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Glycans from Fasciola hepatica Modulate the Host Immune Response and TLR-Induced Maturation of Dendritic Cells.

Rodríguez E, Noya V, Cervi L, Chiribao ML, Brossard N, Chiale C, Carmona C, Giacomini C, Freire T - PLoS Negl Trop Dis (2015)

Bottom Line: Our results indicate that glycans from F. hepatica promote the production of IL-4 and IL-10, suppressing IFNγ production.Inhibition assays using carbohydrates suggest that the immune-modulation is mediated, at least in part, by the recognition of a mannose specific-CLR that signals by recruiting the phosphatase Php2.The results presented here contribute to the understanding of the role of parasite glycosylated molecules in the modulation of the host immunity and might be useful in the design of vaccines against fasciolosis.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Immunomodulation and Vaccine Development, Departamento de Inmunobiología, Facultad de Medicina, UdelaR, Montevideo, Uruguay.

ABSTRACT
Helminths express various carbohydrate-containing glycoconjugates on their surface, and they release glycan-rich excretion/secretion products that can be very important in their life cycles, infection and pathology. Recent evidence suggests that parasite glycoconjugates could play a role in the evasion of the immune response, leading to a modified Th2-polarized immune response that favors parasite survival in the host. Nevertheless, there is limited information about the nature or function of glycans produced by the trematode Fasciola hepatica, the causative agent of fasciolosis. In this paper, we investigate whether glycosylated molecules from F. hepatica participate in the modulation of host immunity. We also focus on dendritic cells, since they are an important target of immune-modulation by helminths, affecting their activity or function. Our results indicate that glycans from F. hepatica promote the production of IL-4 and IL-10, suppressing IFNγ production. During infection, this parasite is able to induce a semi-mature phenotype of DCs expressing low levels of MHCII and secrete IL-10. Furthermore, we show that parasite glycoconjugates mediate the modulation of LPS-induced maturation of DCs since their oxidation restores the capacity of LPS-treated DCs to secrete high levels of the pro-inflammatory cytokines IL-6 and IL-12/23p40 and low levels of the anti-inflammatory cytokine IL-10. Inhibition assays using carbohydrates suggest that the immune-modulation is mediated, at least in part, by the recognition of a mannose specific-CLR that signals by recruiting the phosphatase Php2. The results presented here contribute to the understanding of the role of parasite glycosylated molecules in the modulation of the host immunity and might be useful in the design of vaccines against fasciolosis.

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F. hepatica produces a diverse variety of glycan structures.(A) Lectin reactivity was evaluated on microplates coated with FhTE (2.5 μg/well) using different concentrations of biotin-conjugated lectins and streptavidin-Dylight-800. Mean fluorescence intensity (MFI) was determined on plates with an infrared imaging system. Lectins used in this study were: VV (GalNAc, Tn antigen), WGA ((GlcNAc)2), ConA (αMan>αGlc), PNA (βGal(1–3)GalNAc) and UEA (Fucα(1–2)Gal), ECA (βGal(1–4)GlcNAc), SNA (αNeuAc(2–6)Gal) and HPM (GalNAc). (B) Carbohydrate specificity was demonstrated by performing inhibition assays with specific carbohydrates (50 mM) by pre-incubating with GalNAc (VV), GlcNAc (WGA), Man (ConA), Gal (PNA) or Fuc (UEA). Alternatively, lectin reactivity was evaluated on periodate-oxidized glycans (FhmPox) or control consisting of FhTE only treated with borydrure (FhCB). (C) Parasite lysates were subjected to SDS-PAGE (15%) and stained with silver nitrate. (D) Alternatively, they were transferred to PVDF membranes and incubated for 2 h at RT with the anti-Tn monoclonal antibody 83D4 or a polyclonal anti-cathepsin L1 serum.
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pntd.0004234.g002: F. hepatica produces a diverse variety of glycan structures.(A) Lectin reactivity was evaluated on microplates coated with FhTE (2.5 μg/well) using different concentrations of biotin-conjugated lectins and streptavidin-Dylight-800. Mean fluorescence intensity (MFI) was determined on plates with an infrared imaging system. Lectins used in this study were: VV (GalNAc, Tn antigen), WGA ((GlcNAc)2), ConA (αMan>αGlc), PNA (βGal(1–3)GalNAc) and UEA (Fucα(1–2)Gal), ECA (βGal(1–4)GlcNAc), SNA (αNeuAc(2–6)Gal) and HPM (GalNAc). (B) Carbohydrate specificity was demonstrated by performing inhibition assays with specific carbohydrates (50 mM) by pre-incubating with GalNAc (VV), GlcNAc (WGA), Man (ConA), Gal (PNA) or Fuc (UEA). Alternatively, lectin reactivity was evaluated on periodate-oxidized glycans (FhmPox) or control consisting of FhTE only treated with borydrure (FhCB). (C) Parasite lysates were subjected to SDS-PAGE (15%) and stained with silver nitrate. (D) Alternatively, they were transferred to PVDF membranes and incubated for 2 h at RT with the anti-Tn monoclonal antibody 83D4 or a polyclonal anti-cathepsin L1 serum.

