Fig 1: Binding of CLRs to the recombinant SARS-CoV-2 S.(a–e) Immunoblots with human Fc-fused CLRs DC-SIGN (a), L-SIGN (b), MR (c), Dectin-2 (d) and MGL (e) to detect recombinant S1 and S after mock enzymatic digestion or with Endo H, PNGase F or Neu digestion. As negative controls, these glycosidases were also included in some assays. EBY-100 represents the lysates of yeast strain EBY-100. BSM is the recombinant bovine submaxillary mucin. In all assays 5 mM Ca2+ was included in solutions of CLRs. (f) Schematic presentation of the cleavage sites of Endo H, PNGase F and Neu on N- and O-glycans. Endo H cleaves the oligomannose and hybrid N-glycans, while PNGase F removes all N-glycans including the complex type. Neu removes all sialic acids on N- or O-glycans. (g–k) Affinity constant measurement for DC-SIGN (g), L-SIGN (h), MR (i), MGL (j) and ACE2 (k) by ELISA assay. The plates were coated by recombinant SARS-CoV-2 S trimer. Error bars represent SD of two replicates. The data were plotted as % binding relative to the saturated binding as 100%. In all assays 5 mM Ca2+ and Tween-20 were included in solutions of CLRs.
Fig 2: Binding analysis of cells expressing DC-SIGN and L-SIGN and Vero E6 cells infected by SARS-CoV-2.(a and c) Flow cytometry profiles showing the binding of the full-length SARS-CoV-2 S trimer to parental 3T3 cells (middle), and 3T3-DC-SIGN+ and 3T3-L-SIGN+ cells (bottom). (b and d) Increased binding of SARS-CoV-2 S trimer to the parental 3T3 cells, the 3T3-DC-SIGN+ and 3T3-L-SIGN+ cells relative to the secondary antibody control. Data presented here is the percentage of increase in geometric mean fluorescence intensity (gMFI). (e) Internalization of SARS-CoV-2 S trimer in 3T3-DC-SIGN+ cells compared to the parental 3T3 cells at 10 and 30 min. (f-j) Immunoblots with anti-S mAb 1A9 (f), human Fc-fused CLRs DC-SIGN (g), L-SIGN (h), MR (i), and MGL(j) using lysates of Vero E6 cells mock infected (lane 1) or SARS-CoV-2 infected (lane 2), and the SARS-CoV-2-containing culture supernatant (2X and 20X in lane 3 and 4, respectively). Red arrow indicates the bands corresponding to the SARS-CoV-2 S. In all assays 5 mM Ca2+ was included in solutions of CLRs. The recombinant SARS-CoV-2 S was used as a positive control.
Fig 3: Expression of CLRs in human tissues and CLRs/cytokines/chemokines in BALF from COVID-19 patients.(a and b) Single-cell transcriptomic analysis of CLRs gene expression (DC-SIGN, L-SIGN, MR, MGL) and ACE2 in lung and upper airway and thymus as indicated. NK, natural killer cells; DN, double-negative T cells; DP, double-positive T cells; ETP, early thymic progenitor; Endo, endothelial cells; Ery, erythrocytes; Fb, fibroblasts; Mono, monocyte; Mac, macrophage; Mgk, megakaryocyte; NMP, neutrophil-myeloid progenitor; SP, single-positive T cells; VSMC, vascular smooth muscle cells; TEC, thymic epithelial cells). (c) UMAP showing the gene expression levels of CLRs gene expression (DC-SIGN, L-SIGN, MR, MGL) and ACE2, and selected cytokines and chemokines in BALF immune cells from health controls (HC, n = 3), moderate cases (M, n = 3) and severe/critical cases (S, n = 6).
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