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IL-6: relevance for immunopathology of SARS-CoV-2

E.O. Gubernatorova, E.A. Gorshkova, A.I. Polinova, M.D. Drutskaya

PII: S1359-6101(20)30108-8
DOI: https://doi.org/10.1016/j.cytogfr.2020.05.009
Reference: CGFR 1153

To appear in: Cytokine and Growth Factor Reviews

Received Date: 16 May 2020

Accepted Date: 17 May 2020

Please cite this article as: Gubernatorova EO, Gorshkova EA, Polinova AI, Drutskaya MD,
IL-6: relevance for immunopathology of SARS-CoV-2, Cytokine and Growth Factor Reviews
(2020), doi: https://doi.org/10.1016/j.cytogfr.2020.05.009

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IL-6: relevance for immunopathology of SARS-CoV-2
Gubernatorova E.O.a,b,*, Gorshkova E.A.a,b, Polinova A.I.a,b, Drutskaya M.D.a,**
a
Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Engelhardt
Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia
b
Faculty of Biology, Lomonosov Moscow State University, Moscow, Russia
Corresponding authors at: Center for Precision Genome Editing and Genetic Technologies for
Biomedicine, Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow
119991, Vavilova str. 32, Russia
* ekaterina.gubernatorova412@gmail.com
** marinadru@gmail.com

Highlights

 COVID-19 severe outcomes is associated with the acute respiratory distress syndrome
and cytokine release syndrome with elevated IL-6 as a relevant predicting parameter.
 Chronic inflammation, associated with elevated inflammatory serum cytokines, including
IL-6, may exacerbate SARS-CoV-2 induced pathology.

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 IL-6 is a promising therapeutic target for the anti-cytokine therapy of SARS-CoV-2
severe cases.

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Abstract
COVID-19 mortality is strongly associated with the development of severe pneumonia and acute
respiratory distress syndrome with the worst outcome resulting in cytokine release syndrome
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and multiorgan failure. It is becoming critically important to identify at the early stage of the
infection those patients who are prone to develop the most adverse effects. Elevated systemic
interleukin-6 levels in patients with COVID-19 are considered as a relevant parameter in
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predicting most severe course of disease and the need for intensive care. This review discusses
the mechanisms by which IL-6 may possibly contribute to disease exacerbation and the
potential of therapeutic approaches based on anti-IL-6 biologics.
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Keywords: IL-6, SARS-CoV-2, cytokine release syndrome, anti-cytokine therapeutics.

Introduction
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At the end of 2019 and the beginning of 2020, first, China and then the rest of the world have
been confronted with an outbreak of a new airborne transmitted coronavirus disease 2019
(COVID-19), a viral infection caused by severe acute respiratory syndrome coronavirus 2
(SARS-CoV-2). Just a few months later, there are already millions infected worldwide and with
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borders closed and strict travel bans imposed local transmission patterns are in place in most
countries [1]. COVID-19 mortality rate appears to be in the range of 3,4-5,5% [2], which is
significantly higher than that for seasonal flu caused by the influenza virus (1%), but lower than
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for both severe acute respiratory syndrome (SARS) (15%) [3] and Middle East respiratory
syndrome (MERS) (34%) [4]. At the same time, the COVID-19 transmission rate is much greater
than those of MERS or SARS, and poses a real threat to life for a large number of people
worldwide, especially, in older patients with other co-morbidities [5]. Thus, in the absence of
effective vaccines and treatments, early detection and the ability to define prognostic
parameters predicting the course of the disease appear to be of particular interest.

COVID-19 pathogenesis and IL-6

Coronaviruses represent a large family of zoonotic respiratory viruses [6] that can cause both
seasonal cold symptoms and fatal respiratory failure associated with severe inflammation of
lower respiratory tract [7]. The major clinical symptoms of COVID-19 are similar to those
experienced during a seasonal flu infection and include fever, dry cough, headache, myalgia
and others. Co-morbidities such as cardiovascular diseases, diabetes, respiratory diseases,
hypertension and age may exacerbate the disease manifestations [8, 9]. Both moderate and
severe COVID-19 infections cases may result in pneumonia with fluffy opacities on the chest
computer tomographic scans, lung edema and accumulation of pleural fluid in the lungs [10],
[11]. Severe cases require invasive oxygen supply.
The exact molecular mechanisms of COVID-19-mediated pathogenesis are still under
investigation. However, the lessons learned from SARS-CoV and MERS-CoV infections may
uncover some key features of COVID-19-related pathologies, as well as molecular mediators
and signaling pathways involved. The genetic sequence of SARS-CoV-2 [12], which was
released almost simultaneously with the first reported cases [10] revealed that it belongs to the
β‐coronavirus genus, with 79.0% nucleotide identity to SARS‐CoV and 51.8% identity to MERS-
CoV. Inoculation with SARS-CoV-2 of human airway epithelial cells in vitro causes cytopathic
effects [10] and cessation of the cilium beating of epithelial cells, similar to the cytopathic effect
observed for SARS-CoV infection [13]. The initial step in viral infection was revealed by the
crystal structure of SARS-CoV-2 spike receptor binding domain [14], which, just as SARS-CoV

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[15], binds to the host cell receptor angiotensin-converting enzyme 2 (ACE2). The presence of
ACE2 on the cell membrane is crucial for virus virulence, as HeLa cells, lacking ACE2, are
resistant to SARS-CoV-2 infection [16, 17]. Structural analysis identified residues in the SARS-

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CoV-2 spike receptor binding domain that are critical for ACE2 binding, the majority of which
either are highly conserved or share similar side chain properties with those of SARS-CoV.
These similarities in virus entry between SARS-CoV-2 and SARS-CoV correlate with detectable
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cross-neutralizing activity of serum from SARS-CoV-recovered patients [18]. However, no
available monoclonal antibodies targeting SARS-CoV receptor binding domain were able to
prevent SARS-CoV-2 infecting the cells, highlighting unique intrinsic structure features of SARS-
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CoV-2 S-protein binding domain, including a much higher binding affinity than that of SARS-
CoV S-protein [19]. In line with this, more recent study reports that human recombinant soluble
ACE2 effectively, but not completely prevents SARS-CoV-2 infection, suggesting alternative
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mechanisms for viral entry [20].