Mentions: Carbohydrate structures produced by parasites participate in critical processes such as infection or invasion [25–26]. Although much advance in the area of glycomics has been gained in recent years, the knowledge about the structure and function of F. hepatica glycans is still poor. In order to identify the carbohydrates present in the FhTE used in this study we carried out lectin-reactivity assays with a panel of different vegetal lectins. Glycoconjugates from FhTE strongly reacted with lectins from Vicia Villosa (VV), Triticum vulgaris (WGA), Canavalia ensiformis (ConA), Arachis hpogaea (PNA) and Ulex europaeus (UEA) (Fig 2A), revealing the presence of N-acetil-galactosamine-Ser/Thr (GalNAc-Ser/Thr), N-acetyl-glucosamine (GlcNAc)2, mannose (Man) or glucose (Glc), galactose (Gal) in (βGal(1–3)GalNAc and fucose (Fuc) in Fucα(1–2)Gal, respectively. We also performed inhibition assays with specific monocarbohydrates and with non specific carbohydrates as negative control. More than the 70% of the lectin reactivity was lost when incubating with GalNAc, GlcNAc or Man and VV, WGA or ConA, respectively, confirming the carbohydrate specificity by these lectins (Fig 2B).


Glycans from Fasciola hepatica Modulate the Host Immune Response and TLR-Induced Maturation of Dendritic Cells.

Rodríguez E, Noya V, Cervi L, Chiribao ML, Brossard N, Chiale C, Carmona C, Giacomini C, Freire T - PLoS Negl Trop Dis (2015)

F. hepatica produces a diverse variety of glycan structures.(A) Lectin reactivity was evaluated on microplates coated with FhTE (2.5 μg/well) using different concentrations of biotin-conjugated lectins and streptavidin-Dylight-800. Mean fluorescence intensity (MFI) was determined on plates with an infrared imaging system. Lectins used in this study were: VV (GalNAc, Tn antigen), WGA ((GlcNAc)2), ConA (αMan>αGlc), PNA (βGal(1–3)GalNAc) and UEA (Fucα(1–2)Gal), ECA (βGal(1–4)GlcNAc), SNA (αNeuAc(2–6)Gal) and HPM (GalNAc). (B) Carbohydrate specificity was demonstrated by performing inhibition assays with specific carbohydrates (50 mM) by pre-incubating with GalNAc (VV), GlcNAc (WGA), Man (ConA), Gal (PNA) or Fuc (UEA). Alternatively, lectin reactivity was evaluated on periodate-oxidized glycans (FhmPox) or control consisting of FhTE only treated with borydrure (FhCB). (C) Parasite lysates were subjected to SDS-PAGE (15%) and stained with silver nitrate. (D) Alternatively, they were transferred to PVDF membranes and incubated for 2 h at RT with the anti-Tn monoclonal antibody 83D4 or a polyclonal anti-cathepsin L1 serum.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4697847&req=5

