SELP Asp603Asn and severe thrombosis in COVID-19 males.

Thromboembolism is a frequent cause of severity and mortality in COVID-19. However, the etiology of this phenomenon is not well understood. A cohort of 1186 subjects, from the GEN-COVID consortium, infected by SARS-CoV-2 with different severity was stratified by sex and adjusted by age. Then, common coding variants from whole exome sequencing were mined by LASSO logistic regression. The homozygosity of the cell adhesion molecule P-selectin gene (SELP) rs6127 (c.1807G > A; p.Asp603Asn) which has been already associated with thrombotic risk is found to be associated with severity in the male subcohort of 513 subjects (odds ratio = 2.27, 95% Confidence Interval 1.54-3.36). As the SELP gene is downregulated by testosterone, the odd ratio is increased in males older than 50 (OR 2.42, 95% CI 1.53-3.82). Asn/Asn homozygotes have increased D-dimers values especially when associated with poly Q ≥ 23 in the androgen receptor (OR 3.26, 95% CI 1.41-7.52). These results provide a rationale for the repurposing of antibodies against P-selectin as adjuvant therapy in rs6127 male homozygotes especially if older than 50 or with an impaired androgen receptor.

Downregulation of MALAT1 is a hallmark of tissue and peripheral proliferative T cells in COVID-19.

T cells play key protective but also pathogenic roles in COVID-19. We studied the expression of long non-coding RNAs (lncRNAs) in COVID-19 T-cell transcriptomes by integrating previously published single-cell RNA sequencing datasets. The long intergenic non-coding RNA MALAT1 was the most highly transcribed lncRNA in T cells, with Th1 cells demonstrating the lowest and CD8+ resident memory cells the highest MALAT1 expression, amongst CD4+ and CD8+ T-cells populations, respectively. We then identified gene signatures that covaried with MALAT1 in single T cells. A significantly higher number of transcripts correlated negatively with MALAT1 than those that correlated. Enriched functional annotations of the MALAT1- anti-correlating gene signature included processes associated with T-cell activation such as cell division, oxidative phosphorylation, and response to cytokine. The MALAT1 anti-correlating gene signature shared by both CD4+ and CD8+ T-cells marked dividing T cells in both the lung and blood of COVID-19 patients. Focussing on the tissue, we used an independent patient cohort of post-mortem COVID-19 lung samples and demonstrated that MALAT1 suppression was indeed a marker of MKI67+ proliferating CD8+ T cells. Our results reveal MALAT1 suppression and its associated gene signature are a hallmark of human proliferating T cells.

ApoE4 associated with severe COVID-19 outcomes via downregulation of ACE2 and imbalanced RAS pathway.

BACKGROUND: Recent numerous epidemiology and clinical association studies reported that ApoE polymorphism might be associated with the risk and severity of coronavirus disease 2019 (COVID-19), and yielded inconsistent results. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection relies on its spike protein binding to angiotensin-converting enzyme 2 (ACE2) receptor expressed on host cell membranes. METHODS: A meta-analysis was conducted to clarify the association between ApoE polymorphism and the risk and severity of COVID-19. Multiple protein interaction assays were utilized to investigate the potential molecular link between ApoE and the SARS-CoV-2 primary receptor ACE2, ApoE and spike protein. Immunoblotting and immunofluorescence staining methods were used to access the regulatory effect of different ApoE isoform on ACE2 protein expression. RESULTS: ApoE gene polymorphism (ε4 carrier genotypes VS non-ε4 carrier genotypes) is associated with the increased risk (P = 0.0003, OR = 1.44, 95% CI 1.18-1.76) and progression (P < 0.00001, OR = 1.85, 95% CI 1.50-2.28) of COVID-19. ApoE interacts with both ACE2 and the spike protein but did not show isoform-dependent binding effects. ApoE4 significantly downregulates ACE2 protein expression in vitro and in vivo and subsequently decreases the conversion of Ang II to Ang 1-7. CONCLUSIONS: ApoE4 increases SARS-CoV-2 infectivity in a manner that may not depend on differential interactions with the spike protein or ACE2. Instead, ApoE4 downregulates ACE2 protein expression and subsequently the dysregulation of renin-angiotensin system (RAS) may provide explanation by which ApoE4 exacerbates COVID-19 disease.

