Journal: Immunity

Article Title: SARS-CoV-2 exacerbates proinflammatory responses in myeloid cells through C-type lectin receptors and Tweety family member 2

doi: 10.1016/j.immuni.2021.05.006

Figure Lengend Snippet: Discovery of C-type lectins and TTYH2 as interacting partners of the SARS-CoV-2 S protein (A) Schematic of the myeloid cell receptor discovery approach. Individual plasmids of genes encoding myeloid cell receptors were transfected into HEK293T cells, and a human Fc-tagged SARS-CoV-2 S protein mixture (S-Fc, S1-Fc, and RBD-Fc) and anti-human immunoglobulin G (IgG) Fc detection antibody were added to the cell culture to assess binding (see for more details). S protein subunits and subdomains relative to ACE2 binding (RBD) are also shown. (B) Other than ACE2 (purple), DC-SIGN, L-SIGN, LSECtin, ASGR1, CLEC10A, and TTYH2 (all in red) were identified as binding partners for the SARS-CoV-2 S protein (n = 2). Fc receptors (blue) served as positive controls. (C) Representative images of the binding of the Fc-tagged S protein, its subunits, or Fc control (Fc Ctr) to the indicated receptors, captured by the cellular detection system (CDS) (n = 3). (D) Quantification of the interaction between S protein subdomains/subunits and different receptors, indicated on the x axis. Normalized binding capacity is shown on the y axis (the sum of the total fluorescence intensity to the indicated receptor was set to 100). (E) Binding between HEK293T cells expressing the indicated receptors and HIV-GFP virus pseudotyped with the SARS-CoV-2 S protein was detected by an anti-S polyclonal antibody and analyzed by flow cytometry (n = 4). Data are presented as the mean ± SEM of five pooled independent experiments (D) or a representative experiment (E); ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001 by one-way ANOVA (E). n refers to the number of independent experiments. See also Figure S1 .

Article Snippet: The wells were incubated with recombinant ACE2 (Sino Biological), DC-SIGN (Sino Biological), L-SIGN (Sino Biological), LSECtin (Acro Biosystems), ASGR1 (R&D Systems), and CLEC10A (Sino Biological) with human Fc tag at 37°C for 6 hr.

Techniques: Transfection, Cell Culture, Binding Assay, Fluorescence, Expressing, Flow Cytometry

Journal: Immunity

Article Title: SARS-CoV-2 exacerbates proinflammatory responses in myeloid cells through C-type lectin receptors and Tweety family member 2

doi: 10.1016/j.immuni.2021.05.006

Figure Lengend Snippet: Glycan-dependent SARS-CoV-2 S interaction with C-type lectins via residues distinct from ACE2 (A) Quantification of S-Fc or RBD-Fc binding to HEK293T cells expressing the indicated receptors in the presence or absence of a 100× excess (in the mass ratio) of His-tagged ACE2 ectodomain recombinant protein (ACE2-His) (n = 3). (B) The hydrogen bond formation between the Lys352-Asp353, Tyr41 of human ACE2 (green) and the Thr500-Asn501-Gly502 loop segment of the wild-type (WT) SARS-CoV-2 receptor binding motif (RBM) (PDB: 6M0J ; left , cyan ) or the Ala500-Ala501-Glu502 SARS-CoV-2 RBM mutant (right, cyan). (C) Quantitative comparison of binding to the indicated receptors of Fc-tagged WT S1 and the T500A/N501A/G502A mutant S1 recombinant protein (n = 3). (D) Representative images (left) and quantification (right) of binding of S-Fc or RBD-Fc (for TTYH2) to the indicated receptors in the presence or absence of 20 μg/mL mannan (n = 3). (E) Glycosylated SARS-CoV-2 S protein model highlighting Asn(N)165, N282, N343, and N603 glycans. The SARS-CoV-2 RBD is colored cyan, and the ACE2 N-terminal peptidase domain is shown in green. (F) Quantification of representative S1 Asn mutants that enhanced interaction with ACE2 (N282Q, left) or some of the C-type lectins (N165Q, right) (n = 3). (G) Quantification of representative S1 Asn mutants that reduced interaction with ACE2 (N343Q, left) or C-type lectins (N603Q, right) (n = 3). (H) Schematic of the distribution of N- or O-glycosylation sites in the S1 subunit (top panel) and existence of natural mutations related to some of these sites among ∼5,000 SARS-CoV-2 viral genomes as well as quantitative analysis of the effect of individual mutations of these sites on S protein binding to the indicated receptors (bottom panel). All fluorescence images were captured by CDS and analyzed by CellProfiler software. Data are presented as the mean ± SEM of a representative experiment (A, C, D, F, and G) or three pooled independent experiments (H). ns, not significant. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001 by two-tailed unpaired Student’s t test (A, C, D, F, and G) or one-way ANOVA (H). n refers to the number of independent experiments. See also Figure S2 .

