A Vicious Cycle: In Severe and Critically Ill COVID-19 Patients

Abstract

The coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus, is one of the fastest-evolving viral diseases that has instigated a worldwide pandemic. Severe inflammatory syndrome and venous thrombosis are commonly noted in COVID-19 patients with severe and critical illness, contributing to the poor prognosis. Interleukin (IL)-6, a major complex inflammatory cytokine, is an independent factor in predicting the severity of COVID-19 disease in patients. IL-6 and tumor necrosis factor (TNF)-α participate in COVID-19-induced cytokine storm, causing endothelial cell damage and upregulation of plasminogen activator inhibitor-1 (PAI-1) levels. In addition, IL-6 and PAI-1 form a vicious cycle of inflammation and thrombosis, which may contribute to the poor prognosis of patients with severe COVID-19. Targeted inhibition of IL-6 and PAI-1 signal transduction appears to improve treatment outcomes in severely and critically ill COVID-19 patients suffering from cytokine storms and venous thrombosis. Motivated by studies highlighting the relationship between inflammatory cytokines and thrombosis in viral immunology, we provide an overview of the immunothrombosis and immunoinflammation vicious loop between IL-6 and PAI-1. Our goal is that understanding this ferocious circle will benefit critically ill patients with COVID-19 worldwide.

Full text

A Vicious Cycle: In Severe and
Critically Ill COVID-19 Patients
Peifeng Huang
1‡
, Qingwei Zuo
1‡
, Yue Li
2‡
, Patrick Kwabena Oduro
3‡
, Fengxian Tan
1
,
Yuanyuan Wang
1
, Xiaohui Liu
1
, Jing Li
2
, Qilong Wang
3
, Fei Guo
4
*,
Yue Li
3
*
†
and Long Yang
1,5
*
1
School of Integrative Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin, China,
2
School of Department of
Clinical Training and Teaching of Traditional Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin,
China,
3
State Key Laboratory of Component-Based Chinese Medicine, Tianjin University of Traditional Chinese Medicine,
Tianjin, China,
4
National Health Commission of the People’s Republic of China Key Laboratory of Systems Biology of
Pathogens, Institute of Pathogen Biology and Center for AIDS Research, Chinese Academy of Medical Sciences & Peking
Union Medical College, Beijing, China,
5
Research Center for Infectious Diseases, Tianjin University of Traditional Chinese
Medicine, Tianjin, China
The coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory
syndrome coronavirus 2 (SARS-CoV-2) virus, is one of the fastest-evolving viral
diseases that has instigated a worldwide pandemic. Severe inflammatory syndrome
and venous thrombosis are commonly noted in COVID-19 patients with severe and
critical illness, contributing to the poor prognosis. Interleukin (IL)-6, a major complex
inflammatory cytokine, is an independent factor in predicting the severity of COVID-19
disease in patients. IL-6 and tumor necrosis factor (TNF)-aparticipate in COVID-19-
induced cytokine storm, causing endothelial cell damage and upregulation of
plasminogen activator inhibitor-1 (PAI-1) levels. In addition, IL-6 and PAI-1 form a
vicious cycle of inflammation and thrombosis, which may contribute to the poor
prognosis of patients with severe COVID-19. Targeted inhibition of IL-6 and PAI-1
signal transduction appears to improve treatment outcomes in severely and critically ill
COVID-19 patients suffering from cytokine storms and venous thrombosis. Motivated by
studies highlighting the relationship between inflammatory cytokines and thrombosis in
viral immunology, we provide an overview of the immunothrombosis and
immunoinflammation vicious loop between IL-6 and PAI-1. Our goal is that
understanding this ferocious circle will benefit critically ill patients with COVID-
19 worldwide.
