Am J Physiol Lung Cell Mol Physiol 321: L477–L484, 2021.

First published June 23, 2021; doi:10.1152/ajplung.00223.2021

RAPID REPORT

The Pathophysiology of COVID-19 and SARS-CoV-2 Infection

The SARS-CoV-2 spike protein subunit S1 induces COVID-19-like acute lung

injury in K18-hACE2 transgenic mice and barrier dysfunction in human
endothelial cells
Ruben M. L. Colunga Biancatelli,1* Pavel A. Solopov,1* Elizabeth R. Sharlow,2 John S. Lazo,2
Paul E. Marik,3 and John D. Catravas1,3,4
1
Frank Reidy Research Center for Bioelectrics, Old Dominion University, Norfolk, Virginia; 2Department of Pharmacology,
School of Medicine, University of Virginia, Charlottesville, Virginia; 3Division of Pulmonary Disease and Critical Care Medicine,
Eastern Virginia Medical School, Norfolk, Virginia; and 4School of Medical Diagnostic and Translational Sciences, College of
Health Sciences, Old Dominion University, Norfolk, Virginia

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 ac-
tivator 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 exhib-
ited 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 corona-viruses.

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

INTRODUCTION beneficial pharmaceutical interventions are few (2). It

is thus prudent to accelerate investigations of new ther-
Severe acute respiratory syndrome coronavirus-2 (SARS- apeutic approaches that can reduce mortality world-
CoV-2) is a b-Coronaviridae responsible of the 2020 pan- wide.
demic. Since its first report in the Wuhan city of China (1), it The study of SARS-CoV2 pathogenicity is challenging due
has infected more than 164,000,000 and provoked the death to limited availability of animal models. Whereas hamsters
of over 3.4 million worldwide [COVID-19 Map—Johns and monkeys express the angiotensin-converting enzyme 2
Hopkins Coronavirus Resource Center (www.jhu.edu)]. receptor (ACE2) (3, 4) that mediates viral cellular entry, wild-
Although vaccination programs have started, significant type mice, which are more commonly used in basic research,
delays can result from limited stocks, problems with world- do not (5). Transgenic mice expressing the human ACE2
wide distribution, and limited compliance. In addition, the gene under the control of the cytokeratin 18 promoter (K18-
emergence of new resistant strains of SARS-CoV-2 could hACE2) have been created and they faithfully reproduce the
delay the development of herd immunity and further pathology seen in humans when infected with the live SARS-
reduce the effectiveness of current vaccines. Despite global CoV-2 (5–7). Studies with live SARS-CoV-2 require biosafety
efforts toward the development of randomized clinical trials, level 3 (BSL-3) facilities for personnel protection, thus

* R. M. L. Colunga Biancatelli and P. A. Solopov contributed equally to this work.

Correspondence: J. D. Catravas (jcatrava@odu.edu).
Submitted 20 May 2021 / Revised 13 June 2021 / Accepted 15 June 2021

http://www.ajplung.org 1040-0605/21 Copyright © 2021 the American Physiological Society. L477

