Fasciola hepatica, commonly known as liver fluke, is a trematode that causes Fasciolosis in ruminants and humans. The outer tegumental coat of F. hepatica (FhTeg) is a complex metabolically active biological matrix that is continually exposed to the host immune system and therefore makes a good vaccine target. F. hepatica tegumental coat is highly glycosylated and helminth-derived immunogenic oligosaccharide motifs and glycoproteins are currently being investigated as novel vaccine candidates. This report presents the first systematic characterization of FhTeg glycosylation using lectin microarrays to characterize carbohydrates motifs present, and lectin histochemistry to localize these on the F. hepatica tegument. We discovered that FhTeg glycoproteins are predominantly oligomannose oligosaccharides that are expressed on the spines, suckers and tegumental coat of F. hepatica and lectin blot analysis confirmed the abundance of N- glycosylated proteins. Although some oligosaccharides are widely distributed on the fluke surface other subsets are restricted to distinct anatomical regions. We selectively enriched for FhTeg mannosylated glycoprotein subsets using lectin affinity chromatography and identified 369 proteins by mass spectrometric analysis. Among these proteins are a number of potential vaccine candidates with known immune modulatory properties including proteases, protease inhibitors, paramyosin, Venom Allergen-like II, Enolase and two proteins, nardilysin and TRIL, that have not been previously associated with F. hepatica. Furthermore, we provide a comprehensive insight regarding the putative glycosylation of FhTeg components that could highlight the importance of further studies examining glycoconjugates in host-parasite interactions in the context of F. hepatica infection and the development of an effective vaccine.

FoxP3 + Treg cells and anergic T cells are the two regulatory phenotypes of T-cell responses associated with helminth infection. Here, we examine the T-cell responses in mice duringKeywords: Anergy r C-type lectin receptors r Dendritic cells r Helminth infection r Host-pathogen interactions r Mannose receptor r T-cell responses Additional supporting information may be found in the online version of this article at the publisher's web-site IntroductionHelminth parasites can survive within their hosts for many years by suppressing T-cell driven protective immune responses and Th1/Th2-mediated pathology through the induction of regulatory networks that suppress inflammatory responses [1][2][3][4]. These regulatory networks include regulatory T cells (Treg) that in the context of helminth infection is widely understood. Treg cells supCorrespondence: Dr. Sandra M. O'Neill e-mail: sandra.oneill@dcu.ie press the immune response to helminth-driven Th1/Th2 immune pathology through the induction of IL-10 and TGF-β. Brugia malayi, infection induces a regulatory response with enhanced FoxP3 expression and increased cell surface expression of CTLA4, a negative regulator of T-cell function [5] while Schistosoma haematobium infection in humans has also been associated with FoxP3 + Treg cells with the highest percentage of this cell population observed in younger patients [6]. However, less is currently known about the regulatory response termed in vivo anergy or adaptive tolerance.Few studies have examined the role of anergenic T cells during helminth infection. The induction of this cell population is thought C 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu Eur. J. Immunol. 2016. 46: 1180-1192 Immunity to infection 1181 to be the result of the persistent contact of parasite antigen with immune cells during chronic helminth infection [7]. Anergenic T cells differ from Treg cells in that they do not express FoxP3 or produce IL-10 or TGF-β. Instead anergy is a hyporesponsive state with the failure of T cells to proliferate or produce cytokines when restimulated with antigens in vitro [8]. Anergic T cells do not secrete IL-2, a factor important for T-cell proliferation and effector responses. However, the addition of IL-2 can overcome this hyporesponsiveness, restoring helminth-driven T-cell responses. Gene analysis studies have identified a number of genes including Rnf128 (Grail), Itch, Egr2 and Egr3, and CblB that are involved in the induction and maintenance of T-cell anergy [9,10]. A number of helminth infections have shown T-cell anergy or Tcell hyporesponsiveness to be involved in immune suppression. Studies involving S. mansoni show the enhanced expression of GRAIL is linked to the suppression of Th2 cells and also using a filarial nematode, CD4 + T cells become functionally unresponsive [11,12]. Other studies indicate that anergenic-like T cells are the result of helminth infections, with an increase in anergenic markers and T-cell suppression [13][14][15][16]. Fasciola hepatica causes chronic liver d...

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.

BackgroundMalaria is a major cause of morbidity and mortality worldwide with over one million deaths annually, particularly in children under five years. This study was the first to examine plasma cytokines, chemokines and cellular immune responses in pre-school Nigerian children infected with Plasmodium falciparum from four semi-urban villages near Ile-Ife, Osun State, Nigeria.MethodsBlood was obtained from 231 children (aged 39–73 months) who were classified according to mean P. falciparum density per μl of blood (uninfected (n = 89), low density (<1,000, n = 51), medium density (1,000-10,000, n = 65) and high density (>10,000, n = 22)). IL-12p70, IL-10, Nitric oxide, IFN-γ, TNF, IL-17, IL-4 and TGF-β, C-C chemokine RANTES, MMP-8 and TIMP-1 were measured in plasma. Peripheral blood mononuclear cells were obtained and examined markers of innate immune cells (CD14, CD36, CD56, CD54, CD11c AND HLA-DR). T-cell sub-populations (CD4, CD3 and γδTCR) were intracellularly stained for IL-10, IFN-γ and TNF following polyclonal stimulation or stimulated with malaria parasites. Ascaris lumbricoides was endemic in these villages and all data were analysed taking into account the potential impact of bystander helminth infection. All data were analysed using SPSS 15 for windows and in all tests, p <0.05 was deemed significant.ResultsThe level of P. falciparum parasitaemia was positively associated with plasma IL-10 and negatively associated with IL-12p70. The percentage of monocytes was significantly decreased in malaria-infected individuals while malaria parasitaemia was positively associated with increasing percentages of CD54+, CD11c+ and CD56+ cell populations. No association was observed in cytokine expression in mitogen-activated T-cell populations between groups and no malaria specific immune responses were detected. Although A. lumbricoides is endemic in these villages, an analysis of the data showed no impact of this helminth infection on P. falciparum parasitaemia or on immune responses associated with P. falciparum infection.ConclusionsThese findings indicate that Nigerian children infected with P. falciparum exhibit immune responses associated with active malaria infection and these responses were positively associated with increased P. falciparum parasitaemia.

Summary The M2 subset of macrophages has a critical role to play in host tissue repair, tissue fibrosis and modulation of adaptive immunity during helminth infection. Infection with the helminth, Fasciola hepatica, is associated with M2 macrophages in its mammalian host, and this response is mimicked by its excretory‐secretory products (FhES). The tegumental coat of F. hepatica (FhTeg) is another major source of immune‐modulatory molecules; we have previously shown that FhTeg can modulate the activity of both dendritic cells and mast cells inhibiting their ability to prime a Th1 immune response. Here, we report that FhTeg does not induce Th2 immune responses but can induce M2‐like phenotype in vivo that modulates cytokine production from CD4+ cells in response to anti‐CD3 stimulation. FhTeg induces a RELMα expressing macrophage population in vitro, while in vivo, the expression of Arg1 and Ym‐1/2 but not RELMα in FhTeg‐stimulated macrophages was STAT6 dependent. To support this finding, FhTeg induces RELMα expression in vivo prior to the induction of IL‐13. FhTeg can induce IL‐13‐producing peritoneal macrophages following intraperitoneal injection This study highlights the important role of FhTeg as an immune‐modulatory source during F. hepatica infection and sheds further light on helminth–macrophage interactions.