Transcriptomic study reveals lncRNA-mediated downregulation of innate immune and inflammatory response in the SARS-CoV-2 vaccination breakthrough infections.

Introduction: The emergence of multiple variants of concerns (VOCs) with higher number of Spike mutations have led to enhanced immune escape by the SARS-CoV-2. With the increasing number of vaccination breakthrough (VBT) infections, it is important to understand the possible reason/s of the breakthrough infections. Methods: We performed transcriptome sequencing of 57 VBT and unvaccinated COVID-19 patients, followed by differential expression and co-expression analysis of the lncRNAs and the mRNAs. The regulatory mechanism was highlighted by analysis towards repeat element distribution within the co-expressed lncRNAs, followed by repeats driven homologous interaction between the lncRNAs and the promoter regions of genes from the same topologically associated domains (TAD). Results: We identified 727 differentially expressed lncRNAs (153 upregulated and 574 downregulated) and 338 mRNAs (34 up- and 334 downregulated) in the VBT patients. This includes LUCAT1, MALAT1, ROR1-AS1, UGDH-AS1 and LINC00273 mediated modulation of immune response, whereas MALAT1, NEAT1 and GAS5 regulated inflammatory response in the VBT. LncRNA-mRNA co-expression analysis highlighted 34 lncRNAs interacting with 267 mRNAs. We also observed a higher abundance of Alu, LINE1 and LTRs within the interacting lncRNAs of the VBT patients. These interacting lncRNAs have higher interaction with the promoter region of the genes from the same TAD, compared to the non-interacting lncRNAs with the enrichment of Alu and LINE1 in the gene promoter. Discussion: Significant downregulation and GSEA of the TAD gene suggest Alu and LINE1 driven homologous interaction between the lncRNAs and the TAD genes as a possible mechanism of lncRNA-mediated suppression of innate immune/inflammatory responses and activation of adaptive immune response. The lncRNA-mediated suppression of innate immune/inflammatory responses and activation of adaptive immune response might explain the SARS-CoV-2 breakthrough infections with milder symptoms in the VBT. Besides, the study also highlights repeat element mediated regulation of genes in 3D as another possible way of lncRNA-mediated immune-regulation modulating vaccination breakthroughs milder disease phenotype and shorter hospital stay.

SARS-CoV-2 viral entry and replication is impaired in Cystic Fibrosis airways due to ACE2 downregulation.

As an inherited disorder characterized by severe pulmonary disease, cystic fibrosis could be considered a comorbidity for coronavirus disease 2019. Instead, current clinical evidence seems to be heading in the opposite direction. To clarify whether host factors expressed by the Cystic Fibrosis epithelia may influence coronavirus disease 2019 progression, here we describe the expression of SARS-CoV-2 receptors in primary airway epithelial cells. We show that angiotensin converting enzyme 2 (ACE2) expression and localization are regulated by Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) channel. Consistently, our results indicate that dysfunctional CFTR channels alter susceptibility to SARS-CoV-2 infection, resulting in reduced viral entry and replication in Cystic Fibrosis cells. Depending on the pattern of ACE2 expression, the SARS-CoV-2 spike (S) protein induced high levels of Interleukin 6 in healthy donor-derived primary airway epithelial cells, but a very weak response in primary Cystic Fibrosis cells. Collectively, these data support that Cystic Fibrosis condition may be at least partially protecting from SARS-CoV-2 infection.

Down-regulation of KLF2 in lung fibroblasts is linked with COVID-19 immunofibrosis and restored by combined inhibition of NETs, JAK-1/2 and IL-6 signaling.

