2021Solopov

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 SARSCoV-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 COVID19-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
Severe acute respiratory syndrome coronavirus-2 (SARSCoV-2) is a b-Coronaviridae responsible of the 2020 pandemic. Since its first report in the Wuhan city of China (1), it
has infected more than 164,000,000 and provoked the death
of over 3.4 million worldwide [COVID-19 Map—Johns
Hopkins Coronavirus Resource Center (www.jhu.edu)].
Although vaccination programs have started, significant
delays can result from limited stocks, problems with worldwide distribution, and limited compliance. In addition, the
emergence of new resistant strains of SARS-CoV-2 could
delay the development of herd immunity and further
reduce the effectiveness of current vaccines. Despite global
efforts toward the development of randomized clinical trials,
beneficial pharmaceutical interventions are few (2). It
is thus prudent to accelerate investigations of new therapeutic approaches that can reduce mortality worldwide.
The study of SARS-CoV2 pathogenicity is challenging due
to limited availability of animal models. Whereas hamsters
and monkeys express the angiotensin-converting enzyme 2
receptor (ACE2) (3, 4) that mediates viral cellular entry, wildtype mice, which are more commonly used in basic research,
do not (5). Transgenic mice expressing the human ACE2
gene under the control of the cytokeratin 18 promoter (K18hACE2) have been created and they faithfully reproduce the
pathology seen in humans when infected with the live SARSCoV-2 (5–7). Studies with live SARS-CoV-2 require biosafety
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.
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SARS-CoV-2 SPIKE PROTEIN SUBUNIT 1 INDUCES ACUTE LUNG INJURY
making them more complicated and available to a limited
group of investigators. When infected with live SARS-CoV-2,
K18-hACE2 mice exhibit sex- and time-dependent symptomatology and 100% lethality (5–7).
Patients with COVID-19 develop a “cytokine storm” and a
sepsis-like condition (8, 9); the contribution of surface elements to the activation of these inflammatory pathways
remains unknown. Spike protein (SP) mediates SARS-CoV-2
cell entry by binding to the plasmalemmal ACE2 receptor.
SP, a class I viral fusion protein, requires protease cleavage
for the activation of its fusion potential (10). SP is composed
of two subunits, S1 and S2, that mediate attachment and
membrane fusion, respectively (11). Proteases capable of
cleaving SP include furin, trypsin, cathepsin, transmembrane protease serine protease-2 (TMPRSS-2), TMPRSS-4,
phosphoinositide 5-kinases (PIK5), two pore segment channel 2 (TPC2), and cathepsin L or human airway trypsin-like
protease (HAT) (12–15). Following cleavage, S1SP binds ACE2
receptor and promotes the fusion of the viral membrane to
the host cell and the subsequent release of genomic material
into the cytoplasm (16). The binding to ACE2 also disrupts
the renin-angiotensin signaling (17) and causes ACE2 shedding, which is related to acute lung injury (ALI), increased
vascular permeability (18), and cytokine production (19).
When injected intraperitoneally, the 2003 SARS-CoV SP
exacerbates histologically evident ALI via upregulation of angiotensin II levels (20). Whether SARS-CoV-2 SP or S1SP can
induce local or systemic inflammation in vivo, remains
unknown. To that end, we have tested the hypothesis that intratracheal instillation of SARS-CoV-2 S1SP in K18-hACE2 mice
leads to ALI. For the first time, we report that S1SP, when
instilled intratracheally, produces biochemically, immunologically, and histologically evident COVID-19-like ALI, including
“cytokine storm.” In agreement with these findings, we also
report a direct effect of S1SP on human lung microvascular endothelial cell barrier integrity, in culture. This mouse model is
easily reproducible and can be used for the study of new therapeutic approaches to COVID-19 outside BSL3 environments.
MATERIALS AND METHODS
Ethical Statement
All animal studies were approved by the Old Dominion
University IACUC and adhere to the principles of animal
experimentation as published by the American Physiological
Society and under Biosafety Level 2 (BLS-2) and animal BSL2
(Animal Biosafety Level (ABSL)-2) facilities at Frank Reidy
Center for Bioelectrics in Norfolk, Virginia.
