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1.
FASEB journal : official publication of the Federation of American Societies for Experimental Biology ; 36(Suppl 1), 2022.
Article in English | EuropePMC | ID: covidwho-1971027

ABSTRACT

Severe viral pneumonia caused by the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) is characterized by a hyperinflammatory state typified by elevated circulating pro‐inflammatory cytokines, frequently leading to potentially lethal vascular complications including thromboembolism, disseminated intracellular coagulopathy and vasculitis. Though endothelial infection and subsequent endothelial damage have been described in patients with fatal COVID‐19, the mechanism by which this occurs remains elusive, particularly given that, under naïve conditions, pulmonary endothelial cells demonstrate minimal cell surface expression of the SARS‐CoV‐2 binding receptor ACE2. Herein we describe SARS‐CoV‐2 infection of the pulmonary endothelium in postmortem lung samples from individuals who died of COVID‐19, demonstrating both heterogeneous ACE2 expression and endothelial damage (Figure). In primary endothelial cell cultures, we show that SARS‐CoV‐2 infection is dependent on the induction of ACE2 protein expression and that this process is facilitated by type 1 interferon‐alpha (IFNα) or ‐beta(β) ‐ two of the main anti‐viral cytokines induced in severe SARS‐CoV‐2 infection ‐ but not significantly by other cytokines (including interleukin 6 and interferon g /λ). Our findings suggest that the stereotypical anti‐viral interferon response may paradoxically facilitate the propagation of COVID‐19 from the respiratory epithelium to the vasculature, raising concerns regarding the use of exogenous IFNα/β in the treatment of patients with COVID‐19.

2.
PLoS Pathog ; 18(2): e1010268, 2022 02.
Article in English | MEDLINE | ID: covidwho-1753212

ABSTRACT

Next generation sequencing has revealed the presence of numerous RNA viruses in animal reservoir hosts, including many closely related to known human pathogens. Despite their zoonotic potential, most of these viruses remain understudied due to not yet being cultured. While reverse genetic systems can facilitate virus rescue, this is often hindered by missing viral genome ends. A prime example is Lloviu virus (LLOV), an uncultured filovirus that is closely related to the highly pathogenic Ebola virus. Using minigenome systems, we complemented the missing LLOV genomic ends and identified cis-acting elements required for LLOV replication that were lacking in the published sequence. We leveraged these data to generate recombinant full-length LLOV clones and rescue infectious virus. Similar to other filoviruses, recombinant LLOV (rLLOV) forms filamentous virions and induces the formation of characteristic inclusions in the cytoplasm of the infected cells, as shown by electron microscopy. Known target cells of Ebola virus, including macrophages and hepatocytes, are permissive to rLLOV infection, suggesting that humans could be potential hosts. However, inflammatory responses in human macrophages, a hallmark of Ebola virus disease, are not induced by rLLOV. Additional tropism testing identified pneumocytes as capable of robust rLLOV and Ebola virus infection. We also used rLLOV to test antivirals targeting multiple facets of the replication cycle. Rescue of uncultured viruses of pathogenic concern represents a valuable tool in our arsenal for pandemic preparedness.


Subject(s)
Ebolavirus/genetics , Filoviridae Infections/virology , Filoviridae/genetics , Virus Replication , Animals , Cell Line , Chlorocebus aethiops , Genetic Complementation Test , Genome, Viral , Hemorrhagic Fever, Ebola/virology , Host Microbial Interactions , Humans , Inclusion Bodies/virology , Induced Pluripotent Stem Cells/virology , Macrophages/virology , RNA, Viral , Reverse Genetics , Vero Cells , Virion/genetics
3.
Am J Physiol Lung Cell Mol Physiol ; 322(3): L462-L478, 2022 03 01.
Article in English | MEDLINE | ID: covidwho-1622104

ABSTRACT

There is an urgent need to understand how SARS-CoV-2 infects the airway epithelium and in a subset of individuals leads to severe illness or death. Induced pluripotent stem cells (iPSCs) provide a near limitless supply of human cells that can be differentiated into cell types of interest, including airway epithelium, for disease modeling. We present a human iPSC-derived airway epithelial platform, composed of the major airway epithelial cell types, that is permissive to SARS-CoV-2 infection. Subsets of iPSC-airway cells express the SARS-CoV-2 entry factors angiotensin-converting enzyme 2 (ACE2), and transmembrane protease serine 2 (TMPRSS2). Multiciliated cells are the primary initial target of SARS-CoV-2 infection. On infection with SARS-CoV-2, iPSC-airway cells generate robust interferon and inflammatory responses, and treatment with remdesivir or camostat mesylate causes a decrease in viral propagation and entry, respectively. In conclusion, iPSC-derived airway cells provide a physiologically relevant in vitro model system to interrogate the pathogenesis of, and develop treatment strategies for, COVID-19 pneumonia.