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 SARS-CoV-2 SPIKE PROTEIN SUBUNIT 1 INDUCES ACUTE LUNG INJURY

making them more complicated and available to a limited assay kit from Pierce Co.; and EDTA and Western blot mem-
group of investigators. When infected with live SARS-CoV-2, branes from GE Healthcare. ProtoGel (30% acrylamide mix)
K18-hACE2 mice exhibit sex- and time-dependent symptom- and tetramethylethylenediamine (TEMED) were from National
atology and 100% lethality (5–7). Diagnostics; Tris–HCl buffer from Teknova; and Protein Dual
Patients with COVID-19 develop a “cytokine storm” and a Color Standards and Tricine Sample Buffer from Bio-Rad
sepsis-like condition (8, 9); the contribution of surface ele- Laboratories. All antibodies were purchased from reputable
ments to the activation of these inflammatory pathways commercial sources and have published immunospecificity
remains unknown. Spike protein (SP) mediates SARS-CoV-2 data. Rabbit total and phosphorylated IκBa (No. 9242; No.
cell entry by binding to the plasmalemmal ACE2 receptor. 2859), STAT3 (No. 4904; No. 9145) were obtained from Cell
SP, a class I viral fusion protein, requires protease cleavage Signaling Technology, Inc.; mouse monoclonal anti-b-actin
for the activation of its fusion potential (10). SP is composed from Sigma-Aldrich Corporation; and IRDye 800CW Goat anti-
of two subunits, S1 and S2, that mediate attachment and rabbit and IRDye 680RD Goat anti-mouse from LI-COR
membrane fusion, respectively (11). Proteases capable of Biosciences.
cleaving SP include furin, trypsin, cathepsin, transmem-
brane protease serine protease-2 (TMPRSS-2), TMPRSS-4, Animal Model and Treatment Groups
phosphoinositide 5-kinases (PIK5), two pore segment chan- Male C57BL/6J (No. 000664) and K18-hACE2 transgenic
nel 2 (TPC2), and cathepsin L or human airway trypsin-like (No. 034860) mice (8–10 wk old, 24–28 g body wt; Jackson
protease (HAT) (12–15). Following cleavage, S1SP binds ACE2 Laboratories) were housed under pathogen-free conditions.
receptor and promotes the fusion of the viral membrane to S1SP (400 mg/kg in 2 mL/kg body wt) was given intratra-
the host cell and the subsequent release of genomic material cheally to K18-hACE2 mice. Briefly, mice were anesthetized
into the cytoplasm (16). The binding to ACE2 also disrupts with intraperitoneal injection of xylazine (6 mg/kg) and keta-
the renin-angiotensin signaling (17) and causes ACE2 shed- mine (60 mg/kg). After cleaning and disinfecting the surgical
ding, which is related to acute lung injury (ALI), increased field, a small neck skin incision (1 cm) was made, and the sal-
vascular permeability (18), and cytokine production (19). ivary glands were separated to visualize the trachea. Mice
When injected intraperitoneally, the 2003 SARS-CoV SP were suspended vertically from their incisors and a fine (22 G)
exacerbates histologically evident ALI via upregulation of an- plastic catheter was advanced from the mouth into the trachea
giotensin II levels (20). Whether SARS-CoV-2 SP or S1SP can (2 cm) in such a way that it could be seen through the walls
induce local or systemic inflammation in vivo, remains of the trachea. S1SP was introduced and flushed with 150 mL
unknown. To that end, we have tested the hypothesis that in- air as we have previously described (21). The catheter was
tratracheal instillation of SARS-CoV-2 S1SP in K18-hACE2 mice withdrawn, neck incision closed by surgical adhesive, and the
leads to ALI. For the first time, we report that S1SP, when animal was placed in the ventral position in a small chamber
instilled intratracheally, produces biochemically, immunologi- on top of a heating pad, under supplemental oxygen (slowly
cally, and histologically evident COVID-19-like ALI, including weaned from 100% to 21% O2) and observed for the next 4 h
“cytokine storm.” In agreement with these findings, we also for signs of respiratory distress before being returned to the
report a direct effect of S1SP on human lung microvascular en- regular cage. Mice were monitored daily for abnormal physical
dothelial cell barrier integrity, in culture. This mouse model is appearances. Seventy-two hours later, mice were subjected to
easily reproducible and can be used for the study of new thera- bronchoalveolar lavage (BAL), analysis of lung tissue and BAL
peutic approaches to COVID-19 outside BSL3 environments. fluid (BALF) and histological evaluation. Controls included
administration of SP (400 mg/kg in 2 mL/kg body wt) or saline
MATERIALS AND METHODS to K18-hACE2 mice and S1SP to wild-type (WT) mice. They
were all examined 72 h after saline, SP, or S1SP administration.
Ethical Statement
Histopathology and Lung Injury Score
All animal studies were approved by the Old Dominion
University IACUC and adhere to the principles of animal Mice were euthanized and lungs were fixed in upright posi-
experimentation as published by the American Physiological tion. A small transverse incision was made in the middle of
Society and under Biosafety Level 2 (BLS-2) and animal BSL2 the trachea and the lungs were instilled and inflated with 10%
(Animal Biosafety Level (ABSL)-2) facilities at Frank Reidy formaldehyde solution to a pressure of 15 cmH2O using a 20 G
Center for Bioelectrics in Norfolk, Virginia. catheter. The trachea was then ligated with suture; the lungs
were removed from the thorax and placed in 10% formalde-
Materials hyde solution for 72 h. Mid-transverse slices were made from
Recombinant SARS-CoV-2 Spike Protein, subunit 1 (S1SP) the formalin-fixed lung and embedded in paraffin. Sections 5
was purchased from RayBiotech (No. 230–011101-100), mm thick were stained with hematoxylin-eosin (H&E). Twenty
recombinant SARS-CoV-2 spike protein (SP) was from BEI randomly selected fields from each slide were examined
Resources (No. NR-53257). RIPA (radioimmunoprecipitation under10 and 40 magnification and the lung injury score
assay) buffer and protease inhibitor cocktail were obtained was computed (22).
from Sigma-Aldrich Corporation. Socumb (pentobarbital),
Measurement of Total Protein, Cell, and Cytokine
Anased (xylazine), and Ketaset (ketamine) were supplied by
Concentration in BALF
Henry Schein Animal Health. Wright-Giemsa Stain Kit, 10%
formaldehyde, 10% SDS, and ammonium persulfate were BALF was centrifuged at 2,500 g for 10 min, and the super-
purchased from Thermo Fisher Scientific; the BCA protein natant was collected for total protein and cytokine analysis.

