SARS-CoV-2 Spike triggers barrier dysfunction and vascular leak via integrins and TGF-β signaling

Abstract

Severe COVID-19 is associated with epithelial and endothelial barrier dysfunction within the lung as well as in distal organs. While it is appreciated that an exaggerated inflammatory response is associated with barrier dysfunction, the triggers of this pathology are unclear. Here, we report that cell-intrinsic interactions between the Spike (S) glycoprotein of SARS-CoV-2 and epithelial/endothelial cells are sufficient to trigger barrier dysfunction in vitro and vascular leak in vivo , independently of viral replication and the ACE2 receptor. We identify an S-triggered transcriptional response associated with extracellular matrix reorganization and TGF-β signaling. Using genetic knockouts and specific inhibitors, we demonstrate that glycosaminoglycans, integrins, and the TGF-β signaling axis are required for S-mediated barrier dysfunction. Our findings suggest that S interactions with barrier cells are a contributing factor to COVID-19 disease severity and offer mechanistic insight into SARS-CoV-2 triggered vascular leak, providing a starting point for development of therapies targeting COVID-19 pathogenesis.

Figure 1.. SARS-CoV-2 S triggers endothelial and epithelial barrier hyperpermeability.

(A) Schematic depicting S-triggered barrier dysfunction measured by a trans-endothelial/epithelial electrical resistance assay (TEER; left) and an endo/epithelial glycocalyx layer (EGL) assay (right). (B) Time course TEER assay measuring the barrier function of HPMEC monolayers over time with the indicated treatments, including DENV2 NS1 (5 μg/mL), VEGF (50 ng/mL), and SARS-CoV-2 S (10 μg/mL). Data are from n=2 biological replicates. (C) A TEER assay measuring the barrier of monolayers of HPMEC and HPMEC/ACE2 at 24 hours after the indicated treatments. VEGF positive control (50 ng/mL). Data are from n=3 biological replicates. (D) Same as C but treated with the indicated VSV pseudotyped particles at the indicated dilutions. VSV-bald and VSV-G are diluted 1:30. Data are from n=3 biological replicates. (E) Same as C but treated with the indicated concentrations of SARS-CoV-2 RBD. Data are from n=3 biological replicates. (F) Same as C but measuring the barrier of Calu-3 cell monolayers. Data are from n=2 biological replicates. (G) A TEER inhibition assay measuring the capacity of a cocktail of anti-S antibodies to inhibit S-mediated endothelial hyperpermeability. S (10 μg/mL) and the antibody cocktail (15 μg/mL for each antibody; 1A9 [Genetex] and CR3022 [Absolute Antibody]) were added simultaneously to the upper chamber of transwell inserts to a monolayer of HPMEC or HPMEC/ACE2 and TEER was measured 24 hours post-treatment (hpt). Data are from n=2 biological replicates. In all panels, the dotted line is the normalized TEER value of the untreated control condition. All data are plotted as mean +/− SD. For all panels, values are compared to untreated controls by ANOVA with multiple comparisons with *p<0.05, **p<0.01, ***p<0.001, and n.s. p>0.05.

Figure 2.. SARS-CoV-2 S facilitates disruption of the endothelial and epithelial glycocalyx layer.

(A) An immunofluorescence microscopy-based EGL disruption assay measuring levels of the indicated glycans on the surface of HPMEC. After 24 h of SARS-CoV-2 S (10 μg/mL) treatment, cells were fixed then stained without permeabilization. Displayed are representative images from n=3 biological replicates. (B) Same as A, but cells were permeabilized before staining for the indicated EGL disrupting enzymes. Displayed are representative images from n=3 biological replicates. (C) Quantification of A. (D) Quantification of B. (E) Same as A but measuring EGL disruption of Calu-3 cell monolayers. Displayed are representative images from n=3 biological replicates. (F) Same as B but measuring expression of EGL-disrupting enzymes in Calu-3 cells. Displayed are representative images from n=3 biological replicates. (G) Quantification of E. (H) Quantification of F. For all images, nuclei were probed with Hoechst in blue and the indicated glycans in green with scale bars at 100 μm. Dotted lines are the normalized untreated control conditions. MFI is mean fluorescence intensity. All data are plotted as mean +/− SEM with * p<0.05, ** p<0.01, *** p<0.001, and n.s. p>0.05 by unpaired t-test.