Materials
Recombinant SARS-CoV-2 Spike Protein, subunit 1 (S1SP)
was purchased from RayBiotech (No. 230–011101-100),
recombinant SARS-CoV-2 spike protein (SP) was from BEI
Resources (No. NR-53257). RIPA (radioimmunoprecipitation
assay) buffer and protease inhibitor cocktail were obtained
from Sigma-Aldrich Corporation. Socumb (pentobarbital),
Anased (xylazine), and Ketaset (ketamine) were supplied by
Henry Schein Animal Health. Wright-Giemsa Stain Kit, 10%
formaldehyde, 10% SDS, and ammonium persulfate were
purchased from Thermo Fisher Scientific; the BCA protein
L478
assay kit from Pierce Co.; and EDTA and Western blot membranes from GE Healthcare. ProtoGel (30% acrylamide mix)
and tetramethylethylenediamine (TEMED) were from National
Diagnostics; Tris–HCl buffer from Teknova; and Protein Dual
Color Standards and Tricine Sample Buffer from Bio-Rad
Laboratories. All antibodies were purchased from reputable
commercial sources and have published immunospecificity
data. Rabbit total and phosphorylated IκBa (No. 9242; No.
2859), STAT3 (No. 4904; No. 9145) were obtained from Cell
Signaling Technology, Inc.; mouse monoclonal anti-b-actin
from Sigma-Aldrich Corporation; and IRDye 800CW Goat antirabbit and IRDye 680RD Goat anti-mouse from LI-COR
Biosciences.
Animal Model and Treatment Groups
Male C57BL/6J (No. 000664) and K18-hACE2 transgenic
(No. 034860) mice (8–10 wk old, 24–28 g body wt; Jackson
Laboratories) were housed under pathogen-free conditions.
S1SP (400 mg/kg in 2 mL/kg body wt) was given intratracheally to K18-hACE2 mice. Briefly, mice were anesthetized
with intraperitoneal injection of xylazine (6 mg/kg) and ketamine (60 mg/kg). After cleaning and disinfecting the surgical
field, a small neck skin incision (1 cm) was made, and the salivary glands were separated to visualize the trachea. Mice
were suspended vertically from their incisors and a fine (22 G)
plastic catheter was advanced from the mouth into the trachea
(2 cm) in such a way that it could be seen through the walls
of the trachea. S1SP was introduced and flushed with 150 mL
air as we have previously described (21). The catheter was
withdrawn, neck incision closed by surgical adhesive, and the
animal was placed in the ventral position in a small chamber
on top of a heating pad, under supplemental oxygen (slowly
weaned from 100% to 21% O2) and observed for the next 4 h
for signs of respiratory distress before being returned to the
regular cage. Mice were monitored daily for abnormal physical
appearances. Seventy-two hours later, mice were subjected to
bronchoalveolar lavage (BAL), analysis of lung tissue and BAL
fluid (BALF) and histological evaluation. Controls included
administration of SP (400 mg/kg in 2 mL/kg body wt) or saline
to K18-hACE2 mice and S1SP to wild-type (WT) mice. They
were all examined 72 h after saline, SP, or S1SP administration.
Histopathology and Lung Injury Score
Mice were euthanized and lungs were fixed in upright position. A small transverse incision was made in the middle of
the trachea and the lungs were instilled and inflated with 10%
formaldehyde solution to a pressure of 15 cmH2O using a 20 G
catheter. The trachea was then ligated with suture; the lungs
were removed from the thorax and placed in 10% formaldehyde solution for 72 h. Mid-transverse slices were made from
the formalin-fixed lung and embedded in paraffin. Sections 5
mm thick were stained with hematoxylin-eosin (H&E). Twenty
randomly selected fields from each slide were examined
under10 and 40 magnification and the lung injury score
was computed (22).
Measurement of Total Protein, Cell, and Cytokine
Concentration in BALF
BALF was centrifuged at 2,500 g for 10 min, and the supernatant was collected for total protein and cytokine analysis.
AJP-Lung Cell Mol Physiol doi:10.1152/ajplung.00223.2021 www.ajplung.org
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SARS-CoV-2 SPIKE PROTEIN SUBUNIT 1 INDUCES ACUTE LUNG INJURY
Densitometric quantification of the bands from the blots
was performed using ImageJ software (National Institutes
of Health).