Article Snippet: The wells were incubated with recombinant ACE2 (Sino Biological), DC-SIGN (Sino Biological), L-SIGN (Sino Biological), LSECtin (Acro Biosystems), ASGR1 (R&D Systems), and CLEC10A (Sino Biological) with human Fc tag at 37°C for 6 hr.

Techniques: Binding Assay, Expressing, Recombinant, Mutagenesis, Protein Binding, Fluorescence, Software, Two Tailed Test

Journal: Immunity

Article Title: SARS-CoV-2 exacerbates proinflammatory responses in myeloid cells through C-type lectin receptors and Tweety family member 2

doi: 10.1016/j.immuni.2021.05.006

Figure Lengend Snippet: Myeloid cell receptors mediate ACE2-independent SARS-CoV-2 virus-immune interactions (A) HEK293T cells transfected with the indicated receptors or vector control were co-cultured with HIV-GFP virus pseudotyped with SARS-CoV-2 WT or mutant (T500A/N501A/G502E) S protein (SARS-CoV-2 pseudovirus) in the presence or absence of mannan (100 μg/mL) for 48 h, followed by flow cytometry analysis of GFP expression (n = 3). (B) Human PBMC-derived myeloid cells were co-cultured with SARS-CoV-2 pseudovirus with WT S protein in the presence or absence of mannan (100 μg/mL) for 48 h, followed by flow cytometry analysis (n = 3). (C) HEK293T cells with or without ACE2 overexpression (left) and human PBMC-derived myeloid cells (right) were co-cultured with SARS-CoV-2 pseudovirus in the presence of Fc Ctr or anti-ACE2 antibody (20 μg/mL). GFP-positive cells were quantified by flow cytometry after 48-h incubation (n = 3). (D and E) Human PBMC-derived myeloid cells were co-cultured with SARS-CoV-2 pseudovirus with WT S protein (D) or a clinical isolate of SARS-CoV-2 (MOI = 1) (E), with or without Fc-tagged S-interacting decoy receptor(s) (25 μg/mL of each). Cells were analyzed by flow cytometry after 48-h incubation (D) or lysed for RNA extraction and RT-PCR analysis after 24-h incubation (E). The viral mRNA level was normalized to the host GAPDH level, and the average value of the Fc Ctr group was set to 100 (E) (n = 3). Representative flow cytometry plots (left) and statistical analysis (right) are shown in (A), (B), and (D). Data are presented as mean ± SEM of a representative experiment. ∗ p < 0.05, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001 by two-way ANOVA (A), two-tailed unpaired Student’s t test (B and right panel in C), or one-way ANOVA (left panel in C, D, and E). n refers to the number of independent experiments. See also Figure S4 .

Article Snippet: The wells were incubated with recombinant ACE2 (Sino Biological), DC-SIGN (Sino Biological), L-SIGN (Sino Biological), LSECtin (Acro Biosystems), ASGR1 (R&D Systems), and CLEC10A (Sino Biological) with human Fc tag at 37°C for 6 hr.

Techniques: Transfection, Plasmid Preparation, Cell Culture, Mutagenesis, Flow Cytometry, Expressing, Derivative Assay, Over Expression, Incubation, RNA Extraction, Reverse Transcription Polymerase Chain Reaction, Two Tailed Test

Journal: Immunity

Article Title: SARS-CoV-2 exacerbates proinflammatory responses in myeloid cells through C-type lectin receptors and Tweety family member 2

