SARS-CoV-2 Spike Proteins and Cell-Cell Communication Induce P-Selectin and Markers of Endothelial Injury, NETosis, and Inflammation in Human Lung Microvascular Endothelial Cells and Neutrophils: Implications for the Pathogenesis of COVID-19 Coagulopathy

Abstract

COVID-19 progression often involves severe lung injury, inflammation, coagulopathy, and leukocyte infiltration into pulmonary tissues. The pathogenesis of these complications is unknown. Because vascular endothelium and neutrophils express angiotensin-converting enzyme-2 and spike (S)-proteins, which are present in bodily fluids and tissues of SARS-CoV-2-infected patients, we investigated the effect of S-proteins and cell-cell communication on human lung microvascular endothelial cells and neutrophils expression of P-selectin, markers of coagulopathy, NETosis, and inflammation. Exposure of endothelial cells or neutrophils to S-proteins and endothelial-neutrophils co-culture induced P-selectin transcription and expression, significantly increased expression/secretion of IL-6, von Willebrand factor (vWF, pro-coagulant), and citrullinated histone H3 (cit-H3, NETosis marker). Compared to the SARS-CoV-2 Wuhan variant, Delta variant S-proteins induced 1.4-15-fold higher P-selectin and higher IL-6 and vWF. Recombinant tissue factor pathway inhibitor (rTFPI), 5,5'-dithio-bis-(2-nitrobenzoic acid) (thiol blocker), and thrombomodulin (anticoagulant) blocked S-protein-induced vWF, IL-6, and cit-H3. This suggests that following SARS-CoV-2 contact with the pulmonary endothelium or neutrophils and endothelial-neutrophil interactions, S-proteins increase adhesion molecules, induce endothelial injury, inflammation, NETosis and coagulopathy via the tissue factor pathway, mechanisms involving functional thiol groups, and/or the fibrinolysis system. Using rTFPI, effectors of the fibrinolysis system and/or thiol-based drugs could be viable therapeutic strategies against SARS-CoV-2-induced endothelial injury, inflammation, NETosis, and coagulopathy.

Keywords: DTNB; IL-6; P-selectin; SARS-CoV-2 spike proteins; TFPI; citrullinated histone H3; human lung endothelial cells; neutrophils; neutrophils extracellular traps; thrombomodulin; von willebrand factor.

Figure 1

S-proteins and endothelial–neutrophil interactions induce P-selectin transcriptional upregulation in HLMEC and neutrophils. (A): HLMEC treated (for 6–24 h) with 1 nM S-protein Wuhan (SW) or Delta (SD) variants. (B,C): HLMEC were treated (6 h) with SW or SD, washed, and co-cultured (6–24 h) with neutrophils. (D,E): neutrophils treated (6 h) with SW or SD, washed, and co-cultured (6–24 h) with HLMEC. P-selectin mRNA in endothelial cells (A,B,D) and neutrophils (C,E) was quantified by real-time PCR. Data presented as mean ± standard deviation. Control: untreated cells; ACE2: cells treated with recombinant human (rh) ACE2 (1 µg/mL). Hi: cells treated with heat-inactivated S-proteins. * p < 0.015; ** p < 0.007; *** p < 0.0007; # p < 0.0001.

Figure 2

S-proteins induce P-selectin expression in HLMEC. HLMEC were treated with 1 nM S-proteins (SW or SD) for 12 h and P-selectin expression was quantified by immunofluorescence (A,B) and Western blot (C,D) analysis. DAPI (blue) was used for nuclear counterstaining. ImageJ software was used for densitometry quantification. For immunofluorescence images, five fields of view (FOV) were analyzed for each sample (B). For Western blot analysis, densitometry values were normalized to the sample’s β-actin levels (D). For panel (A), all images were at 20×. PS: P-selectin; control: untreated cells; ACE2: cells treated with rhACE2; Hi: cells treated with heat-inactivated S-proteins ** p = 0.01; # p < 0.0001.