Surface molecule CD147 (EMMPRIN or Basigin) is being considered as one alternative
pathway for the virus to enter the host cells, since SARS-CoV-2 S-protein can bind CD147 as
suggested by preliminary data [21]. CD147 is expressed on hematopoietic cells, including red
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blood cells, epithelial and neuronal cells [22]. Basigin is viewed as the molecular gate for
plasmodium P.malariae [23]. Not surprisingly, CD147 is also associated with the HIV1 [24] and
SARS-CoV [25] infections. In both cases, viral protein binds to the CD147 in complex with
cyclophilin A (CyPA). SARS-CoV N-protein conjugates with CyPA inside ACE2-expressing
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infected host cells [25]. With this modification, newly assembled viral particles can infect CD147-
positive cells. However, further studies are needed to evaluate possible impact of SARS-CoV-2
interaction with Basigin in the context of COVID-19.
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ACE2, crucial for SARS-CoV-2 virulence, is expressed in the upper and the lower
respiratory tract, most remarkably on lung alveolar epithelial cells, arterial and venous
endothelial cells, as well as enterocytes of the small intestine, epithelial cells in the kidney.
ACE2 expression is also detected in heart, pancreas, testis and brain [26]. Interestingly, ACE2
expression is not the highest in the upper respiratory tract [27, 26], once again, supporting the
hypothesis that increased transmissibility of SARS-CoV-2 as compared to SARS-CoV may be
attributed to yet to be identified co-receptors or auxiliary factors adopted by SARS-CoV-2 [28].
Also, the fact that ACE2 is widely expressed in other tissues and organs explains a broad
spectrum of adverse effects not limited just to the lungs. In addition, it was shown that SARS-
CoV-2 directly infects ACE2-expressing tissue-resident CD169+ macrophages in the spleens
and LNs, causing lymph follicle depletion, splenic nodule atrophy, histiocyte hyperplasia and
lymphocyte reduction [29]. Thus, virus infection of macrophages, firstly, enhances viral spread
and, secondly, triggers destructive events in the immune organs such as spleen and LNs.
Recent data from the experiments using SARS-CoV-2 capsid or live virus infection of cultured T
cell lines (MT-2 and A3.01) provides first evidence that SARS-CoV-2 could also infects T cells.
However, expression level of ACE2 on T cells is low and, thus, CD147 is considered to mediate
SARS-CoV-2 entry into T cells [17].
After virus binds to ACE2 (Fig.1), serine protease, TMPRSS2, cleaves the viral spike
protein. Then another protease, Furin, subsequently releases spike fusion peptide, and the virus
enters the host cell through an endosomal pathway [18, 30, 31]. Following viral replication,
assembly and release, the infected cells may undergo apoptosis or necrosis, triggering the
inflammatory response with production of pro-inflammatory cytokines and activation of
macrophages and Th1 cells, as well as production of IFNγ, IL-17A, IL-21, and IL-22 by
neutrophils, Th17 and CD8+ cells. In turn, SARS-CoV-2 infection of recruited immune cells may
increase their apoptosis and exacerbate lymphocytosis [32, 33], and, finally, may lead in some
patients to life-threatening conditions, such as respiratory distress syndrome, cytokine storm,
and secondary hemophagocytic lymphohistiocytosis.

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COVID-19 mortality is strongly associated with the development of severe respiratory
distress syndrome, which requires invasive ventilation. It is important to identify those patients
who are most likely to develop a severe form of the disease as early as possible. Based on the