Kruppel-like factor 2 (KLF2) has been linked with fibrosis and neutrophil-associated thromboinflammation; however, its role in COVID-19 remains elusive. We investigated the effect of disease microenvironment on the fibrotic potential of human lung fibroblasts (LFs) and its association with KLF2 expression. LFs stimulated with plasma from severe COVID-19 patients down-regulated KLF2 expression at mRNA/protein and functional level acquiring a pre-fibrotic phenotype, as indicated by increased CCN2/collagen levels. Pre-incubation with the COMBI-treatment-agents (DNase I and JAKs/IL-6 inhibitors baricitinib/tocilizumab) restored KLF2 levels of LFs to normal abolishing their fibrotic activity. LFs stimulated with plasma from COMBI-treated patients at day-7 expressed lower CCN2 and higher KLF2 levels, compared to plasma prior-to-treatment, an effect not observed in standard-of-care treatment. In line with this, COMBI-treated patients had better outcome than standard-of-care group. These data link fibroblast KLF2 with NETosis and JAK/IL-6 signaling, suggesting the potential of combined therapeutic strategies in immunofibrotic diseases, such as COVID-19.

Downregulation of the Protein C Signaling System Is Associated with COVID-19 Hypercoagulability-A Single-Cell Transcriptomics Analysis.

Because of the interface between coagulation and the immune response, it is expected that COVID-19-associated coagulopathy occurs via activated protein C signaling. The objective was to explore putative changes in the expression of the protein C signaling network in the liver, peripheral blood mononuclear cells, and nasal epithelium of patients with COVID-19. Single-cell RNA-sequencing data from patients with COVID-19 and healthy subjects were obtained from the COVID-19 Cell Atlas database. A functional protein-protein interaction network was constructed for the protein C gene. Patients with COVID-19 showed downregulation of protein C and components of the downstream protein C signaling cascade. The percentage of hepatocytes expressing protein C was lower. Part of the liver cell clusters expressing protein C presented increased expression of ACE2. In PBMC, there was increased ACE2, inflammatory, and pro-coagulation transcripts. In the nasal epithelium, PROC, ACE2, and PROS1 were expressed by the ciliated cell cluster, revealing co-expression of ACE-2 with transcripts encoding proteins belonging to the coagulation and immune system interface. Finally, there was upregulation of coagulation factor 3 transcript in the liver and PBMC. Protein C could play a mechanistic role in the hypercoagulability syndrome affecting patients with severe COVID-19.

SARS-CoV-2 escapes direct NK cell killing through Nsp1-mediated downregulation of ligands for NKG2D.

Natural killer (NK) cells are cytotoxic effector cells that target and lyse virally infected cells; many viruses therefore encode mechanisms to escape such NK cell killing. Here, we interrogate the ability of SARS-CoV-2 to modulate NK cell recognition and lysis of infected cells. We find that NK cells exhibit poor cytotoxic responses against SARS-CoV-2-infected targets, preferentially killing uninfected bystander cells. We demonstrate that this escape is driven by downregulation of ligands for the activating receptor NKG2D (NKG2D-L). Indeed, early in viral infection, prior to NKG2D-L downregulation, NK cells are able to target and kill infected cells; however, this ability is lost as viral proteins are expressed. Finally, we find that SARS-CoV-2 non-structural protein 1 (Nsp1) mediates downregulation of NKG2D-L and that Nsp1 alone is sufficient to confer resistance to NK cell killing. Collectively, our work demonstrates that SARS-CoV-2 evades direct NK cell cytotoxicity and describes a mechanism by which this occurs.

Wnt5a/ß-catenin axis is involved in the downregulation of AT2 lineage by PAI-1.

Failure to regenerate injured alveoli functionally and promptly causes a high incidence of fatality in coronavirus disease 2019 (COVID-19). How elevated plasminogen activator inhibitor-1 (PAI-1) regulates the lineage of alveolar type 2 (AT2) cells for re-alveolarization has not been studied. This study aimed to examine the role of PAI-1-Wnt5a-ß catenin cascades in AT2 fate. Dramatic reduction in AT2 yield was observed in Serpine1Tg mice. Elevated PAI-1 level suppressed organoid number, development efficiency, and total surface area in vitro. Anti-PAI-1 neutralizing antibody restored organoid number, proliferation and differentiation of AT2 cells, and ß-catenin level in organoids. Both Wnt family member 5A (Wnt5a) and Wnt5a-derived N-butyloxycarbonyl hexapeptide (Box5) altered the lineage of AT2 cells. This study demonstrates that elevated PAI-1 regulates AT2 proliferation and differentiation via the Wnt5a/ß catenin cascades. PAI-1 could serve as autocrine signaling for lung injury repair.