Keywords: COVID-19, PAI-1, IL-6, inflammatory reaction, venous thrombosis, tocilizumab, endothelial cells
Abbreviations: ACE, Angiotensin-converting enzyme; COVID-19, Coronavirus disease 2019; ECs, Endothelial cells; EGFR,
Epidermal growth factor receptor; HFD, High-fat diet; HPMECs, Human pulmonary microvascular endothelial cells; ICU,
Intensive care unit; IL, Interleukin; IL-6R, Interleukin-6 receptor; JAK, Janus kinase; LPS, Lipopolysaccharide; MD2, Myeloid
differentiation protein 2; NF-kB, Nuclear factor of kappa B; PAI-1, Plasminogen activator inhibitor 1; STAT3, Signal
transducer and activator of transcription 3; TCZ, Tocilizumab; TLR, Toll-like receptors; TNF, Tumor necrosis factor; tPA,
Tissue plasminogen activator; uPA, Urokinase-type plasminogen activator.
Frontiers in Immunology | www.frontiersin.org June 2022 | Volume 13 | Article 9306731
Edited by:
Chang Li,
Chinese Academy of Agricultural
Sciences (CAAS), China
Reviewed by:
Zhanbo Zhu,
Heilongjiang Bayi Agricultural
University, China
Wentao Qiao,
Nankai University, China
*Correspondence:
Fei Guo
guofei@ipb.pumc.edu.cn
Yue Li
liyue2018@tjutcm.edu.cn
Long Yang
long.yang@tjutcm.edu.cn
†
ORCID:
Yue Li
orcid.org/0000-0001-8198-9911
‡
These authors have contributed
equally to this work and share
first authorship
Specialty section:
This article was submitted to
Molecular Innate Immunity,
a section of the journal
Frontiers in Immunology
Received: 28 April 2022
Accepted: 12 May 2022
Published: 15 June 2022
Citation:
Huang P, Zuo Q, Li Y, Oduro PK,
Tan F, Wang Y, Liu X, Li J, Wang Q,
Guo F, Li Y and Yang L (2022)
A Vicious Cycle: In Severe and
Critically Ill COVID-19 Patients.
Front. Immunol. 13:930673.
doi: 10.3389/fimmu.2022.930673
REVIEW
published: 15 June 2022
doi: 10.3389/fimmu.2022.930673

INTRODUCTION
Since late December, coronavirus disease 2019 (COVID-19) (1)
has spread worldwide and instigated a pandemic. Globally, as of
April 12, 2022, more than five hundred million people have been
diagnosed with COVID-19 disease, including more than 6
million deaths from the disease (WHO, https://covid19.who.
int/), posing a great challenge to the health system around the
world. The causative agent of the disease is the SARS-CoV-2
virus. Based on the clinical presentation of the COVID-19
disease, the mild-to-moderate disease accounts for 81% of
COVID-19 infections and is accompanied by symptoms such
as cough, fever, fatigue, and others. Meanwhile, only about 14%
of cases have severe symptoms such as dyspnea and hypoxemia,
while 5% present with respiratory failure, shock failure, multiple
organ failure, and other severe conditions that can result in
death. In addition, 14.8% of patients are classified as severe or
critically ill patients (Table 1)(2). Emerging laboratory and
pathological examination data indicate that cytokine storms and
thrombosis were closely related to the disease progression,
accounting for the poor prognosis in COVID-19 patients (3–8).
A significant reduction in spontaneous clot dissolution after
activation of the external clotting pathway and increased resistance
to tissue plasminogen activator (tPA) suggests a potential link
between fibrinolytic disorder and thrombosis (9). Serum
proteomics studies in patients with COVID-19 have found that
abnormal increases in IL-6 correlate with increases in the
coagulation and complement cascade components (10). PAI-1 is
a serine protease inhibitor that acts as a principal inhibitor of tPA
and urokinase-type plasminogen activator (uPA) to inhibit
fibrinolysis. Based on PAI-1’s primary function, diseases, or
disorders that increase PAI-1 levels appear to result in high
coagulation states (11–13). Interestingly, in patients with mild-
to-moderate disease, plasma levels of PAI-1 were normal
compared to critically ill COVID-19 patients (14,15). However,
reports from studies suggested that PAI-1 levels significantly
increase in critically ill (14) and hospitalized COVID-19 patients
(Figure 1). In addition, previous analyses on the detection of
inflammatory and prethrombotic biomarkers in the blood showed
significant differences between IL-6 and PAI-1 levels. The mean
concentration of IL-6 in the non-severe COVID-19 group was
430.3 pg/ml, whereas that of the control group was 419.5 pg/ml.