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existing statistics, a number of clinical predictors of the disease deterioration and mortality,
including the presence of secondary infections, lymphopenia [34], comorbidities such as
cardiovascular and chronic respiratory diseases, diabetes, hypertension, obesity and cancer
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[35], increased cytokine production and serum ferritin [36] have been reported. The other study
showed that the elevated serum creatinine, D-dimers, lactate dehydrogenase, C-reactive
protein, procalcitonin, as well as increased white blood cell counts may indicate impending
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respiratory failure and the need for invasive oxygen supply [37]. Interestingly, in recently
published reports elevated interleukin-6 (IL-6) level was proposed as a relevant parameter
predicting the unwanted course of the disease and the need for mechanical ventilation [38-40].
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These assumptions are consistent with data from a clinical trial in China that demonstrated on a
small number of patients the efficacy of IL-6 neutralization during exacerbation of COVID-19
associated pneumonia [41] (Supplementary Table 1).
IL-6 is a cytokine with pleotropic functions ranging from hematopoiesis and metabolic
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regulation to inflammation, autoimmunity and acute phase response. IL-6 modulates host
defense through a number of immune stimulating mechanisms: control of monocytes and their
differentiation into macrophages [42], modulation of antigen-dependent B cell differentiation
[43], increased IgG production by B cells [44], and promotion of Th2 response by inhibiting Th1
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polarization [45]. Several studies revealed strong correlation between IL-6 levels in the serum
and the upcoming respiratory failure [46-48]. It was shown, that even moderately elevated IL-6
levels above 80 pg/ml were sufficient to identify COVID-19 infected patients with a high risk of
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respiratory failure [37]. Moreover, serum SARS-CoV-2 nucleic acid (RNAaemia), which is
strongly associated with cytokine storm, is closely correlated with extremely high IL-6 serum
levels [47]. It was also suggested that serial measurement of circulating IL-6 might be important
in identifying disease progression or, when evaluated immediately after COVID-19 diagnosis
confirmation, may predict the upcoming respiratory failure or, alternatively, asymptomatic
disease among SARS-CoV-2 infected patients [49].
Therefore, it would be valuable to explore the potential contribution of IL-6 in the events
occurring during SARS-CoV-2 infection. The next section will discuss the mechanisms by which
IL-6 may possibly affect the disease exacerbation and the potential of therapeutic approaches
based on anti-IL-6 biologics.
Controversy concerning IL-6 functions during viral lung infections
IL-6 is one of the key proinflammatory cytokines during infection onset, especially, at the
mucosal sites [50]. However, the impact of IL-6 on the disease outcome may vary significantly
(Fig. 2). On one hand, IL-6-dependent Th17 activation and differentiation are important for
effective neutrophil migration, IL-6 together with IL-15 modulate cytolytic capacity of CD8+ T
cells [51, 52], and, finally, IL-6, as a pyrogenic cytokine, contributes to thermostatic regulation
which is important for effective anti-viral response [53, 54]. Studies, using IL-6-deficient mice,
showed that IL-6 ameliorates acute lung injury in influenza A virus (IAV) infection [55]. IL-6-
deficient mice infected with influenza virus exhibited higher lethality, as well as increased weight
loss and fibroblast accumulation in the lungs associated with virus-induced apoptosis of lung
epithelial cells and neutrophils [56] indicating that IL-6 may promote repair following virus-
induced lung injury [57]. Finally, early IL-6 signaling promotes IL-27-dependent maturation of
Treg cells in the lungs and resolution of viral immunopathology in mouse model of respiratory
syncytial and influenza virus infection [58].
On the other hand, IL-6 has been implicated in the progression of viral infections. In this
regard, IL-6 synergizes with IL-1b and TNF to upregulate trypsin expression, which activates

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matrix metalloproteinases and causes the breakdown of basal membrane and extracellular
matrix, which, in turn, results in increased tissue permeability and edema [59]. Th1 cell-derived
IFNγ is essential for effective antiviral immune response. However, IL-6 may inhibit Th1

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polarization by stimulating CD4+ cells to differentiate into Th2 cells or by suppressing IFNγ
expression [60, 61]. Moreover, IL-6 promotes Th17 cell differentiation and IL-17A secretion,
which, in turn, activates the expression of anti-apoptotic molecules, such as Bcl-XL, favoring
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survival of virus-infected cells in the model of persistent viral infection [62]. At the same time, IL-
17 promotes the migration [63], and survival [64] of neutrophils which, in turn, contribute to the
pathogenesis of COVID-19-driven ARDS [65]. Finally, in contrast to influenza virus infection, IL-
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6-deficient mice infected with murine pneumonia virus are characterized by better survival than
WT mice with decreased lung edema and diminished neutrophil recruitment [66].
Taking into account that in several experimental models of viral lung infections IL-6
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demonstrates either pathogenic [66] or protective [55] effects in vivo, the role of this cytokine in
SARS-CoV-2 infection should be carefully evaluated. Moreover, the consequences of IL-6
induction in COVID-19 may vary depending on the infection stage and on the host immune
status.
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SARS-CoV-2-associated cytokine release syndrome

Patients with the most severe outcome of SARS-CoV-2 displayed high serum levels of
proinflammatory cytokines responsible for cytokine storm induction [36, 67]. Interestingly, the
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majority of severe COVID-19 cases with respiratory distress syndrome were associated with
high systemic IL-1b, TNF and IL-6 levels, suggesting a direct connection between
proinflammatory cytokine induction and adverse effects of COVID-19 [34]. As SARS-CoV-2
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shows 79% nucleotide identity to SARS-CoV, major pathogenic mechanisms, including cytokine
storm and extensive lung damage during SARS and COVID-19 infection may be similar.
Cytokine storm is a condition caused by extensive activation of the immune system, and, as a
result, extremely high production of cytokines and chemokines. Since cytokine storm can lead to
multiple organ failure, it is important to understand the mechanisms driving this condition.
In lower respiratory tract SARS-CoV-2 infects predominantly type 2 alveolar
epitheliocytes, the cell type which does not participate in active gas exchange. Following
replication and disengagement of the lung epithelial layer, virus enters the underlying tissues
and infects or is being captured by macrophages, dendritic cells and neutrophils, resulting in
further viral spread [29] (Fig. 3). At the same time, damaged epitheliocytes release danger
molecules which drive the activation of lung epithelium and resident immune cells. Thus, even
at the early onset of viral infection, its penetration of the mucosa to the epithelial layer activates
the innate immune response. Adaptive immunity is also activated during COVID-19, mainly by
antigen presenting dendritic cells, which produce large amounts of cytokines, including IL-6, IL-
1b and TNF and migrate to the regional lymph nodes to present viral antigen to naïve T cells,
pushing their differentiation and migration into the affected tissue [68]. Specific humoral
response represented by viral-specific antibody-producing B cells is also in place [69].
Replication of the virus results in further damage of affected organs together with self-boosting
immune response manifesting to uncontrolled overexpression of inflammatory mediators in
severe cases. Another mechanism driving cytokine storm during viral infections is increased
blood vessel permeability which enables infiltration of effector cells, producing additional
amounts of inflammatory molecules and exacerbating hyper cytokine production [70]. In
addition, leakiness of blood vessels allows virus to spread to other tissues and organs,
compromising their functions. Finally, under persistence of inflammatory factors, an increased
number of inflammatory exudates and erythrocytes enter the alveoli, resulting in lung damage
and respiratory failure syndrome.
As mentioned above, severe COVID-19 induced pneumonia is marked by