Total protein concentration was determined using the bicinchoninic acid (BCA) protein assay kit according to manufacturer’s instructions. Cell numbers were measured with a
hemocytometer and differential analysis was performed with
the Wright-Giemsa stain kit. Quantitative analysis of BALF
and serum chemokines and cytokines were performed by the
University of Virginia Flow Cytometry Core Facility, using a
MILLIPLEX map murine cytokine/chemokine premixed
panel (EMD Millipore) using a Luminex MAGPIX system.
Ingenuity Pathway Analysis
Chemokine and cytokine genes of interest in BALF were
identified using a P < 0.05 threshold as assessed using the
unpaired-samples t test. Ingenuity Pathway Analysis (Qiagen)
was used for subsequent bioinformatics analysis, which
included canonical pathway analysis, disease and function,
regulator effects, upstream regulators, and molecular networks. Analyses were restricted accordingly by species (i.e.,
mouse) and tissue type (i.e., lung).
Western Blotting
Lung homogenates from frozen lung tissues were prepared by sonication in ice-cold RIPA buffer with protease
inhibitor cocktail (100:1 -EMD, Millipore Corporation).
Protein lysates were agitated for 3 h at 4 C, and then centrifuged twice at 14,000 g for 10 min. The supernatants were
separated from the pellets and total protein concentration
was determined by the bicinchoninic acid method (BCA
Assay). Equal amounts of protein from the lysates were first
mixed with tricine sample buffer 1:1, heat denatured at 95 C
for 10 min and separated on a 12% SDS-PAGE gel by electrophoresis. Proteins were then transferred on a nitrocellulose
membrane, incubated with primary and secondary antibodies, and detected by digital fluorescence imaging (LICOR Odyssey CLx). b-actin was used as a loading control.
A
In-house harvested and identified human lung microvascular endothelial cells (HLMVECs) were isolated and
maintained in M199 media supplemented with 20% FBS
and antibiotics/antimycotics as described previously (23).
The barrier function of confluent endothelial cell monolayers was estimated using electric cell-substrate impedance sensing (ECIS) model 1600R fh (Applied Biophysics)
as previously described (24). Experiments were conducted with cells that had reached a steady-state resistance of at least 800 X.
B
Body weight changes
Total BALF protein
100
****
90
80
0
24
48
***
***
4000
Total protein μg/ml
Changes in weight
from baseline (%)
Endothelial Cell Culture and Barrier Measurements
***
3000
***
2000
1000
0
72
Hours
D
K18-hACE2 +VEH
K18-hACE2 + S1SP
K18-hACE2 + SP
WT + S1SP
*
150000
***
100000
50000
****
0
es
ls
yt
hi
oc
op
m
ph
tr
eu
A
lv
M
ac
ro
ph
ag
es
0
200000
es
100000
***
****
****
250000
N
200000
**
yt
300000
***
M
on
oc
400000
BALF differentials
***
Ly
WBC in BALF
WBC in BALF, cells/ml
WBC in BALF, cells/ml
C
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.
AJP-Lung Cell Mol Physiol doi:10.1152/ajplung.00223.2021 www.ajplung.org
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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,
and extensive leukocyte infiltrates when compared with
saline or SP controls and, to a lesser extent, —WT mice
receiving S1SP (Fig. 3A); these observations were confirmed by quantification in the Lung Injury Index (Fig. 3B).
Western analysis of lung homogenates demonstrated a
dramatical increase in the phosphorylation of STAT3 in
K18-hACE2 mice instilled with S1SP compared with all
other groups (Fig. 3C), whereas phosphorylation of IκBa (IkappaB alpha - cytosolic inhibitor of NF-κB) increased in
both K18-hACE2 and WT mice instilled with S1SP (Fig. 3C).
