Fig 2: DC-SIGN present on macrophages contributes to phagocytosis of Vi capsulated S. Typhi. (A and C) Transcription levels of CD209 were determined by quantitative real-time PCR in THP-1 cells (A) and human monocytes (C). The graphs show fold changes in CD209 transcription levels prior to stimulation with phorbol 12-myristate-13-acetate (PMA) (A) or macrophage colony-stimulating factor (M-CSF) (C) compared to transcription levels detected 3 days after differentiation into macrophage-like THP-1 cells (A) or monocyte-derived macrophages (C). Bars indicate geometric means ± standard error from 7 (A) or 3 (C) biological repeats. (B and D) Synthesis of DC-SIGN on the surfaces of THP-1 cells (B) and human monocytes (D) was detected by flow cytometry using APC-labeled anti-DC-SIGN antibody. (B) DC-SIGN levels before stimulation with PMA (left panel) and after 3 days of stimulation with PMA (right panel). (D) DC-SIGN levels before stimulation with macrophage colony-stimulating factor (day 0 M-CSF, dotted line) and after 3 (blue line) or 6 days (orange line) of stimulation with M-CSF. MFI, mean fluorescence intensity. (E) Macrophage-like THP-1 cells were infected with Vi capsulated S. Typhi (wild type) or a noncapsulated S. Typhi tviB-vexE mutant in the presence of an anti-DC-SIGN blocking antibody or an isotype control antibody. Recovery of CFU from a gentamicin protection assay 1 h after infection. Bars indicate geometric means ± standard error from 6 biological repeats. NS, P > 0.05; *, P < 0.05; **, P < 0.01; ****, P < 0.001.

Fig 3: Purified Vi capsular polysaccharide binds the C-type lectin receptor (CLR) DC-SIGN. (A) Binding of biotinylated Vi capsular polysaccharide (Biotin-Vi) or biotin to macrophage-like THP-1 cells was visualized using FITC (fluorescein isothiocyanate)-labeled avidin (FITC-Avidin, green fluorescence). Macrophage-like THP-1 cells were counterstained with Hoechst nuclear stain (blue fluorescence). (B) Streptavidin-coated microplates were coated with Biotin-Vi or biotin, and binding to the indicated murine (m) or human (h) CLR proteins fused to the Fc portion of human immunoglobulin G1 (IgG1) (hFc) was assessed by enzyme-linked immunosorbent assay (ELISA). (C) Streptavidin-coated microplates were coated with increasing concentrations of biotin or biotin-Vi, and binding to the indicated CLR fusion proteins was measured by ELISA. (B and C) Graphs show signal (optical density at 450 nm [OD450]) generated by binding to biotin-Vi-coated wells which was higher than the background levels generated by binding to biotin-coated wells. Bars indicate geometric means ± standard error from 6 biological repeats. *, P < 0.05; **, P < 0.01.

Fig 4: DC-SIGN interacts with both SP-D and SARS-CoV-2 spike. Docked poses of (A) complex A, and (B) complex B selected for docking and MD simulations respectively. In complex B, spike interacts with DC-SIGN (CRD) through the NTD domain (orange).

Fig 5: rfhSP-D promotes interaction between SARS-CoV-2 Spike Pseudotypes and DC-SIGN expressing cells. DC-HEK cells (A) and DC-THP-1 cells (B) were treated with rfhSP-D and SARS-CoV-2 Spike-Pseudotypes. The cell binding was analysed using Alexa Fluor 488 (FTIC) and Alexa Fluor 647 (APC); the fluorescence intensity was measured using a GloMax 96 Microplate Luminometer (Promega). An increased fluorescence intensity was observed in DC-HEK and DC-THP-1 cells treated with 20 µg/ml of rfhSP-D compared to cells challenged with Spike pseudotypes alone. Experiments were conducted in triplicates, and error bars represent ± SEM. Unpaired t-test was used to calculate the significance (*p < 0.05, **p < 0.01, and ***p < 0.001) (n = 3). (0, untreated sample; 20, treated sample).