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hyperactivation of the immune system and excessive production of IL-6 [36, 67]. The main
source of pathogenic IL-6 appears to be myeloid cells as their number is significantly increased
among PBMC [69] and in BALF of severe COVID-19 patients [71]. Myeloid cell recruitment and

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activation in the lungs is associated with increased mortality and morbidity in SARS-CoV mouse
model [72]. Human primary macrophages and dendritic cells can be infected, but they fail to
support productive replication of SARS-CoV [73, 74], unlike alveocytes, in which virus can
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replicate very quickly. At the same time, SARS-CoV and, apparently, SARS-CoV-2-infected
macrophages demonstrate up-regulation of IL-6 production and low expression of interferons
[29, 73]. Interestingly, patients requiring intensive care and invasive lung ventilation display
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negative correlation between IL-6, TNF and IL-1b concentrations and CD4+ and CD8+ T cell
counts [75], confirming previous studies in animal models, which suggested that cytokine storm
actually dampens adaptive immunity against SARS-CoV infection [76]. Moreover, production of
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IFNγ by CD4+ T cells tended to be lower in severe as compared to moderate cases, suggesting
that SARS-CoV-2 infection induces lymphopenia with the suppression of antiviral IFNγ
production [36]. Furthermore, it has been suggested that this pathogenic IL-6 is likely to be
produced by highly inflammatory macrophages [71], but not by alveolar macrophages, which
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are almost absent in the lung aspirates of severely affected patients [77]. At the same time, it
should be noted that complete ablation of IL-6 at the very early stages of viral infection may
result in depressed Tfh cell maturation and delayed antiviral antibody response [78].
It is known that acute inflammation and cytokine storm, as its extreme manifestation, can
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have a significant effect on hemodynamics and also on platelets and erythrocytes. The
dysregulated coagulation cascade and the subsequent formation of intra-alveolar or systemic
fibrin clots are prominent findings in previous zoonotic coronavirus infections (SARS and MERS)
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orchestrated by CRS [79-81]. COVID-19 patients also show significant changes in coagulative
status (i.e. low platelet count, increased D-dimer, fibrinogen levels) and dysfunction of micro-
vessels in pulmonary circulation [82, 83], thus, thrombosis of the pulmonary capillaries is
believed to contribute to rapidly developing hypoxia. The role of IL-6 in inflammatory
hypercoagulation is controversial: IL-6 can increase the rate of fibrotic clot formation in whole
blood, but the effect is less pronounced than it is shown for IL-1 or IL-8 [84]. On the other hand,
IL-6 is important for thrombus formation being a regulator of MMPs and other thrombolysis-
associated genes in macrophages [85].
Other characteristic feature of cytokine release syndrome is change in iron homeostasis
and hyperferritinemia as indicated by high levels of serum ferritin [86]. In COVID-19 patients
ferritin elevation correlated with poor prognosis [36], as it was an indication of hemolytic events
caused by hypercoagulation and hemophagocytosis. In general, inadequate macrophage
activation leads to pathologic phagocytosis of erythrocytes and reutilization of heme iron using
ferritin [87]. In line with this, high systemic ferritin may serve as an indicator of developing
macrophage activation syndrome, rather than a factor driving COVID-19 pathogenesis.
Another key proinflammatory cytokine, TNF, is also released abundantly during cytokine
storm, caused by SARS-CoV-2 infection. Serum TNF levels were found to be elevated in
severely affected COVID-19 patients [34]. Previously, it was shown for SARS-CoV that its spike
protein is able to modulate TNF converting enzyme (TACE)-dependent shedding of the ACE2
ectodomain [88]. TACE expression is coupled to TNF production. Therefore, it is hypothesized
that the use of TNF inhibitors may also show some efficacy in preventing viral entry [89], as well
as in decreasing immunopathology associated mortality, as it was shown in preclinical studies in
mice infected with severe respiratory syncytial virus [90]. Moreover, anti-TNF therapy was
effective in decreasing the IL-6 and IL-1b serum levels in lethal bacteremia, suggesting that
such treatment may provide complex anti-inflammatory effect [91]. Data obtained from COVID-
19 infected patients with inflammatory bowel diseases undergoing anti-TNF therapy support
safety of such intervention in the context of SARS-CoV-2 treatment [92, 93]. These findings

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argue for a potentially protective effect of TNF inhibition in COVID-19 and further studies are
needed to address this possibility.