K18-hACE2 mice instilled with S1SP displayed a rapid and
sustained decline in body weight, unlike mice receiving SP
or saline. WT type instilled with S1SP exhibited a significant
but less pronounced decrease (Fig. 1A). Strong alveolar
inflammation was also shown by increased concentrations
of leukocytes and proteins in BALF of transgenic mice
instilled with the S1SP, and significantly less in WT type
receiving S1SP, whereas similar baseline values were
observed in K18-hACE2 mice receiving SP or saline (Fig. 1,
B and C). Monocytes, macrophages, and neutrophils were
primarily increased in S1SP-instilled K18-hACE2, but only
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 upregulated and downregulated cytokines and chemokines in BALF
computationally revealed a prominent association of our in
vivo model system with diseases and biofunctions aligned
with inflammatory disease, and most specifically, lung infection, inflammation, and permeability of microvascular endothelial cells (Fig. 4A).
Mice instilled with S1SP displayed a cytokine storm in
BALF (Fig. 2A) and serum (Fig. 2B), in agreement with the
observed neutrophil, monocyte, and macrophage recruitment (Fig. 1D). Minimal cytokine levels were observed in
mice exposed to either saline or SP.
IFN γ
5
0
30
20
pg/ml
250
200
50
20
15
10
100
50
0
0
MIG
*
4000
pg/ml
*
***
**
3000
2000
5
1000
0
0
**
**
150
5
IL 1β
pg/ml
pg/ml
0
****
IP 10
***
600
pg/ml
IFN γ
10
****
100
50
MIP 1α
TNFα
****
0
B
10
0
15
0
20
500
15
0
15
30
1000
5
****
***
20
0
250
50
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10
150
150
1500
KC
pg/ml
pg/ml
****
****
***
2000
10
MCP-1
250
****
pg/ml
500
***
40
pg/ml
***
****
pg/ml
pg/ml
1000
IL-17
IL-6
IL-1β
pg/ml
A
K18-hACE2 + Saline
K18-hACE2 + S1SP
K18-hACE2 + SP
WT + S1SP
400
*
***
200
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 protein; 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
A
K18-hACE2 +VEH
K18-hACE2 + S1SP
K18-hACE2 + SP
WT + S1SP
B
***
Lung Injury Score
K18-hACE2 + VEH
***
10
8
***
6
4
2
0
C
K18-hACE2 + S1SP
pSTAT3/STAT3
***
***
1
2
3
4
5
***
0
Fold of Control
6
K18-hACE2 + SP
pIKBα/IKBα
**
WT + S1SP
Fold of control
4
*
3
2
1
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 pathways 72 h after exposure. A: H&E staining of lung sections demonstrates septal thickening, neutrophil infiltration, and edema in S1SP-instilled K18hACE2 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
Endothelial Barrier Function
Because S1SP produced alveolar inflammation and edema,
we also examined possible direct effects on endothelial barrier dysfunction. In primary human lung microvascular endothelial cells that naturally express ACE2, S1SP—but not
the entire, intact SP—produced a profound, concentrationdependent decrease in transendothelial resistance (TER),
indicative of concentration dependence increases in permeability and barrier dysfunction (Fig. 4B).
DISCUSSION
The live SARS-CoV-2 can be lethal in K18-hACE2 but
not in WT mice and that, like what is observed in patients
with COVID-19, infected K18-hACE2 mice exhibit strong
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SARS-CoV-2 SPIKE PROTEIN SUBUNIT 1 INDUCES ACUTE LUNG INJURY
A
B
1.2
Normalized TER
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-CoV2 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-CoV2, severe acute respiratory syndrome coronavirus-2; CCL, C-C motif chemokine.
****
1.0
0.8
****
0.6
Control
10 nM S1SP
20 nM S1SP
50 nM S1SP
50 nM SP
0.4
0.2
0
5
10
15
20
25
Time (Hours)
inflammation and acute respiratory distress syndrome
(ARDS)-like pathology (5). Indeed, critically ill patients with
COVID-19 develop a strong inflammatory response, with
“cytokine storm” (25). The contribution of individual viral
elements to the pathology of COVID-19 remains unclear. The
SP of SARS-CoV is proinflammatory in vitro (26). Although
inactive by itself, SARS-CoV SP exacerbates lung injury (20).
Similarly, S1SP of SARS-CoV-2 is proinflammatory in isolated
peritoneal macrophages (27) and in human bronchial epithelial cells (28). However, these data do not necessarily predict
COVID-19-like lung injury or systemic inflammation.
