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1.
JCI Insight ; 6(24)2021 12 22.
Article in English | MEDLINE | ID: covidwho-1598468

ABSTRACT

mRNA vaccines for SARS-CoV-2 have shown exceptional clinical efficacy, providing robust protection against severe disease. However, our understanding of transcriptional and repertoire changes following full vaccination remains incomplete. We used scRNA-Seq and functional assays to compare humoral and cellular responses to 2 doses of mRNA vaccine with responses observed in convalescent individuals with asymptomatic disease. Our analyses revealed enrichment of spike-specific B cells, activated CD4+ T cells, and robust antigen-specific polyfunctional CD4+ T cell responses following vaccination. On the other hand, although clonally expanded CD8+ T cells were observed following both vaccination and natural infection, CD8+ T cell responses were relatively weak and variable. In addition, TCR gene usage was variable, reflecting the diversity of repertoires and MHC polymorphism in the human population. Natural infection induced expansion of CD8+ T cell clones that occupy distinct clusters compared to those induced by vaccination and likely recognize a broader set of viral antigens of viral epitopes presented by the virus not seen in the mRNA vaccine. Our study highlights a coordinated adaptive immune response in which early CD4+ T cell responses facilitate the development of the B cell response and substantial expansion of effector CD8+ T cells, together capable of contributing to future recall responses.


Subject(s)
/immunology , COVID-19/immunology , Immunity, Cellular/immunology , Immunity, Humoral/immunology , /therapeutic use , Adaptive Immunity/genetics , Adaptive Immunity/immunology , Adult , Aged , Antigens, Viral , B-Lymphocytes , CD4-Positive T-Lymphocytes , CD8-Positive T-Lymphocytes , COVID-19/prevention & control , COVID-19 Vaccines/immunology , COVID-19 Vaccines/therapeutic use , Carrier State , Convalescence , Epitopes , Female , Humans , Immunity, Cellular/genetics , Immunity, Humoral/genetics , Immunogenicity, Vaccine , Immunologic Memory , Male , Middle Aged , RNA-Seq , SARS-CoV-2 , Single-Cell Analysis , Spike Glycoprotein, Coronavirus/immunology , Th1 Cells , Th17 Cells , Vaccines, Synthetic/immunology , Vaccines, Synthetic/therapeutic use , Young Adult , /therapeutic use
2.
Cells ; 10(12)2021 12 10.
Article in English | MEDLINE | ID: covidwho-1572376

ABSTRACT

To assess the biology of the lethal endpoint in patients with SARS-CoV-2 infection, we compared the transcriptional response to the virus in patients who survived or died during severe COVID-19. We applied gene expression profiling to generate transcriptional signatures for peripheral blood mononuclear cells (PBMCs) from patients with SARS-CoV-2 infection at the time when they were placed in the Intensive Care Unit of the Pavlov First State Medical University of St. Petersburg (Russia). Three different bioinformatics approaches to RNA-seq analysis identified a downregulation of three common pathways in survivors compared with nonsurvivors among patients with severe COVID-19, namely, low-density lipoprotein (LDL) particle receptor activity (GO:0005041), important for maintaining cholesterol homeostasis, leukocyte differentiation (GO:0002521), and cargo receptor activity (GO:0038024). Specifically, PBMCs from surviving patients were characterized by reduced expression of PPARG, CD36, STAB1, ITGAV, and ANXA2. Taken together, our findings suggest that LDL particle receptor pathway activity in patients with COVID-19 infection is associated with poor disease prognosis.


Subject(s)
COVID-19/genetics , Down-Regulation/genetics , Gene Expression Profiling , Receptors, LDL/genetics , Aged , COVID-19/virology , Gene Ontology , Gene Regulatory Networks , Humans , Leukocytes, Mononuclear/metabolism , Male , Middle Aged , RNA-Seq , SARS-CoV-2/physiology
3.
Nat Commun ; 12(1): 4502, 2021 07 23.
Article in English | MEDLINE | ID: covidwho-1550282