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ACE2 downregulation may induce IL-6 production in Angiotensin II-dependent manner
Hypertension has been suggested as a risk factor for severe COVID-19 outcomes [94]. Early
data from the literature already assumed an association between blood pressure and circulating
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IL-6 levels in otherwise healthy individuals [95]. Moreover, several studies reported an
increased IL-6 expression in patients with hypertension [96, 97]. IL-6-transgenic mice,
characterized by elevated plasma IL-6 levels, displayed pulmonary hypertension [98, 99]. Taken
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together, this data suggest that IL-6 might be connected with increased risk of respiratory failure
in hypertensive patients with COVID-19.
One of the mechanisms regulating blood pressure and electrolyte balance is
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angiotensin-renin system. It is comprised of two pathways, which are normally functioning in the
state of dynamic equilibrium: ACE2-Angiotensin II–Angiotensin 1 Receptor axis, activating
vasoconstriction and inflammatory response, and ACE2-Angiotensin 1–7-Mas receptor axis,
supporting vasodilation and suppression of inflammation (Fig. 4, A) [100]. Changes in renin-
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angiotensin system activity are related to the pathogenesis of hypertension and of inflammatory
lung diseases, in particular, acute lung injury caused by viral infections [15, 101].
SARS-CoV2 uses ACE2 as an entry site, thus, reducing ACE2 availability, promoting an
imbalance between the ACE2-Angiotensin II–Angiotensin 1 receptor axis and the ACE2-
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Angiotensin 1–7-Mas receptor axis [15, 102]. In line with this, recent studies have reported that
COVID-19 patients have increased Angiotensin II compared to healthy individuals [103].
Altogether, the abovementioned data suggests that COVID-19 infection leads to imbalanced
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angiotensin conversion and a shift towards pro-inflammatory signaling mediated by Angiotensin

1 receptor axis [104] (Fig. 4, B).
Angiotensin II is known to significantly increase the expression of IL-6 in a dose-
dependent manner [105] through pleiotropic activation of NF-kB transcription factors [106],
[107]. Angiotensin 1 receptor downstream activation of NADPH oxidase results in elevated
oxidative stress, in particularly, superoxide that activates transcriptional mechanisms that
directly promotes IL-6 gene expression. In turn, plasma IL-6 concentrations correlate with blood
pressure, plasma Angiotensin II levels and vascular hypertrophy, suggesting an important role
of IL-6 in the development and maintenance of hypertension, specifically, that mediated by
Angiotensin II [108, 109]. IL-6 promotes Angiotensin 1 receptor expression sensitizing the
vascular wall to angiotensin II-dependent signaling mechanisms. Activated cells can release
cytokines, including IL-6, which promote expression of adhesion molecules and induce
endothelial activation, inflammatory cell infiltration and vascular inflammation [110]. This further
drives oxidative stress and IL-6 expression. Therefore, it is plausible that IL-6 and Angiotensin II
activate each other through a positive feedback mechanism. In support of this hypothesis,
ACE2 protects against lethal avian influenza A H5N1 infection [111] through prevention of
oxidative stress and acute respiratory distress syndrome. Moreover, it was recently reported
that in COVID-19 patients with hypertension Angiotensin receptor blockade attenuated the
inflammatory response, possibly through IL-6 inhibition [102, 112]. Taking together, Angiotensin
II accumulation due to SARS-CoV-2-mediated ACE2 downregulation may cause Angiotensin 1
receptor downstream activation of NADPH oxidase, which, in turn, leads to elevated ROS
production and to induction of transcriptional mechanisms that directly promote IL-6 expression,
implicated in inflammation-induced injury and immunopathology.

Risk factors associated with COVID-19

Chronic inflammation is the one common feature of hypertension, diabetes, obesity and age –
the risk factors associated with COVID-19 severe outcomes. Elderly, obese and diabetic

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persons are affected by a low-grade chronic inflammation and are characterized by systemically
increased levels of proinflammatory cytokines, which may contribute to and facilitate the
cytokine storm, the main cause of COVID-19 mortality.

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Among conditions, predisposing patients infected with SARS-CoV-2 to severe outcomes,
diabetes is the most prominent. It is estimated that mortality rate of those with COVID-19 and
diabetes is about 16% [8] to 35% [113]. IL-6 is known for its dual role in autoimmune diabetes,
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as it is involved in the development of insulin resistance and β-cell dysfunction and, at the same
time, has anti-inflammatory effects and improves glucose metabolism [114]. Recent clinical data
demonstrates significantly elevated systemic IL-6 in COVID-19 patients with diabetes as
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compared to those without diabetes [115]. COVID-19 may exacerbate the pre-existing diabetes,
since its entry site, ACE2, is expressed in the pancreas, with a potential role in the development
of insulin resistance and impaired insulin secretion [116]. IL-6 may be the link between diabetes
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and severe COVID-19 outcomes through its ability to modulate T cell response. Some indirect
evidence to support this comes from mouse studies of closely related MERS-CoV infection
demonstrating that abnormal elevation of IL-17A produced by IL-6-dependent Th17 cells
exacerbates disease severity in diabetic mice [117]. Consistent with that finding, patients with
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COVID-19 have a higher proportion of Th17 cells in the blood [118], suggesting that diabetics
due to improper activation of Th1 and Th17 cells may have a compromised anti-viral immune
response [31].
Obese patients with COVID-19 are more likely to develop severe respiratory distress
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syndrome [119, 120]. Just as diabetics, obese patients have higher concentration of
proinflammatory cytokines both in steady state [121] and, apparently, during COVID-19 infection
[8]. In addition to decreased anti-viral T cell response, enhanced IL-6 production by adipose
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tissue in obese patients [122] may alter innate anti-viral immune response by neutrophils [121]
and cause uncontrolled replication of the virus at the early stages of infection.
From the very beginning of COVID-19 pandemic, age was identified as a risk factor [38],
supporting the idea that ACE2 may be a marker of cell senescence [123]. Myofibroblasts, which
increase in number with age, express high levels of ACE2 [124], thus, myofibroblasts are more
susceptible to SARS-CoV-2, and elderly people, bearing higher proportion of myofibroblasts,
might be more susceptible to COVID-19 infection (reviewed in [125]). IL-6 is a critical
inflammatory regulator of senescence-associated secretory phenotype (SASP) [126]. Therefore,
IL-6 overexpression may correlate both with senescent status of cells and tissues, as well as
with increased expression of ACE2. In line with this idea, hydroxychloroquine, an anti-malaria
drug, which is now approved in some countries for COVID-19 treatment, effectively reduced the
salivary and serum levels of IL-6 in patients with Sjorgen’s syndrome [127]. Moreover,
azithromycin, prescribed together with hydroxychloroquine, can target and selectively remove
senescent cells [128] and, thus, provide anti-fibrotic activity by protecting the lungs from
inflammation-induced injury [129, 130].
Another issue facing elderly patients with COVID-19 is a compromised activation status
of their effector T cells. These cells may display limited function during prolonged infection as a
result of their exhaustion, associated with the surface expression of such immune-inhibitory
factors as PD-1 [75]. Since aging itself is a process known to promote reorganization of the T
cell landscape [131] and IL-6 is a SASP predictor and regulator, T cell exhaustion in the elderly
patients with COVID-19 is much more evident due to higher basal concentration of IL-6, which
can enhance the exhaustion process [132, 133] .
Smoking is another potential risk factor for SARS-CoV-2 infection. Smokers are more
vulnerable to influenza virus infections [134, 135] and are more likely to develop chronic
obstructive pulmonary disease (COPD) [136]. Enhanced ACE2 expression had been reported in
the lungs of healthy smokers [137, 138]. In line with that, studies in mice have established that
cigarette smoke can trigger the expansion of a subpopulation of respiratory epithelial cells that

