Host factors secreted by activated innate immune cells inhibit SARS-CoV-2 induced cell-cell fusion.

(A) Schematics of the cell-cell fusion model used to quantify spike-mediated syncytium formation upon treatment with cell culture supernatants from TLR ligands stimulated innate immune cells. Cells co-expressing SARS-CoV-2 spike and Cre, were co-cultured with ACE2 and Stop-luc co-expressing HEK293T cells for 16 hours, before cell lysates were collected for bioluminescence assay and immunoblotting. Cells co-expressing SARS-CoV-2 spike and Zsgreen, were co-cultured with ACE2 expressing HEK293T cells for 16 hours before fluorescence imaging. (B) Luciferase activity (RLU) measured from HEK293T cell lysates collected from THP-1 supernatants treated HEK293T-S and HEK293T-ACE2 described in (A) for 16 hours. FBS free RPMI 1640 served as medium control group. Data are representative of six individual repeats and displayed as individual points with mean ± standard error of the mean (SEM). (C) Immunoblots showing full-length spike, S2, cleaved S2’ and ACE2 collected from THP-1 supernatants treated HEK293T-S and HEK293T-ACE2 described in (A) for 16 hours. Blots are representative of three independent experiments. Numbers below the blots indicated the ratio of S2’ versus Tubulin. (D) Representative fluorescent image captured at 488 nm from THP-1 supernatants treated HEK293T-S-Zsgreen and HEK293T-ACE2 for 16 hours. (E) Schematic presentation of THP-1 supernatants pre-treatment on authentic SARS-CoV-2 infected cells. Pre-treatment of HEK293T-ACE2 cells with THP-1 supernatants for 1 hour, then inoculated with 0.5 multiplicity of infection (MOI) Delta or WT authentic SARS-CoV-2 (B.1.617.2 and Wuhan-Hu-1) virus. Imaging was performed at 24 hours post-infection (hpi) before cell lysates were harvested for immunoblotting. (F) Immunoblots of Delta SARS-CoV-2 S, S2, cleaved S2’, N and ACE2 proteins collected from HEK293T-ACE2 cells 24 hpi as described in (E). Blots are representative of three individual experiments. Numbers below the blots indicated the ratio of S2’ or N versus Tubulin. (G) Immunoblots of WT SARS-CoV-2 S, S2, cleaved S2’ and N proteins collected from Caco-2 cells 24 hpi as described in (E). Blots are representative of three individual experiments. Numbers below the blots indicated the ratio of S2’ or N versus β-Actin. (H) Immunofluorescent images showing morphology of SARS-CoV-2 infected Caco-2 cells pre-treated with THP-1 supernatants. Anti-SARS-CoV-2 N was stained with Alexa fluor 555 and nuclei were counterstained with DAPI respectively. White arrow heads in (D and H) indicate syncytia formation or infected cells, scale bars are indicative of 50 μm and images are representative of three independent experiments.

IL-1β inhibits SARS-CoV-2 induced cell-cell fusion.