ABSTRACT

Cells in many tissues, such as bone, muscle, and placenta, fuse into syncytia to acquire new functions and transcriptional programs. While it is known that fused cells are specialized, it is unclear whether cell-fusion itself contributes to programmatic-changes that generate the new cellular state. Here, we address this by employing a fusogen-mediated, cell-fusion system to create syncytia from undifferentiated cells. RNA-Seq analysis reveals VSV-G-induced cell fusion precedes transcriptional changes. To gain mechanistic insights, we measure the plasma membrane surface area after cell-fusion and observe it diminishes through increases in endocytosis. Consequently, glucose transporters internalize, and cytoplasmic glucose and ATP transiently decrease. This reduced energetic state activates AMPK, which inhibits YAP1, causing transcriptional-reprogramming and cell-cycle arrest. Impairing either endocytosis or AMPK activity prevents YAP1 inhibition and cell-cycle arrest after fusion. Together, these data demonstrate plasma membrane diminishment upon cell-fusion causes transient nutrient stress that may promote transcriptional-reprogramming independent from extrinsic cues.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Cell Membrane/metabolism , Cell Nucleus/metabolism , Membrane Glycoproteins/metabolism , Transcription Factors/metabolism , Transcription, Genetic/genetics , Viral Envelope Proteins/metabolism , AMP-Activated Protein Kinases/genetics , AMP-Activated Protein Kinases/metabolism , Adaptor Proteins, Signal Transducing/genetics , Animals , Biological Transport , Cell Fusion , Cell Line , Cell Line, Tumor , Cells, Cultured , Giant Cells/metabolism , HEK293 Cells , Humans , Membrane Glycoproteins/genetics , Mice , RNA-Seq/methods , Signal Transduction/genetics , Transcription Factors/genetics , Viral Envelope Proteins/genetics
4.
JCI Insight ; 6(22)2021 11 22.
Article in English | MEDLINE | ID: covidwho-1528615

ABSTRACT

BACKGROUNDInfluenza A virus (IAV) and SARS-CoV-2 are pandemic viruses causing millions of deaths, yet their clinical manifestations are distinctly different.METHODSWith the hypothesis that upper airway immune and epithelial cell responses are also distinct, we performed single-cell RNA sequencing (scRNA-Seq) on nasal wash cells freshly collected from adults with either acute COVID-19 or influenza or from healthy controls. We focused on major cell types and subtypes in a subset of donor samples.ResultsNasal wash cells were enriched for macrophages and neutrophils for both individuals with influenza and those with COVID-19 compared with healthy controls. Hillock-like epithelial cells, M2-like macrophages, and age-dependent B cells were enriched in COVID-19 samples. A global decrease in IFN-associated transcripts in neutrophils, macrophages, and epithelial cells was apparent in COVID-19 samples compared with influenza samples. The innate immune response to SARS-CoV-2 appears to be maintained in macrophages, despite evidence for limited epithelial cell immune sensing. Cell-to-cell interaction analyses revealed a decrease in epithelial cell interactions in COVID-19 and highlighted differences in macrophage-macrophage interactions for COVID-19 and influenza.ConclusionsOur study demonstrates that scRNA-Seq can define host and viral transcriptional activity at the site of infection and reveal distinct local epithelial and immune cell responses for COVID-19 and influenza that may contribute to their divergent disease courses.FundingMassachusetts Consortium on Pathogen Readiness, the Mathers Foundation, and the Department of Defense (W81XWH2110029) "COVID-19 Expansion for AIRe Program."


Subject(s)
COVID-19 , Immunity, Innate , Influenza A virus , Influenza, Human , Macrophages , RNA-Seq , SARS-CoV-2 , Adult , COVID-19/genetics , COVID-19/immunology , Female , Humans , Influenza A virus/genetics , Influenza A virus/immunology , Influenza, Human/genetics , Influenza, Human/immunology , Macrophages/immunology , Macrophages/virology , Male , Nasal Lavage , SARS-CoV-2/genetics , SARS-CoV-2/immunology
5.
Brief Bioinform ; 22(5)2021 09 02.
Article in English | MEDLINE | ID: covidwho-1528156

ABSTRACT

The low capture rate of expressed RNAs from single-cell sequencing technology is one of the major obstacles to downstream functional genomics analyses. Recently, a number of imputation methods have emerged for single-cell transcriptome data, however, recovering missing values in very sparse expression matrices remains a substantial challenge. Here, we propose a new algorithm, WEDGE (WEighted Decomposition of Gene Expression), to impute gene expression matrices by using a biased low-rank matrix decomposition method. WEDGE successfully recovered expression matrices, reproduced the cell-wise and gene-wise correlations and improved the clustering of cells, performing impressively for applications with sparse datasets. Overall, this study shows a potent approach for imputing sparse expression matrix data, and our WEDGE algorithm should help many researchers to more profitably explore the biological meanings embedded in their single-cell RNA sequencing datasets. The source code of WEDGE has been released at https://github.com/QuKunLab/WEDGE.