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 SARS-CoV-2 SPIKE PROTEIN SUBUNIT 1 INDUCES ACUTE LUNG INJURY

Total protein concentration was determined using the bicin- Densitometric quantification of the bands from the blots
choninic acid (BCA) protein assay kit according to manufac- was performed using ImageJ software (National Institutes
turer’s instructions. Cell numbers were measured with a of Health).
hemocytometer and differential analysis was performed with
the Wright-Giemsa stain kit. Quantitative analysis of BALF Ingenuity Pathway Analysis
and serum chemokines and cytokines were performed by the Chemokine and cytokine genes of interest in BALF were
University of Virginia Flow Cytometry Core Facility, using a identified using a P < 0.05 threshold as assessed using the
MILLIPLEX map murine cytokine/chemokine premixed unpaired-samples t test. Ingenuity Pathway Analysis (Qiagen)
panel (EMD Millipore) using a Luminex MAGPIX system. was used for subsequent bioinformatics analysis, which
included canonical pathway analysis, disease and function,
Western Blotting regulator effects, upstream regulators, and molecular net-
works. Analyses were restricted accordingly by species (i.e.,
Lung homogenates from frozen lung tissues were pre- mouse) and tissue type (i.e., lung).
pared by sonication in ice-cold RIPA buffer with protease
inhibitor cocktail (100:1 -EMD, Millipore Corporation).
Endothelial Cell Culture and Barrier Measurements
Protein lysates were agitated for 3 h at 4 C, and then centri-
fuged twice at 14,000 g for 10 min. The supernatants were In-house harvested and identified human lung micro-
separated from the pellets and total protein concentration vascular endothelial cells (HLMVECs) were isolated and
was determined by the bicinchoninic acid method (BCA maintained in M199 media supplemented with 20% FBS
Assay). Equal amounts of protein from the lysates were first and antibiotics/antimycotics as described previously (23).
mixed with tricine sample buffer 1:1, heat denatured at 95 C The barrier function of confluent endothelial cell mono-
for 10 min and separated on a 12% SDS-PAGE gel by electro- layers was estimated using electric cell-substrate imped-
phoresis. Proteins were then transferred on a nitrocellulose ance sensing (ECIS) model 1600R fh (Applied Biophysics)
membrane, incubated with primary and secondary anti- as previously described (24). Experiments were con-
bodies, and detected by digital fluorescence imaging (LI- ducted with cells that had reached a steady-state resist-
COR Odyssey CLx). b-actin was used as a loading control. ance of at least 800 X.

A Body weight changes

B Total BALF protein

***
4000
*** ***
Changes in weight
from baseline (%)

Total protein μg/ml

100
3000
**** ***
2000
90

1000

80 0
0 24 48 72
Hours

C WBC in BALF D BALF differentials

*** ****
WBC in BALF, cells/ml

*** ** 250000
****
WBC in BALF, cells/ml

400000

200000 K18-hACE2 +VEH

300000 ***
* K18-hACE2 + S1SP
150000
200000
*** K18-hACE2 + SP
100000 WT + S1SP

100000
50000 ****
0 0
es

ls

es
es

hi
yt

yt
ag

op
oc

oc
ph

tr
on

ph
ro

eu
M

m
ac

Ly
M
lv
A

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.

