Natural Killer cell activation, reduced ACE2, TMPRSS2, cytokines G-CSF, M-CSF and SARS-CoV-2-S pseudovirus infectivity by MEK inhibitor treatment of human cells

Abstract

COVID-19 affects vulnerable populations including elderly individuals and patients with cancer. Natural Killer (NK) cells and innate-immune TRAIL suppress transformed and virally-infected cells. ACE2, and TMPRSS2 protease promote SARS-CoV-2 infectivity, while inflammatory cytokines IL-6, or G-CSF worsen COVID-19 severity. We show MEK inhibitors (MEKi) VS-6766, trametinib and selumetinib reduce ACE2 expression in human cells. Chloroquine or hydroxychloroquine increase cleaved active SP-domain of TMPRSS2, and this is potentiated by MEKi. In some human cells, remdesivir increases ACE2-promoter luciferase-reporter expression, ACE2 mRNA and protein, and ACE2 expression is attenuated by MEKi. We show elevated cytokines in COVID-19-(+) patient plasma (N=9) versus control (N=11). TMPRSS2, inflammatory cytokines G-CSF, M-CSF, IL-1alpha, IL-6 and MCP-1 are suppressed by MEKi alone or in combination with remdesivir. MEKi enhance NK cell (but not T-cell) killing of target-cells, without suppressing TRAIL-mediated cytotoxicity. We generated a pseudotyped SARS-CoV-2 virus with a lentiviral core but with the SARS-CoV-2 D614 or G614 SPIKE (S) protein on its envelope and used VSV-G lentivirus as a negative control. Our results show infection of human bronchial epithelial cells or lung cancer cells and that MEKi suppress infectivity of the SARS-CoV-2-S pseudovirus following infection. We show a drug class-effect with MEKi to promote immune responses involving NK cells, inhibit inflammatory cytokines and block host-factors for SARS-CoV-2 infection leading also to suppression of SARS-CoV-2-S pseudovirus infection of human cells in a model system. MEKi may attenuate coronavirus infection to allow immune responses and antiviral agents to control COVID-19 disease progression and severity.

Establishment of a well-characterized SARS-CoV-2 lentiviral pseudovirus neutralization assay using 293T cells with stable expression of ACE2 and TMPRSS2

“…A previous study reported higher infectivity of VSV-based pseudoviruses in Huh7, 293T, and Vero cells compared to CHO, MDCK, and HepG2 cells [39], but other studies reported poor or no infectivity of 293T cells due to the absence of ACE2 receptor [33,43]. Other cell types, including stably engineered cells (293T, 293T17, HT1080, BHK21), transiently transfected cells (293T, Caco-2, Huh7, HepG2, MDCK), and various continuous cell lines (Vero-E6, A549, BEAS2B, Calu-3, H1299, MRC5, Caco-2, HeLa, K562) that express ACE2, TMPRSS2, or both, have been widely reported to support pseudovirus infectivity to different degrees [20,24,[31][32][33][34][35][36][37][38][39][40][41][42][43][44]. We therefore assessed a panel of cell types, including Vero, Vero E6, A549, Caco-2, Calu-3, Huh7, 293T, and 293T cells transiently expressing ACE2 (293T-ACE2 t ), TMPRSS2 (293T-TMPRSS2 t ), or both (293T-ACE2.TMPRSS2 t ), to identify the cells that supported the highest levels of infectivity for our pseudoviruses.…”

“…Pseudoviruses bearing viral envelope proteins provide safe surrogates for highly pathogenic viruses in MN assays. Several groups have generated SARS-CoV-2 pseudoviruses with glycoprotein defective murine leukemia virus (MLV)-, human immunodeficiency virus (HIV)-, and vesicular stomatitis virus (VSV)-based systems and used them in neutralization assays based on fluorescence (monomeric Neon Green or mNG) or enzymatic activity (nano-, gaussia-, and firefly luciferases) read outs in a variety of target cell types [20,24,[31][32][33][34][35][36][37][38][39][40][41][42][43][44]. In the present study, we describe our optimized conditions for an HIV-based lentiviral SARS CoV-2 pseudovirus neutralization assay.…”

Potential Clinical Benefits of Quercetin in the Early Stage of COVID-19: Results of a Second, Pilot, Randomized, Controlled and Open-Label Clinical Trial

“…19 This is likely of critical importance in patients with COVID-19 infection as SARS-CoV-2 inhibits NK cell activity at multiple steps. 20 Finally, although quercetin's precise antiviral mechanisms are not yet completely understood, there is a considerable body of evidence supporting the broad antiviral properties of quercetin, demonstrated both in in vitro and in vivo studies through inhibition of viral replication of several respiratory viruses, including influenza virus, parainfluenza virus, respiratory syncytial virus, adenovirus, and rhinovirus. 21 As for most of polyphenols, the practical use of quercetin is limited by its low solubility and reduced oral absorption.…”