Post-COVID pulmonary injury in K18-hACE2 mice shows persistent neutrophils and neutrophil extracellular trap formation.

The involvement of neutrophils in the lungs during the recovery phase of coronavirus disease 2019 (COVID-19) is not well defined mainly due to the limited accessibility of lung tissues from COVID-19 survivors. The lack of an appropriate small animal model has affected the development of effective therapeutic strategies. We here developed a long COVID mouse model to study changes in neutrophil phenotype and association with lung injury. Our data shows persistent neutrophil recruitment and neutrophil extracellular trap formation in the lungs for up to 30 days post-infection which correlates with lung fibrosis and inflammation.

Inflammation in the COVID-19 airway is due to inhibition of CFTR signaling by the SARS-CoV-2 spike protein.

SARS-CoV-2-contributes to sickness and death in COVID-19 patients partly by inducing a hyper-proinflammatory immune response in the host airway. This hyper-proinflammatory state involves activation of signaling by NFκB, and unexpectedly, ENaC, the epithelial sodium channel. Post-infection inflammation may also contribute to "Long COVID"/PASC. Enhanced signaling by NFκB and ENaC also marks the airway of patients suffering from cystic fibrosis, a life-limiting proinflammatory genetic disease due to inactivating mutations in the CFTR gene. We therefore hypothesized that inflammation in the COVID-19 airway might similarly be due to inhibition of CFTR signaling by SARS-CoV-2 spike protein, and therefore activation of both NFκB and ENaC signaling. We used western blot and electrophysiological techniques, and an organoid model of normal airway epithelia, differentiated on an air-liquid-interface (ALI). We found that CFTR protein expression and CFTR cAMP-activated chloride channel activity were lost when the model epithelium was exposed to SARS-CoV-2 spike proteins. As hypothesized, the absence of CFTR led to activation of both TNFα/NFκB signaling and α and γ ENaC. We had previously shown that the cardiac glycoside drugs digoxin, digitoxin and ouabain blocked interaction of spike protein and ACE2. Consistently, addition of 30 nM concentrations of the cardiac glycoside drugs, prevented loss of both CFTR protein and CFTR channel activity. ACE2 and CFTR were found to co-immunoprecipitate in both basal cells and differentiated epithelia. Thus spike-dependent CFTR loss might involve ACE2 as a bridge between Spike and CFTR. In addition, spike exposure to the epithelia resulted in failure of endosomal recycling to return CFTR to the plasma membrane. Thus, failure of CFTR recovery from endosomal recycling might be a mechanism for spike-dependent loss of CFTR. Finally, we found that authentic SARS-CoV-2 virus infection induced loss of CFTR protein, which was rescued by the cardiac glycoside drugs digitoxin and ouabain. Based on experiments with this organoid model of small airway epithelia, and comparisons with 16HBE14o- and other cell types expressing normal CFTR, we predict that inflammation in the COVID-19 airway may be mediated by inhibition of CFTR signaling by the SARS-CoV-2 spike protein, thus inducing a cystic fibrosis-like clinical phenotype. To our knowledge this is the first time COVID-19 airway inflammation has been experimentally traced in normal subjects to a contribution from SARS-CoV-2 spike-dependent inhibition of CFTR signaling.

TurboID-mediated proximity labeling technologies to identify virus co-receptors.

Virus receptors determine the tissue tropism of viruses and have a certain relationship with the clinical outcomes caused by viral infection, which is of great importance for the identification of virus receptors to understand the infection mechanism of viruses and to develop entry inhibitor. Proximity labeling (PL) is a new technique for studying protein-protein interactions, but it has not yet been applied to the identification of virus receptors or co-receptors. Here, we attempt to identify co-receptor of SARS-CoV-2 by employing TurboID-catalyzed PL. The membrane protein angiotensin-converting enzyme 2 (ACE2) was employed as a bait and conjugated to TurboID, and a A549 cell line with stable expression of ACE2-TurboID was constructed. SARS-CoV-2 pseudovirus were incubated with ACE2-TurboID stably expressed cell lines in the presence of biotin and ATP, which could initiate the catalytic activity of TurboID and tag adjacent endogenous proteins with biotin. Subsequently, the biotinylated proteins were harvested and identified by mass spectrometry. We identified a membrane protein, AXL, that has been functionally shown to mediate SARS-CoV-2 entry into host cells. Our data suggest that PL could be used to identify co-receptors for virus entry.