Meanwhile, the concentration of IL-6 in severe COVID-19 and
death group was 1,463 and 2,200 pg/ml, respectively (14). PAI-1 is
a widely recognized biomarker of endothelial dysfunction and has
been shown that increased concentration is associated with the
severity of the disease (16,17). The expression of PAI-1 may
reflect the severity of SARS-CoV-2 infection to some extent (18).
The plasma concentration of PAI-1 detected in patients with
severe COVID-19 was 713.3 ng/ml, while in the COVID-19
death group, it was 1,223.5 ng/ml. Then again, in the non-severe
COVID-19 group, the plasma concentration of PAI-1 was 465.2
ng/ml and that of healthy donors was 183.7 ng/ml (14). It is
important to note that severe and critically ill patients with
COVID-19oftensufferfromunderlyingdiseases(19,20).
Evidence has also suggested that most of the underlying diseases
present with elevated levels of PAI-1 (21). For example, among
diabetes and acute cerebral infarction patients without COVID-19,
PAI-1 levels averaged 36.5 and 63.95 ng/ml (22,23). Nonetheless,
COVID-19-infected individuals have significantly higher levels of
PAI-1 than those with diabetes or acute cerebral infarction,
providing indirect evidence that COVID-19 could increase PAI-
1levels(Table 2).
Studies on coexpression-induced IL-6 and PAI-1 through the
nuclear factor-kappa B (NF-kB) pathway and ligand-dependent
epidermal growth factor receptor (EGFR) activation confirmed a
significant correlation between IL-6 and PAI-1 (26). The same
phenomenon has revealed significant differences between IL-6
and PAI-1 levels in severe and mild-to-moderate COVID-19
FIGURE 1 | SARS-Co-2 upregulates plasma IL-6, TNF- a, and PAI-1 levels. The levels of IL-6, PAI-1, and TNF-ain the serum of severely and critically ill COVID-19
patients with SARS-CoV-2 pulmonary infection via the respiratory tract were significantly increased.
TABLE 1 | The distribution of age, degree, and fatality rate of COVID-19 (2).
Categories Subgroup Cases Distribution
Age ≥80 years 1,408 3%
30–79 years 38,680 87%
10–29 years 4,168 6%
<10 years 416 1%
Degree Mild 36,160 81%
Severe 6,168 4%
Critically ill 2,087 5%
Fatality rate 44,672 confirmed cases 1,023 2.3%
Aged ≥80 years 208 14.8%
70–79 years 312 8.0%
Critically cases 1,023 49.0%
Bold values highlight the proportion and mortality of critically ill patients and emphasize the
lethality of COVID-19.
Huang et al. COVID-19 Patients Suffer Vicious Cycle
Frontiers in Immunology | www.frontiersin.org June 2022 | Volume 13 | Article 9306732

patients (14). Treatment with anti-TNFs can reduce the death
rate and poor outcomes of COVID-19 patients (27). Below, we
review the possible relationship between inflammatory levels and
thrombosis in severe and critically ill COVID-19 patients.