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express ACE2 in a dose-dependent manner [139]. Patients with COPD, as well as active
smokers, have increased ACE2 expression in the lungs [140], which may potentially explain a
fourfold increased risk of adverse development and severe outcome of COVID-19 [141, 142].

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Taken together, these recent reports suggest that smokers may be at higher risk of infection.
Examination of factors, predisposing to severe COVID-19 in Wuhan, China, revealed, that
19.2% of patients with severe disease were smokers [143]. On the other hand, preliminary
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meta-analysis based on Chinese patients suggest that active smoking was not associated with
the severity of COVID-19 [144], indicating that impact of smoking on COVID-19 progression is
controversial and requires further assessment.
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Intuitively, patients with pre-existing asthma should display higher proportion of COVID-
19 severe cases. However, the prevalence of asthma in COVID-19 patients in Wuhan study was
markedly lower (0,9%) than that reported in the adult population of Wuhan (6,4%) [143]. One
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possible explanation is a shift toward Th2-type immune response in asthmatics as opposed to

Th1-associated inflammatory response induced by SARS-CoV-2 infection [118]. Recent study
reveals that TMRSS2 expression in upper respiratory tract correlates positively with type 2
response genes, especially with IL-13, whereas ACE2 expression is suppressed by IL-13.
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Considering low numbers of cells co-expressing these proteases in the upper respiratory tract, it
has been proposed that in the context of IL-13-dependent reduction in ACE2 expression allergy
should promote resistance to infection [145]. In line with this, it was shown that ACE2
expression is decreased in nasal and bronchial epithelium of allergic individuals following
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allergen challenge, suggesting, that asthma-associated immune response actually protects from
COVID-19-induced pathology [146]. Nevertheless, further studies are required to reveal the
impact of COVID-19 severity in patients with preexisting asthma.
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Meta-analysis studies of COVID-19 cases in China from December 2019 to February

2020 report lower incidence of the disease [147] and risk of death [35] in women than in men.
Similar data on association of gender with poorer prognosis for males were obtained earlier for
SARS-CoV [148], [149] and MERS-CoV [150, 151]. In connection to SARS-CoV-2, the X-linked
ACE2 gene is of a particular interest. Significant fraction of genes encoded on the X
chromosome undergo dosage compensation, however, ACE2 is located at Xp22, i.e. the area in
which genes may escape from X chromosome inactivation [152], possibly contributing to gender
disparity in disease susceptibility. Gender differences in immune responses are known to
depend not only on genetic, but also on endocrine factors [153, 154]. High doses of estradiol
acting through estrogen receptors decrease production of proinflammatory cytokines via
suppressing NF-kB signaling pathway [155]. In relation to COVID-19 the effects of estrogens on
IL-6 and on disease progression is of particular interest. Estrogen may suppress LPS-mediated
IL-6 expression in mouse macrophages by both blocking of NF-kB activation [155, 156] and
inhibiting p38 MAPK phosphorylation [156]. Furthermore, downregulation of IL-6 expression in
Kupffer cells appears to be ERα-dependent [157]. Overall, immunomodulatory effects of
estrogens may contribute to decreased susceptibility to SARS-CoV-2 infection in women and
this question requires further study.

The aftermath of COVID-19 infection

One of the common complications from the respiratory viral infections, including SARS [158]
and COVID-19 [159], is lung fibrosis. This is characterized by alveolar epithelial cell injury,
recruitment and activation of fibroblasts and subsequent production of extracellular matrix
(ECM) in the presence of specifically activated immune cells. Fibrosis develops as an
exaggerated repair process following injury associated with abnormal immune response leading
to chronic progressive disease [160]. Histological examination of lung biopsies from COVID-19
patients show massive apoptosis of pneumocytes, hyaline membrane formation, fibromyxoid
exudates [161] which are manifestations that are characteristic for ARDS and represent key

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events preceding development of fibrosis (reviewed in [162]). Significant pro-fibrotic M2-like
SPP1+macrophage infiltration was also described for COVID-19 correlating with the disease
severity [71]. Damage to the epithelial layer caused by the virus further attracts various immune