Submandibular Gland Pathogenesis Following SARS-CoV-2 Infection and Implications for Xerostomia.

Although SARS-CoV-2 induces mucin hypersecretion in the respiratory tract, hyposalivation/xerostomia has been reported by COVID-19 patients. We evaluate the submandibular gland (SMGs) pathogenesis in SARS-CoV-2-infected K18-hACE2 mice, focusing on the impact of infection on the mucin production and structural integrity of acini, ductal system, myoepithelial cells (MECs) and telocytes. The spike protein, the nucleocapsid protein, hACE2, actin, EGF, TNF-α and IL-1ß were detected by immunofluorescence, and the Egfr and Muc5b expression was evaluated. In the infected animals, significant acinar hypertrophy was observed in contrast to ductal atrophy. Nucleocapsid proteins and/or viral particles were detected in the SMG cells, mainly in the nuclear membrane-derived vesicles, confirming the nuclear role in the viral formation. The acinar cells showed intense TNF-α and IL-1ß immunoexpression, and the EGF-EGFR signaling increased, together with Muc5b upregulation. This finding explains mucin hypersecretion and acinar hypertrophy, which compress the ducts. Dying MECs and actin reduction were also observed, indicating failure of contraction and acinar support, favoring acinar hypertrophy. Viral assembly was found in the dying telocytes, pointing to these intercommunicating cells as viral transmitters in SMGs. Therefore, EGF-EGFR-induced mucin hypersecretion was triggered by SARS-CoV-2 in acinar cells, likely mediated by cytokines. The damage to telocytes and MECs may have favored the acinar hypertrophy, leading to ductal obstruction, explaining xerostomia in COVID-19 patients. Thus, acinar cells, telocytes and MECs may be viral targets, which favor replication and cell-to-cell viral transmission in the SMG, corroborating the high viral load in saliva of infected individuals.

Suppression of interferon α and γ response by Huwe1-mediated Miz1 degradation promotes SARS-CoV-2 replication.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has been demonstrated to limit the host interferon response; however, the underlying mechanism remains unclear. Here, we found that SARS-CoV-2 infection upregulated the E3 ubiquitin ligase Huwe1, which in turn facilitated the degradation of the transcription factor Miz1. The degradation of Miz1 hampered interferon alpha and gamma responses, consequently fostering viral replication and impeding viral clearance. Conversely, silencing or inhibiting Huwe1 enhanced the interferon responses, effectively curbing viral replication. Consistently, overexpressing Miz1 augmented the interferon responses and limited viral replication, whereas silencing Miz1 had the opposite effect. Targeting Huwe1 or overexpressing Miz1 elicited transcriptomic alterations characterized by enriched functions associated with bolstered antiviral response and diminished virus replication. Further study revealed Miz1 exerted epigenetic control over the transcription of specific interferon signaling molecules, which acted as common upstream regulators responsible for the observed transcriptomic changes following Huwe1 or Miz1 targeting. These findings underscore the critical role of the Huwe1-Miz1 axis in governing the host antiviral response, with its dysregulation contributing to the impaired interferon response observed during COVID-19.

Neuropathogenesis of SARS-CoV-2 in human neuronal, microglial and glial cells.