pntd.0004234.g002: F. hepatica produces a diverse variety of glycan structures.(A) Lectin reactivity was evaluated on microplates coated with FhTE (2.5 μg/well) using different concentrations of biotin-conjugated lectins and streptavidin-Dylight-800. Mean fluorescence intensity (MFI) was determined on plates with an infrared imaging system. Lectins used in this study were: VV (GalNAc, Tn antigen), WGA ((GlcNAc)2), ConA (αMan>αGlc), PNA (βGal(1–3)GalNAc) and UEA (Fucα(1–2)Gal), ECA (βGal(1–4)GlcNAc), SNA (αNeuAc(2–6)Gal) and HPM (GalNAc). (B) Carbohydrate specificity was demonstrated by performing inhibition assays with specific carbohydrates (50 mM) by pre-incubating with GalNAc (VV), GlcNAc (WGA), Man (ConA), Gal (PNA) or Fuc (UEA). Alternatively, lectin reactivity was evaluated on periodate-oxidized glycans (FhmPox) or control consisting of FhTE only treated with borydrure (FhCB). (C) Parasite lysates were subjected to SDS-PAGE (15%) and stained with silver nitrate. (D) Alternatively, they were transferred to PVDF membranes and incubated for 2 h at RT with the anti-Tn monoclonal antibody 83D4 or a polyclonal anti-cathepsin L1 serum.
Mentions: Carbohydrate structures produced by parasites participate in critical processes such as infection or invasion [25–26]. Although much advance in the area of glycomics has been gained in recent years, the knowledge about the structure and function of F. hepatica glycans is still poor. In order to identify the carbohydrates present in the FhTE used in this study we carried out lectin-reactivity assays with a panel of different vegetal lectins. Glycoconjugates from FhTE strongly reacted with lectins from Vicia Villosa (VV), Triticum vulgaris (WGA), Canavalia ensiformis (ConA), Arachis hpogaea (PNA) and Ulex europaeus (UEA) (Fig 2A), revealing the presence of N-acetil-galactosamine-Ser/Thr (GalNAc-Ser/Thr), N-acetyl-glucosamine (GlcNAc)2, mannose (Man) or glucose (Glc), galactose (Gal) in (βGal(1–3)GalNAc and fucose (Fuc) in Fucα(1–2)Gal, respectively. We also performed inhibition assays with specific monocarbohydrates and with non specific carbohydrates as negative control. More than the 70% of the lectin reactivity was lost when incubating with GalNAc, GlcNAc or Man and VV, WGA or ConA, respectively, confirming the carbohydrate specificity by these lectins (Fig 2B).

Bottom Line: Our results indicate that glycans from F. hepatica promote the production of IL-4 and IL-10, suppressing IFNγ production.Inhibition assays using carbohydrates suggest that the immune-modulation is mediated, at least in part, by the recognition of a mannose specific-CLR that signals by recruiting the phosphatase Php2.The results presented here contribute to the understanding of the role of parasite glycosylated molecules in the modulation of the host immunity and might be useful in the design of vaccines against fasciolosis.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Immunomodulation and Vaccine Development, Departamento de Inmunobiología, Facultad de Medicina, UdelaR, Montevideo, Uruguay.

ABSTRACT
Helminths express various carbohydrate-containing glycoconjugates on their surface, and they release glycan-rich excretion/secretion products that can be very important in their life cycles, infection and pathology. Recent evidence suggests that parasite glycoconjugates could play a role in the evasion of the immune response, leading to a modified Th2-polarized immune response that favors parasite survival in the host. Nevertheless, there is limited information about the nature or function of glycans produced by the trematode Fasciola hepatica, the causative agent of fasciolosis. In this paper, we investigate whether glycosylated molecules from F. hepatica participate in the modulation of host immunity. We also focus on dendritic cells, since they are an important target of immune-modulation by helminths, affecting their activity or function. Our results indicate that glycans from F. hepatica promote the production of IL-4 and IL-10, suppressing IFNγ production. During infection, this parasite is able to induce a semi-mature phenotype of DCs expressing low levels of MHCII and secrete IL-10. Furthermore, we show that parasite glycoconjugates mediate the modulation of LPS-induced maturation of DCs since their oxidation restores the capacity of LPS-treated DCs to secrete high levels of the pro-inflammatory cytokines IL-6 and IL-12/23p40 and low levels of the anti-inflammatory cytokine IL-10. Inhibition assays using carbohydrates suggest that the immune-modulation is mediated, at least in part, by the recognition of a mannose specific-CLR that signals by recruiting the phosphatase Php2. The results presented here contribute to the understanding of the role of parasite glycosylated molecules in the modulation of the host immunity and might be useful in the design of vaccines against fasciolosis.

Show MeSH
Related in: MedlinePlus