(A) Schematics of the cell-cell fusion model used to quantify spike-mediated syncytium formation upon treatment with different cytokines. Cells co-expressing SARS-CoV-2 spike and Cre, were co-cultured with ACE2 and Stop-luc co-expressing HEK293T cells for 16 hours, before cell lysates were collected for bioluminescence assay and immunoblotting. Cells co-expressing SARS-CoV-2 spike and Zsgreen, were co-cultured with ACE2 expressing HEK293T cells for 16 hours before fluorescence imaging. (B) Luciferase activity (RLU) measured from HEK293T cell lysates collected from different cytokines treated HEK293T-S and HEK293T-ACE2 described in (A) for 16 hours. IL-1α (10 ng/mL), IL-1β (1 ng/mL), IL-6 (100 ng/mL) or IL-8 (100 ng/mL) were added into the cell-cell fusion system. Data are representative of six individual repeats and displayed as individual points with mean ± standard error of the mean (SEM). (C) Luciferase activity (RLU) measured from HEK293T cell lysates collected from different concentrations of IL-1β treated HEK293T-S and HEK293T-ACE2 for 16 hours. Data are representative of six individual repeats and displayed as individual points with mean ± standard error of the mean (SEM). (D) Immunoblots showing full-length spike, S2, cleaved S2’ and ACE2 collected from different concentrations of IL-1β treated HEK293T-S and HEK293T-ACE2 for 16 hours. Blots are representative of three independent experiments. Numbers below the blots indicated the ratio of S2’ versus Tubulin. (E) Schematic presentation of IL-1β pre-treatment on authentic SARS-CoV-2 infected cells. Pre-treatment of HEK293T-ACE2 cells with different concentrations of IL-1β for 1 hour, then inoculated with 0.5 MOI Delta or WT authentic SARS-CoV-2 (B.1.617.2 and Wuhan-Hu-1) virus. Brightfield images were captured at 24 hours post-infection (hpi) before cell lysates were harvested for immunoblotting. (F) Immunoblots of Delta SARS-CoV-2 S, S2, cleaved S2’, N and ACE2 proteins collected from HEK293T-ACE2 cells 24 hpi as described in (E). Blots are representative of three individual experiments. Numbers below the blots indicated the ratio of S2’ or N versus Tubulin. (G) Immunoblots of WT SARS-CoV-2 S, S2, cleaved S2’ and N proteins collected from Caco-2 cells 24 hpi as described in (E). Blots are representative of three individual experiments. Numbers below the blots indicated the ratio of S2’ or N versus β-Actin. (H) Immunofluorescent images showing morphology of SARS-CoV-2 infected Caco-2 cells pre-treated with or without IL-1β. Anti-SARS-CoV-2 N was stained with Alexa fluor 555 and nuclei were counterstained with DAPI respectively. White arrow heads indicate syncytia formation or infected cells, scale bars are indicative of 50 μm and images are representative of three independent experiments.

IL-1β inhibits SARS-CoV-2 induced cell-cell fusion through IL-1R1/MyD88/IRAK/TRAF6 pathway.

(A) Schematics of gene knockout or inhibitor treatment in the IL-1 receptor pathway. (B) Luciferase activity (RLU) measured from HEK293T cell lysates and immunoblots showing full-length spike, S2, cleaved S2’ and ACE2 collected from HEK293T-S and HEK293T-ACE2 pre-treated with 1000 ng/mL IL-1RA for 30 min, then treated with 1 ng/mL IL-1β for 16 hours. Data and blots are representative of five individual repeats. Numbers below the blots indicated the ratio of S2’ versus Tubulin. (C) Luciferase activity (RLU) measured from cell lysates collected from 10 ng/mL IL-1α or 1 ng/mL IL-1β treated sgControl or sgMyD88 HEK293T cell-cell fusion system for 16 hours. Data are representative of four individual repeats and displayed as individual points with mean ± standard error of the mean (SEM). (D) Luciferase activity (RLU) measured from HEK293T cell lysates and immunoblots showing full-length spike, S2, cleaved S2’ and ACE2 collected from HEK293T-S and HEK293T-ACE2 pre-treated with 2μM IRAK1/4 inhibitor for 30 min, then treated with 1 ng/mL IL-1β for 16 hours. Data and blots are representative of four individual repeats. Numbers below the blots indicated the ratio of S2’ versus Tubulin. (E) Luciferase activity (RLU) measured from cell lysates collected from 10 ng/mL IL-1α or 1 ng/mL IL-1β treated sgControl or sgTRAF6 HEK293T cell-cell fusion system for 16 hours. Data are representative of four individual repeats and displayed as individual points with mean ± standard error of the mean (SEM). (F) Immunoblots showing full-length spike, S2, cleaved S2’ and ACE2 collected from Caco-2 cells, which pre-treated with 2μM IRAK1/4 inhibitor and 10 ng/mL IL-1β for 1 hour, then infected with authentic SARS-CoV-2 for 24 hours. Blots are representative of three independent experiments. Numbers below the blots indicated the ratio of S2’ or N versus β-Actin. (G) Immunoblots showing full-length spike, S2, cleaved S2’ and ACE2 collected from Calu-3 cells, which infected with authentic SARS-CoV-2 for 1 hour, then washed with PBS before treated with 2μM IRAK1/4 inhibitor and 10 ng/mL IL-1β for 24 hours. Blots are representative of three independent experiments. Numbers below the blots indicated the ratio of S2’ or N versus β-Actin.

IL-1β inhibits SARS-CoV-2 induced cell-cell fusion through RhoA/ROCK mediated actin bundles at cell-cell junction.