SARS-COV-2 RAISES THE EXPRESSION
OF PAI-1, IL-6, AND TNF-Α
The SARS-CoV-2 infection has a devastating effect on immune
regulation, leading to a life-threatening systemic inflammatory
syndrome called the cytokine storm. This systemic inflammatory
syndrome involves abnormal immune-cell hyperactivation and
uncontrolled release of circulatory cytokines. Elegant evidence
from the COVID-19 pandemic shows that IL-6 and TNF-aare
involved in the COVID-19-induced cytokine storm (28). In
severe disease, IL-6 and TNF-aare major contributing factors
that worsen the condition and cause poor clinical outcomes and
even death (29–31). IL-6 is a multifunctional cytokine capable of
transmitting cell signals. It is the main trigger of endothelial
cytokine storm and an intervention target for clinical therapy
(32,33). Almost all stromal cells and immune system cells can
produce IL-6, and the primary activator is IL-1bor TNF-a(34).
Toll-like receptor (TLR)-stimulated monocytes and
macrophages can also promote the expression of IL-6 (35).
During propagation of the SARS-CoV-2 virus, the envelope
spike glycoprotein of the SARS-CoV-2 virus attaches to the
angiotensin-converting enzyme (ACE)-2 on the target cell
surface, resulting in ACE-2 loss (36). ACE-2 is a negative
regulator that functions by activating tPA. ACE-2 deficiency
disrupts the effective ACE-2/angiotensin (1–7)/Mas receptor
axis, making Ang II more active and decreasing tPA activity,
prompting endothelial and smooth muscle cells to synthesize
and release PAI-1, leading to the balance of PAI-1/tPA to revert
to its prethrombotic state (37,38). Studies on intensive care unit
(ICU) patients with critically ill COVID-19 found that low
fibrinolysis was mainly associated with elevated PAI-1 levels
(39). The action of recombinant SARS-CoV-2 on the ACE-2
receptor is comparable to that of live viruses, and its spiking
glycoprotein induces the expression of PAI-1 in human
pulmonary microvascular endothelial cells (HPMECs) (40). In
individuals with severe COVID-19 illness, increased PAI-1
expression reduces tPA activity and increases thrombosis while
perhaps worsening the inflammatory response (Table 3).
PAI-1 Upregulates the Expressions of IL-6
and TNF-a
In several studies, PAI-1 has been found at the inflammatory site
after tissue damage (47,48). PAI-1 inhibitors reduce TNF-a
expression and, at the same time, decrease PAI-1 expression in
diabetic mice (49). PAI-1 upregulation may be related to its
capacity to activate macrophages. PAI-1 helps to regulate the
lipopolysaccharide (LPS)-induced inflammatory response in
NR8383 cells, possibly by influencing the TLR4-myeloid
differentiation protein 2 (MD-2)/NF-kBsignalingtransfer
pathway (50). PAI-1-induced TLR4 activation causes monocyte
macrophages to release significant quantities of IL-6 and TNF-a,
exacerbating the inflammatory response (51,52). This shows that
TLR4 is an essential medium for PAI-1 to activate macrophages
and promote TNF-aexpression. The expression spectrum of
macrophages stimulated by PAI-1 occurs 2 h after the peak
transcription of PAI-1 (53). PAI-1 can promote macrophage
activation and may also be an initial response gene
for predicting inflammation. PAI-1 promotes the recruitment
of monocytes/macrophages in tumor cells. Its lipoprotein-
receptor-related protein 1 interaction domain regulates
macrophage migration, whereas its C-terminal uPA interaction
domain auto-secretes IL-6 by activating the p38MAPK and NF-kB
pathway and inducing macrophage polarization (54). There was a
considerable increase in the expression of M1 macrophages in
obese mice caused by a high-fat diet (HFD), but PAI-1 deficiency
and PAI-039therapy prevented the development of these markers,
demonstrating that PAI-1 is required for macrophage
polarization. Meanwhile, PAI-1 activates TLR4, triggering a
robust inflammatory response in endothelial cells (ECs),
allowing ECs to continuously secrete IL-6 (55). PAI-1 may
interact with TLR4 to activate NF-kB, leading ECs to generate
TABLE 3 | The expressions of PAI-1 and IL-6 in severe COVID-19 patients.