Figure 3.. SARS-CoV-2 S triggers vascular leak in vivo.

(A) A representative mouse back from a dermal leak experiment. The dorsal dermises of mice were injected intradermally with the treatments and doses indicated. Mice then received a dextran-680 tracer molecule intravenously. Mouse dermises were collected 2 h post-treatment and quantification of local dermal leak was assessed by a fluorescent scanner. (B) Quantification of A from mice treated with PBS (n=27), DENV2 NS1 (15 μg; n=5), S (10 μg; n=25), and S (25 μg; n=5). (C) Representative lung images from a SARS-CoV-2 S systemic vascular leak assay. Mice were administered 50 μg of SARS-CoV-2 S or ovalbumin intranasally as indicated, and 22 hpt were administered a dextran-680 tracer intravenously as in A. Organs of mice were collected 2 hours post dextran-680 administration (24 hours post-S treatment), and accumulation of dextran-680 was measured with a fluorescent scanner. (D) Quantification of C from n=6 mice. (E) Same as C except representative images of spleens. (F) Quantification of E from n=6 mice. (G) Same as C except representative images of small intestine. (H) Quantification of G from n=5 mice. MFI is mean fluorescence intensity. All data are plotted as mean +/− SEM with *p<0.05, **p<0.01, and ***p<0.001 by unpaired t-test.

Figure 4.. Glycosaminoglycans and EGL modulating enzymes are required for SARS-CoV-2 S-mediated barrier dysfunction.

(A) TEER inhibition assay on monolayers of HPMEC or HPMEC/ACE2 treated with heparin (10 μg/mL), S (10 μg/mL), or both simultaneously. TEER readings were taken 24 hpt. Data are from n=2 biological replicates. (B) A TEER inhibition assay where monolayers of HPMEC were treated with recombinant hyaluronidase (10 μg/mL), heparin lyases I and III (5 mU/mL each), neuraminidase (1 U/mL), or chondroitinase (25 mU/mL) simultaneously with S (10 μg/mL) treatment. TEER readings were taken 24 hpt. Data are from n=2 biological replicates. (C) EGL inhibition assay on HPMEC treated with hyaluronidase (10 μg/mL) or heparin lyases I and III (5 mU/mL each) and simultaneously treated with S (10 μg/mL) and fixed 24 hpt. (D) Quantification of C from n=2 biological replicates. Statistics are comparisons of indicated conditions to the S-only control condition. (E) Representative images from an EGL disruption assay of HPMEC transduced with lentiviruses encoding the indicated gene-targeting guide RNA. Cells were treated with 10 μg/mL S, and sialic acid was visualized by IFA 24 hpt. (F) Quantification of E from n=3 biological replicates. Control guide data from this panel are from the same experiment as Figure S1G. (G) Same as E and F but using the indicated guide RNAs. Non-target (NT) data are pooled from two cell line replicates. Control guide data from this panel are from the same experiments as Figure 7G. For all panels, sialic acid is stained with Wheat Germ Agglutinin in green and nuclei are stained with Hoechst in blue with scale bars at 100 μm. MFI is mean fluorescence intensity. Dotted lines are the normalized untreated control conditions. All data are plotted as mean +/− SD (TEER) or SEM (EGL) with *p<0.05, **p<0.01, ***p<0.001, and n.s. p>0.05 by ANOVA with multiple comparisons.

Figure 5.. RNA-Sequencing analysis of SARS-CoV-2 S-treated HPMEC and HPMEC/ACE2.

(A) A volcano plot of differentially expressed genes (DEGs) detected in HPMEC treated with 10 μg/mL of SARS-CoV-2 S at 24 hpt. (B) Same as A but displaying DEGs from HPMEC/ACE2 treated with 10 μg/mL SARS-CoV-2 S. Dotted lines indicate the threshold for significance. (C) A STRING protein-protein interaction network of DEGs identified between S-treated and untreated conditions for HPMEC.

Figure 6.. Integrins are required for SARS-CoV-2 S-mediated endothelial barrier dysfunction.