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cells participating in inflammation-repair balance (monocytes, macrophages, granulocytes and T
cells). In addition, some viral components, such as N-protein and papain-like protease, can
directly stimulate production of TGF-β, one of the pro-fibrotic hallmarks in respiratory epithelium,
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similar to what has previously been reported for SARS-CoV [163, 164].
The role of IL-6 had been extensively studied in bleomycin model of lung fibrosis in mice.
Bleomycin is a chemotherapeutic antibiotic which causes damage of tissues lacking bleomycin
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hydrolase, predominantly, in the lungs [165]. Mice with genetic ablation of IL-6 were
characterized by impaired fibrosis [166]. However, in the same model mice with
pharmacological rather than genetic inhibition of IL-6 demonstrated opposing phenotypes
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depending on the phase of inflammation-repair cycle. Specifically, IL-6 neutralization at the

early inflammatory phase (day 2 following bleomycin-induced lung injury) resulted in apoptosis
of pneumocytes, which further contributed to pulmonary fibrosis, whereas IL-6 blockade at the
early fibrotic stage (day 8 following bleomycin-induced lung injury) significantly ameliorated lung
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fibrosis [167]. Interestingly, treatment of mice with recombinant soluble gp130Fc at the late
stage of chronic bleomycin model attenuated lung fibrosis, emphasizing the importance of sIL-
6Ra and IL-6 trans-signaling in mediating this condition [168]. It was further suggested that M2-
like infiltrating macrophages are the major source of soluble IL-6Ra in fibrotic foci, whereas
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pulmonary fibroblasts are the recipient cells, which proliferate and produce extracellular matrix
under trans-IL-6 activation. Finally, overexpression of IL-6 in respiratory tract mediated by
adenoviral vectors also resulted in the exacerbation of fibrosis and was associated with
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increased levels of M2-macrophages with pro-fibrotic features [169]. These pro-fibrotic

macrophages are also characterized by increased IL-6Ra expression [168]. ACE2 expression is
reported for lung, heart, kidney, intestine, liver and brain. Thus, similar fibrosis-associated
complications should be expected, not just for respiratory tract, but also for other tissues and
organs in correlation with viral load and severity of cytokine release syndrome. Summarizing the
data available at the moment from infection-free experimental models of fibrosis, one can
conclude that IL-6 may affect fibroblast proliferation and functions directly or through
potentiation of a distinct macrophage phenotype. Taking into account the importance of this
cytokine at the repair stage, anti-IL-6 therapy may prove beneficial not only in acute phase, but
also in subacute proliferative phase of COVID-19-related ARDS.
 SARS-CoV-2 tropism to neural system is of particular interest while neurological
symptoms, such as dizziness, headache, ataxia and anosmia, are often found in mild COVID-19
patients and develop early in the infection [170]. ACE2 expression in CNS is attributed to the
nuclei in brain stem and is associated with cardio‐respiratory control [171, 172]. Recent study
based on scRNA sequencing suggests co-expression of ACE2 and TMPRSS2 in olfactory
epithelial cells, but not in mature olfactory sensory neurons, which were initially considered as
the most obvious target population for the virus in CNS [173]. Even without the understanding of
the exact mechanisms of SARS-CoV-2 penetration and distribution in CNS, it should be noted
that as a result of the disseminated intravascular coagulopathy and cytokine storm caused by
infection, blood-brain-barrier permeability is significantly increased, which can lead to negative
consequences for CNS functioning [174, 175].

Therapeutic potential of IL-6 inhibitors

An effective therapeutic approach should include a combination of specific antiviral drugs, which
inhibit viral dissemination and limit direct virus-induced cytopathic effects coupled with anti-
inflammatory therapeutics, directed against immunopathology induced by a viral infection [176].

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SARS-CoV-2-induced immunopathology exhibits features of systemic hyper-inflammation
reminiscent both of cytokine storm or macrophage activation syndrome and secondary
haemophagocytic lymphohistocytosis [177]. Since overexpression of IL-6 appears to be

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associated with severe COVID-19 outcomes, neutralizing antibodies used to treat a number of
autoimmune diseases by targeting exacerbated inflammatory immune response of the host may
provide a life-saving approach by preventing the cytokine release syndrome. In line with that,
-p
patients responding to CD19 CAR T cell therapy experience cytokine release syndrome [178],
which is well managed by IL-6 neutralizing antibodies [179]. In a non-randomized, open-label
clinical trial, 21 patients with severe or critical COVID-19 were treated with a single dose of
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Tocilizumab, in addition to routine therapy [180]. 90% of patients recovered, suggesting that
anti-IL-6 might be a powerful potential rescue therapy in managing acute respiratory distress
syndrome of COVID-19 and inhibiting the most adverse outcome. Peripheral mononuclear cell
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scRNA-seq profiling data obtained from these patients indicate that treatment with Tocilizumab
does not reduce the number of cytotoxic CD8 + and plasma B cells, thus, does not interfere with
robust adaptive immune response [69]. Preliminary data from 21 patients with COVID-19 who
developed acute respiratory distress syndrome and participated in a compassionate-use
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program at Papa Giovanni XXIII hospital in Bergamo, Italy, indicate that intravenous introduction
of Siltuximab reduced C-reactive protein level in all patients and significantly improved clinical
conditions with a reduced need for ventilation in 33% of patients. 43% of patients showed
stabilization of their conditions, and 24% of patients experienced a worsening of the disease
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and required intubation [181].

Sarilumab is a monoclonal antibody that works by inhibiting the IL-6 pathway through
binding and blocking the IL-6 receptor. Recently, another clinical trial was initiated, which will
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evaluate the effects of Sarilumab in patients with severe COVID-19. This U.S.-based multi-
center, double-blind, phase 2/3 trial will begin at medical centers in New York and is anticipated
to enroll up to 400 patients [182].
The overview of initiated clinical trials of anti-IL-6 therapeutics in COVID-19 patients is
presented in Supplementary Table 1. In summary, strong scientific evidence of the beneficial
impact of IL-6 inhibitors in the modulation of the COVID-19 infection requires further clinical
data.

