Natural killer cells are important in the control of viral infections. However, the role of NK cells during severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has previously not been identified. Peripheral blood NK cells from SARS-CoV and SARS-CoV-2 naïve subjects were evaluated for their activation, degranulation, and interferon-gamma expression in the presence of SARS-CoV and SARS-CoV-2 spike proteins. K562 and lung epithelial cells were transfected with spike proteins and co-cultured with NK cells. The analysis was performed by flow cytometry and immune fluorescence. SARS-CoV and SARS-CoV-2 spike proteins did not alter NK cell activation in a K562 in vitro model. On the contrary, SARS-CoV-2 spike 1 protein (SP1) intracellular expression by lung epithelial cells resulted in NK cell-reduced degranulation. Further experiments revealed a concomitant induction of HLA-E expression on the surface of lung epithelial cells and the recognition of an SP1-derived HLA-E-binding peptide. Simultaneously, there was increased modulation of the inhibitory receptor NKG2A/CD94 on NK cells when SP1 was expressed in lung epithelial cells. We ruled out the GATA3 transcription factor as being responsible for HLA-E increased levels and HLA-E/NKG2A interaction as implicated in NK cell exhaustion. We show for the first time that NK cells are affected by SP1 expression in lung epithelial cells via HLA-E/NKG2A interaction. The resulting NK cells’ exhaustion might contribute to immunopathogenesis in SARS-CoV-2 infection.

(1) Background: Acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent for the coronavirus disease (COVID-19) that has led to a pandemic that began in March 2020. The role of the SARS-CoV-2 components on innate and adaptive immunity is still unknown. We investigated the possible implication of pathogen-associated molecular patterns (PAMPs)–pattern recognition receptors (PRRs) interaction. (2) Methods: We infected Calu-3/MRC-5 multicellular spheroids (MTCSs) with a SARS-CoV-2 clinical strain and evaluated the activation of RNA sensors, transcription factors, and cytokines/interferons (IFN) secretion, by quantitative real-time PCR, immunofluorescence, and ELISA. (3) Results: Our results showed that the SARS-CoV-2 infection of Calu-3/MRC-5 multicellular spheroids induced the activation of the TLR3 and TLR7 RNA sensor pathways. In particular, TLR3 might act via IRF3, producing interleukin (IL)-1α, IL-1β, IL-4, IL-6, and IFN-α and IFN-β, during the first 24 h post-infection. Then, TLR3 activates the NFκB transduction pathway, leading to pro-inflammatory cytokine secretion. Conversely, TLR7 seems to mainly act via NFκB, inducing type 1 IFN, IFN-γ, and IFN-λ3, starting from the 48 h post-infection. (4) Conclusion: We showed that both TLR3 and TLR7 are involved in the control of innate immunity during lung SARS-CoV-2 infection. The activation of TLRs induced pro-inflammatory cytokines, such as IL-1α, IL-1β, IL-4, and IL-6, as well as interferons. TLRs could be a potential target in controlling the infection in the early stages of the disease.

Several evidence suggests that, in addition to the respiratory tract, also the gastrointestinal tract is a main site of severe acute respiratory syndrome CoronaVirus 2 (SARS‐CoV‐2) infection, as an example of a multi‐organ vascular damage, likely associated with poor prognosis. To assess mechanisms SARS‐CoV‐2 responsible of tissue infection and vascular injury, correlating with thrombotic damage, specimens of the digestive tract positive for SARS‐CoV‐2 nucleocapsid protein were analyzed deriving from three patients, negative to naso‐oro‐pharyngeal swab for SARS‐CoV‐2. These COVID‐19–negative patients came to clinical observation due to urgent abdominal surgery that removed different sections of the digestive tract after thrombotic events. Immunohistochemical for the expression of SARS‐CoV‐2 combined with a panel of SARS‐CoV‐2 related proteins angiotensin‐converting enzyme 2 receptor, cluster of differentiation 147 (CD147), human leukocyte antigen‐G (HLA‐G), vascular endothelial growth factor (VEGF) and matrix metalloproteinase‐9 was performed. Tissue samples were also evaluated by electron microscopy for ultrastructural virus localization and cell characterization. The damage of the tissue was assessed by ultrastructural analysis. It has been observed that CD147 expression levels correlate with SARS‐CoV‐2 infection extent, vascular damage and an increased expression of VEGF and thrombosis. The confirmation of CD147 co‐localization with SARS‐CoV‐2 Spike protein binding on gastrointestinal tissues and the reduction of the infection level in intestinal epithelial cells after CD147 neutralization, suggest CD147 as a possible key factor for viral susceptibility of gastrointestinal tissue. The presence of SARS‐CoV‐2 infection of gastrointestinal tissue might be consequently implicated in abdominal thrombosis, where VEGF might mediate the vascular damage.

Human leukocyte antigen (HLA) is a group of molecules involved in inflammatory and infective responses. We evaluated blood sHLA-E and sHLA-G levels in hospitalized COVID-19 patients with respiratory failure and their relationship with clinical evolution, changes in endothelial activation biomarker profile, and neutrophil adhesion. sHLA-E, sHLA-G, and endothelial activation biomarkers were quantified by ELISA assay in plasma samples. Neutrophil adhesion to endothelium was assessed in the presence/absence of patients’ plasma samples. At admission, plasma levels of sHLA-G and sHLA-E were significantly higher in COVID-19 patients with respiratory failure compared to controls. COVID-19 clinical improvement was associated with increased sHLA-G plasma levels. In COVID-19, but not in control patients, an inverse correlation was found between serum sICAM-1 and E-selectin levels and plasma sHLA-G values. The in vitro analysis of activated endothelial cells confirmed the ability of HLA-G molecules to control sICAM-1 and sE-selectin expression via CD160 interaction and FGF2 induction and consequently neutrophil adhesion. We suggest a potential role for sHLA-G in improving COVID-19 patients’ clinical condition related to the control of neutrophil adhesion to activated endothelium.

The peptide nociceptin/orphanin FQ (N/OFQ) is the natural ligand of the N/OFQ receptor (NOP) which is widely expressed in the central and peripheral nervous system. Selective NOP antagonists are worthy of testing as innovative drugs to treat depression, Parkinson's disease, and drug abuse. The aim of this study was to perform a detailed in vitro characterization of BTRX-246040 (also known as LY2940094, [2-[4-[(2-chloro-4,4-difluoro-spiro[5H-thieno[2,3-c]pyran-7,4'-piperidine]-1'-yl)methyl]-3-methylpyrazol-1-yl]-3-pyridyl]methanol)a novel NOP antagonist that has been already studied in humans. BTRX-246040 has been tested in vitro in the following assays: calcium mobilization in cells expressing NOP and classical opioid receptors and chimeric G proteins, BRET assay measuring NOP interaction with G proteins and -arrestins, the label free dynamic mass redistribution assay, and the electrically stimulated mouse vas deferens. BTRX-246040 was systematically compared to the standard NOP antagonist SB-612111. In all assays BTRX-246040 behaves as a pure and selective antagonist at human recombinant and murine native NOP receptors displaying 3-10 fold higher potency than the standard antagonist SB-612111. BTRX-246040 is as an essential pharmacological tool to further investigate the therapeutic potential of NOP antagonists in preclinical and clinical studies. Significance Statement NOP antagonists may be innovative antidepressant drugs. In this research the novel clinically viable NOP antagonist BTRX-246040 has been deeply characterized in vitro in a panel of assays. BTRX-246040 resulted a pure, potent and selective NOP antagonist.