Neurological complications, both acute and chronic, are reported commonly in COVID-19 affected individuals. In this context, the understanding of pathogenesis of SARS-CoV-2 in specific cells of central nervous system (CNS) origin is relevant. The present study explores infection biology of a clinical isolate of SARS-CoV-2 in human cell lines of neural origin such as the glioblastoma (U87-MG), neuroblastoma (SHSY5Y) and microglia (C20). Despite showing clear evidence of infection by immunofluorescence with an anti-spike protein antibody, all the three neural cell lines were observed to be highly restrictive to the replication of the infecting virus. While the U87-MG glioblastoma cells demonstrated no cytopathic effects and a low viral titre with no signs of replication, the SHSY5Y neuroblastoma cells exhibited cytopathic effects with bleb formation but no evidence of viable virus. The C20 microglial cells showed neither signs of cytopathic effects nor viable virus. Ultrastructural studies demonstrated intracellular virions in infected neural cells. The presence of lipid droplets in infected SHSY5Y cells suggested an impact on host cell metabolism. The decrease in viral RNA levels over time in all the neural cell lines suggested restricted viral replication. In conclusion, this study highlights the limited susceptibility of neural cells to SARS-CoV-2 infection. This reduced permissibility of neural cell lines to SARS-CoV-2 may point to their inherent lower expression of receptors that support viral entry in addition to the intracellular factors that potently inhibit viral replication. The study findings prompt further investigation into the mechanisms of SARS-CoV-2 infection of neural cells.

SARS-CoV-2 infects neurons, astrocytes, choroid plexus epithelial cells and pericytes of the human central nervous system in vitro.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection is associated with neurological sequelae including haemorrhage, thrombosis and ischaemic necrosis and encephalitis. However, the mechanism by which this occurs is unclear. Neurological disease associated with COVID-19 has been proposed to occur following direct infection of the central nervous system and/or indirectly by local or systemic immune activation. We evaluated the expression of angiotensin-converting enzyme-2 and transmembrane protease, serine 2 (TMPRSS2) in brain tissue from five healthy human donors and observed low-level expression of these proteins in cells morphologically consistent with astrocytes, neurons and choroidal ependymal cells within the frontal cortex and medulla oblongata. Primary human astrocytes, neurons, choroid plexus epithelial cells and pericytes supported productive SARS-CoV-2 infection with ancestral, Alpha, Delta and Omicron variants. Infected cells supported the full viral life cycle, releasing infectious virus particles. In contrast, primary brain microvascular endothelial cells and microglia were refractory to SARS-CoV-2 infection. These data support a model whereby SARS-CoV-2 can infect human brain cells, and the mechanism of viral entry warrants further investigation.

Potential Role of APOBEC3 Family Proteins in SARS-CoV-2 Replication.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has acquired multiple mutations since its emergence. Analyses of the SARS-CoV-2 genomes from infected patients exhibit a bias toward C-to-U mutations, which are suggested to be caused by the apolipoprotein B mRNA editing enzyme polypeptide-like 3 (APOBEC3, A3) cytosine deaminase proteins. However, the role of A3 enzymes in SARS-CoV-2 replication remains unclear. To address this question, we investigated the effect of A3 family proteins on SARS-CoV-2 replication in the myeloid leukemia cell line THP-1 lacking A3A to A3G genes. The Wuhan, BA.1, and BA.5 variants had comparable viral replication in parent and A3A-to-A3G-null THP-1 cells stably expressing angiotensin-converting enzyme 2 (ACE2) protein. On the other hand, the replication and infectivity of these variants were abolished in A3A-to-A3G-null THP-1-ACE2 cells in a series of passage experiments over 20 days. In contrast to previous reports, we observed no evidence of A3-induced SARS-CoV-2 mutagenesis in the passage experiments. Furthermore, our analysis of a large number of publicly available SARS-CoV-2 genomes did not reveal conclusive evidence for A3-induced mutagenesis. Our studies suggest that A3 family proteins can positively contribute to SARS-CoV-2 replication; however, this effect is deaminase-independent.

PI3Kγ inhibition circumvents inflammation and vascular leak in SARS-CoV-2 and other infections.