Subject(s)
Algorithms , Computational Biology/methods , Gene Expression Profiling/methods , RNA-Seq/methods , Single-Cell Analysis/methods , COVID-19/blood , COVID-19/genetics , COVID-19/virology , Cluster Analysis , Computer Simulation , Genomics/methods , Humans , Leukocytes, Mononuclear/classification , Leukocytes, Mononuclear/metabolism , Reproducibility of Results , SARS-CoV-2/physiology , Severity of Illness Index
6.
J Am Soc Nephrol ; 32(1): 41-51, 2021 01.
Article in English | MEDLINE | ID: covidwho-1496668

ABSTRACT

BACKGROUND: Mutations in PKD1 and PKD2, which encode the transmembrane proteins polycystin-1 and polycystin-2, respectively, cause autosomal dominant polycystic kidney disease (ADPKD). Polycystins are expressed in the primary cilium, and disrupting cilia structure significantly slows ADPKD progression following inactivation of polycystins. The cellular mechanisms of polycystin- and cilia-dependent cyst progression in ADPKD remain incompletely understood. METHODS: Unbiased transcriptional profiling in an adult-onset Pkd2 mouse model before cysts formed revealed significant differentially expressed genes (DEGs) in Pkd2 single-knockout kidneys, which were used to identify candidate pathways dysregulated in kidneys destined to form cysts. In vivo studies validated the role of the candidate pathway in the progression of ADPKD. Wild-type and Pkd2/Ift88 double-knockout mice that are protected from cyst growth served as controls. RESULTS: The RNASeq data identified cell proliferation as the most dysregulated pathway, with 15 of 241 DEGs related to cell cycle functions. Cdk1 appeared as a central component in this analysis. Cdk1 expression was similarly dysregulated in Pkd1 models of ADPKD, and conditional inactivation of Cdk1 with Pkd1 markedly improved the cystic phenotype and kidney function compared with inactivation of Pkd1 alone. The Pkd1/Cdk1 double knockout blocked cyst cell proliferation that otherwise accompanied Pkd1 inactivation alone. CONCLUSIONS: Dysregulation of Cdk1 is an early driver of cyst cell proliferation in ADPKD due to Pkd1 inactivation. Selective targeting of cyst cell proliferation is an effective means of slowing ADPKD progression caused by inactivation of Pkd1.


Subject(s)
CDC2 Protein Kinase/metabolism , Polycystic Kidney, Autosomal Dominant/genetics , Polycystic Kidney, Autosomal Dominant/metabolism , TRPP Cation Channels/metabolism , Animals , Apoptosis , CDC2 Protein Kinase/genetics , Catalytic Domain , Cell Proliferation , Crosses, Genetic , DNA Replication , Female , Gene Expression Profiling , Gene Expression Regulation , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mutation , Phenotype , Pyruvate Dehydrogenase Acetyl-Transferring Kinase/genetics , RNA-Seq , TRPP Cation Channels/genetics , Transcription, Genetic , Whole Exome Sequencing
7.
J Am Soc Nephrol ; 32(1): 86-97, 2021 01.
Article in English | MEDLINE | ID: covidwho-1496654

ABSTRACT

BACKGROUND: Cultured cell lines are widely used for research in the physiology, pathophysiology, toxicology, and pharmacology of the renal proximal tubule. The lines that are most appropriate for a given use depend upon the genes expressed. New tools for transcriptomic profiling using RNA sequencing (RNA-Seq) make it possible to catalog expressed genes in each cell line. METHODS: Fourteen different proximal tubule cell lines, representing six species, were grown on permeable supports under conditions specific for the respective lines. RNA-Seq followed standard procedures. RESULTS: Transcripts expressed in cell lines variably matched transcripts selectively expressed in native proximal tubule. Opossum kidney (OK) cells displayed the highest percentage match (45% of proximal marker genes [TPM threshold =15]), with pig kidney cells (LLC-PK1) close behind (39%). Lower-percentage matches were seen for various human lines, including HK-2 (26%), and lines from rodent kidneys, such as NRK-52E (23%). Nominally, identical OK cells from different sources differed substantially in expression of proximal tubule markers. Mapping cell line transcriptomes to gene sets for various proximal tubule functions (sodium and water transport, protein transport, metabolic functions, endocrine functions) showed that different lines may be optimal for experimentally modeling each function. An online resource (https://esbl.nhlbi.nih.gov/JBrowse/KCT/) has been created to interrogate cell line transcriptome data. Proteomic analysis of NRK-52E cells confirmed low expression of many proximal tubule marker proteins. CONCLUSIONS: No cell line fully matched the transcriptome of native proximal tubule cells. However, some of the lines tested are suitable for the study of particular metabolic and transport processes seen in the proximal tubule.