Here, we show that the activated (cleaved) form of the
SP, S1SP, elicits strong pulmonary and systemic inflammatory responses in K18-hACE2 mice and milder responses in
WT mice, whereas the intact SP provokes minimal or no
responses.
The cytokine storm observed in this study exhibits critical
similarities with the inflammatory response elicited by the
live virus in K18-hACE2 mice (5, 29). Hamsters, intratracheally instilled with a pseudovirus expressing SP, display
ALI, endothelial dysfunction, and mitochondrial damage
mediated by the downregulation of ACE2 (30).
Our results indicate that S1SP can induce a milder form of
ALI in WT mice that do not express the human ACE2.
Recently, Asandei et al. (31) characterized a non-receptormediated mechanism for SARS-CoV-2 S1SP to destabilize cell
membranes. Furthermore, the human and mouse peptidase
M2 domain of ACE2 that binds S1SP share 85% homology
(32). Either or both observations could explain the milder
form of COVID-19 induced by S1SP in WT mice. This data
suggest that, despite the strong biological activity of S1SP in
wild-type animals, the hACE2 receptor is an important
L482
requirement for the pathogenesis of COVID-19 and thus, k18hACE2 mice represent a more valid and trustable model for
COVID-19. They further suggest that the SARS-CoV-2 S1SP
could play the role of a “toxin” and, when released in the vascular compartment after cell lysis, participate in the pathogenesis of systemic inflammation.
Additional work is required to determine the precise doseresponse relationships to S1SP in vivo, define S1SP pathogenicity in different cell lines, and further characterize the
diverse pathways of activity between hACE2 expressing and
WT mice.
HLMVEC exposed to S1SP displayed a rapid concentration-dependent decrease in TER, whereas the highest dose
of SP evoked a late and minimal decrease in resistance. As
the SP requires to be cleaved into its subunits to bind the
ACE2 receptor, it is not surprising that the intact SP evokes a
minimal and late response, which could be delayed due to
the time required for the cleavage, and lesser because of the
small amounts of proteases present in this in vitro model.
Here, we demonstrate that intratracheal instillation of a
single element of SARS-CoV-2, S1SP, in K18-hACE2 transgenic mice induces COVID-19-like local (lung) and systemic
inflammatory responses. This model will allow the preclinical investigation of potential countermeasures and of the
long-term effects of the inflammatory response provoked by
SARS-CoV-2.
ACKNOWLEDGMENTS
We thank the Eastern Virginia Medical School (EVMS),
Department of Anatomy and Pathology Laboratory for lung tissue
processing and staining. We thank Betsy Gregory for outstanding
technical assistance.
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SARS-CoV-2 SPIKE PROTEIN SUBUNIT 1 INDUCES ACUTE LUNG INJURY
GRANTS
10.
This work was supported by the Old Dominion Research
Foundation and the Fiske Drug Discovery Laboratory at the
University of Virginia.
DISCLOSURES
11.
12.
No conflicts of interest, financial or otherwise, are declared by
the authors.
13.
AUTHOR CONTRIBUTIONS
J.S.L., P.E.M., and J.D.C. conceived and designed research;
R.M.L.C.B., P.A.S., and E.R.S. performed experiments; R.M.L.C.B.,
P.A.S., E.R.S., and J.D.C. analyzed data; E.R.S. and J.D.C. interpreted results of experiments; R.M.L.C.B., P.A.S., and E.R.S. prepared figures; R.M.L.C.B. and P.A.S. drafted manuscript; E.R.S.,
J.S.L., P.E.M., and J.D.C. edited and revised manuscript; R.M.L.C.B.,
P.A.S., E.R.S., J.S.L., P.E.M., and J.D.C. approved final version of
manuscript.
15.
REFERENCES
1.
2.
3.
4.
5.
6.
7.
8.
9.
14.
Zhou P, Yang X-L, Wang X-G, Hu B, Zhang L, Zhang W, Si HR, Zhu
Y, Li B, Huang CL, Chen HD, Chen J, Luo Y, Guo H, Jiang RD, Liu
MQ, Chen Y, Shen XR, Wang X, Zheng XS, Zhao K, Chen QJ, Deng
F, Liu LL, Yan B, Zhan FX, Wang YY, Xiao GF, Shi ZL. A pneumonia
outbreak associated with a new coronavirus of probable bat origin.