Factors Expressing and working Reference
PAI-1 rSARS-CoV-2-S1 infect HPMECs exhibited robust
induction of PAI-1
(40)
Circulating levels of PAI-1 upregulate and function as an
independent predictor of the severity of COVID-19
disease in patients
(41)
Decreased the PAI-1 levels and alleviated critical illness in
severe COVID-19 patients
(42)
Significant expression of PAI-1 exists only in severe
COVID-19 patients and promotes patient thrombosis
(14)
Hypercoagulability and hypofibrinolysis are connected to
the elevated level of PAI-1 in COVID-19
(39)
IL-6 IL-6 can serve as an independent factor predictor of the
severity of COVID-19 disease in patients
(43–46)
Seroproteomics studies found IL-6 significant
upregulation, and IL-6 signal transduction is the most
upstream upregulation pathway in severe patients with
COVID-19 patients
(10)
IL-6 is the main trigger of endothelial cytokine storms in
COVID-19 patients
(32)
TABLE 2 | The expression of IL-6 and PAI-1 in COVID-19 and underlying diseases.
Disease IL-6 (mean
pg/ml)
PAI-1 (mean
ng/ml)
COVID-19 Healthy donors 419.5 183.7
Non-severe
COVID-19 group
430.3 465.2
Severe COVID-19
group
1463 713.3
Death group 2200 1,223.5
Type 2
diabetes
–<20 (24) 36.5
Acute cerebral
infarction
–<1,000 (25) 63.95
Huang et al. COVID-19 Patients Suffer Vicious Cycle
Frontiers in Immunology | www.frontiersin.org June 2022 | Volume 13 | Article 9306733

cytokines such as IL-6 (56,57). This shows that PAI-1 can
stimulate macrophages and endothelial cells in various ways,
promoting inflammatory responses (Table 4).
There is no clinical use of PAI-1 inhibitors in COVID-19
patients. However, it is worth noting that bortezomib upregulates
KLF2 to suppress PAI-1 expression and reduce EC damage in
HPME cells stimulated with rSARS-CoV-2-S1 glycoprotein (58).
The IL-6 Increases the Expression
of the PAI-1
Severe clotting disorder in patients with COVID-19 is closely
related to the increased risk of death (59–62). Venous
thromboembolism was prevalent in COVID-19 patients, with a
total incidence of 31% in 184 patients with severe COVID-19 (63),
and a preliminary autopsy on 11 of the COVID-19 patients revealed
thrombus in the pulmonary arterioles (64). The D-dimer is a fibrin
degradation product used as an alternative marker of fibrinolysis
and is often elevated in thrombotic events (65). Relevant studies on
COVID-19 report that D-dimer elevation is a prevalent feature (66).
Low fibrinolysis is the primary cause of increased blood viscosity
and is associated with elevated PAI-1 levels (39). PAI-1 circulating
levels may be used as an independent predictor of severity in
COVID-19 patients (41), and regulating PAI-1 expression can
benefitpatientswithCOVID-19(42).
Alongside PAI-1, IL-6 is an independent predictor of COVID-
19 severity (43–46). IL-6 levels have a substantial predictive value
for mortality in COVID-19 ICUs (67). Patients with severe
COVID-19 have considerable IL-6 overexpression, and IL-6
signal transduction is the most upregulated pathway in COVID-
19 patients (10). IL-6 may have a significant role in the progression
of severe COVID-19 disease in patients. PAI-1 expression is only
found in severe COVID-19 patients and increases thrombosis
(14). PAI-1 is linked to elevated levels of IL-6 in critically ill
COVID-19 patients. IL-6 signals through two central pathways.