Virulent infectious agents such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and methicillin-resistant Staphylococcus aureus (MRSA) induce tissue damage that recruits neutrophils, monocyte, and macrophages, leading to T cell exhaustion, fibrosis, vascular leak, epithelial cell depletion, and fatal organ damage. Neutrophils, monocytes, and macrophages recruited to pathogen-infected lungs, including SARS-CoV-2-infected lungs, express phosphatidylinositol 3-kinase gamma (PI3Kγ), a signaling protein that coordinates both granulocyte and monocyte trafficking to diseased tissues and immune-suppressive, profibrotic transcription in myeloid cells. PI3Kγ deletion and inhibition with the clinical PI3Kγ inhibitor eganelisib promoted survival in models of infectious diseases, including SARS-CoV-2 and MRSA, by suppressing inflammation, vascular leak, organ damage, and cytokine storm. These results demonstrate essential roles for PI3Kγ in inflammatory lung disease and support the potential use of PI3Kγ inhibitors to suppress inflammation in severe infectious diseases.

A therapy for suppressing canonical and noncanonical SARS-CoV-2 viral entry and an intrinsic intrapulmonary inflammatory response.

The prevalence of "long COVID" is just one of the conundrums highlighting how little we know about the lung's response to viral infection, particularly to syndromecoronavirus-2 (SARS-CoV-2), for which the lung is the point of entry. We used an in vitro human lung system to enable a prospective, unbiased, sequential single-cell level analysis of pulmonary cell responses to infection by multiple SARS-CoV-2 strains. Starting with human induced pluripotent stem cells and emulating lung organogenesis, we generated and infected three-dimensional, multi-cell-type-containing lung organoids (LOs) and gained several unexpected insights. First, SARS-CoV-2 tropism is much broader than previously believed: Many lung cell types are infectable, if not through a canonical receptor-mediated route (e.g., via Angiotensin-converting encyme 2(ACE2)) then via a noncanonical "backdoor" route (via macropinocytosis, a form of endocytosis). Food and Drug Administration (FDA)-approved endocytosis blockers can abrogate such entry, suggesting adjunctive therapies. Regardless of the route of entry, the virus triggers a lung-autonomous, pulmonary epithelial cell-intrinsic, innate immune response involving interferons and cytokine/chemokine production in the absence of hematopoietic derivatives. The virus can spread rapidly throughout human LOs resulting in mitochondrial apoptosis mediated by the prosurvival protein Bcl-xL. This host cytopathic response to the virus may help explain persistent inflammatory signatures in a dysfunctional pulmonary environment of long COVID. The host response to the virus is, in significant part, dependent on pulmonary Surfactant Protein-B, which plays an unanticipated role in signal transduction, viral resistance, dampening of systemic inflammatory cytokine production, and minimizing apoptosis. Exogenous surfactant, in fact, can be broadly therapeutic.

A glimpse into viral warfare: decoding the intriguing role of highly pathogenic coronavirus proteins in apoptosis regulation.

Coronaviruses employ various strategies for survival, among which the activation of endogenous or exogenous apoptosis stands out, with viral proteins playing a pivotal role. Notably, highly pathogenic coronaviruses such as SARS-CoV-2, SARS-CoV, and MERS-CoV exhibit a greater array of non-structural proteins compared to low-pathogenic strains, facilitating their ability to induce apoptosis via multiple pathways. Moreover, these viral proteins are adept at dampening host immune responses, thereby bolstering viral replication and persistence. This review delves into the intricate interplay between highly pathogenic coronaviruses and apoptosis, systematically elucidating the molecular mechanisms underpinning apoptosis induction by viral proteins. Furthermore, it explores the potential therapeutic avenues stemming from apoptosis inhibition as antiviral agents and the utilization of apoptosis-inducing viral proteins as therapeutic modalities. These insights not only shed light on viral pathogenesis but also offer novel perspectives for cancer therapy.

A versatile automated pipeline for quantifying virus infectivity by label-free light microscopy and artificial intelligence.

