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\begin{document}

\title{A rationale for targeting the P2X7 receptor in Coronavirus disease 19
(Covid-19)}



\author[1]{Francesco Di Virgilio}%
\author[2]{Yong Tang}%
\author[1]{Alba Sarti}%
\author[3]{Marco Rossato}%
\affil[1]{Univ. of Ferrara}%
\affil[2]{Chengdu University of Traditional Chinese Medicine}%
\affil[3]{University of Padova}%


\vspace{-1em}



  
  \date{April 28, 2024}


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\selectlanguage{english}
\begin{abstract}
Acute respiratory distress syndrome (ARDS) is the main cause of
morbidity and mortality in Coronavirus disease 19 (Covid-19) for which
as of now there is no effective treatment. ARDS is caused and sustained
by an uncontrolled inflammatory activation characterized by a massive
release of cytokines (cytokine storm), diffuse lung edema, inflammatory
cell infiltraton and disseminated coagulation. Macrophage and T
lymphocyte dysfunction plays a central role in this syndrome. In several
experimental in vitro and in vivo models, many of these
pathophysiological changes are triggered by stimulation of the P2X7
receptor. We hypothesize that this receptor might be an ideal candidate
to target in Covid-19-associated ARDS.%
\end{abstract}%



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\par\null

An effective treatment for Covid-19 is still lacking, although anecdotal
evidence suggests that IL-6 blockade is beneficial (Mehta \emph{et al}
., 2020), and blockers of other pro-inflammatory cytokines such as IL-1\selectlanguage{greek}β
\selectlanguage{english}are being considered in Covid-19 patients showing hyperinflammation
(Monteagudo \emph{et al} ., 2020). In such a dramatic situation, it
might be necessary to explore unconventional, but rationale, therapeutic
options. There is evidence that severely ill Covid-19 patients develop a
cytokine storm syndrome causing acute respiratory distress syndrome
(ARDS), the main cause of morbility and mortality (Mehta \emph{et
al}2020). This is thought to be due to uncontrolled, virus-mediated,
direct activation of lung macrophages in a process reminiscent of the
macrophage activation syndrome (MAS) observed in some rheumatologic
diseases such as systemic juvenile idiopathic arthritis (sJIA), adult
onset Still's disease and systemic lupus erithematosus (LES), although
it is debated wheter hyperinflammation in Covid-19 should be considered
MAS sensu strictu (Crayne \emph{et al} , 2019; Henderson \emph{et al} .,
2020). Covid-19 patients show massive infiltration by inflammatory cells
(neutrophils, monocyte/macrophages) in their lungs, and increased blood
levels of IL-1\selectlanguage{greek}β, \selectlanguage{english}IL-6, IL-2, IL-7, TNF-\selectlanguage{greek}α \selectlanguage{english}and several other
pro-inflammatory cytokines and chemokines (Guo \emph{et al} ., 2019).
While main target of coronaviruses are lung epithelial cells, they also
infect macrophages and dendritic cells, where they cause abortive
infection and sustained activation, that in turn drives
hyperinflammation (Fehr and Perlman, 2015). Thus, macrophage inhibition
is likely to be a crucial step to prevent the extensive lung injury
caused by Sars Cov-2. A promising target receptor to down-modulate
macrophage responses might be the P2X7 receptor (P2X7R).