Figure 3

S-proteins and endothelial–neutrophil interactions induce/increase the expression and secretion of cit-H3. Human neutrophils were treated with 1 nM S-proteins (SW or SD) for 6–24 h (A). In separate experiments, neutrophils (B) and HLMEC (C) were treated with S-proteins for 6 h, washed, and co-cultured (for 6–24 h) with HLMEC (B) or neutrophils (C). Levels of cit-H3 in culture supernatants were quantified by ELISA. Data presented as mean ± standard deviation. Control: untreated cells; ACE2: cells treated with rhACE2; Hi: cells treated with heat-inactivated S-proteins. *** p = 0.0003; # p < 0.0001.

Figure 4

S-proteins and endothelial–neutrophil interactions induce vWF expression and release in HLMEC. (A,B) HLMEC were treated (for 6–24 h) with SW or SD (1 nM). HLMEC (C) and neutrophils (D) were treated (for 6 h) with SW or SD, washed, and co-cultured (for 6–24 h) with neutrophils (C) or endothelial cells (D). vWF levels in culture supernatants (A,C,D) and endothelial cell lysates (B) were quantified by ELISA. Data presented as mean ± standard deviation. Control: untreated cells; Hi: cells treated with heat-inactivated S-proteins; ACE2: cells treated with rhACE2. * p < 0.03; ** p < 0.01; *** p = 0.0002; # p < 0.0001.

Figure 5

S-proteins and endothelial–neutrophil interactions increased IL-6 expression. (A) HLMEC treated (6–24 h) with SW or SD. HLMEC (B) and neutrophils (C) were treated (for 6 h) with SW or SD and co-cultured (for 6–24 h) with untreated neutrophils (B) or endothelial cells (C). IL-6 levels in culture supernatants were quantified by ELISA. Data presented as mean ± standard deviation. Control: untreated cells; ACE2: cells treated with rhACE2. Hi: cells treated with heat-inactivated S-proteins. * p < 0.02; ** p < 0.005; # p < 0.0001.

Figure 6

rTFPI, thrombomodulin (BDCA3), and thiol blockers (DTNB) prevent S-protein-induced H3 citrullination. (A): human neutrophils treated for 24 h with SW or SD, with or without rTFPI, DTNB, and BDCA3 (200 ng/mL). Neutrophils (B) and HLMEC (C) were treated (6 h) with SW or SD, with or without rTFPI, DTNB, and BDCA3, washed, and co-cultured (for 24 h) with HLMEC (B) or neutrophils (C). cit-H3 levels in culture supernatants quantified by ELISA. Data presented as mean ± standard deviation. Control: untreated cells; Hi: heat-inactivated (SW, SD, rTFPI, DTNB, BDCA3). # p < 0.0001.

Figure 7

rTFPI, thrombomodulin, and thiol blockers prevent S-protein-induced vWF expression. (A,B) HLMEC were treated (24 h) with SW or SD, with or without rTFPI, DTNB, and BDCA3. HLMEC (C) and neutrophils (D) were treated (6 h) with SW or SD, with or without rTFPI, DTNB, and BDCA3, washed, and co-cultured (for 24 h) with neutrophils (C) or HLMEC (D). vWF levels in culture supernatants (A,C,D) and endothelial cell lysates (B) were quantified by ELISA. Data presented as mean ± standard deviation. Control: untreated cells; Hi: heat-inactivated (SW, SD, rTFPI, DTNB, BDCA3). # p < 0.0001.

Figure 8

rTFPI, thrombomodulin, and thiol blockers prevent S-protein-induced IL-6 expression. (A) HLMEC were treated (24 h) with SW or SD, with or without rTFPI, DTNB, and BDCA3. HLMEC (B) and neutrophils (C) were treated (6 h) with SW or SD, with or without rTFPI, DTNB, and BDCA3, washed, and co-cultured (for 24 h) with neutrophils (B) or HLMEC (C). IL-6 levels in culture supernatants were quantified by ELISA. Data presented as mean ± standard deviation. Control: untreated cells; Hi: heat-inactivated (SW, SD, rTFPI, DTNB, BDCA3). ** p < 0.007; *** p = 0.0009; # p < 0.0001.

Figure 9

Model illustrating S-protein-induced P-selectin, vWF, IL-6 and cit-H3. Arrows indicate direct activation. The red arrows indicate upregulation; the red perpendicular symbol (⊥) indicates pharmacological inhibitors.