Virus infectivity is traditionally determined by endpoint titration in cell cultures, and requires complex processing steps and human annotation. Here we developed an artificial intelligence (AI)-powered automated framework for ready detection of virus-induced cytopathic effect (DVICE). DVICE uses the convolutional neural network EfficientNet-B0 and transmitted light microscopy images of infected cell cultures, including coronavirus, influenza virus, rhinovirus, herpes simplex virus, vaccinia virus, and adenovirus. DVICE robustly measures virus-induced cytopathic effects (CPE), as shown by class activation mapping. Leave-one-out cross-validation in different cell types demonstrates high accuracy for different viruses, including SARS-CoV-2 in human saliva. Strikingly, DVICE exhibits virus class specificity, as shown with adenovirus, herpesvirus, rhinovirus, vaccinia virus, and SARS-CoV-2. In sum, DVICE provides unbiased infectivity scores of infectious agents causing CPE, and can be adapted to laboratory diagnostics, drug screening, serum neutralization or clinical samples.

Neuropathological lesions in intravenous BCG-stimulated K18-hACE2 mice challenged with SARS-CoV-2.

In the wake of the COVID-19 pandemic caused by SARS-CoV-2, questions emerged about the potential effects of Bacillus Calmette-Guérin (BCG) vaccine on the immune response to SARS-CoV-2 infection, including the neurodegenerative diseases it may contribute to. To explore this, an experimental study was carried out in BCG-stimulated and non-stimulated k18-hACE2 mice challenged with SARS-CoV-2. Viral loads in tissues determined by RT-qPCR, histopathology in brain and lungs, immunohistochemical study in brain (IHC) as well as mortality rates, clinical signs and plasma inflammatory and coagulation biomarkers were assessed. Our results showed BCG-SARS-CoV-2 challenged mice presented higher viral loads in the brain and an increased frequency of neuroinvasion, with the greatest differences observed between groups at 3-4 days post-infection (dpi). Histopathological examination showed a higher severity of brain lesions in BCG-SARS-CoV-2 challenged mice, mainly consisting of neuroinflammation, increased glial cell population and neuronal degeneration, from 5 dpi onwards. This group also presented higher interstitial pneumonia and vascular thrombosis in lungs (3-4 dpi), BCG-SARS-CoV-2 mice showed higher values for TNF-α and D-dimer values, while iNOS values were higher in SARS-CoV-2 mice at 3-4 dpi. Results presented in this study indicate that BCG stimulation could have intensified the inflammatory and neurodegenerative lesions promoting virus neuroinvasion and dissemination in this experimental model. Although k18-hACE2 mice show higher hACE2 expression and neurodissemination, this study suggests that, although the benefits of BCG on enhancing heterologous protection against pathogens and tumour cells have been broadly demonstrated, potential adverse outcomes due to the non-specific effects of BCG should be considered.

Exploring the protective association between COVID-19 infection and laryngeal cancer: insights from a Mendelian randomization study.

Introduction: Viral infections have been implicated as a risk factor for laryngeal cancer. Given the possible effects of Corona virus disease 2019 (COVID-19) on the laryngeal tissue, we investigated the causal link between COVID-19 and laryngeal cancer using a two-sample Mendelian randomization (MR) approach. Methods: We utilized genetic data from the 5th Genome-wide association studies (GWAS) edition of the COVID-19 Host Genetics Initiative (published on January 18, 2021) and a large-scale laryngeal cancer GWAS comprising 180 cases and 218,612 controls of European ancestry. We applied inverse variance weighting, MR Egger, and weighted median methods to infer causality. We performed sensitivity analysis using the "leave-one-out" method to verify robustness. Results: We found no evidence of a causal association between gene-predicted COVID-19 and laryngeal cancer [Odds ratio (OR)=0.24 (95% Confidence intervals (CI), 0.05-1.26), P=0.09]. However, we observed significant inverse associations between gene-predicted COVID-19 hospitalization [OR=0.51 (95% CI, 0.28-0.95), P=0.03] and severe patients [OR=0.62 (95% CI, 0.43-0.90), P=0.01] and laryngeal cancer. Notably, the study detected important genetic variants, such as rs13050728, that modulate the expression of interferon alpha receptor 2 (IFNAR2), indicating possible roles for immune response pathways in both COVID-19 and cancer. Discussion: This study reveals a potential interaction between COVID-19 severity, genetic factors, and laryngeal cancer, underscoring the importance of investigating the immune response mechanisms in both conditions. These findings contribute to the understanding of the complex interactions between COVID-19 and laryngeal cancer and may guide future research on the role of immune response, particularly involving IFNAR2.