doi: 10.1016/j.immuni.2021.05.006

Figure Lengend Snippet: Identification of nanobodies capable of blocking SARS-CoV-2-induced hyperinflammatory responses (A) Schematic of the nanobody screening program to develop bispecific nanobodies that block S protein interaction with ACE2 and myeloid cell receptors (see for details). (B) Clustered heatmap of the relative blocking score for each VHH nanobody. Clones A8 and G11 are highlighted in the rectangular areas, and non-RBD interacting nanobodies were labeled with an asterisk (n = 3). (C) Probelife kinetics analysis of binding of A8-Fc, G11-Fc, and A8-G11-Fc to the S protein. K D , K on , and K off values of individual nanobodies are shown in the table. A representative experiment is shown. (D) Human PBMC-derived myeloid cells were incubated with HIV-GFP virus pseudotyped with SARS-CoV-2 S protein in the presence of Fc Ctr, A8-Fc, G11-Fc, or A8-G11-Fc (50 μg/mL) for 48 h, followed by flow cytometry (n = 3). (E) Neutralization of mNeonGreen SARS-CoV-2 reporter virus (MOI = 1) infection of HEK293T cells expressing ACE2 by an S-interacting decoy receptor cocktail (ACE2-Fc/L-SIGN-Fc, 25 μg/mL of each) or a bi-specific nanobody (A8-G11-Fc, 50 μg/mL). (F and G) Human PBMC-derived myeloid cells from two healthy cohorts were incubated with a clinical isolate of SARS-CoV-2 (MOI = 0.5) in the presence of Fc Ctr protein (50 μg/mL), an ACE2-Fc/L-SIGN-Fc cocktail (25 μg/mL of each), or an A8-G11-Fc bi-specific nanobody (50 μg/mL). Mock control was performed using conditioned medium. Cells were lysed for RNA extraction, and the supernatant was harvested 24 h after incubation. mRNA levels of the indicated cytokines and chemokines were measured by RT-PCR and normalized to that of GAPDH (F), and cytokines in the supernatant were quantified by a multiplex magnetic bead assay (G). Data are presented as mean ± SEM of a representative experiment (D and E) or four independent pooled experiments (F and G). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001 by one-way ANOVA (D, F, and G). n refers to the number of independent experiments. See also Figure S6 .

Article Snippet: The wells were incubated with recombinant ACE2 (Sino Biological), DC-SIGN (Sino Biological), L-SIGN (Sino Biological), LSECtin (Acro Biosystems), ASGR1 (R&D Systems), and CLEC10A (Sino Biological) with human Fc tag at 37°C for 6 hr.

Techniques: Blocking Assay, Clone Assay, Labeling, Binding Assay, Derivative Assay, Incubation, Flow Cytometry, Neutralization, Infection, Expressing, RNA Extraction, Reverse Transcription Polymerase Chain Reaction, Multiplex Assay

Journal: Immunity

Article Title: SARS-CoV-2 exacerbates proinflammatory responses in myeloid cells through C-type lectin receptors and Tweety family member 2

doi: 10.1016/j.immuni.2021.05.006

Figure Lengend Snippet:

Article Snippet: The wells were incubated with recombinant ACE2 (Sino Biological), DC-SIGN (Sino Biological), L-SIGN (Sino Biological), LSECtin (Acro Biosystems), ASGR1 (R&D Systems), and CLEC10A (Sino Biological) with human Fc tag at 37°C for 6 hr.

Techniques: Staining, Recombinant, Enzyme-linked Immunosorbent Assay, Western Blot, SYBR Green Assay, Multiplex Assay, Infection, shRNA, cDNA Library Assay, Software, Luminex

Journal: Immunity

Article Title: SARS-CoV-2 exacerbates proinflammatory responses in myeloid cells through C-type lectin receptors and Tweety family member 2

doi: 10.1016/j.immuni.2021.05.006

Figure Lengend Snippet: Discovery of C-type lectins and TTYH2 as interacting partners of the SARS-CoV-2 S protein (A) Schematic of the myeloid cell receptor discovery approach. Individual plasmids of genes encoding myeloid cell receptors were transfected into HEK293T cells, and a human Fc-tagged SARS-CoV-2 S protein mixture (S-Fc, S1-Fc, and RBD-Fc) and anti-human immunoglobulin G (IgG) Fc detection antibody were added to the cell culture to assess binding (see for more details). S protein subunits and subdomains relative to ACE2 binding (RBD) are also shown. (B) Other than ACE2 (purple), DC-SIGN, L-SIGN, LSECtin, ASGR1, CLEC10A, and TTYH2 (all in red) were identified as binding partners for the SARS-CoV-2 S protein (n = 2). Fc receptors (blue) served as positive controls. (C) Representative images of the binding of the Fc-tagged S protein, its subunits, or Fc control (Fc Ctr) to the indicated receptors, captured by the cellular detection system (CDS) (n = 3). (D) Quantification of the interaction between S protein subdomains/subunits and different receptors, indicated on the x axis. Normalized binding capacity is shown on the y axis (the sum of the total fluorescence intensity to the indicated receptor was set to 100). (E) Binding between HEK293T cells expressing the indicated receptors and HIV-GFP virus pseudotyped with the SARS-CoV-2 S protein was detected by an anti-S polyclonal antibody and analyzed by flow cytometry (n = 4). Data are presented as the mean ± SEM of five pooled independent experiments (D) or a representative experiment (E); ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001 by one-way ANOVA (E). n refers to the number of independent experiments. See also Figure S1 .