The first is the classic cis signaling, and the second is the trans-
signaling. In the classic cis pathway, IL-6 attaches to cells, mainly
immune cells, expressing the membrane-bound interleukin-6
receptor (IL-6R) to initiate a downstream signaling response (68,
69). On the other hand, in trans-signaling, IL-6 binds to the
soluble form of IL-6R, which is released from IL-6R expressing cell
surfaces by proteolysis and IL-6R mRNA to form an exciting
complex that associates with membrane-bound gp130 (70–72). In
the presence of high circulating levels of IL-6, trans-signaling
typically occurs. For instance, ECs express the membrane-bound
gp130 but not the membrane-bound IL-6R (73–76), allowing for
IL-6/soluble-IL-6R/gp130 downstream signaling activation.
The detection of PAI-1 expression before and after tocilizumab
(TCZ) treatment demonstrates that IL-6 signaling transduction
can promote PAI-1 expression in ECs (18,42). LPS stimulates the
NF-kB classical pathway to increase the PAI-1 expression and
promote alveolar hypercoagulation and fibrinolysis inhibitory
states. PAI-1 expression is dramatically reduced following NF-
kB knockout (77,78), indicating that the NF-kB pathway can
control PAI-1 expression to some extent. At the same time,
elevated plasma IL-6 levels promote NF-kB activation (79),
resulting in EC-induced PAI-1 overexpression. In hepatocytes,
IL-6 signals via the Janus kinase (JAK) pathway to promote C/
EBPd-induced PAI-1 expression (80). In addition, IL-6 signals and
activates the IL-6R/signal transducer and activator of transcription
3 (STAT3) pathway (54), which can indirectly upregulate PAI-1
via miR-34a (81). TNF-acan also upregulate PAI-1 (82).
However, it is less commonly documented in the literature, and
the mechanism remains unknown (Table 5).
According to the preceding discussion, elevated and
persistent IL-6, TNF-a, and PAI-1 levels in severe COVID-19
patients potentially generate a vicious cycle of inflammatory
response and thrombosis (Figure 2).
CLINICAL SIGNIFICANCE
The probable inflammatory response and thrombus interaction
mechanisms are first described in critically ill COVID-19
patients. TCZ is a recombinant human-resistant human IL-6R
IgG1 monoclonal antibody (83). The use of TCZ in critically ill
COVID-19 patients can decrease PAI-1 levels and improve the
condition of severe COVID-19 patients (42). TCZ is authorized
for the treatment of rheumatoid arthritis (84) and systemic
juvenile idiopathic arthritis (85) because it selectively binds
soluble and membrane-bound IL-6 receptors and inhibits IL-6-
mediated classic cis and trans-signaling (86). IL-6 levels in severe
COVID-19 patients are significantly higher than in other
patients, prompting several researchers to recommend TCZ to
TABLE 4 | PAI-1 upregulate the expressions of IL-6 and TNF-a.
Targets Cell/host Model Mechanism Reference
PAI-1 upregulates
TNF-ɑ
NR8383 cells Inflammatory model induced by LPS TLR4-MD-2/NF-kB signaling transduction pathway (50)
Mouse Type 2 diabetes mellitus PAItrap3 decreases the levels of both PAI-1 and TNF-a(49)
Mouse Systemic inflammation model PAI-1 regulates inflammatory responses through TLR4
mediated macrophage activation
(53)
PAI-1 upregulates
IL-6
C57 mouse/HT-1080 fibrosarcoma
cancer cell line
Rag1
−/−
PAI1
−/−
/Rag1
−/−
PAI-1 mice PAI-1 promotes the recruitment and polarization of
