Fasciola hepatica Surface Coat Glycoproteins Contain Mannosylated and Phosphorylated N-glycans and Exhibit Immune Modulatory Properties Independent of the Mannose Receptor

Abstract

Fascioliasis, caused by the liver fluke Fasciola hepatica, is a neglected tropical disease infecting over 1 million individuals annually with 17 million people at risk of infection. Like other helminths, F. hepatica employs mechanisms of immune suppression in order to evade its host immune system. In this study the N-glycosylation of F. hepatica’s tegumental coat (FhTeg) and its carbohydrate-dependent interactions with bone marrow derived dendritic cells (BMDCs) were investigated. Mass spectrometric analysis demonstrated that FhTeg N-glycans comprised mainly of oligomannose and to a lesser extent truncated and complex type glycans, including a phosphorylated subset. The interaction of FhTeg with the mannose receptor (MR) was investigated. Binding of FhTeg to MR-transfected CHO cells and BMDCs was blocked when pre-incubated with mannan. We further elucidated the role played by MR in the immunomodulatory mechanism of FhTeg and demonstrated that while FhTeg’s binding was significantly reduced in BMDCs generated from MR knockout mice, the absence of MR did not alter FhTeg’s ability to induce SOCS3 or suppress cytokine secretion from LPS activated BMDCs. A panel of negatively charged monosaccharides (i.e. GlcNAc-4P, Man-6P and GalNAc-4S) were used in an attempt to inhibit the immunoregulatory properties of phosphorylated oligosaccharides. Notably, GalNAc-4S, a known inhibitor of the Cys-domain of MR, efficiently suppressed FhTeg binding to BMDCs and inhibited the expression of suppressor of cytokine signalling (SOCS) 3, a negative regulator the TLR and STAT3 pathway. We conclude that F. hepatica contains high levels of mannose residues and phosphorylated glycoproteins that are crucial in modulating its host’s immune system, however the role played by MR appears to be limited to the initial binding event suggesting that other C-type lectin receptors are involved in the immunomodulatory mechanism of FhTeg.

Fasciola and fasciolosis in ruminants in Europe: Identifying research needs

“…The importance of these carbohydrate residues is re-enforced by the finding that tegumental antigens (Teg) induce angeric T-cells via dendritic cells (DCs) in a mannose receptor (MR)-dependent fashion . A second study from the same group, however, demonstrated that despite the role of the mannose receptor binding components in Teg not all of its effects were MR-dependent (Ravid a et al, 2016).…”

“…The importance of these carbohydrate residues is re‐enforced by the finding that tegumental antigens (Teg) induce angeric T‐cells via dendritic cells (DCs) in a mannose receptor (MR)‐dependent fashion (Aldridge & O'Neill, ). A second study from the same group, however, demonstrated that despite the role of the mannose receptor binding components in Teg not all of its effects were MR‐dependent (Ravidà et al., ). Indeed the effects of Teg on DCs are known to TLR4‐independent (Hamilton et al., ), while those of excretory/secretory (ES) antigen on the same cell type is partially TLR4‐dependent (Dowling et al., ).…”

Advances in Fasciola hepatica research using ‘omics’ technologies

“…To date F. hepatica glycomic analyses have focussed on the outer surface of the parasite, the glycocalyx, that is rich in glycoproteins and glycolipids (Threadgold, 1976). Analysis has shown that the tegumental surface is highly glycosylated, with an abundance of mannose-rich N-linked glycoproteins present on the surface, spines and suckers (Garcia-Campos et al, 2016;Ravida et al, 2016a;Ravida et al, 2016b). The exact role these N-glycoproteins play at the host-parasite interface is currently unknown, though parasite glyco-conjugates that been implicated in evading the host immune response (van Die and Cummings, 2010).…”

Fasciola hepatica glycoconjugates immuneregulate dendritic cells through the Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin inducing T cell anergy

“…Very recent reports3334, including our own32, have brought insights about the role of F. hepatica glycans in mediating the regulation of DC-maturation through CLR recognition. Our group has recently described that glycan structures produced by F. hepatica participate in the modulation of bone marrow-derived DCs (BMDCs) and induce/mediate the production of IL-10 and IL-4 during infection32.…”