Downregulation of ACE, AGTR1, and ACE2 genes mediating SARS-CoV-2 pathogenesis by gut microbiota members and their postbiotics on Caco-2 cells.

INTRODUCTION: Coronavirus disease-2019 (COVID-19) is a complex infection caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that can cause also gastrointestinal symptoms. There are various factors that determine the host susceptibility and severity of infection, including the renin-angiotensin system, the immune response, and the gut microbiota. In this regard, we aimed to investigate the gene expression of ACE, AGTR1, ACE2, and TMPRSS2, which mediate SARS-CoV-2 pathogenesis by Akkermansia muciniphila, Faecalibacterium prausnitzii, Bacteroides thetaiotaomicron, and Bacteroides fragilis on Caco-2 cells. Also, the enrichment analysis considering the studied genes was analyzed on raw data from the microarray analysis of COVID-19 patients. MATERIALS AND METHODS: Caco-2 cells were treated with live, heat-inactivated form and cell free supernatants of A. muciniphila, F. prausnitzii, B. thetaiotaomicron and B. fragilis for overnight. After RNA extraction and cDNA synthesis, the expression of studied genes was assessed by RT-qPCR. DNA methylation of studied genes was analyzed by Partek® Genomics Suite® software on the GSE174818 dataset. We used GSE164805 and GSE166552 datasets from COVID-19 patients to perform enrichment analysis by considering the mentioned genes via GEO2R, DAVID. Finally, the related microRNAs to GO terms concerned on the studied genes were identified by miRPath. RESULTS: The downregulation of ACE, AGTR1, and ACE2 genes by A. muciniphila, F. prausnitzii, B. thetaiotaomicron, and B. fragilis in live, heat-inactivated, and cell-free supernatants was reported for the first time. These genes had hypomethylated DNA status in COVID-19 patients' raw data. The highest fold enrichment in upregulated RAS pathways and immune responses belonged to ACE, AGTR1, and ACE2 by considering the protein-protein interaction network. The common miRNAs targeting the studied genes were reported as miR-124-3p and miR-26b-5p. CONCLUSION: In combination with our experimental data and bioinformatic analysis, we showed the potential of A. muciniphila, F. prausnitzii, B. thetaiotaomicron, and B. fragilis and their postbiotics to reduce ACE, ATR1, and ACE2 expression, which are essential genes that drive upregulated biological processes in COVID-19 patients. Accordingly, due to the potential of studied bacteria on the alteration of ACE, AGTR1, ACE2 genes expression, understanding their correlation with demonstrated miRNAs expression could be valuable. These findings suggest the importance of considering targeted gut microbiota intervention when designing the possible therapeutic strategy for controlling the COVID-19.

Effects of tocilizumab and dexamethasone on the downregulation of proinflammatory cytokines and upregulation of antioxidants in the lungs in oleic acid-induced ARDS.