The P2X7R is a plasma membrane receptor gated by extracellular ATP, the
earliest and most ubiquitous damage-associated molecular pattern (DAMP)
released at all inflammatory sites, the lungs included (Tolle and
Standiford, 2013; Di Virgilio \emph{et al} ., 2020). Very interestingly,
increased ATP levels are also found in bronchoalveolar lavage fluid
(BALF) from patients with ARDS, or from mice with lipopolysaccharide
(LPS)-induced acute lung injury (ALI) (Cicko \emph{et al} , 2018).
Increased extracellular ATP levels have also been direclty
demonstrated\emph{in vivo} in the lungs of mice inhaled with LPS thanks
to the bioluminescent luciferase probe pmeLUC (Cicko \emph{et al} .
2018). The P2X7R has a central role in inflammation as it is one of the
most potent stimulator of the NLRP3 inflammasome, and therefore of
caspase-1 activation and IL-1\selectlanguage{greek}β \selectlanguage{english}and IL-18 release (Di Virgilio \emph{et
al} ., 2017) (Figure 1). P2X7R stimulation also promotes release of
other cytokines and chemokines, e.g. IL-6, TNF-\selectlanguage{greek}α, \selectlanguage{english}monocyte chemotactic
protein-1 (MCP-1), IL-8, CC-chemokine ligand 3 (CCL3) and CXCL2, of
pro-fibrotic factors such as TGF-\selectlanguage{greek}β, \selectlanguage{english}and extracellular matrix remodelling
factors such as metalloproteinase-9 and tissue inhibitor of
metalloproteinase (TIMP)-1 (Di Virgilio \emph{et al} ., 2017; Riteau
\emph{et al} , 2010). The P2X7R is a potent trigger of reactive oxygen
species (ROS) production, therefore its overactivation (as it is the
case at sites of heavy inflammation) may inhibit lymphocyte functions by
impairing mitochondrial metabolism, as it has been shown in monocytes
during sepsis (Martinez-Garcia \emph{et al} 2019). Dysfunctional
mitochondria are a feature of exhausted T lymphocytes (Desd\selectlanguage{ngerman}ín-Micó
\emph{et al} ., 2018). Although no specific data are available in
Covid-19, reduced T cell function, as documented by the increased
expression of T lymphocyte exhaustion markers, is a feature of
coronavirus-induced lung infection (Diao \emph{et al} ., 2019). It is
known that inability of cytotoxic T lymphocyte and NK cells to lyse
virus-infected cells occurs in MAS, and a is main cause of the sustained
cytokine release driving the cytokine storm typical of this syndrome and
of Covid-19 as well (Crayne \emph{et al} ., 2019). In addition, a key
feature of ARDS is the extensive pulmonary edema which is largely
dependent on release of vascular endothelial growth factor (VEGF).
Although the pathogenic role of VEGF in ARDS is controversial, blood
VEGF levels are reported to be increased in Covid-19 patients, and this
has prompted a clinical study aimed at testing the efficacy of
bevacizumab administration (NCT04275414)
(\emph{https://clinicaltrials.gov/ct2/results?cond=\&term=NCT04275414)}
. While mostly known for its pro-inflammatory activity, it is also well
documented that the P2X7R is a potent inducer of VEGF release and
neo-angiogenesis \emph{in vivo} , and accordingly it has been shown that
its blockade inhibits VEGF-dependent increase in vascular permeability
(Clapp \emph{et al} ., 2019). Thus P2X7R targeting might prove
beneficial to fight the early exudative phase in ARDS. Furthemore,
thromboembolic complications are common among critically ill Covid-19
patients (Tang et al. 2020a), which has led to explore the protective
effect of heparine administration Tang \emph{et al} ., 2020b).
Inhibition of the P2X7R might be beneficial also to treat
thromboembolism since its stimulation promotes massive release of tissue
factor (Baroni \emph{et al} , 2007; Furlan-Freguia \emph{et al} .,
2011).

A commonly used experimental model for ARDS, is the intratracheal
application of LPS in mice. This causes ALI, an acute lung inflammation
reminiscent of human ARDS (Cicko \emph{et al} ., 2018). Early work
showed that pharmacological blockade of the P2X7R, or its genetic
ablation, substantially reduced inflammatory cell infiltration, cytokine
levels and lung damage in acute inflammatory response (Wang \emph{et al}
., 2015; Cicko \emph{et al} ., 2018). P2X7R deficiency was also shown to
reduce alveolar macrophage death and pro-IL-1\selectlanguage{greek}α \selectlanguage{english}release in the lungs of
LPS-treated mice (Dagvadori \emph{et al} ., 2015). Monitoring the P2X7R
as an inflammatory biomarker in Covid-19 by recently developed
radiopharmaceuticals has been recently proposed (Juengling \emph{et al.}
, 2020)

On the basis of these convergent observations, we suggest that the P2X7R
could be an ideal candidate receptor for pharmacological targeting in
ARDS.

Small molecule drugs targeting the P2X7R have been developed by most
Pharma Industries and have undergone extensive Phase I Clinical Trials
revealing an excellent safety profile (Arulkumaran \emph{et al} .,
2011). However, therapeutic efficacy in a number of chronic inflammatory
diseases investigated in Phase II turned out to be limited, which led
major Pharma Industries to drop or outsource P2X7R clinical research. A
relevant exception is Johnson \& Johnson that in late 2019 started a new
Phase II clinical trial to test the efficacy of P2X7R blockade in
depression (Cully, 2020). Therapeutic efficacy of P2X7R blockade has
never been tested in a disease condition characterized by uncontrolled
hyperinflammation, as in Covid-19-dependent ARDS. We propose that P2X7R
antagonists should be given a chance at least for the compassionate
treatment of Covid-19 patients with rapid evolutive ARDS.

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\textbf{Legend to Figure 1}

Central role of the P2X7R in Sars-Cov-2-dependent lung inflammation.
Sars-Cov-2 infects lung mononuclear phagocytes, besides epithelial
cells, triggering massive ATP release which feeds-back on the P2X7R
expressed on macrophages and antigen-presenting cells (APC). P2X7R
activation down-modulates MHC-I expression, and at the same time
promotes IL-6 release, NLRP3-mediated IL-1\selectlanguage{greek}β \selectlanguage{english}and IL-18 release, VEGF
secretion and thus lung edema. Release of several other cytokines and
chemokines is also promoted by P2X7R activation. Stimulation of P2X7R on
APC-stimulated T lymphocytes causes exhaustion and T cell polarization.
The combined effect of all these factors promotes the pathophysiological
changes typical of ARDS.\selectlanguage{english}
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