(A) GTP-RhoA pull-down assay to detect the active level of RhoA in sgControl, sgMyD88 and sgTRAF6 HEK293T cells after 1 ng/mL IL-1β treatment for 30 min. Immunoblots showing activated RhoA and total RhoA. Blots are representative of three independent experiments. Numbers below the blots indicated the ratio of active RhoA versus total RhoA. (B) Representative confocal images of GFP-AHPH after 1 ng/mL IL-1β treatment for 30 min in sgControl, sgMyD88 and sgTRAF6 HEK293T cells. Scale bars, 10 μm. (C) Quantification of fluorescence intensity of GFP-AHPH in (B). Data are representative of eight individual repeats. (D) Representative confocal images of GFP-AHPH localization with or without 1 ng/mL IL-1β treatment at different time points of syncytia formation in HEK293T-S-HA and HEK293T-ACE2 cells. Schematics with green dots in the white dashed line boxes representing GFP-AHPH, red cycles representing S-expressing cells, and magenta cycles representing ACE2-expressing cells. White arrow heads indicate the localization of GFP-AHPH, scale bars, 10 μm. Images are representative of three independent experiments. (E) Representative confocal images of F-actin stained with phalloidin-488 in the presence or absence of 1 ng/mL IL-1β treatment at different time points of syncytia formation in HEK293T-S-HA and HEK293T-ACE2 cells. Schematics with green lines in the white dashed line boxes representing actin bundles, red cycles representing S-expressing cells, and magenta cycles representing ACE2-expressing cells. White arrow heads indicate the enrichment or disappearance of F-actin, scale bars, 10 μm. Images are representative of three independent experiments. (F) Representative confocal images of GFP-AHPH localization with or without 1 ng/mL IL-1β treatment in 0.5 MOI WT authentic SARS-CoV-2 infected HEK293T-ACE2 cells at 6 hpi and 24 hpi. Schematics with green dots in the white dashed line boxes representing GFP-AHPH, red cycles representing SARS-CoV-2 infected cells, and white cycles representing neighboring cells. White arrow heads indicate the localization of GFP-AHPH, scale bars, 10 μm. Images are representative of three independent experiments. (G, H) Representative confocal images of F-actin stained with phalloidin-488 in the presence or absence of 1 ng/mL IL-1β treatment in 0.5 MOI WT authentic SARS-CoV-2 infected HEK293T-ACE2 cells at 6 hpi and 24 hpi (G) or Caco-2 cells at 24 hpi (H). Schematics with green lines in the white dashed line boxes representing actin bundles, red cycles representing SARS-CoV-2 infected cells, and white cycles representing neighboring cells. Scale bars, 10 μm. Images are representative of three independent experiments. White arrow heads in (G and H) indicate the enrichment or disappearance of F-actin.

Activation of RhoA/ROCK pathway prevents authentic SARS-CoV-2 induced cell-cell fusion by forming actin bundles.