Article Snippet: The wells were incubated with recombinant ACE2 (Sino Biological), DC-SIGN (Sino Biological), L-SIGN (Sino Biological), LSECtin (Acro Biosystems), ASGR1 (R&D Systems), and CLEC10A (Sino Biological) with human Fc tag at 37°C for 6 hr.

Techniques: Transfection, Cell Culture, Binding Assay, Fluorescence, Expressing, Flow Cytometry

Journal: Immunity

Article Title: SARS-CoV-2 exacerbates proinflammatory responses in myeloid cells through C-type lectin receptors and Tweety family member 2

doi: 10.1016/j.immuni.2021.05.006

Figure Lengend Snippet: Identification of nanobodies capable of blocking SARS-CoV-2-induced hyperinflammatory responses (A) Schematic of the nanobody screening program to develop bispecific nanobodies that block S protein interaction with ACE2 and myeloid cell receptors (see for details). (B) Clustered heatmap of the relative blocking score for each VHH nanobody. Clones A8 and G11 are highlighted in the rectangular areas, and non-RBD interacting nanobodies were labeled with an asterisk (n = 3). (C) Probelife kinetics analysis of binding of A8-Fc, G11-Fc, and A8-G11-Fc to the S protein. K D , K on , and K off values of individual nanobodies are shown in the table. A representative experiment is shown. (D) Human PBMC-derived myeloid cells were incubated with HIV-GFP virus pseudotyped with SARS-CoV-2 S protein in the presence of Fc Ctr, A8-Fc, G11-Fc, or A8-G11-Fc (50 μg/mL) for 48 h, followed by flow cytometry (n = 3). (E) Neutralization of mNeonGreen SARS-CoV-2 reporter virus (MOI = 1) infection of HEK293T cells expressing ACE2 by an S-interacting decoy receptor cocktail (ACE2-Fc/L-SIGN-Fc, 25 μg/mL of each) or a bi-specific nanobody (A8-G11-Fc, 50 μg/mL). (F and G) Human PBMC-derived myeloid cells from two healthy cohorts were incubated with a clinical isolate of SARS-CoV-2 (MOI = 0.5) in the presence of Fc Ctr protein (50 μg/mL), an ACE2-Fc/L-SIGN-Fc cocktail (25 μg/mL of each), or an A8-G11-Fc bi-specific nanobody (50 μg/mL). Mock control was performed using conditioned medium. Cells were lysed for RNA extraction, and the supernatant was harvested 24 h after incubation. mRNA levels of the indicated cytokines and chemokines were measured by RT-PCR and normalized to that of GAPDH (F), and cytokines in the supernatant were quantified by a multiplex magnetic bead assay (G). Data are presented as mean ± SEM of a representative experiment (D and E) or four independent pooled experiments (F and G). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001 by one-way ANOVA (D, F, and G). n refers to the number of independent experiments. See also Figure S6 .

Article Snippet: The wells were incubated with recombinant ACE2 (Sino Biological), DC-SIGN (Sino Biological), L-SIGN (Sino Biological), LSECtin (Acro Biosystems), ASGR1 (R&D Systems), and CLEC10A (Sino Biological) with human Fc tag at 37°C for 6 hr.

Techniques: Blocking Assay, Clone Assay, Labeling, Binding Assay, Derivative Assay, Incubation, Flow Cytometry, Neutralization, Infection, Expressing, RNA Extraction, Reverse Transcription Polymerase Chain Reaction, Multiplex Assay

Journal: Immunity

Article Title: SARS-CoV-2 exacerbates proinflammatory responses in myeloid cells through C-type lectin receptors and Tweety family member 2

doi: 10.1016/j.immuni.2021.05.006

Figure Lengend Snippet:

Article Snippet: The wells were incubated with recombinant ACE2 (Sino Biological), DC-SIGN (Sino Biological), L-SIGN (Sino Biological), LSECtin (Acro Biosystems), ASGR1 (R&D Systems), and CLEC10A (Sino Biological) with human Fc tag at 37°C for 6 hr.

Techniques: Staining, Recombinant, Enzyme-linked Immunosorbent Assay, Western Blot, SYBR Green Assay, Multiplex Assay, Infection, shRNA, cDNA Library Assay, Software, Luminex