Subject(s)
Cell Culture Techniques/methods , Kidney Tubules, Proximal/metabolism , Transcriptome , Animals , Biological Transport , Cell Line , Chromatography, Liquid , Gene Expression Profiling , Humans , Internet , Mice , Opossums , Proteomics , RNA-Seq , Rats , Sequence Analysis, RNA , Species Specificity , Swine , Tandem Mass Spectrometry
8.
Int J Mol Sci ; 22(21)2021 Oct 30.
Article in English | MEDLINE | ID: covidwho-1488618

ABSTRACT

The inflammatory response plays a central role in the complications of congenital pulmonary airway malformations (CPAM) and severe coronavirus disease 2019 (COVID-19). The aim of this study was to evaluate the transcriptional changes induced by SARS-CoV-2 exposure in pediatric MSCs derived from pediatric lung (MSCs-lung) and CPAM tissues (MSCs-CPAM) in order to elucidate potential pathways involved in SARS-CoV-2 infection in a condition of exacerbated inflammatory response. MSCs-lung and MSCs-CPAM do not express angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TRMPSS2). SARS-CoV-2 appears to be unable to replicate in MSCs-CPAM and MSCs-lung. MSCs-lung and MSCs-CPAM maintained the expression of stemness markers MSCs-lung show an inflammatory response (IL6, IL1B, CXCL8, and CXCL10), and the activation of Notch3 non-canonical pathway; this route appears silent in MSCs-CPAM, and cytokine genes expression is reduced. Decreased value of p21 in MSCs-lung suggested no cell cycle block, and cells did not undergo apoptosis. MSCs-lung appears to increase genes associated with immunomodulatory function but could contribute to inflammation, while MSCs-CPAM keeps stable or reduce the immunomodulatory receptors expression, but they also reduce their cytokines expression. These data indicated that, independently from their perilesional or cystic origin, the MSCs populations already present in a patient affected with CPAM are not permissive for SARS-CoV-2 entry, and they will not spread the disease in case of infection. Moreover, these MSCs will not undergo apoptosis when they come in contact with SARS-CoV-2; on the contrary, they maintain their staminality profile.


Subject(s)
Mesenchymal Stem Cells/metabolism , Respiratory System Abnormalities , SARS-CoV-2/physiology , Transcriptome , COVID-19/genetics , COVID-19/metabolism , COVID-19/pathology , Case-Control Studies , Cells, Cultured , Gene Expression Profiling , Host-Pathogen Interactions/genetics , Humans , Infant , Lung/abnormalities , Lung/metabolism , Lung/pathology , Male , Mesenchymal Stem Cells/pathology , Mesenchymal Stem Cells/virology , RNA-Seq , Respiratory System Abnormalities/genetics , Respiratory System Abnormalities/pathology , Respiratory System Abnormalities/virology
9.
Sci Rep ; 11(1): 20833, 2021 10 21.
Article in English | MEDLINE | ID: covidwho-1479812

ABSTRACT

Several single-cell RNA sequencing (scRNA-seq) studies analyzing immune response to COVID-19 infection have been recently published. Most of these studies have small sample sizes, which limits the conclusions that can be made with high confidence. By re-analyzing these data in a standardized manner, we validated 8 of the 20 published results across multiple datasets. In particular, we found a consistent decrease in T-cells with increasing COVID-19 infection severity, upregulation of type I Interferon signal pathways, presence of expanded B-cell clones in COVID-19 patients but no consistent trend in T-cell clonal expansion. Overall, our results show that the conclusions drawn from scRNA-seq data analysis of small cohorts of COVID-19 patients need to be treated with some caution.