macrophages in cancer
(54)
Microvascular (MIC) and
macrovascular (MAC) endothelial
cells (ECs)
Inflammatory model induced by LPS PAI-1 was necessary for macrophage polarization (55)
Mice/human aortic endothelial cells
(HAECs)
Endotoxemia of mouse/Inflammatory
model induced by LPS
PAI-1 combines with TLR4 to promote NF-kB
activation so that ECs produce chemokines, such as
IL-6
(56,57)
Huang et al. COVID-19 Patients Suffer Vicious Cycle
Frontiers in Immunology | www.frontiersin.org June 2022 | Volume 13 | Article 9306734

inhibit IL-6 signaling in patients with severe COVID-19 to
improve patient symptoms (35,87). According to reports, TCZ
can be used as an alternative therapy for COVID-19 patients who
are at risk of cytokine storms (88). It is advised that in critically ill
patients with elevated IL-6 levels, a repeated dose of TCZ will be
necessary to reduce IL-6 levels significantly (88). However, TCZ
is ineffective for patients with moderate COVID-19 (89) but can
improve clinical symptoms in severely and critically ill COVID-
19 patients (90). Breathing and bilateral diffuse turbidity
disappear by intravenous TCZ in severe COVID-19 patients
with pneumonia and acute respiratory distress syndrome
(ARDS) (91). Unfortunately, thrombosis in severe COVID-19
patients was not mentioned. PAI-1 inhibition can improve the
level of IL-6 and the damage to ECs. Treatment with TM5614
(PAI-1 inhibitor) eliminates the elevated circulating levels of
PAI-1 and thrombin in plasma produced by particulate matter
(PM) 2.5 (92). Meanwhile, TM5614 significantly reduces the
elevated level of IL-6 (92). Bortezomib, a proteasomal
degradation inhibitor, enhances KLF2, decreases PAI-1
expression, and reduces EC damage in HPMECs stimulated
with rSARS-CoV-2-S1 glycoprotein (58). PAI-1 may have a
role in prothrombotic events and inflammation in COVID-19
patients. This asserts the vicious cycle of PAI-1 and IL-6 in
COVID-19.
CONCLUSION AND
FUTURE PERSPECTIVE
In this review, we briefly discussed the possible link between
elevated IL-6 levels and thrombosis in COVID-19 patients. From
non-viral contexts, the link between PAI-1 and IL-6 forms an
inflammatory–thrombus circuit (42). PAI-1 and IL-6 were not
shown to be strongly connected in COVID-19 case reports,
although autopsy demonstrated substantial damage to ECs (93).
In COVID-19patients, inflammation and thrombosis are two of the
FIGURE 2 | Relationship between PAI-1 and IL-6 after SARS-Co-2 infection. SARS-CoV-2 binds to ACE-2 on the target cell surface, resulting in the loss of ACE-2.
ACE-2 is a negative regulator that works by activating tPA. ACE-2 deficiency loses the effective ACE-2/angiotensin (1–7)/Mas receptor axis and increases the level of
Ang1. ACE converts Ang I to Ang II and decreases tPA activity, causing endothelial cells and smooth muscle cells to synthesize and release PAI-1. Ang II binds to
AT1/AT2 to break the balance of PAI-1/tPA to its prethrombotic state. Elevated levels of PAI-1 in severely and critically ill COVID-19 patients may upregulate IL-6
expression through TLR4/NF-kB pathway and activate macrophages to upregulate IL-6 and TNF-aexpression. At the same time, TNF-acan also upregulate PAI-1
expression. IL-6 upregulates the expression of PAI-1 via STAT3/miR-29a.
TABLE 5 | IL-6 and TNF-apromote the expression of PAI-1.
