Discussion
In this study we tested the hypothesis that PTP4A3 phosphatase could
represent a new therapeutic target for COVID-19-related ALI/ARDS.
Protein tyrosine phosphatases are overexpressed in many types of cancer
and are associated with a poor prognosis (Narasaraju et al., 2011;
Vainonen et al., 2021). The PTP4A family includes three members: PTP4A1,
PTP4A2, and PTP4A3 that display diverse subcellular localization and
activity within different systems, including cell surface receptors, the
actin cytoskeleton, intracellular signaling effectors and ion channels;
when highly expression contributes to pathological states (Hardy et al.,
2018; McQueeney et al., 2017; Yu & Zhang, 2018). PTPs are involved in
multiple physiological processes, including cell proliferation,
migration, metabolism, survival, and immune responses (Yu & Zhang,
2018) and all these processes play a direct role in ARDS symptomatology.
Neutrophils induce non-specific epithelial cell necrosis, with a
two-edge sword effect on killing cells infected by viruses, but also
contributing to lung injury (Narasaraju et al., 2011). Pulmonary immune
responses in severe COVID-19 patients are characterized by dysregulated
myeloid cell expansion, which include the infiltration and activation of
polymorphonuclear neutrophils (Hazeldine & Lord, 2021). Neutrophils are
also present in lung infections associated with ARDS caused by influenza
and SARS-CoV-1 (Camp & Jonsson, 2017). We previously reported that
increased levels of neutrophils were observed in K18-hACE2 transgenic
mice, instilled with SARS-CoV-2 S1SP (Colunga Biancatelli et al., 2021;
Solopov et al., 2022). Here, treatment with KVX-053 after S1SP
instillation significantly decreased the number of immune cells in the
BALF. We also observed the decrease in alveolar and parenchymal
neutrophils, that is reflected in reduced lung injury scoring (Fig.2,
4). Neutrophils, besides their main immune functions – i.e.,
phagocytosis, generation of toxic molecules, enzyme production, and
formation of extracellular traps- regulate inflammatory and immune
responses, via the release of a large variety of cytokines and
chemokines (Tecchio et al., 2014). The dramatic release of Interleukins,
Interferons, growth factors, and other cytokines and chemokines was
observed in the BALF and serum of S1SP-treated mice. Here we
demonstrated that KVX-053 successfully blocked most of the cytokines and
chemokines in both the lavage and serum, thereby modulating the
“cytokine storm” caused by S1SP. STAT3 is expressed in the cytoplasm
in an inactive form and can be activated by numerous types of cytokines,
chemokines, and growth factors related with COVID-19 inflammation
(Jafarzadeh et al., 2021). Serine phosphorylation of STAT3 is commonly
activated in the kidney of patients with COVID-19 (Salem et al., 2022).
Abnormal activation of the NF-κB signaling pathway resulting from
SARS-CoV-2 infection is also associated with the pathogenic profile of
immune cells, cytokine storm and multiorgan defects (Kandasamy, 2021). A
natural host cell response to SARS-COV-2 infection, as a result of the
activation of the NLRP3 inflammasome, protects the host from viral
infections, but at the same time leads to chronic inflammation and
pulmonary fibrosis (Amin et al., 2022; R. M. L. Colunga Biancatelli et
al., 2022). We previously demonstrated the effectiveness of PTP4A3
inhibitors in blocking phosphorylation of NF-kB and STAT3 in ovarian
cancer via SHP-2 and RAP1 (McQueeney et al., 2017). Here we have shown
that KVX-053 blocks activation (phosphorylation) of STAT3 and IkBα, with
consequent reduction of the cytokine storm. The reduction of
interstitial and perivascular overexpression of PTP4A3 phosphatase by
KVX-053 is likely due to an inhibition of IL-6, which controls a feed
forward loop regulating PTP4A3 expression (Chong et al., 2019).
Pulmonary dysfunction is an important criterion to the diagnosis of
severe COVID-19 and ARDS. Failure of the adaptive function of the immune
system, damage of epithelial and endothelial cells, coagulation issues
and cytokine storm impact on respiratory dynamics (Ackermann et al.,
2020; Laveneziana et al., 2021). Clinical data shows that even a few
months post hospital discharge, the transfer capacity of the lung (TLCO
test) remains <80% and TLC is at <80% of predicted
value in 33% and 16% of patients, respectively (Anastasio et al.,
2021; Huang et al., 2021). Pulmonary anomalies in DLSO, TLC, FEV and FVC
are also reduced in patients at time of hospital discharge (Mo et al.,
2020). Recently, K18h-ACE2 transgenic mice were infected with SARS-CoV-2
virus and a decline in pulmonary function was observed, expressed as
changes in Rn, Ers, Cst, IC, G, and downward shift of PV-loops (Winkler
et al., 2020). In this study, for the first time, we characterized
S1SP-induced lung dysfunction and detected findings similar to those
observed with the live SARS-CoV-2 virus. Importantly, the PTP4A3
inhibitor KVX-053 was highly beneficial in preventing S1SP-induced lung
dysfunction and bronchial hyperresponsiveness, suggesting its potential
clinical relevance in preventing the development of COVID-19-related
ARDS.
COVID-19 patients have a high incidence of vascular events, such as
stroke, myocardial infarction, venous thrombosis; lung microthrombi were
found in the lungs of COVID-19 non-survivors (Chen & Pan, 2021; Siddiqi
et al., 2021). Consistent with our previous work (Colunga Biancatelli et
al., 2021), others have documented that SARS-CoV-2 and the surface
elements of the virus, i.e., the Spike protein, have a direct effect on
endothelial monolayer permeability, mitochondrial function and ACE2
expression (Buzhdygan et al., 2020; Lei et al., 2021). Besides ACE2,
genes SPECC1L and EPPK1, which encode cell junction proteins (Saadi et
al., 2011; Shimada et al., 2013), could be also targeted by the S
protein (Zhou et al., 2023). S1SP can dose- and time-dependently affect
the permeability of HLMVEC (Colunga Biancatelli et al., 2021) via strong
activation of the inflammatory pathways STAT3 and NF-κB and consequent
reduced expression of tight junction proteins Occludin and VE-Cadherin
and cytoskeletal modulator Cofilin (Ruben Manuel Luciano Colunga
Biancatelli et al., 2022). We previously demonstrated that KVX-053
prevents LPS-induced loss of barrier function in HLMVEC (McQueeney et
al., 2018). Here, we confirmed its protective effects on the endothelium
against SARS-CoV-2 S1SP in both pre-treatment and post-treatment assays.
These protective effects may result from the ability of KVX-053 to
suppress the activation of STAT3 and AKT and protect the expression of
multiple intercellular focal structures (Fig. 9, 10). Indeed, another
member of the Phosphatase family, PTP-PEST, has been previously
associated with membrane function and the formation of focal complexes
necessary for migration via the Rho GTPase downstream signaling (Garton
& Tonks, 1994; Ushio-Fukai, 2006). Thus, dual specificity phosphatases
in addition to their relevant role in inflammation, may also represent a
valid target for wound-healing, migration and proliferation.