Subject(s)
Biomarkers/metabolism , COVID-19/immunology , COVID-19/metabolism , RNA, Small Cytoplasmic , Single-Cell Analysis , Bronchoalveolar Lavage Fluid , Computational Biology , Databases, Factual , Gene Expression Profiling/methods , Genome, Human , Genome, Viral , Humans , Immunity , Leukocytes, Mononuclear/cytology , RNA-Seq , Reproducibility of Results , SARS-CoV-2 , Sequence Analysis, RNA/methods , Signal Transduction , Up-Regulation
10.
Nat Microbiol ; 6(11): 1337-1338, 2021 11.
Article in English | MEDLINE | ID: covidwho-1475300
11.
J Clin Invest ; 131(20)2021 10 15.
Article in English | MEDLINE | ID: covidwho-1470549

ABSTRACT

Multisystem inflammatory syndrome in children (MIS-C) manifests as a severe and uncontrolled inflammatory response with multiorgan involvement, occurring weeks after SARS-CoV-2 infection. Here, we utilized proteomics, RNA sequencing, autoantibody arrays, and B cell receptor (BCR) repertoire analysis to characterize MIS-C immunopathogenesis and identify factors contributing to severe manifestations and intensive care unit admission. Inflammation markers, humoral immune responses, neutrophil activation, and complement and coagulation pathways were highly enriched in MIS-C patient serum, with a more hyperinflammatory profile in severe than in mild MIS-C cases. We identified a strong autoimmune signature in MIS-C, with autoantibodies targeted to both ubiquitously expressed and tissue-specific antigens, suggesting autoantigen release and excessive antigenic drive may result from systemic tissue damage. We further identified a cluster of patients with enhanced neutrophil responses as well as high anti-Spike IgG and autoantibody titers. BCR sequencing of these patients identified a strong imprint of antigenic drive with substantial BCR sequence connectivity and usage of autoimmunity-associated immunoglobulin heavy chain variable region (IGHV) genes. This cluster was linked to a TRBV11-2 expanded T cell receptor (TCR) repertoire, consistent with previous studies indicating a superantigen-driven pathogenic process. Overall, we identify a combination of pathogenic pathways that culminate in MIS-C and may inform treatment.


Subject(s)
Autoimmunity , COVID-19/complications , Systemic Inflammatory Response Syndrome/immunology , Adaptive Immunity , Adolescent , Biomarkers/metabolism , COVID-19/genetics , COVID-19/immunology , COVID-19/metabolism , Case-Control Studies , Child , Child, Preschool , Cohort Studies , Cytokine Release Syndrome/immunology , Female , Humans , Infant , Inflammation/immunology , Male , Mucocutaneous Lymph Node Syndrome/genetics , Mucocutaneous Lymph Node Syndrome/immunology , Mucocutaneous Lymph Node Syndrome/metabolism , Neutrophil Activation , Proteomics , RNA-Seq , Receptors, Antigen, B-Cell/genetics , Severity of Illness Index , Systemic Inflammatory Response Syndrome/genetics , Systemic Inflammatory Response Syndrome/metabolism
12.
Nat Commun ; 12(1): 5695, 2021 09 28.
Article in English | MEDLINE | ID: covidwho-1442780

ABSTRACT

The dynamics of SARS-CoV-2 RNA structure and their functional relevance are largely unknown. Here we develop a simplified SPLASH assay and comprehensively map the in vivo RNA-RNA interactome of SARS-CoV-2 genome across viral life cycle. We report canonical and alternative structures including 5'-UTR and 3'-UTR, frameshifting element (FSE) pseudoknot and genome cyclization in both cells and virions. We provide direct evidence of interactions between Transcription Regulating Sequences, which facilitate discontinuous transcription. In addition, we reveal alternative short and long distance arches around FSE. More importantly, we find that within virions, while SARS-CoV-2 genome RNA undergoes intensive compaction, genome domains remain stable but with strengthened demarcation of local domains and weakened global cyclization. Taken together, our analysis reveals the structural basis for the regulation of replication, discontinuous transcription and translational frameshifting, the alternative conformations and the maintenance of global genome organization during the whole life cycle of SARS-CoV-2, which we anticipate will help develop better antiviral strategies.


Subject(s)
Frameshifting, Ribosomal/genetics , Genome, Viral/genetics , RNA, Viral/genetics , SARS-CoV-2/genetics , Animals , COVID-19/virology , Chlorocebus aethiops , Humans , RNA-Seq , Transcription, Genetic , Vero Cells , Virus Replication/genetics
13.
Immunity ; 54(11): 2650-2669.e14, 2021 11 09.
Article in English | MEDLINE | ID: covidwho-1442406

ABSTRACT

Longitudinal analyses of the innate immune system, including the earliest time points, are essential to understand the immunopathogenesis and clinical course of coronavirus disease (COVID-19). Here, we performed a detailed characterization of natural killer (NK) cells in 205 patients (403 samples; days 2 to 41 after symptom onset) from four independent cohorts using single-cell transcriptomics and proteomics together with functional studies. We found elevated interferon (IFN)-α plasma levels in early severe COVD-19 alongside increased NK cell expression of IFN-stimulated genes (ISGs) and genes involved in IFN-α signaling, while upregulation of tumor necrosis factor (TNF)-induced genes was observed in moderate diseases. NK cells exert anti-SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) activity but are functionally impaired in severe COVID-19. Further, NK cell dysfunction may be relevant for the development of fibrotic lung disease in severe COVID-19, as NK cells exhibited impaired anti-fibrotic activity. Our study indicates preferential IFN-α and TNF responses in severe and moderate COVID-19, respectively, and associates a prolonged IFN-α-induced NK cell response with poorer disease outcome.