Electrophysiological Impact of SARS-CoV-2 Envelope Protein in U251 Human Glioblastoma Cells: Possible Implications in Gliomagenesis?

SARS-CoV-2 is the causative agent of the COVID-19 pandemic, the acute respiratory disease which, so far, has led to over 7 million deaths. There are several symptoms associated with SARS-CoV-2 infections which include neurological and psychiatric disorders, at least in the case of pre-Omicron variants. SARS-CoV-2 infection can also promote the onset of glioblastoma in patients without prior malignancies. In this study, we focused on the Envelope protein codified by the virus genome, which acts as viroporin and that is reported to be central for virus propagation. In particular, we characterized the electrophysiological profile of E-protein transfected U251 and HEK293 cells through the patch-clamp technique and FURA-2 measurements. Specifically, we observed an increase in the voltage-dependent (Kv) and calcium-dependent (KCa) potassium currents in HEK293 and U251 cell lines, respectively. Interestingly, in both cellular models, we observed a depolarization of the mitochondrial membrane potential in accordance with an alteration of U251 cell growth. We, therefore, investigated the transcriptional effect of E protein on the signaling pathways and found several gene alterations associated with apoptosis, cytokines and WNT pathways. The electrophysiological and transcriptional changes observed after E protein expression could explain the impact of SARS-CoV-2 infection on gliomagenesis.

Peptide S4 is an entry inhibitor of SARS-CoV-2 infection.

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a significant socioeconomic burden, and combating COVID-19 is imperative. Blocking the SARS-CoV-2 RBD-ACE2 interaction is a promising therapeutic approach for viral infections, as SARS-CoV-2 binds to the ACE2 receptors of host cells via the RBD of spike proteins to infiltrate these cells. We used computer-aided drug design technology and cellular experiments to screen for peptide S4 with high affinity and specificity for the human ACE2 receptor through structural analysis of SARS-CoV-2 and ACE2 interactions. Cellular experiments revealed that peptide S4 effectively inhibited SARS-CoV-2 and HCoV-NL63 viruses from infecting host cells and was safe for cells at effective concentrations. Based on these findings, peptide S4 may be a potential pharmaceutical agent for clinical application in the treatment of the ongoing SARS-CoV-2 pandemic.

Self-inhibiting percolation and viral spreading in epithelial tissue.

SARS-CoV-2 induces delayed type-I/III interferon production, allowing it to escape the early innate immune response. The delay has been attributed to a deficiency in the ability of cells to sense viral replication upon infection, which in turn hampers activation of the antiviral state in bystander cells. Here, we introduce a cellular automaton model to investigate the spatiotemporal spreading of viral infection as a function of virus and host-dependent parameters. The model suggests that the considerable person-to-person heterogeneity in SARS-CoV-2 infections is a consequence of high sensitivity to slight variations in biological parameters near a critical threshold. It further suggests that within-host viral proliferation can be curtailed by the presence of remarkably few cells that are primed for IFN production. Thus, the observed heterogeneity in defense readiness of cells reflects a remarkably cost-efficient strategy for protection.

Virological characteristics of a SARS-CoV-2-related bat coronavirus, BANAL-20-236.

