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

Abstract

Dendritic cell-specific ICAM-3 grabbing non-integrin (DC-SIGN) expressed on a variety of DCs, is a C-type lectin receptor that recognizes glycans on a diverse range of pathogens, including parasites. The interaction of DC-SIGN with pathogens triggers specific signaling events that modulate DC-maturation and activity and regulate T-cell activation by DCs. In this work we evaluate whether F. hepatica glycans can immune modulate DCs via DC-SIGN. We demonstrate that DC-SIGN interacts with F. hepatica glycoconjugates through mannose and fucose residues. We also show that mannose is present in high-mannose structures, hybrid and trimannosyl N-glycans with terminal GlcNAc. Furthermore, we demonstrate that F. hepatica glycans induce DC-SIGN triggering leading to a strong production of TLR-induced IL-10 and IL-27p28. In addition, parasite glycans induced regulatory DCs via DC-SIGN that decrease allogeneic T cell proliferation, via the induction of anergic/regulatory T cells, highlighting the role of DC-SIGN in the regulation of innate and adaptive immune responses by F. hepatica. Our data confirm the immunomodulatory properties of DC-SIGN triggered by pathogen-derived glycans and contribute to the identification of immunomodulatory glyans of helminths that might eventually be useful for the design of vaccines against fasciolosis.

Figure 1. F. hepatica glycoconjugates favor the production of IL-10 by TLR-triggered mo-DCs.

(A) IL-6, IL-10, TNFα and IL-12p70 levels determined by ELISA on supernatants from Pam3CSK4- and LPS-stimulated mo-DC cultures incubated with and without FhTE. (B) IL-10, IL-27p28, IL-27 EBI3 and IL-12p35 levels determined by qRT-PCR of purified RNA of Pam3CSK4- and LPS-sitmulated mo-DC cultures incubated with and without FhTE. (C) IL-10 levels determined by ELISA on supernatants of TLR-triggered mo-DCs incubated in the presence of FhTE, FhCB (oxidation negative control) or FhmPox (oxidized FhTE). A representative Figure of four independent experiments is shown (±SD, indicated by error bars). Asterisks indicate statistically significant differences (p < 0.05).

Figure 2. F. hepatica glycoconjugates are uptaken by MR and DC-SIGN.

To evaluate FhTE-uptake by mo-DCs, cells were incubated with Alexa 647-labeled FhTE at 37 °C. (A) Internalization of FhTE was followed in time and co-localization scores with the routing markers EEA1 and LAMP1 were calculated using imaging flow cytometry. Plotted values were normalized to the condition obtained at 0 min. (B) To identify the receptors that mediate the internalization, moDCs were incubated with Alexa 647-labeled FhTE for 1 h at 37 °C or 4 °C as a control in presence of EGTA, MR-, DC-SIGN, DCIR- and MGL-specific antibodies, and analyzed by FACS. Internalization was calculated as the difference between the MFI at 37 °C and MFI at 4 °C. (C) MR and DC-SIGN bindings were evaluated on FhTE-coated plates with MR-Fc and DC-SIGN-CLR in presence or absence of EGTA. A representative Figure of four independent experiments is shown (±SD, indicated by error bars). Asterisks indicate statistically significant differences (p < 0.05).

Figure 3. DC-SIGN favors the production of IL-10 and IL-27p28 by LPS-stimulated mo-DCs.

(A) IL-10 production levels on supernatants from LPS-stimulated mo-DC overnight cultures incubated with and without FhTE in presence of MR-, DC-SIGN, DCIR- and MGL-specific antibodies. (B) IL-10 and IL-27p28 production on LPS-stimulated mo-DC cultures incubated with and without FhTE together with anti-DC-SIGN antibodies or isotype control. A representative Figure of four independent experiments is shown (±SD, indicated by error bars). Asterisks indicate statistically significant differences (p < 0.05).

Figure 4. DC-SIGN on mo-DCs interacts with FhTE via Man and Fuc residues.

(A) Analysis of FhTE internalization using Imaging Flow Cytometry. The co-localization of Alexa 647-labeled FhTE, EEA1, LAMP1 and DC-SIGN was studied. (B) Co-localization scores of Alexa 647-labeled FhTE with DC-SIGN were calculated over time using imaging flow cytometry. Plotted values were normalized to the condition obtained at 0 min. (C) DC-SIGN binding was evaluated on FhTE-coated plates with DC-SIGN-Fc in presence of EGTA, mannan, GalNAc, anti-DC-SIGN antibody or isotype control. (D) DC-SIGN binding was evaluated on Fucosidase (Fuc), mannosidase (Man) or GalNAcase (GalNAc) treatments of FhTE with DC-SIGN-Fc. A representative Figure of four independent experiments is shown (±SD, indicated by error bars). Asterisks indicate statistically significant differences (p < 0.05).

Figure 5. Simultaneous DC-SIGN and TLR4 triggering by FhTE and LPS on mo-DCs decreases allogeneic T cell proliferation.

(A) Mo-DCs were incubated overnight with FhTE with or without LPS (10 µg/ml) and exposed to allogeneic naïve CD4⁺ T cells. IFN-γ was analyzed by ELISA. (B) The same experiment in presence of DC-SIGN specific antibodies and isotype control. (C) T cells were allowed to rest, and then restimulated with LPS-matured DCs. T-cell proliferation was measured by ³H-Thymidine incorporation in presence or absence of DC-SIGN specific antibodies and isotype control. A representative Figure of four independent experiments is shown (±SD, indicated by error bars). Asterisks indicate statistically significant differences (p < 0.05).

Figure 6. Schematic illustration summarizing the main findings in the present study.

Fucosylated and mannosylated glycoconjugates on F. hepatica interact with DC-SIGN expressed on the DC surface, triggering a tolerogenic program together with TLR signaling that induces enhanced expression of IL-10 and IL-27 by DCs and differentiates naïve CD4⁺ T cells into anergic/regulatory T cells.