Subject(s)
COVID-19/immunology , Interferon-alpha/immunology , Killer Cells, Natural/immunology , SARS-CoV-2/immunology , Tumor Necrosis Factor-alpha/metabolism , Base Sequence , Humans , Immunity, Innate/immunology , Inflammation/immunology , Interferon-alpha/blood , Pulmonary Fibrosis/pathology , RNA-Seq , Severity of Illness Index , Transcriptome/genetics , United Kingdom , United States
14.
Sci Rep ; 11(1): 19140, 2021 09 27.
Article in English | MEDLINE | ID: covidwho-1440481

ABSTRACT

Despite the reported low expression of the primary SARS-CoV-2 receptor ACE2 in distinct ocular tissues, some clinical evidence suggests that SARS-CoV-2 can infect the eye. In this study, we explored potential entry sites for SARS-CoV-2 by viral S protein histochemistry on various ocular tissues and compared the staining patterns with RNA and protein expression of TMPRSS2 and ACE2. Potential viral entry sites were investigated by histochemistry using tagged recombinant viral S protein on 52 ocular tissue samples including specimens of the cornea, conjunctiva, lid margin, lacrimal gland tissue, retina, choroid, and RPE. In addition, ACE2 and TMPRSS2 immunohistochemistry were performed on the same ocular tissue, each with distinct antibodies binding to different epitopes. Lung tissue samples were used as positive controls. Finally, bulk RNA sequencing (RNA-Seq) was used to determine the expression of ACE2 and its auxiliary factors in the tissues mentioned above. S protein histochemistry revealed a positive staining in lung tissue but absent staining in the cornea, the conjunctiva, eye lid samples, the lacrimal glands, the retina and the optic nerve which was supported by hardly any immunoreactivity for ACE2 and TMPRSS2 and scarce ACE2 and TMPRSS2 RNA expression. Negligible staining with antibodies targeting ACE2 or TMPRSS2 was seen in the main and accessory lacrimal glands. In contrast, ocular staining (S protein, ACE2, TMPRSS2) was distinctly present in pigmented cells of the RPE and choroid, as well as in the ciliary body and the iris stroma. S protein histochemistry revealed hardly any SARS-CoV-2 entry sites in all ocular tissues examined. Similarly, no significant ACE2 or TMPRSS2 expression was found in extra- and intraocular tissue. While this study suggest a rather low risk of ocular infection with SARS-CoV-2, it should be noted, that potential viral entry sites may increase in response to inflammation or in certain disease states.


Subject(s)
COVID-19/prevention & control , Conjunctiva/metabolism , Cornea/metabolism , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , COVID-19/virology , Conjunctiva/virology , Cornea/virology , Gene Expression Profiling/methods , Humans , Immunohistochemistry/methods , RNA-Seq/methods , SARS-CoV-2/physiology , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , Virus Internalization
15.
Sci Rep ; 11(1): 19161, 2021 09 27.
Article in English | MEDLINE | ID: covidwho-1440480

ABSTRACT

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is associated with fatal pulmonary fibrosis. Small interfering RNAs (siRNAs) can be developed to induce RNA interference against SARS-CoV-2, and their susceptible target sites can be inferred by Argonaute crosslinking immunoprecipitation sequencing (AGO CLIP). Here, by reanalysing AGO CLIP data in RNA viruses, we delineated putative AGO binding in the conserved non-structural protein 12 (nsp12) region encoding RNA-dependent RNA polymerase (RdRP) in SARS-CoV-2. We utilised the inferred AGO binding to optimise the local RNA folding parameter to calculate target accessibility and predict all potent siRNA target sites in the SARS-CoV-2 genome, avoiding sequence variants. siRNAs loaded onto AGO also repressed seed (positions 2-8)-matched transcripts by acting as microRNAs (miRNAs). To utilise this, we further screened 13 potential siRNAs whose seed sequences were matched to known antifibrotic miRNAs and confirmed their miRNA-like activity. A miR-27-mimicking siRNA designed to target the nsp12 region (27/RdRP) was validated to silence a synthesised nsp12 RNA mimic in lung cell lines and function as an antifibrotic miR-27 in regulating target transcriptomes related to TGF-ß signalling. siRNA sequences with an antifibrotic miRNA-like activity that could synergistically treat COVID-19 are available online ( http://clip.korea.ac.kr/covid19 ).


