AM3 modulates dendritic cell pathogen recognition capabilities by targeting DC-SIGN

Diego Serrano-Gómez, Rocío T Martínez-Nuñez, Elena Sierra-Filardi, Nuria Izquierdo, María Colmenares, Jesús Pla, Luis Rivas, Javier Martinez-Picado, Jesús Jimenez-Barbero, José Luis Alonso-Lebrero, Salvador González, Angel L Corbí, Diego Serrano-Gómez, Rocío T Martínez-Nuñez, Elena Sierra-Filardi, Nuria Izquierdo, María Colmenares, Jesús Pla, Luis Rivas, Javier Martinez-Picado, Jesús Jimenez-Barbero, José Luis Alonso-Lebrero, Salvador González, Angel L Corbí

Abstract

AM3 (Inmunoferon) is an orally effective immunomodulator that influences the regulatory and effector functions of the immune system whose molecular mechanisms of action are mostly unknown. We hypothesized that the polysaccharide moiety of AM3 (IF-S) might affect immune responses by modulating the lectin-dependent pathogen recognition abilities of human dendritic cells. IF-S inhibited binding of viral, fungal, and parasite pathogens by human monocyte-derived dendritic cells in a dose-dependent manner. IF-S specifically impaired the pathogen recognition capabilities of DC-SIGN, as it reduced the attachment of Candida, Aspergillus, and Leishmania to DC-SIGN transfectants. IF-S also inhibited the interaction of DC-SIGN with both its cellular counterreceptor (intercellular adhesion molecule 3) and the human immunodeficiency virus (HIV) type 1 gp120 protein and blocked the DC-SIGN-dependent capture of HIV virions and the HIV trans-infection capability of DC-SIGN transfectants. IF-S promoted DC-SIGN internalization in DCs without affecting mannose receptor expression, and (1)D saturation transfer difference nuclear magnetic resonance demonstrated that IF-S directly interacts with DC-SIGN on the cell surface. Therefore, the polysaccharide moiety of AM3 directly influences pathogen recognition by dendritic cells by interacting with DC-SIGN. Our results indicate that DC-SIGN is the target for an immunomodulator and imply that the adjuvant and immunomodulatory actions of AM3 are mediated, at least in part, by alteration of the DC-SIGN functional activities.