Nature 579: 270–273, 2020. doi:10.1038/s41586-020-2012-7.
Horby P, Lim WS, Emberson J, Mafham M, Bell J, Linsell L, Staplin
N, Brightling C, Ustianowski A, Elmahi E, Prudon B, Green C,
Felton T, Chadwick D, Rege K, Fegan C, Chappell LC, Faust SN,
Jaki T, Jeffery K, Montgomery A, Rowan K, Juszczak E, Baillie
JK, Haynes R, Landray MJ; RECOVERY Collaborative Group.
Effect of dexamethasone in hospitalized patients with COVID-19:
preliminary report (Preprint). medRxiv, 2020. doi:10.1101/2020.
06.22.20137273.
rezMunster VJ, Feldmann F, Williamson BN, van Doremalen N, Pe
rez L, Schulz J, Meade-White K, Okumura A, Callison J,
Pe
Brumbaugh B, Avanzato VA, Rosenke R, Hanley PW, Saturday G,
Scott D, Fischer ER, de Wit E. Respiratory disease in rhesus macaques inoculated with SARS-CoV-2. Nature 585: 268–272, 2020.
doi:10.1038/s41586-020-2324-7.
Sia SF, Yan L-M, Chin AWH, Fung K, Choy K-T, Wong AYL,
Kaewpreedee P, Perera RAPM, Poon LLM, Nicholls JM, Peiris M,
Yen H-L. Pathogenesis and transmission of SARS-CoV-2 in golden
hamsters. Nature 583: 834–838, 2020. doi:10.1038/s41586-0202342-5.
Oladunni FS, Park J-G, Pino PA, Gonzalez O, Akhter A, Allue
Guardia A, et al. Lethality of SARS-CoV-2 infection in K18 human angiotensin-converting enzyme 2 transgenic mice. Nat Commun 11:
6122, 2020. doi:10.1038/s41467-020-19891-7.
Bao L, Deng W, Huang B, Gao H, Liu J, Ren L, et al. The pathogenicity of SARS-CoV-2 in hACE2 transgenic mice. Nature 583: 830–833,
2020. doi:10.1038/s41586-020-2312-y.
Winkler ES, Bailey AL, Kafai NM, Nair S, McCune BT, Yu J, Fox JM,
Chen RE, Earnest JT, Keeler SP, Ritter JH, Kang L-I, Dort S,
Robichaud A, Head R, Holtzman MJ, Diamond MS. SARS-CoV-2
infection of human ACE2-transgenic mice causes severe lung
inflammation and impaired function. Nat Immunol 21: 1327–1335,
2020 [Erratum in Nat Immunol 21: 1470, 2020]. doi:10.1038/s41590020-0778-2.
Karki R, Sharma BR, Tuladhar S, Williams EP, Zalduondo L, Samir P,
Zheng M, Sundaram B, Banoth B, Malireddi RKS, Schreiner P,
Neale G, Vogel P, Webby R, Jonsson CB, Kanneganti T-D.
Synergism of TNF-a and IFN-c triggers inflammatory cell death, tissue
damage, and mortality in SARS-CoV-2 infection and cytokine shock
syndromes. Cell 184: 149–168.e17, 2021. doi:10.1016/j.cell.2020.11.025.
Zafer MM, El-Mahallawy HA, Ashour HM. Severe COVID-19 and sepsis: immune pathogenesis and laboratory markers. Microorganisms 9:
159, 2021. doi:10.3390/microorganisms9010159.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
Ou X, Zheng W, Shan Y, Mu Z, Dominguez SR, Holmes KV, Qian Z.
Identification of the fusion peptide-containing region in betacoronavirus spike glycoproteins. J Virol 90: 5586–5600, 2016. doi:10.1128/
JVI.00015-16.
Li F, Li W, Farzan M, Harrison SC. Structure of SARS coronavirus
spike receptor-binding domain complexed with receptor. Science
309: 1864–1868, 2005. doi:10.1126/science.1116480.