Conclusion
In the face of a pandemic, affecting millions of people worldwide, the need to develop new
approaches or reassess the existing ones in the treatment of severe viral infections is more
pressing than ever. Despite the fact that humanity experienced viral infections causing deadly
pneumonia twice in the past 20 years, there are still no drugs specific for coronaviruses. The
evidence discussed in this review suggests that for patients with risk factors, especially those
with high systemic levels of IL-6, clinically approved neutralizing antibodies against IL-6 or its
receptor, IL-6R, in combination with standard treatment protocols may provide benefit. Gaining
a deeper understanding of the factors that affect the immune response and mechanistic link of
these factors to disease severity is crucial for effective clinical management of COVID-19 in
severely affected patients.

Funding
This work was supported by the Ministry of Science and Higher Education of the Russian
Federation (grant 075-15-2019-1660).
Conflict of interest
The authors declare that there is no conflict of interest.

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Acknowledgments
We thank Prof. S. A. Nedospasov and Dr. K.-S. N. Atretkhany for critical reading of manuscript.

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We thank P.V. Matveev for assistance with figure preparation.

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Kevzara-R-sarilumab-clinical-trial-program-in-patients-with-severe-COVID-19, 2020, March 16.

Ekaterina O. Gubernatorova is PhD student in Immunology at Lomonosov Moscow State
University (MSU) and junior staff scientist in Laboratory of Molecular Mechanisms of Immunity,
Center for Precision Genome Editing and Genetic Technologies for Biomedicine Engelhardt
Institute of Molecular Biology, Russian Academy of Sciences (EIMB RAS). Ekaterina is working

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on several research projects in the areas of mucosal immunology. She is focused on the role of
cell-specific IL-6 signaling in lungs and gut homeostasis and inflammation.

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Ekaterina A. Gorshkova is PhD student in Immunology at Lomonosov MSU and junior staff
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scientist in Laboratory of Molecular Mechanisms of Immunity, Center for Precision Genome

Editing and Genetic Technologies for Biomedicine, EIMB RAS. Ekaterina is involved in several
research projects, including studying the role of IL-6 overexpression in mouse model of
inflammatory disease.
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Almina I. Polinova is bachelor student in Immunology in Lomonosov MSU. Almina received her
bachelor training in the laboratory of Molecular Mechanisms of Immunity, participating in a
project to study the role of IL-6 in gut inflammation.

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Marina S. Drutskaya, PhD, is Leading Staff Scientist in Laboratory of Molecular Mechanisms of
Immunity, Center for Precision Genome Editing and Genetic Technologies for Biomedicine,
EIMB RAS. Marina is studying proinflammatory cytokines, mainly TNF and IL-6, in several

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relevant autoimmune and inflammatory disease models in mice.

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Figure 1. Schematic representation of SARS-CoV-2 interaction with a target cell

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Binding of virion to ACE2 is followed by serine protease, TMPRSS2, cleavage of the viral spike
protein and induction of internalization of the complex with subsequent reduction of ACE2
surface availability. Then another protease, Furin, releases spike fusion peptide, and the virus
enters the host cell through an endosomal pathway.
Figure 2. Role of IL-6 in respiratory viral infections.

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IL-6 demonstrates opposing effects during the immune response to viral infections in the lungs.
IL-6 promotes highly specific reaction of adaptive immunity by stimulating of CD8+ T cells and B

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cells, which is balanced by T regulatory cells. Also, IL-6 facilitates survival of phagocytic
neutrophils. On the other hand, IL-6 can provide unfavorable Th2 and Th17 over Th1 helper
differentiation and facilitate tissue injury by dysregulation of extracellular matrix and attraction of
neutrophils and pro-inflammatory macrophages. -p
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Figure 3. Cytokine release syndrome in COVID-19.

SARS-CoV-2 replicates in alveolar epithelial cells. After the virions are released, they can infect
or are being captured by macrophages, dendritic cells and neutrophils. At the same time,
damaged epithelial cells produce alarmins, which are sensed by the neighboring epithelial and
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myeloid cells. All these events lead to release of proinflammatory cytokines, increase of alveolar
vessel permeability and cell recruitment to the site of infection, forming positive loop for
pathological activation of IL-6, IL-1, TNF and other proinflammatory cytokines. Due to high
permeability of pulmonary vessels under inflammatory conditions, SARS-CoV-2 can inseminate
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distant ACE2-expressing tissues (intestine, kidney, pancreas), and trigger burst of inflammation
at distant sites.
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Figure 4. ACE2 downregulation may drive IL-6 production in Angiotensin II-dependent

manner.
A – in healthy state pro-inflammatory signaling of Angiotensin II via Angiotensin 1 receptor
(AT1R) is stabilized by Angiotensin 1-7 (ACE2-Angiotensin I proteolysis product) signaling via
Mas receptor (MasR) that has anti-inflammatory effect.
B – virus-mediated down-regulation of ACE2 leads to Angiotensin II accumulation in the
absence of Angiotensin 1-7 and acceleration of its proinflammatory effect. AT1R downstream
activation of NADPH oxidase results in elevated ROS directly promoting IL-6 expression. In its
turn, IL-6 can positively regulate AT1R levels. Thus, by acting together these processes
exacerbate hypertension, edema, infiltration of immune cells and inflammation in COVID-19
affected lungs.

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