BACKGROUND: Acute respiratory distress syndrome (ARDS) is a life-threatening disease caused by the induction of inflammatory cytokines and chemokines in the lungs. There is a dearth of drug applications that can be used to prevent cytokine storms in ARDS treatment. This study was designed to investigate the effects of tocilizumab and dexamethasone on oxidative stress, antioxidant parameters, and cytokine storms in acute lung injury caused by oleic acid in rats. METHODS: Adult male rats were divided into five groups: the CN (healthy rats, n = 6), OA (oleic acid administration, n = 6), OA + TCZ-2 (oleic acid and tocilizumab at 2 mg/kg, n = 6), OA + TCZ-4 (oleic acid and tocilizumab at 4 mg/kg, n = 6), and OA + DEX-10 (oleic acid and dexamethasone at 10 mg/kg, n = 6) groups. All animals were euthanized after treatment for histopathological, immunohistochemical, biochemical, PCR, and SEM analyses. RESULTS: Expressions of TNF-α, IL-1ß, IL-6, and IL-8 cytokines in rats with acute lung injury induced by oleic acid were downregulated in the TCZ and DEX groups compared to the OA group (P < 0.05). The MDA level in lung tissues was statistically lower in the OA + TCZ-4 group compared to the OA group. It was further determined that SOD, GSH, and CAT levels were decreased in the OA group and increased in the TCZ and DEX groups (P < 0.05). Histopathological findings such as thickening of the alveoli, hyperemia, and peribronchial cell infiltration were found to be similar when lung tissues of the TCZ and DEX groups were compared to the control group. With SEM imaging of the lung tissues, it was found that the alveolar lining layer had become indistinct in the OA, OA + TCZ-2, and OA + TCZ-4 groups. CONCLUSIONS: In this model of acute lung injury caused by oleic acid, tocilizumab and dexamethasone were effective in preventing cytokine storms by downregulating the expression of proinflammatory cytokines including TNF-α, IL-1ß, IL-6, and IL-8. Against the downregulation of antioxidant parameters such as SOD and GSH in the lung tissues caused by oleic acid, tocilizumab and dexamethasone upregulated them and showed protective effects against cell damage.

Down regulation of defensin genes during SARS-CoV-2 infection.

Defensins, crucial components of the innate immune system, play a vital role against infection as part of frontline immunity. Association of SARS-CoV-2 infection with defensins has not been investigated. In this study, we have investigated the expression of defensin genes in the buccal cavity from patients with COVID-19 infection along with negative control samples. Nasopharyngeal/oropharyngeal swab samples collected for screening SARS-CoV-2 infection in early 2020 from Hyderabad, India, were analyzed for the expression of major defensin genes by the quantitative real-time reverse transcription polymerase chain reaction, qRT-PCR. Forty SARS-CoV-2 infected positive and 40 negative swab samples were selected for this study. Based on the qRT-PCR analysis involving gene specific primers for defensin genes, 9 defensin genes were found to be expressed in the nasopharyngeal/oropharyngeal cavity. Four defensin genes were found to be significantly down regulated in SARS-CoV-2 infected patients in comparison with the control samples based on differential expression analysis. The significantly down regulated genes were defensin beta 4A/B, 106B, 107B, and 103A. Down regulation of human beta defensin 2, 3, 6 and 7 suggests that antiviral innate immune response provided by defensins may be compromised in SARS-CoV-2 infection resulting in progression of the disease. Correction of the down regulation process through appropriate defensin peptide-based therapy could be an attractive method of treatment. Keywords: host defense; defensins; COVID-19; gene regulation; SARS-CoV-2.

Spike-mediated ACE2 down-regulation was involved in the pathogenesis of SARS-CoV-2 infection.

The ongoing global pandemic of Coronavirus disease 2019 (COVID-19) poses a serious threat to human health, with patients reportedly suffering from thrombus, vascular injury and coagulation in addition to acute and diffuse lung injury and respiratory diseases. Angiotensin converting enzyme 2 (ACE2) as the receptor for SARS-CoV-2 entry, is also an important regulator of renin-angiotensin system (RAS) homeostasis, which plays an unsettled role in the pathogenesis of COVID-19. Here, we demonstrated that SARS-CoV-2 Spike protein activated intracellular signals to degrade ACE2 mRNA. The decrease of ACE2 and higher level of angiotensin (Ang) II were verified in COVID-19 patients. High dose of Ang II induced pulmonary artery endothelial cell death in vitro, which was also observed in the lung of COVID-19 patients. Our finding indicates that the downregulation of ACE2 potentially links COVID-19 to the imbalance of RAS.

Increased Tacrolimus Exposure in Kidney Transplant Recipients With COVID-19: Inflammation-Driven Downregulation of Metabolism as a Potential Mechanism.