Subject(s)
Argonaute Proteins/genetics , COVID-19/prevention & control , MicroRNAs/genetics , RNA, Small Interfering/genetics , SARS-CoV-2/genetics , A549 Cells , Argonaute Proteins/metabolism , Base Sequence , Binding Sites/genetics , COVID-19/virology , Cell Line , Coronavirus RNA-Dependent RNA Polymerase/genetics , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Gene Expression Profiling/methods , HeLa Cells , Humans , Pulmonary Fibrosis/genetics , Pulmonary Fibrosis/metabolism , RNA Interference , RNA-Seq/methods , SARS-CoV-2/physiology , Sequence Homology, Nucleic Acid
16.
Int J Mol Sci ; 22(18)2021 Sep 16.
Article in English | MEDLINE | ID: covidwho-1409704

ABSTRACT

Autotaxin (ATX; ENPP2) is a secreted lysophospholipase D catalyzing the extracellular production of lysophosphatidic acid (LPA), a pleiotropic signaling phospholipid. Genetic and pharmacologic studies have previously established a pathologic role for ATX and LPA signaling in pulmonary injury, inflammation, and fibrosis. Here, increased ENPP2 mRNA levels were detected in immune cells from nasopharyngeal swab samples of COVID-19 patients, and increased ATX serum levels were found in severe COVID-19 patients. ATX serum levels correlated with the corresponding increased serum levels of IL-6 and endothelial damage biomarkers, suggesting an interplay of the ATX/LPA axis with hyperinflammation and the associated vascular dysfunction in COVID-19. Accordingly, dexamethasone (Dex) treatment of mechanically ventilated patients reduced ATX levels, as shown in two independent cohorts, indicating that the therapeutic benefits of Dex include the suppression of ATX. Moreover, large scale analysis of multiple single cell RNA sequencing datasets revealed the expression landscape of ENPP2 in COVID-19 and further suggested a role for ATX in the homeostasis of dendritic cells, which exhibit both numerical and functional deficits in COVID-19. Therefore, ATX has likely a multifunctional role in COVID-19 pathogenesis, suggesting that its pharmacological targeting might represent an additional therapeutic option, both during and after hospitalization.


Subject(s)
COVID-19/diagnosis , Dendritic Cells/immunology , Phosphodiesterase Inhibitors/therapeutic use , Phosphoric Diester Hydrolases/blood , SARS-CoV-2/immunology , Adult , Aged , Aged, 80 and over , Biomarkers/blood , COVID-19/blood , COVID-19/immunology , COVID-19/therapy , Cohort Studies , Datasets as Topic , Dendritic Cells/drug effects , Dexamethasone/pharmacology , Dexamethasone/therapeutic use , Endothelium, Vascular/immunology , Endothelium, Vascular/pathology , Female , Humans , Interleukin-6/blood , Interleukin-6/metabolism , Male , Middle Aged , Phosphodiesterase Inhibitors/pharmacology , Phosphoric Diester Hydrolases/metabolism , RNA-Seq , Respiration, Artificial , SARS-CoV-2/isolation & purification , Severity of Illness Index , Signal Transduction/drug effects , Signal Transduction/immunology , Single-Cell Analysis
17.
Int J Med Sci ; 18(12): 2561-2569, 2021.
Article in English | MEDLINE | ID: covidwho-1389722

ABSTRACT

SARS-CoV-2 infection poses a global challenge to human health. Upon viral infection, host cells initiate the innate antiviral response, which primarily involves type I interferons (I-IFNs), to enable rapid elimination of the invading virus. Previous studies revealed that SARS-CoV-2 infection limits the expression of I-IFNs in vitro and in vivo, but the underlying mechanism remains incompletely elucidated. In the present study, we performed data mining and longitudinal data analysis using SARS-CoV-2-infected normal human bronchial epithelial (NHBE) cells and ferrets, and the results confirmed the strong inhibitory effect of SARS-CoV-2 on the induction of I-IFNs. Moreover, we identified genes that are negatively correlated with IFNB1 expression in vitro and in vivo based on Pearson correlation analysis. We found that SARS-CoV-2 activates numerous intrinsic pathways, such as the circadian rhythm, phosphatidylinositol signaling system, peroxisome, and TNF signaling pathways, to inhibit I-IFNs. These intrinsic inhibitory pathways jointly facilitate the successful immune evasion of SARS-CoV-2. Our study elucidates the underlying mechanism by which SARS-CoV-2 evades the host innate antiviral response in vitro and in vivo, providing theoretical evidence for targeting these immune evasion-associated pathways to combat SARS-CoV-2 infection.