Millet JK, Whittaker GR. Host cell entry of Middle East respiratory
syndrome coronavirus after two-step, furin-mediated activation of
the spike protein. Proc Natl Acad Sci USA 111: 15214–15219, 2014.
doi:10.1073/pnas.1407087111.
Bertram S, Dijkman R, Habjan M, Heurich A, Gierer S, Glowacka I,
Welsch K, Winkler M, Schneider H, Hofmann-Winkler H, Thiel V,
€ hlmann S. TMPRSS2 activates the human coronavirus 229E for
Po
cathepsin-independent host cell entry and is expressed in viral target cells in the respiratory epithelium. J Virol 87: 6150–6160, 2013.
doi:10.1128/JVI.03372-12.
Bertram S, Glowacka I, M€
uller MA, Lavender H, Gnirss K,
Nehlmeier I, Niemeyer D, He Y, Simmons G, Drosten C, Soilleux
€ hlmann S. Cleavage and activation of the
EJ, Jahn O, Steffen I, Po
severe acute respiratory syndrome coronavirus spike protein by
human airway trypsin-like protease. J Virol 85: 13363–13372, 2011.
doi:10.1128/JVI.05300-11.
€mer-K€
Gierer S, Bertram S, Kaup F, Wrensch F, Heurich A, Kra
uhl
A, Welsch K, Winkler M, Meyer B, Drosten C, Dittmer U, von Hahn
€ hlmann S. The spike protein of the
T, Simmons G, Hofmann H, Po
emerging betacoronavirus EMC uses a novel coronavirus receptor
for entry, can be activated by TMPRSS2, and is targeted by neutralizing antibodies. J Virol 87: 5502–5511, 2013. doi:10.1128/JVI.00128-13.
Walls AC, Park Y-J, Tortorici MA, Wall A, McGuire AT, Veesler D.
Structure, function, and antigenicity of the SARS-CoV-2 spike glycoprotein. Cell 181: 281–292.e6, 2020 [Erratum in Cell 183: 1735, 2020].
doi:10.1016/j.cell.2020.02.058.
Gaddam RR, Chambers S, Bhatia M. ACE and ACE2 in inflammation: a tale of two enzymes. Inflamm Allergy Drug Targets 13: 224–
234, 2014. doi:10.2174/1871528113666140713164506.
Imai Y, Kuba K, Rao S, Huan Y, Guo F, Guan B, Yang P, Sarao R,
Wada T, Leong-Poi H, Crackower MA, Fukamizu A, Hui CC, Hein L,
Uhlig S, Slutsky AS, Jiang C, Penninger JM. Angiotensin-converting
enzyme 2 protects from severe acute lung failure. Nature 436: 112–
116, 2005. doi:10.1038/nature03712.
Haga S, Yamamoto N, Nakai-Murakami C, Osawa Y, Tokunaga K,
Sata T, Yamamoto N, Sasazuki T, Ishizaka Y. Modulation of TNFalpha-converting enzyme by the spike protein of SARS-CoV and ACE2
induces TNF-alpha production and facilitates viral entry. Proc Natl Acad
Sci USA 105: 7809–7814, 2008. doi:10.1073/pnas.0711241105.
Kuba K, Imai Y, Rao S, Gao H, Guo F, Guan B, Huan Y, Yang P,
Zhang Y, Deng W, Bao L, Zhang B, Liu G, Wang Z, Chappell M, Liu
Y, Zheng D, Leibbrandt A, Wada T, Slutsky AS, Liu D, Qin C, Jiang
C, Penninger JM. A crucial role of angiotensin converting enzyme 2
(ACE2) in SARS coronavirus-induced lung injury. Nat Med 11: 875–
879, 2005. doi:10.1038/nm1267.
Marinova M, Solopov P, Dimitropoulou C, Colunga Biancatelli
RML, Catravas JD. Acute exposure of mice to hydrochloric acid
leads to the development of chronic lung injury and pulmonary fibrosis. Inhal Toxicol 31: 147–160, 2019. doi:10.1080/08958378.
2019.1624895.
Matute-Bello G, Downey G, Moore BB, Groshong SD, Matthay MA,
Slutsky AS, Kuebler WM; Acute Lung Injury in Animals Study
Group. An official American Thoracic Society workshop report: features and measurements of experimental acute lung injury in animals. Am J Respir Cell Mol Biol 44: 725–738, 2011. doi:10.1165/
rcmb.2009-0210ST.