Figures

FIG. 1.
FIG. 1.
IF-S inhibits the binding of C. albicans and A. fumigatus to MDDCs. (A) Flow cytometry analysis of the binding of FITC-labeled C. albicans to MDDCs in the presence of IF-S or the indicated antibodies. The percentage of cells with bound C. albicans is indicated. MOI, multiplicity of infection. (B) Mean fluorescence intensities (MFI) of MDDCs in the binding assay whose results are shown in panel A. (C) Phase-contrast images (left panels) and epifluorescence visualization (right panels) of the experiment whose results are shown in panel A. (D) Flow cytometry analysis of the binding of FITC-labeled A. fumigatus to MDDCs in the presence of S. cerevisiae mannan, IF-S, or the indicated antibodies. The percentage of cells with bound Aspergillus is indicated in each case.
FIG. 2.
FIG. 2.
IF-S inhibits the binding of C. albicans and A. fumigatus to K562-CD209 cells. (A) Flow cytometry analysis of the binding of FITC-labeled C. albicans to K562 and K562-CD209 cells in the presence of IF-S or the indicated antibodies. The percentage of cells with bound C. albicans is indicated in each case. MOI, multiplicity of infection. (B) Mean fluorescence intensity (MFI) of K562-CD209 cells after the binding assay whose results are shown in panel A. (C) Flow cytometry analysis of the binding of FITC-labeled A. fumigatus conidia to K562-CD209 cells in the presence of S. cerevisiae mannan, IF-S, or the indicated antibodies. The percentage of cells with bound A. fumigatus is indicated in each case.
FIG. 3.
FIG. 3.
IF-S inhibits the binding of Leishmania pifanoi amastigotes to MDDCs and K562-CD209 cells. (A and B) Flow cytometry analysis of the binding of CFSE-labeled L. pifanoi amastigotes to MDDCs in the presence of IF-S or the indicated antibodies. The percentage of cells with bound amastigotes is indicated. MOI, multiplicity of infection. (C and D) DC-SIGN cell surface expression (percentage of marker-positive cells and mean fluorescence intensity) on the MDDCs analyzed and for which the results are shown in panels A and B, as determined by flow cytometry. (E) Flow cytometry analysis of the binding of CFSE-labeled L. pifanoi amastigotes to K562-CD209 cells (multiplicity of infection [MOI], 5:1) in the presence of IF-S or an anti-DC-SIGN antibody. The percentage of cells with bound amastigotes is indicated. (F) Phase-contrast images (left panels) and epifluorescence visualization (right panels) of the experiment whose results are shown in panel E.
FIG. 4.
FIG. 4.
IF-S inhibits DC-SIGN-dependent recognition of HIV-1. (A and B) Binding of gp120-Fc to K562 and K562-CD209 cells (A) or MDDCs (B) in the presence of S. cerevisiae mannan (Man), laminarin (Lam), IF-S, anti-DC-SIGN, or an irrelevant antibody, as determined by flow cytometry. The percentage of marker-positive cells (B) and the mean fluorescence intensity (MFI) (A and B) are indicated. (C) Binding of HIVNFN-SX to Raji and Raji DC-SIGN cells in the presence of DC-SIGN blocking agents, as determined by a p24gag ELISA. (D) HIVJRFL/NL43-Luc transmission from Raji DC-SIGN or Raji cells to Hut CCR5 target cells in the presence of DC-SIGN blocking agents. As a control of direct infection, pulsed Raji DC-SIGN cells were also cultured with Hut CXCR4 target cells. Dotted line, background levels of luciferase (RLU, relative light units) observed with nonpulsed Raji DC-SIGN cells.
FIG. 5.
FIG. 5.
IF-S inhibits DC-SIGN-dependent adhesive functions. (A) Flow cytometry determination of the DC-SIGN cell surface expression on immature MDDCs, K562 cells, and K562-CD209 cells. The percentage of marker-positive cells and the mean fluorescence intensity are indicated in each case. (B) Adhesion of MDDCs, K562 cells, and K562-CD209 cells to recombinant ICAM-3-Fc in the presence of mannan, IF-S, an anti-DC-SIGN antibody (MR1), or an irrelevant antibody. Each condition was assayed in triplicate, and the data shown are the means ± standard deviations. (C) Flow cytometry determination of the DC-SIGN cell surface expression on Jurkat and Jurkat-CD209 cells. The percentage of marker-positive cells and the mean fluorescence intensity are indicated in each case. (D) Adhesion of Jurkat-CD209 cells to recombinant ICAM-3-Fc performed as described in the legend to panel B.
FIG. 6.
FIG. 6.
IF-S inhibits the DC-SIGN-dependent homotypic aggregation of K562-CD209 cells. K562-CD209 cells were resuspended and allowed to aggregate for 20 min in the presence of A. fumigatus galactomannan (Gal-Man), IF-S, or the indicated antibodies.
FIG. 7.
FIG. 7.
IF-S promotes DC-SIGN internalization in MDDCs. (A to C) MDDCs (2.5 × 105 cells per time point) were incubated with IF-S at 4°C for 1 h, washed, and placed at 37°C for the indicated time points to allow internalization. The cell surface expression of DC-SIGN (A and B), CD29 (B), or mannose receptor (C) was then determined by flow cytometry. Each experiment was performed twice, with similar results each time, and the results of representative experiments are shown. (D) Production of TNF-α by MDDCs exposed for 18 h to LPS, IF-S, anti-DC-SIGN antibody (MR1), or both MR1 and IF-S. Each treatment was assayed in triplicate, and the data shown are the means ± standard deviations.
FIG. 8.
FIG. 8.
IF-S binds and directly contacts DC-SIGN on the cell surface. (A) Adhesion of K562 K562-CD209 cells to IF-S-, mannan-, and ICAM-3-coated wells. (B) Adhesion of K562 and K562-CD209 cells to IF-S-coated wells in the presence of mannan or the indicated antibodies. Each adhesion condition was assayed in triplicate, and the data shown are the means ± standard deviations. (C) Binding of IF-S to cell surface DC-SIGN as determined by one-dimensional STD NMR. (Upper left panel) 1H NMR spectrum of IF-S with K562-CD209 cells in PBS at 298 K; (lower left panel) STD reference spectrum of IF-S with K562 cells (signal enhancement = 32×, NS [number of scans] = 128, on-resonance frequency = −0.3 ppm, off-resonance frequency = 100 ppm, total saturation time = 2 s); (upper right panel) STD spectrum of IF-S with K562-CD209 cells (signal enhancement = 16×, NS = 64, on-resonance frequency = −0.3 ppm, off-resonance frequency = 100 ppm); (lower right panel) STD spectrum of IF-S with K562-CD209 cells (signal enhancement = 32×, NS = 64, on-resonance frequency = 6.8 ppm, off-resonance frequency = 100 ppm).

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