhibitor PD-L1 in LPM, SPM, and CPM and induced the co-inhibitor PD-L2 in SPM and CPM. Therefore, local macrophages acquire M2-like phenotypes with probable suppressive capacities. Regarding T cells, infection induced an increase in the percentage of CD4+ cells that are PD-1+, which represent a potential target of suppression by PD-L1+/PD-L2+ macrophages. In possible agreement, CD4+ T cells from infected animals showed blunted proliferative responses to in vitro stimulation with anti-CD3. Further evidence of immune suppression in the parasite vicinity arose from the observation of an expansion in FoxP3+ CD4+ regulatory T cells and increases in the local concentrations of the anti-inflammatory cytokines TGF-ß and IL-1Ra.

Mucins Shed from the Laminated Layer in Cystic Echinococcosis Are Captured by Kupffer Cells via the Lectin Receptor Clec4F.

Cystic echinococcosis is caused by the larval stages (hydatids) of cestode parasites belonging to the species cluster Echinococcus granulosus sensu lato, with E. granulosus sensu stricto being the main infecting species. Hydatids are bladderlike structures that attain large sizes within various internal organs of livestock ungulates and humans. Hydatids are protected by the massive acellular laminated layer (LL), composed mainly of mucins. Parasite growth requires LL turnover, and abundant LL-derived particles are found at infection sites in infected humans, raising the question of how LL materials are dealt with by the hosts. In this article, we show that E. granulosus sensu stricto LL mucins injected into mice are taken up by Kupffer cells, the liver macrophages exposed to the vascular space. This uptake is largely dependent on the intact mucin glycans and on Clec4F, a C-type lectin receptor which, in rodents, is selectively expressed in Kupffer cells. This uptake mechanism operates on mucins injected both in soluble form intravenously (i.v.) and in particulate form intraperitoneally (i.p.). In mice harboring intraperitoneal infections by the same species, LL mucins were found essentially only at the infection site and in the liver, where they were taken up by Kupffer cells via Clec4F. Therefore, shed LL materials circulate in the host, and Kupffer cells can act as a sink for these materials, even when the parasite grows in sites other than the liver.

Immunology of a unique biological structure: the Echinococcus laminated layer.

The larval stages of the cestode parasites belonging to the genus Echinococcus grow within internal organs of humans and a range of animal species. The resulting diseases, collectively termed echinococcoses, include major neglected tropical diseases of humans and livestock. Echinococcus larvae are outwardly protected by the laminated layer (LL), an acellular structure that is unique to this genus. The LL is based on a fibrillar meshwork made up of mucins, which are decorated by galactose-rich O-glycans. In addition, in the species cluster termed E. granulosus sensu lato, the LL features nano-deposits of the calcium salt of myo-inositol hexakisphosphate (Insp6). The main purpose of our article is to update the immunobiology of the LL. Major recent advances in this area are (i) the demonstration of LL "debris" at the infection site and draining lymph nodes, (ii) the characterization of the decoy activity of calcium Insp6 with respect to complement, (iii) the evidence that the LL mucin carbohydrates interact specifically with a lectin receptor expressed in Kupffer cells (Clec4F), and (iv) the characterization of what appear to be receptor-independent effects of LL particles on dendritic cells and macrophages. Much information is missing on the immunology of this intriguing structure: we discuss gaps in knowledge and propose possible avenues for research.

Inefficient and abortive classical complement pathway activation by the calcium inositol hexakisphosphate component of the Echinococcus granulosus laminated layer.

Persistent extracellular tissue-dwelling pathogens face the challenge of antibody-dependent activation of the classical complement pathway (CCP). A prime example of this situation is the larva of the cestode Echinococcus granulosus sensu lato, causing cystic echinococcosis. This tissue-dwelling, bladder-like larva is bounded by a cellular layer protected by the outermost acellular "laminated layer" (LL), to which host antibodies bind. The LL is made up of a mucin meshwork and interspersed nano-deposits of calcium inositol hexakisphosphate (calcium InsP6). We previously reported that calcium InsP6 bound C1q, apparently initiating CCP activation. The present work dissects CCP activation on the LL. Most of the C1 binding activity in the LL corresponded to calcium InsP6, and this binding was enhanced by partial proteolysis of the mucin meshwork. The remaining C1 binding activity was attributable to host antibodies, which included CCP-activating IgG isotypes. Calcium InsP6 made only a weak contribution to early CCP activation on the LL, suggesting inefficient C1 complex activation as reported for other polyanions. CCP activation on calcium InsP6 gave rise to a dominant population of C3b deposited onto calcium InsP6 itself that appeared to be quickly inactivated. Apparently as a result of inefficient initiation plus C3b inactivation, calcium InsP6 made no net contribution to C5 activation. We propose that the LL protects the underlying parasite cells from CCP activation through the combined effects of inefficient permeation of C1 through the mucins and C1 retention on calcium InsP6. This mechanism does not result in C5 activation, which is known to drive parasite-damaging inflammation.

Particles from the Echinococcus granulosus laminated layer inhibit IL-4 and growth factor-driven Akt phosphorylation and proliferative responses in macrophages.

Proliferation of macrophages is a hallmark of inflammation in many type 2 settings including helminth infections. The cellular expansion is driven by the type 2 cytokine interleukin-4 (IL-4), as well as by M-CSF, which also controls homeostatic levels of tissue resident macrophages. Cystic echinococcosis, caused by the tissue-dwelling larval stage of the cestode Echinococcus granulosus, is characterised by normally subdued local inflammation. Infiltrating host cells make contact only with the acellular protective coat of the parasite, called laminated layer, particles of which can be ingested by phagocytic cells. Here we report that a particulate preparation from this layer (pLL) strongly inhibits the proliferation of macrophages in response to IL-4 or M-CSF. In addition, pLL also inhibits IL-4-driven up-regulation of Relm-α, without similarly affecting Chitinase-like 3 (Chil3/Ym1). IL-4-driven cell proliferation and up-regulation of Relm-α are both known to depend on the phosphatidylinositol (PI3K)/Akt pathway, which is dispensable for induction of Chil3/Ym1. Exposure to pLL in vitro inhibited Akt activation in response to proliferative stimuli, providing a potential mechanism for its activities. Our results suggest that the E. granulosus laminated layer exerts some of its anti-inflammatory properties through inhibition of PI3K/Akt activation and consequent limitation of macrophage proliferation.