Catravas JD, Snead C, Dimitropoulou C, Chang ASY, Lucas R,
Verin AD, Black SM. Harvesting, identification and barrier function
of human lung microvascular endothelial cells. Vascul Pharmacol
52: 175–181, 2010. doi:10.1016/j.vph.2009.12.009.
Barabutis N, Dimitropoulou C, Gregory B, Catravas JD. Wild-type
p53 enhances endothelial barrier function by mediating RAC1 signalling and RhoA inhibition. J Cell Mol Med 22: 1792–1804, 2018.
doi:10.1111/jcmm.13460.
Rumende CM, Susanto EC, Sitorus TP. The management of cytokine storm in COVID-19. Acta Med Indones 52: 306–313, 2020.
AJP-Lung Cell Mol Physiol doi:10.1152/ajplung.00223.2021 www.ajplung.org
Downloaded from journals.physiology.org/journal/ajplung (201.160.193.051) on August 16, 2021.
L483
SARS-CoV-2 SPIKE PROTEIN SUBUNIT 1 INDUCES ACUTE LUNG INJURY
26.
27.
28.
29.
L484
Dosch SF, Mahajan SD, Collins AR. SARS coronavirus spike protein-induced innate immune response occurs via activation of the
NF-kappaB pathway in human monocyte macrophages in vitro.
Virus Res 142: 19–27, 2009. doi:10.1016/j.virusres.2009.01.005.
Shirato K, Kizaki T. SARS-CoV-2 spike protein S1 subunit induces
pro-inflammatory responses via toll-like receptor 4 signaling in murine and human macrophages. Heliyon 7: e06187, 2021. doi:10.1016/
j.heliyon.2021.e06187.
Hsu AC-Y, Wang G, Reid AT, Veerati PC, Pathinayake PS, Daly K,
Mayall JR, Hansbro PM, Horvat JC, Wang F, Wark PA. SARS-CoV-2
Spike protein promotes hyper-inflammatory response that can be
ameliorated by Spike-antagonistic peptide and FDA-approved ER
stress and MAP kinase inhibitors in vitro (Preprint). bioRxiv, 2020.
doi:10.1101/2020.09.30.317818.
Yinda CK, Port JR, Bushmaker T, Offei Owusu I, Purushotham JN,
Avanzato VA, Fischer RJ, Schulz JE, Holbrook MG, Hebner MJ,
Rosenke R, Thomas T, Marzi A, Best SM, de Wit E, Shaia C, van
30.
31.
32.
Doremalen N, Munster VJ. K18-hACE2 mice develop respiratory
disease resembling severe COVID-19. PLoS Pathog 17: e1009195,
2021. doi:10.1371/journal.ppat.1009195.
Lei Y, Zhang J, Schiavon CR, He M, Chen L, Shen H, Zhang Y, Yin
Q, Cho Y, Andrade L, Shadel GS, Hepokoski M, Lei T, Wang H,
Zhang J, Yuan JX-J, Malhotra A, Manor U, Wang S, Yuan Z-Y, Shyy
JY-J. SARS-CoV-2 spike protein impairs endothelial function via
downregulation of ACE 2. Circ Res 128: 1323–1326, 2021. doi:10.1161/
CIRCRESAHA.121.318902.
Asandei A, Mereuta L, Schiopu I, Park J, Seo CH, Park Y, Luchian
T. Non-receptor-mediated lipid membrane permeabilization by the
SARS-CoV-2 spike protein S1 subunit. ACS Appl Mater Interfaces 12:
55649–55658, 2020. doi:10.1021/acsami.0c17044.
Soldatov VO, Kubekina MV, Silaeva YY, Bruter AV, Deykin AV. On
the way from SARS-CoV-sensitive mice to murine COVID-10 model.
Res Results Pharmacol 6: 1–7, 2020. doi:10.3897/rrpharmacology.
6.53633.
AJP-Lung Cell Mol Physiol doi:10.1152/ajplung.00223.2021 www.ajplung.org
Downloaded from journals.physiology.org/journal/ajplung (201.160.193.051) on August 16, 2021.