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.