Subject(s)
COVID-19/immunology , Host-Pathogen Interactions/immunology , Interferon-gamma/metabolism , SARS-CoV-2/immunology , Animals , Bronchi/cytology , COVID-19/virology , Cell Line , Datasets as Topic , Disease Models, Animal , Epithelial Cells , Ferrets , Gene Expression Regulation/immunology , Host-Pathogen Interactions/genetics , Humans , Immunity, Innate , Interferon-gamma/immunology , RNA-Seq , Respiratory Mucosa/cytology , Signal Transduction/genetics , Signal Transduction/immunology
18.
Sci Adv ; 7(1)2021 01.
Article in English | MEDLINE | ID: covidwho-1388432

ABSTRACT

Using AI, we identified baricitinib as having antiviral and anticytokine efficacy. We now show a 71% (95% CI 0.15 to 0.58) mortality benefit in 83 patients with moderate-severe SARS-CoV-2 pneumonia with few drug-induced adverse events, including a large elderly cohort (median age, 81 years). An additional 48 cases with mild-moderate pneumonia recovered uneventfully. Using organotypic 3D cultures of primary human liver cells, we demonstrate that interferon-α2 increases ACE2 expression and SARS-CoV-2 infectivity in parenchymal cells by greater than fivefold. RNA-seq reveals gene response signatures associated with platelet activation, fully inhibited by baricitinib. Using viral load quantifications and superresolution microscopy, we found that baricitinib exerts activity rapidly through the inhibition of host proteins (numb-associated kinases), uniquely among antivirals. This reveals mechanistic actions of a Janus kinase-1/2 inhibitor targeting viral entry, replication, and the cytokine storm and is associated with beneficial outcomes including in severely ill elderly patients, data that incentivize further randomized controlled trials.


Subject(s)
Antiviral Agents/pharmacology , Azetidines/pharmacology , COVID-19/mortality , Enzyme Inhibitors/pharmacology , Janus Kinases/antagonists & inhibitors , Liver/virology , Purines/pharmacology , Pyrazoles/pharmacology , SARS-CoV-2/pathogenicity , Sulfonamides/pharmacology , Adult , Aged , Aged, 80 and over , COVID-19/drug therapy , COVID-19/metabolism , COVID-19/virology , Cytokine Release Syndrome , Cytokines/metabolism , Drug Evaluation, Preclinical , Female , Gene Expression Profiling , Humans , Interferon alpha-2/metabolism , Italy , Janus Kinases/metabolism , Liver/drug effects , Male , Middle Aged , Patient Safety , Platelet Activation , Proportional Hazards Models , RNA-Seq , Spain , Virus Internalization/drug effects
20.
PLoS One ; 15(8): e0238490, 2020.
Article in English | MEDLINE | ID: covidwho-1388885

ABSTRACT

SARS-CoV-2 is still rampaging throughout the world while the many evolutionary studies on it are simultaneously springing up. Researchers have simply utilized the public RNA-seq data to find out the so-called SNPs in the virus genome. The evolutionary analyses were largely based on these mutations. Here, we claim that we reliably detected A-to-G RNA modifications in the RNA-seq data of SARS-CoV-2 with high signal to noise ratios, presumably caused by the host's deamination enzymes. Intriguingly, since SARS-CoV-2 is an RNA virus, it is technically impossible to distinguish SNPs and RNA modifications from the RNA-seq data alone without solid evidence, making it difficult to tell the evolutionary patterns behind the mutation spectrum. Researchers should clarify their biological significance before they automatically regard the mutations as SNPs or RNA modifications. This is not a problem for DNA organisms but should be seriously considered when we are investigating the RNA viruses.


Subject(s)
Betacoronavirus/genetics , Evolution, Molecular , Polymorphism, Single Nucleotide , RNA, Viral/genetics , Base Sequence , COVID-19 , Coronavirus Infections , Humans , Mutation Rate , Pandemics , Pneumonia, Viral , RNA-Seq , SARS-CoV-2
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