BACKGROUND: Although several SARS-CoV-2-related coronaviruses (SC2r-CoVs) were discovered in bats and pangolins, the differences in virological characteristics between SARS-CoV-2 and SC2r-CoVs remain poorly understood. Recently, BANAL-20-236 (B236) was isolated from a rectal swab of Malayan horseshoe bat and was found to lack a furin cleavage site (FCS) in the spike (S) protein. The comparison of its virological characteristics with FCS-deleted SARS-CoV-2 (SC2ΔFCS) has not been conducted yet. METHODS: We prepared human induced pluripotent stem cell (iPSC)-derived airway and lung epithelial cells and colon organoids as human organ-relevant models. B236, SARS-CoV-2, and artificially generated SC2ΔFCS were used for viral experiments. To investigate the pathogenicity of B236 in vivo, we conducted intranasal infection experiments in hamsters. FINDINGS: In human iPSC-derived airway epithelial cells, the growth of B236 was significantly lower than that of the SC2ΔFCS. A fusion assay showed that the B236 and SC2ΔFCS S proteins were less fusogenic than the SARS-CoV-2 S protein. The infection experiment in hamsters showed that B236 was less pathogenic than SARS-CoV-2 and even SC2ΔFCS. Interestingly, in human colon organoids, the growth of B236 was significantly greater than that of SARS-CoV-2. INTERPRETATION: Compared to SARS-CoV-2, we demonstrated that B236 exhibited a tropism toward intestinal cells rather than respiratory cells. Our results are consistent with a previous report showing that B236 is enterotropic in macaques. Altogether, our report strengthens the assumption that SC2r-CoVs in horseshoe bats replicate primarily in the intestinal tissues rather than respiratory tissues. FUNDING: This study was supported in part by AMED ASPIRE (JP23jf0126002, to Keita Matsuno, Kazuo Takayama, and Kei Sato); AMED SCARDA Japan Initiative for World-leading Vaccine Research and Development Centers "UTOPIA" (JP223fa627001, to Kei Sato), AMED SCARDA Program on R&D of new generation vaccine including new modality application (JP223fa727002, to Kei Sato); AMED SCARDA Hokkaido University Institute for Vaccine Research and Development (HU-IVReD) (JP223fa627005h0001, to Takasuke Fukuhara, and Keita Matsuno); AMED Research Program on Emerging and Re-emerging Infectious Diseases (JP21fk0108574, to Hesham Nasser; JP21fk0108493, to Takasuke Fukuhara; JP22fk0108617 to Takasuke Fukuhara; JP22fk0108146, to Kei Sato; JP21fk0108494 to G2P-Japan Consortium, Keita Matsuno, Shinya Tanaka, Terumasa Ikeda, Takasuke Fukuhara, and Kei Sato; JP21fk0108425, to Kazuo Takayama and Kei Sato; JP21fk0108432, to Kazuo Takayama, Takasuke Fukuhara and Kei Sato; JP22fk0108534, Terumasa Ikeda, and Kei Sato; JP22fk0108511, to Yuki Yamamoto, Terumasa Ikeda, Keita Matsuno, Shinya Tanaka, Kazuo Takayama, Takasuke Fukuhara, and Kei Sato; JP22fk0108506, to Kazuo Takayama and Kei Sato); AMED Research Program on HIV/AIDS (JP22fk0410055, to Terumasa Ikeda; and JP22fk0410039, to Kei Sato); AMED Japan Program for Infectious Diseases Research and Infrastructure (JP22wm0125008 to Keita Matsuno); AMED CREST (JP21gm1610005, to Kazuo Takayama; JP22gm1610008, to Takasuke Fukuhara; JST PRESTO (JPMJPR22R1, to Jumpei Ito); JST CREST (JPMJCR20H4, to Kei Sato); JSPS KAKENHI Fund for the Promotion of Joint International Research (International Leading Research) (JP23K20041, to G2P-Japan Consortium, Keita Matsuno, Takasuke Fukuhara and Kei Sato); JST SPRING (JPMJSP2108 to Shigeru Fujita); JSPS KAKENHI Grant-in-Aid for Scientific Research C (22K07103, to Terumasa Ikeda); JSPS KAKENHI Grant-in-Aid for Scientific Research B (21H02736, to Takasuke Fukuhara); JSPS KAKENHI Grant-in-Aid for Early-Career Scientists (22K16375, to Hesham Nasser; 20K15767, to Jumpei Ito); JSPS Core-to-Core Program (A. Advanced Research Networks) (JPJSCCA20190008, to Kei Sato); JSPS Research Fellow DC2 (22J11578, to Keiya Uriu); JSPS Research Fellow DC1 (23KJ0710, to Yusuke Kosugi); JSPS Leading Initiative for Excellent Young Researchers (LEADER) (to Terumasa Ikeda); World-leading Innovative and Smart Education (WISE) Program 1801 from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) (to Naganori Nao); Ministry of Health, Labour and Welfare (MHLW) under grant 23HA2010 (to Naganori Nao and Keita Matsuno); The Cooperative Research Program (Joint Usage/Research Center program) of Institute for Life and Medical Sciences, Kyoto University (to Kei Sato); International Joint Research Project of the Institute of Medical Science, the University of Tokyo (to Terumasa Ikeda and Takasuke Fukuhara); The Tokyo Biochemical Research Foundation (to Kei Sato); Takeda Science Foundation (to Terumasa Ikeda and Takasuke Fukuhara); Mochida Memorial Foundation for Medical and Pharmaceutical Research (to Terumasa Ikeda); The Naito Foundation (to Terumasa Ikeda); Hokuto Foundation for Bioscience (to Tomokazu Tamura); Hirose Foundation (to Tomokazu Tamura); and Mitsubishi Foundation (to Kei Sato).