(A) Luciferase activity (RLU) measured from HEK293T cell lysates and immunoblots showing full-length spike, S2, cleaved S2’, ACE2 and Myc-RhoA collected from transfected vector, 10 ng or 20 ng constitutively active RhoA mutant (RhoA-CA) both in HEK293T-S and HEK293T-ACE2. Data and blots are representative of four individual repeats. Numbers below the blots indicated the ratio of S2’ versus Tubulin. (B) Immunoblots of WT SARS-CoV-2 S, S2, cleaved S2’, N and Myc-RhoA collected from Caco-2 cells, which transfected with vector, 10 ng or 20 ng RhoA-CA before infected with 0.5 MOI WT authentic SARS-CoV-2 for 24 hours. Blots are representative of three individual experiments. Numbers below the blots indicated the ratio of S2’ or N versus β-Actin. (C) Immunoblots of WT SARS-CoV-2 S, S2, cleaved S2’, N and Myc-RhoA collected from lentivirus-transduced Calu-3 cells expressing vector or RhoA-CA, infected with WT authentic SARS-CoV-2 for 24 hours. Blots are representative of three individual experiments. Numbers below the blots indicated the ratio of S2’ or N versus β-Actin. (D) Representative confocal images of F-actin stained with phalloidin-488 from Caco-2 cells described in (B). Schematics with green lines in the white dashed line boxes representing actin bundles, red cycles representing S-expressing cells. Scale bars, 10 μm. Images are representative of four independent experiments. (E) Representative confocal images of F-actin stained with phalloidin-488 from Calu-3 cells described in (C). Schematics with green lines in the white dashed line boxes representing actin bundles, red cycles representing S-expressing cells. White arrow heads in (D and E) indicate the enrichment or disappearance of F-actin, scale bars, 10 μm. Images are representative of four independent experiments. (F) Immunoblots of WT SARS-CoV-2 S, S2, cleaved S2’ and N collected from Caco-2 cells, which treated with different concentrations of Y-27632 and 10 ng/mL IL-1β for 1 hour, then infected with 0.5 MOI WT authentic SARS-CoV-2 for 24 hours. Blots are representative of three individual experiments. Numbers below the blots indicated the ratio of S2’ or N versus β-Actin. (G) Immunoblots of WT SARS-CoV-2 S, S2, cleaved S2’ and N collected from Calu-3 cells, which infected with authentic SARS-CoV-2 for 1 hour, then washed with PBS before treated with different concentrations of Y-27632 and 10 ng/mL IL-1β for 24 hours. Blots are representative of three independent experiments. Numbers below the blots indicated the ratio of S2’ or N versus β-Actin. (H) Immunoblots of WT SARS-CoV-2 S, S2, cleaved S2’ and N collected from human lung cells, which infected with authentic SARS-CoV-2 for 1 hour, then washed with PBS before treated with 10 μM Y-27632 and 10 ng/mL IL-1β for 24 hours. Blots are representative of three independent experiments. Numbers below the blots indicated the ratio of S2’ or N versus β-Actin. (I) Representative confocal images of F-actin stained with phalloidin-488 in Caco-2 cells described in (G). Schematics with green lines in the white dashed line boxes representing actin bundles, red cycles representing S-expressing cells. Scale bars, 10 μm. Images are representative of four independent experiments. (J) Representative confocal images of F-actin stained with phalloidin-488 in Calu-3 cells described in (H). Schematics with green lines in the white dashed line boxes representing actin bundles, red cycles representing S-expressing cells. White arrow heads in (I and J) indicate the enrichment or disappearance of F-actin, scale bars, 10 μm. Images are representative of four independent experiments.

IL-1β restricts SARS-CoV-2 transmission via induction of actin bundles in lung tissue.

(A) Schematic for a murine model of authentic SARS-CoV-2 infection, PBS control (n=8); 1 μg/kg mIL-1β (n=8); 150 μg/kg mIL-1RA + 1 μg/kg mIL-1β (n=8) were administered 1 hour before intranasal challenge with 5 × 104 FFU of SARS-CoV-2 B.1.351; mice were then intraperitoneal injection with PBS, mIL-1β and mIL-1RA + mIL-1β at 1 dpi and 2 dpi, before sacrificed at 4 dpi. (B) The body weights were assessed daily for weight loss after SARS-CoV-2 infection. (C) qPCR analysis of SARS-CoV-2 N mRNA collected from infected lung tissues at 4 dpi. (D) Immunohistochemistry analysis of SARS-CoV-2 N staining in the lung tissue slices at 4 dpi, scale bars are indicative of 500 μm (top panel), 50 μm (bottom panel) and images are representative of eight independent experiments. (E) The percentages of SARS-CoV-2 infected area in (D) were quantified. (F) Representative confocal images of F-actin stained with phalloidin-488 and SARS-CoV-2 N in in the lung tissue slices at 4 dpi. White arrow heads indicate syncytia formation or infected cells, scale bars are indicative of 10 μm and images are representative of three independent experiments (Top). White lines indicate SARS-CoV-2 cell-cell transmission and quantify with fluorescence intensity of F-actin and SARS-CoV-2 N (Bottom). (G) Immunoblots of SARS-CoV-2 S, S2, cleaved S2’ and N proteins collected from authentic SARS-CoV-2 BF.7 infected lung tissue cells, which isolated from BALB/c mice treated with PBS, 1 μg/kg mIL-1β or 1mg/kg Y-26732 + 1 μg/kg mIL-1β at day 2. Blots are representative of three individual experiments. Numbers below the blots indicated the ratio of S2’ or N versus β-Actin.