The SARS-CoV-2 spike protein subunit S1 induces COVID-19-like acute lung injury in Κ18-hACE2 transgenic mice and barrier dysfunction in human endothelial cells

Abstract

Acute lung injury (ALI) leading to acute respiratory distress syndrome is the major cause of COVID-19 lethality. Cell entry of SARS-CoV-2 occurs via the interaction between its surface spike protein (SP) and angiotensin-converting enzyme-2 (ACE2). It is unknown if the viral spike protein alone is capable of altering lung vascular permeability in the lungs or producing lung injury in vivo. To that end, we intratracheally instilled the S1 subunit of SARS-CoV-2 spike protein (S1SP) in K18-hACE2 transgenic mice that overexpress human ACE2 and examined signs of COVID-19-associated lung injury 72 h later. Controls included K18-hACE2 mice that received saline or the intact SP and wild-type (WT) mice that received S1SP. K18-hACE2 mice instilled with S1SP exhibited a decline in body weight, dramatically increased white blood cells and protein concentrations in bronchoalveolar lavage fluid (BALF), upregulation of multiple inflammatory cytokines in BALF and serum, histological evidence of lung injury, and activation of signal transducer and activator of transcription 3 (STAT3) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathways in the lung. K18-hACE2 mice that received either saline or SP exhibited little or no evidence of lung injury. WT mice that received S1SP exhibited a milder form of COVID-19 symptoms, compared with the K18-hACE2 mice. Furthermore, S1SP, but not SP, decreased cultured human pulmonary microvascular transendothelial resistance (TER) and barrier function. This is the first demonstration of a COVID-19-like response by an essential virus-encoded protein by SARS-CoV-2 in vivo. This model of COVID-19-induced ALI may assist in the investigation of new therapeutic approaches for the management of COVID-19 and other coronaviruses.

Keywords: COVID-19 murine model; SARS-CoV-2; acute lung injury; endothelial permeability; spike protein.

Figure 1.

Exposure to SARS-CoV-2 subunit 1 of spike protein (S1SP) induces body weight loss and alveolar inflammation. K18-hACE2 and WT S1SP-instilled mice exhibit weight loss compared with saline controls or SP-instilled K18-hACE2 mice (A). S1SP-instilled mice have elevated protein (B) and leukocyte (C) concentrations in BALF, 72 h after instillation, especially increased monocyte and neutrophil levels that are maximal in the K18-hACE2 strain (D). means ± SE; n = 5 mice/group; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 with one-way ANOVA followed by Tukey’s. BALF, bronchoalveolar lavage fluid; Alv, alveolar; SARS-CoV-2, severe acute respiratory syndrome coronavirus-2; VEH, vehicle; SP, spike protein; WBC, white blood cell(s); WT, wild type.

Figure 2.

Exposure to S1SP of SARS-CoV-2 induces alveolar and systemic “cytokine storm” in BALF (A) and serum (B) 72 h after instillation.means ± SE; n = 5 mice/group; *P < 0.05; **P < 0.01, ***P < 0.001; ****P < 0.0001 with one-way ANOVA followed by Tukey’s in log-transformed values. BALF, bronchoalveolar lavage fluid; MCP, monocyte chemoattractant protein; SARS-CoV-2, severe acute respiratory syndrome coronavirus-2; SP, spike protein; VEH, vehicle; S1SP, subunit 1 of spike protein; KC, keratinocytes-derived chemokine; MIP1α, macrophage inflammatory protein 1-alpha; MIG, gamma interferon; IP 10, interferon gamma-induced protein 10.

Figure 3.

The S1 subunit of the SARS-CoV-2 spike protein (S1SP) causes acute lung injury and the activation of the STAT3 and NF-κB inflammatory pathways 72 h after exposure. A: H&E staining of lung sections demonstrates septal thickening, neutrophil infiltration, and edema in S1SP-instilled K18-hACE2 mice, minimal edema and leucocyte infiltration in S1SP-instilled WT, whereas SP-instilled K18-hACE2 display minimal changes compared with controls. B: the Lung Injury Score quantifies maximal injury in S1SP-instilled K18-hACE2 mice, a milder pathology in S1SP-instilled WT and no significant changes in SP- or saline-instilled K18-hACE2 mice. C: Western blot analysis of lung homogenates revealed increased phosphorylation of I-kappaB alpha (IκBα) and STAT3. Bands were normalized to β-actin and are shown as fold of control. (means ± SE; n = 5 mice/group; *P < 0.05; **P < 0.01; ***P < 0.001 with one-way ANOVA and Tukey’s). H&E, hematoxylin-eosin; SARS-CoV-2, severe acute respiratory syndrome coronavirus-2; VEH, vehicle; SP, spike protein; S1SP, subunit 1 of spike protein; WT, wild type.

Figure 4.

A: Ingenuity Pathway Analysis of BALF cytokine and chemokine in mice after S1SP or SP instillation. The statistically significant cytokines were computed into a database for known pathways of inflammation, infection, apoptosis and permeability. B: the S1 subunit of the SARS-CoV-2 spike protein (S1SP) causes a fast concentration- and time-dependent decrease in transendothelial electrical resistance (TER), whereas the intact SP provokes a late and milder decrease in TER of human lung microvascular endothelial cells. n = 4 replicates/time point. ****P < 0.0001 by two-way ANOVA for repeated measures and Tukey’s. Values were normalized to time = 0 for easier comparisons. Starting resistance was >800 Ω. BALF, bronchoalveolar lavage fluid; SP, spike protein; SARS-CoV-2, severe acute respiratory syndrome coronavirus-2; CCL, C-C motif chemokine.