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 SARS-CoV-2 SPIKE PROTEIN SUBUNIT 1 INDUCES ACUTE LUNG INJURY

RESULTS Spike Protein Induces Morphologically Evident ALI and

Activates the NF-κB and STAT3 Pathways in the Lungs
Spike Protein Induces Alveolar Inflammation and Acute
Lung Injury K18-hACE2 mice instilled with S1SP revealed alveolar
septal thickening, alveolar edema, hyaline membranes,
K18-hACE2 mice instilled with S1SP displayed a rapid and
and extensive leukocyte infiltrates when compared with
sustained decline in body weight, unlike mice receiving SP
or saline. WT type instilled with S1SP exhibited a significant saline or SP controls and, to a lesser extent, —WT mice
but less pronounced decrease (Fig. 1A). Strong alveolar receiving S1SP (Fig. 3A); these observations were con-
inflammation was also shown by increased concentrations firmed by quantification in the Lung Injury Index (Fig. 3B).
of leukocytes and proteins in BALF of transgenic mice Western analysis of lung homogenates demonstrated a
instilled with the S1SP, and significantly less in WT type dramatical increase in the phosphorylation of STAT3 in
receiving S1SP, whereas similar baseline values were K18-hACE2 mice instilled with S1SP compared with all
observed in K18-hACE2 mice receiving SP or saline (Fig. 1, other groups (Fig. 3C), whereas phosphorylation of IκBa (I-
B and C). Monocytes, macrophages, and neutrophils were kappaB alpha - cytosolic inhibitor of NF-κB) increased in
primarily increased in S1SP-instilled K18-hACE2, but only both K18-hACE2 and WT mice instilled with S1SP (Fig. 3C).
neutrophils increased in WT-type mice (Fig. 1D).
Ingenuity Pathway Analysis
Spike Protein Elicits “Cytokine Storm” in BALF and
Serum Ingenuity Pathway Analysis of the significantly upregu-
lated and downregulated cytokines and chemokines in BALF
Mice instilled with S1SP displayed a cytokine storm in computationally revealed a prominent association of our in
BALF (Fig. 2A) and serum (Fig. 2B), in agreement with the vivo model system with diseases and biofunctions aligned
observed neutrophil, monocyte, and macrophage recruit- with inflammatory disease, and most specifically, lung infec-
ment (Fig. 1D). Minimal cytokine levels were observed in tion, inflammation, and permeability of microvascular endo-
mice exposed to either saline or SP. thelial cells (Fig. 4A).

K18-hACE2 + Saline
K18-hACE2 + S1SP
K18-hACE2 + SP
A IFN γ IL-1β IL-6 IL-17 WT + S1SP

**** **** *** ***

*** *** ***
2000 20
1000 40
1500
30 15
pg/ml

pg/ml
pg/ml

pg/ml

500 1000
20 500 10

5 10 5
10
0 0 0 0

MCP-1 KC TNFα MIP 1α

**** **** **** **

250
****
250
**** 100 **** 250
**
150 200
pg/ml

pg/ml

pg/ml

150
pg/ml

50 150
50
50 100
15
15 5 50
0 0 0 0

B IFN γ IL 1β MIG IP 10

30 *** 20
* *** 600 ***
* *
4000
15
3000 **
pg/ml

pg/ml

400
pg/ml

20
pg/ml

10 2000
10 200
5 1000

0 0 0 0

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 pro-
tein; VEH, vehicle; S1SP, subunit 1 of spike protein; KC, keratinocytes-derived chemokine; MIP1a, macrophage inflammatory protein 1-alpha; MIG, gamma
interferon; IP 10, interferon gamma-induced protein 10.

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 SARS-CoV-2 SPIKE PROTEIN SUBUNIT 1 INDUCES ACUTE LUNG INJURY

K18-hACE2 +VEH
K18-hACE2 + S1SP
K18-hACE2 + SP
A B WT + S1SP
***
K18-hACE2 + VEH
***

Lung Injury Score

10

6
***
4

C
K18-hACE2 + S1SP

pSTAT3/STAT3
K18-hACE2 + SP ***
*** ***

6
Fold of Control
5
4
3
2
1
0

pIKBα/IKBα
WT + S1SP **
4
*
Fold of control

0
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 path-
ways 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
(IκBa) and STAT3. Bands were normalized to b-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.

Spike Protein Disrupts Pulmonary Microvascular indicative of concentration dependence increases in perme-
Endothelial Barrier Function ability and barrier dysfunction (Fig. 4B).

Because S1SP produced alveolar inflammation and edema,

we also examined possible direct effects on endothelial bar- DISCUSSION
rier dysfunction. In primary human lung microvascular en-
dothelial cells that naturally express ACE2, S1SP—but not The live SARS-CoV-2 can be lethal in K18-hACE2 but
the entire, intact SP—produced a profound, concentration- not in WT mice and that, like what is observed in patients
dependent decrease in transendothelial resistance (TER), with COVID-19, infected K18-hACE2 mice exhibit strong