Loss of CFTR Reverses Senescence Hallmarks in SARS-CoV-2 Infected Bronchial Epithelial Cells.

SARS-CoV-2 infection has been recently shown to induce cellular senescence in vivo. A senescence-like phenotype has been reported in cystic fibrosis (CF) cellular models. Since the previously published data highlighted a low impact of SARS-CoV-2 on CFTR-defective cells, here we aimed to investigate the senescence hallmarks in SARS-CoV-2 infection in the context of a loss of CFTR expression/function. We infected WT and CFTR KO 16HBE14o-cells with SARS-CoV-2 and analyzed both the p21 and Ki67 expression using immunohistochemistry and viral and p21 gene expression using real-time PCR. Prior to SARS-CoV-2 infection, CFTR KO cells displayed a higher p21 and lower Ki67 expression than WT cells. We detected lipid accumulation in CFTR KO cells, identified as lipolysosomes and residual bodies at the subcellular/ultrastructure level. After SARS-CoV-2 infection, the situation reversed, with low p21 and high Ki67 expression, as well as reduced viral gene expression in CFTR KO cells. Thus, the activation of cellular senescence pathways in CFTR-defective cells was reversed by SARS-CoV-2 infection while they were activated in CFTR WT cells. These data uncover a different response of CF and non-CF bronchial epithelial cell models to SARS-CoV-2 infection and contribute to uncovering the molecular mechanisms behind the reduced clinical impact of COVID-19 in CF patients.

Impact of in vitro SARS-CoV-2 infection on breast cancer cells.

The pandemic of coronavirus disease 19 (COVID-19), caused by severe respiratory syndrome coronavirus 2 (SARS-CoV-2), had severe repercussions for breast cancer patients. Increasing evidence indicates that SARS-CoV-2 infection may directly impact breast cancer biology, but the effects of SARS-CoV-2 on breast tumor cells are still unknown. Here, we analyzed the molecular events occurring in the MCF7, MDA-MB-231 and HCC1937 breast cancer cell lines, representative of the luminal A, basal B/claudin-low and basal A subtypes, respectively, upon SARS-CoV-2 infection. Viral replication was monitored over time, and gene expression profiling was conducted. We found that MCF7 cells were the most permissive to viral replication. Treatment of MCF7 cells with Tamoxifen reduced the SARS-CoV-2 replication rate, suggesting an involvement of the estrogen receptor in sustaining virus replication in malignant cells. Interestingly, a metagene signature based on genes upregulated by SARS-CoV-2 infection in all three cell lines distinguished a subgroup of premenopausal luminal A breast cancer patients with a poor prognosis. As SARS-CoV-2 still spreads among the population, it is essential to understand the impact of SARS-CoV-2 infection on breast cancer, particularly in premenopausal patients diagnosed with the luminal A subtype, and to assess the long-term impact of COVID-19 on breast cancer outcomes.