Kidney transplant recipients (KTRs) are at increased risk of severe COVID-19 disease compared to the general population. This is partly driven by their use of immunosuppressive therapy, which influences inflammatory responses and viral loads. Current guidelines suggest to withdraw mycophenolate while calcineurin inhibitors are often continued during a COVID-19 infection. However, clinical signs of calcineurin toxicity have been described in multiple COVID-19 positive KTRs. In this report we describe the course of tacrolimus exposure prior to, during, and post COVID-19 in observations from three clinical cases as well as four KTRs from a controlled trial population. We postulate inflammation driven downregulation of the CYP3A metabolism as a potential mechanism for higher tacrolimus exposure. To mitigate the risk of tacrolimus overexposure and toxicity therapeutic drug monitoring is recommended in KTRs with COVID-19 both in the in-, out-patient and home monitoring setting.

Anti-IFN-α/-ω neutralizing antibodies from COVID-19 patients correlate with downregulation of IFN response and laboratory biomarkers of disease severity.

A significant number of COVID-19 patients were shown to have neutralizing antibodies (NAB) against IFN; however, NAB specificity, fluctuation over time, associations with biochemical and hematological parameters, and IFN gene expression are not well characterized. Binding antibodies (BAB) to IFN-α/-ß were screened in COVID-19 patients' serum. All BAB positive sera, and a subset of respiratory samples, were tested for NAB against IFN-α/-ß/-ω, using an antiviral bioassay. Transcript levels of IFN-α/-ß/-ω and IFN-stimulated genes (ISGs) were quantified. Anti-IFN-I BAB were found in 61 out of 360 (17%) of patients. Among BAB positive sera, 21.3% had a high NAB titer against IFN-α. A total of 69.2% of anti-IFN-α NAB sera displayed cross-reactivity to IFN-ω. Anti-IFN-I NAB persisted in all patients. NAB to IFN-α were also detected in 3 out of 17 (17.6%) of respiratory samples. Anti-IFN-I NAB were higher in males (p = 0.0017), patients admitted to the ICU (p < 0.0001), and patients with a fatal outcome (p < 0.0001). NAB were associated with higher levels of CRP, LDH, d-Dimer, and higher counts of hematological parameters. ISG-mRNAs were reduced in patients with persistently NAB titer. NAB are detected in a significant proportion of severe COVID-19. NAB positive patients presented a defective IFN response and increased levels of laboratory biomarkers of disease severity.

Dopamine Reduces SARS-CoV-2 Replication In Vitro through Downregulation of D2 Receptors and Upregulation of Type-I Interferons.

Recent evidence suggests that SARS-CoV-2 hinders immune responses via dopamine (DA)-related mechanisms. Nonetheless, studies addressing the specific role of DA in the frame of SARS-CoV-2 infection are still missing. In the present study, we investigate the role of DA in SARS-CoV-2 replication along with potential links with innate immune pathways in CaLu-3 human epithelial lung cells. We document here for the first time that, besides DA synthetic pathways, SARS-CoV-2 alters the expression of D1 and D2 DA receptors (D1DR, D2DR), while DA administration reduces viral replication. Such an effect occurs at non-toxic, micromolar-range DA doses, which are known to induce receptor desensitization and downregulation. Indeed, the antiviral effects of DA were associated with a robust downregulation of D2DRs both at mRNA and protein levels, while the amount of D1DRs was not significantly affected. While halting SARS-CoV-2 replication, DA, similar to the D2DR agonist quinpirole, upregulates the expression of ISGs and Type-I IFNs, which goes along with the downregulation of various pro-inflammatory mediators. In turn, administration of Type-I IFNs, while dramatically reducing SARS-CoV-2 replication, converges in downregulating D2DRs expression. Besides configuring the CaLu-3 cell line as a suitable model to study SARS-CoV-2-induced alterations at the level of the DA system in the periphery, our findings disclose a previously unappreciated correlation between DA pathways and Type-I IFN response, which may be disrupted by SARS-CoV-2 for host cell invasion and replication.