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 SARS-CoV-2 SPIKE PROTEIN SUBUNIT 1 INDUCES ACUTE LUNG INJURY

Figure 4. A: Ingenuity Pathway Analysis of

BALF cytokine and chemokine in mice af-
ter S1SP or SP instillation. The statistically
significant cytokines were computed into
a database for known pathways of inflam-
mation, infection, apoptosis and perme-
ability. B: the S1 subunit of the SARS-CoV-
2 spike protein (S1SP) causes a fast con-
centration- and time-dependent decrease
in transendothelial electrical resistance
(TER), whereas the intact SP provokes a
late and milder decrease in TER of human B 1.2
lung microvascular endothelial cells. n = 4
replicates/time point. P < 0.0001 by ****
Normalized TER

1.0
two-way ANOVA for repeated measures Control
and Tukey’s. Values were normalized to 10 nM S1SP
time = 0 for easier comparisons. Starting re- 0.8
20 nM S1SP
sistance was >800 Ω. BALF, bronchoalveo-
lar lavage fluid; SP, spike protein; SARS-CoV- 0.6
**** 50 nM S1SP
50 nM SP
2, severe acute respiratory syndrome coro-
navirus-2; CCL, C-C motif chemokine.
0.4

0.2
0 5 10 15 20 25
Time (Hours)

inflammation and acute respiratory distress syndrome requirement for the pathogenesis of COVID-19 and thus, k18-
(ARDS)-like pathology (5). Indeed, critically ill patients with hACE2 mice represent a more valid and trustable model for
COVID-19 develop a strong inflammatory response, with COVID-19. They further suggest that the SARS-CoV-2 S1SP
“cytokine storm” (25). The contribution of individual viral could play the role of a “toxin” and, when released in the vas-
elements to the pathology of COVID-19 remains unclear. The cular compartment after cell lysis, participate in the patho-
SP of SARS-CoV is proinflammatory in vitro (26). Although genesis of systemic inflammation.
inactive by itself, SARS-CoV SP exacerbates lung injury (20). Additional work is required to determine the precise dose-
Similarly, S1SP of SARS-CoV-2 is proinflammatory in isolated response relationships to S1SP in vivo, define S1SP pathoge-
peritoneal macrophages (27) and in human bronchial epithe- nicity in different cell lines, and further characterize the
lial cells (28). However, these data do not necessarily predict diverse pathways of activity between hACE2 expressing and
COVID-19-like lung injury or systemic inflammation. WT mice.
Here, we show that the activated (cleaved) form of the HLMVEC exposed to S1SP displayed a rapid concentra-
SP, S1SP, elicits strong pulmonary and systemic inflamma- tion-dependent decrease in TER, whereas the highest dose
tory responses in K18-hACE2 mice and milder responses in of SP evoked a late and minimal decrease in resistance. As
WT mice, whereas the intact SP provokes minimal or no the SP requires to be cleaved into its subunits to bind the
responses. ACE2 receptor, it is not surprising that the intact SP evokes a
The cytokine storm observed in this study exhibits critical minimal and late response, which could be delayed due to
similarities with the inflammatory response elicited by the the time required for the cleavage, and lesser because of the
live virus in K18-hACE2 mice (5, 29). Hamsters, intratra- small amounts of proteases present in this in vitro model.
cheally instilled with a pseudovirus expressing SP, display Here, we demonstrate that intratracheal instillation of a
ALI, endothelial dysfunction, and mitochondrial damage single element of SARS-CoV-2, S1SP, in K18-hACE2 trans-
mediated by the downregulation of ACE2 (30). genic mice induces COVID-19-like local (lung) and systemic
Our results indicate that S1SP can induce a milder form of inflammatory responses. This model will allow the preclini-
ALI in WT mice that do not express the human ACE2. cal investigation of potential countermeasures and of the
Recently, Asandei et al. (31) characterized a non-receptor- long-term effects of the inflammatory response provoked by
mediated mechanism for SARS-CoV-2 S1SP to destabilize cell SARS-CoV-2.
membranes. Furthermore, the human and mouse peptidase
M2 domain of ACE2 that binds S1SP share 85% homology ACKNOWLEDGMENTS
(32). Either or both observations could explain the milder We thank the Eastern Virginia Medical School (EVMS),
form of COVID-19 induced by S1SP in WT mice. This data Department of Anatomy and Pathology Laboratory for lung tissue
suggest that, despite the strong biological activity of S1SP in processing and staining. We thank Betsy Gregory for outstanding
wild-type animals, the hACE2 receptor is an important technical assistance.

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 SARS-CoV-2 SPIKE PROTEIN SUBUNIT 1 INDUCES ACUTE LUNG INJURY

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preted results of experiments; R.M.L.C.B., P.A.S., and E.R.S. pre- 14. Bertram S, Glowacka I, M€ uller MA, Lavender H, Gnirss K,
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