Promote expres-
sion
Cell/host Model Possible mechanism Reference
TNF-aupregulates
PAI-1
Clinic patients Atherosclerosis TNF-ainhibition with infliximab decreases PAI-1 Ag level (82)
IL-6 upregulates
PAI-1
Clinic patients/HUVECs Patients diagnosed with
CRS from sepsis
Tocilizumab treatment decreased the PAI-1 levels and alleviated
critical illness in severe COVID-19 patients
(18,42)
Human hepatoma/primary mouse
hepatocytes
–IL-6 induces PAI-1 expression through JAK signaling pathways
converging on C/EBPd
(80)
Human colorectal cancer/breast
cancer/prostate cancer
Rag1
−/−
PAI1
−/−
/Rag1
−/
−
PAI-1 mice (54)
IL-6 activates the IL-6/STAT3 pathway and, through miR-34a,
upregulates PAI-1
(54,81)
Huang et al. COVID-19 Patients Suffer Vicious Cycle
Frontiers in Immunology | www.frontiersin.org June 2022 | Volume 13 | Article 9306735

most significant deleterious responses (94,95). The development of
blood clotsin the heart can be explainedby the distributionof ECs in
the heart and by the above process (96). In critically ill COVID-19
patients, EC dysfunction increases PAI-1 expression (17) and
promotes macrophage recruitment and activation (54). This
raises the amount of IL-6 and TNF-ain the blood, increasing the
odds of a “cytokine storm”(28). TCZ can decrease IL-6 signal
transduction via IL-6R and soluble IL-6R. TNF-a, on the other
hand, stimulates endothelial PAI-1 production and activates
macrophages, exposing ECs to prominent levels of IL-6 and TNF-
aand causing sustained tissue and organ damage. In thrombosis,
therapeutic use of thrombolytic treatment merely lowers fibrin
production. Inability to directly suppress PAI-1 expression and
break the vicious cycle between PAI-1 and IL-6 results in serum
PAI-1 and IL-6 buildup, facilitating tissue damage and thrombosis
development. IL-6 trans-signaling has been shown to increase PAI-
1 expression. When IL-6 is coupled with soluble IL-6R and gp130, it
activates the downstream JAK/STAT signal pathway and promotes
the expression of IL-6 and PAI-1 (54,97,98)(Figure 3). STAT3-
dependent transcription inhibition significantly reduces VEGF-
induced vascular permeability in zebrafish, mouse, and human
endothelial cells (99). Increased endothelial cell permeability can
aggravate pulmonary edema and dyspnea in COVID-19 patients
(100). Although the connectionbetween PAI-1 and IL-6 has not yet
been shown,the possibility of a malignantinteraction between PAI-
1 and IL-6 in critically ill COVID-19 patients should not be
overlooked. PAI-1 and IL-6 may produce a vicious cycle in which
their expression is mutually induced, but the mechanism involved
remains unclear. Thrombosis and inflammatory responses in
patients with severe COVID-19 are discussed from a new
perspective, which provides innovative ideas for future studies.
AUTHOR CONTRIBUTIONS
All authors have read and approved the manuscript. FG, YL
(11th author), and LY supervised and edited the final
manuscript with comments from co-authors. PH, QZ, YL
(3rd author), and PO conceptualized and wrote the initial
draft, which was further reviewed and edited by FT, YW, XL,
JL, and QW for intellectual content. All authors provided
crucial revisions in subsequent drafts.
FUNDING
This work was supported by the Tianjin Municipal Education
Commission Scientific Research Project (Natural Science, Grant
No. 2019ZD11 to LY), Science and Technology Program of
Tianjin (21ZYJDJC00070), the National Key Research and
Development Program of China (2019YFC1708803), and
Innovation Team and Talents Cultivation Program of National
Administration of Traditional Chinese Medicine (ZYYCXTD-
C-202203).
ACKNOWLEDGMENTS
We thank LY, YL, and FG for their assistance with
conceptualization and helpful discussion. We are also grateful
to the Tianjin Municipal Education Commission Scientific
Research Project for the funding support.
FIGURE 3 | IL-6 promotes PAI-1 expression via trans signaling. High concentration of IL-6 combined with soluble IL-6R can activate the JAK/STAT3 signal pathway
through gp130 and upregulate the expression of PAI-1 and IL-6. TCZ canreduce the expression of PAI-1 and IL-6 by inhibiting the binding of IL-6 and soluble IL-6R.
Huang et al. COVID-19 Patients Suffer Vicious Cycle
Frontiers in Immunology | www.frontiersin.org June 2022 | Volume 13 | Article 9306736

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Huang et al. COVID-19 Patients Suffer Vicious Cycle
Frontiers in Immunology | www.frontiersin.org June 2022 | Volume 13 | Article 9306739