(A) TEER inhibition assay of HPMEC and HPMEC/ACE2 monolayers treated with S (10 μg/mL) and the indicated concentrations of the integrin inhibitor ATN-161. TEER readings were taken 24 hpt with ATN-161 and S added simultaneously to cells. Data are from n=2 biological replicates. (B) EGL inhibition assay detecting sialic acid on the surface of HPMEC monolayers treated with S and ATN-161 as in A. Data are from at least n=2 biological replicates. (C) Representative back from an intradermal leak assay of mice with the indicated treatments; S (10 μg/mL) and ATN-161 (1 μM) injected simultaneously. (D) Quantification of C from n=7 mice. (E) TEER assay of HPMEC and HPMEC/ACE2 monolayers treated with the indicated peptides at 0.4 μM. TEER readings were taken 24 hpt. Data are from n=2 biological replicates. (F) Same as E, but an EGL assay detecting sialic acid on the surface of HPMEC monolayers. Data are from n=3 biological replicates. (G) Representative back from an intradermal leak assay of mice with the indicated treatments with S (10 μg/mL) and the indicated doses of RGD peptide. (H) Quantification of G from n=4 mice. (I) Western blot analysis of HPMEC transduced with the indicated lentivirus-encoding guide RNA. Actin was used as a loading control. (J) TEER assay of HPMEC transduced with lentivirus-encoding guide RNAs targeting the indicated genes as in I. Cell were treated with 10 μg/mL of S and TEER was read 24 hpt. Data are from n=2 biological replicates. (K) EGL assay detecting sialic acid on the cell surface of transduced HPMECs as in J. Data are from n=3 biological replicates. MFI is mean fluorescence intensity. Dotted lines are the normalized untreated control conditions. All data are as plotted as mean +/− SD (TEER) or SEM (EGL) with *p<0.05, **p<0.01, ***p<0.001, and n.s. p>0.05 by ANOVA with multiple comparisons.

Figure 7.. SARS-CoV-2 S triggers production of TGF-β, and TGF-β signaling is required for S-mediated barrier dysfunction

(A) Commercial ELISA detecting TGF-β in medium without cell conditioning (Media), medium from untreated HPMEC, and medium from HPMEC treated with 10 μg/mL SARS-CoV-2 S. Data are from n=3 biological replicates. (B) TEER assay measuring the effect of recombinant TGF-β on barrier function of HPMEC at the indicated concentrations. TEER readings were taken 24 hpt. Data are from n=2 biological replicates. (C) TEER assay measuring the capacity of an anti-TGFBR antibody, at the indicated concentrations, to abrogate S-mediated (10 μg/mL) endothelial hyperpermeability of HPMEC and HPMEC/ACE2. TEER readings were taken 24 hpt. Data are from n=2 biological replicates. (D) TEER assay measuring the capacity of TGFBR inhibitor SB431542 (1 μM) to inhibit S (10 μg/mL) function. Data are from n=2 biological replicates. (E) Same as D, except an EGL assay measuring sialic acid. Data are from n=3 biological replicates. (F) Western blot analysis of HPMEC transduced with lentivirus-encoding guide RNAs targeting the indicated genes. Actin was used as a loading control. Data are one representative experiment from n=3 biological replicates. (G) TEER assay on the same HPMEC as in F treated with 10 μg/mL of S and measured 24 hpt. Data are from n=3 biological replicates. (H) EGL disruption assay on HPMECs from F, treated with 10 μg/mL S and imaged 24 hpt. Control guide data from this panel are from the same experiment as Figure 4G. Data are from n=3 biological replicates. (I) Graphical abstract summarizing the ACE2-independent pathway by which SARS-CoV-2 S triggers barrier dysfunction. Interactions with proteoglycans, glycosaminoglycans, and integrins are required for S-mediated release of TGF-β, which is required for S-mediated barrier dysfunction via TGFBR signaling. Solid lines represent steps with direct experimental evidence while dotted lines represent hypothesized steps. For all figures, dotted lines in graphs are the normalized untreated control conditions. MFI is mean fluorescence intensity. All data are plotted as mean +/− SD (TEER) and SEM (EGL) with *p<0.05, **p<0.01, ***p<0.001, and n.s. p>0.05 by ANOVA with multiple comparisons.