Subject(s)
COVID-19 , Induced Pluripotent Stem Cells , Pluripotent Stem Cells , Epithelial Cells , Humans , SARS-CoV-2
5.
Proc Natl Acad Sci U S A ; 119(1)2022 01 04.
Article in English | MEDLINE | ID: covidwho-1595265

ABSTRACT

Infection by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) provokes a potentially fatal pneumonia with multiorgan failure, and high systemic inflammation. To gain mechanistic insight and ferret out the root of this immune dysregulation, we modeled, by in vitro coculture, the interactions between infected epithelial cells and immunocytes. A strong response was induced in monocytes and B cells, with a SARS-CoV-2-specific inflammatory gene cluster distinct from that seen in influenza A or Ebola virus-infected cocultures, and which reproduced deviations reported in blood or lung myeloid cells from COVID-19 patients. A substantial fraction of the effect could be reproduced after individual transfection of several SARS-CoV-2 proteins (Spike and some nonstructural proteins), mediated by soluble factors, but not via transcriptional induction. This response was greatly muted in monocytes from healthy children, perhaps a clue to the age dependency of COVID-19. These results suggest that the inflammatory malfunction in COVID-19 is rooted in the earliest perturbations that SARS-CoV-2 induces in epithelia.


Subject(s)
COVID-19/immunology , Epithelial Cells/immunology , Monocytes/immunology , SARS-CoV-2/pathogenicity , Adult , B-Lymphocytes/immunology , COVID-19/pathology , Child , Coculture Techniques , Ebolavirus/pathogenicity , Epithelial Cells/virology , Gene Expression Profiling , Humans , Inflammation , Influenza A virus/pathogenicity , Lung/immunology , Myeloid Cells/immunology , Species Specificity , Viral Proteins/immunology
6.
Non-conventional in English | [Unspecified Source], Grey literature | ID: grc-750471

ABSTRACT

The most severe and fatal infections with SARS-CoV-2 result in the acute respiratory distress syndrome, a clinical phenotype of coronavirus disease 2019 (COVID-19) that is associated with virions targeting the epithelium of the distal lung, particularly the facultative progenitors of this tissue, alveolar epithelial type 2 cells (AT2s). Little is known about the initial responses of human lung alveoli to SARS-CoV-2 infection due in part to inability to access these cells from patients, particularly at early stages of disease. Here we present an in vitro human model that simulates the initial apical infection of the distal lung epithelium with SARS-CoV-2, using AT2s that have been adapted to air-liquid interface culture after their derivation from induced pluripotent stem cells (iAT2s). We find that SARS-CoV-2 induces a rapid global transcriptomic change in infected iAT2s characterized by a shift to an inflammatory phenotype predominated by the secretion of cytokines encoded by NF-kB target genes, delayed epithelial interferon responses, and rapid loss of the mature lung alveolar epithelial program. Over time, infected iAT2s exhibit cellular toxicity that can result in the death of these key alveolar facultative progenitors, as is observed in vivo in COVID-19 lung autopsies. Importantly, drug testing using iAT2s confirmed the efficacy of TMPRSS2 protease inhibition, validating putative mechanisms used for viral entry in human alveolar cells. Our model system reveals the cell-intrinsic responses of a key lung target cell to infection, providing a platform for further drug development and facilitating a deeper understanding of COVID-19 pathogenesis.

7.
Angiogenesis ; 25(2): 225-240, 2022 05.
Article in English | MEDLINE | ID: covidwho-1491183

ABSTRACT

Severe viral pneumonia caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is characterized by a hyperinflammatory state typified by elevated circulating pro-inflammatory cytokines, frequently leading to potentially lethal vascular complications including thromboembolism, disseminated intracellular coagulopathy and vasculitis. Though endothelial infection and subsequent endothelial damage have been described in patients with fatal COVID-19, the mechanism by which this occurs remains elusive, particularly given that, under naïve conditions, pulmonary endothelial cells demonstrate minimal cell surface expression of the SARS-CoV-2 binding receptor ACE2. Herein we describe SARS-CoV-2 infection of the pulmonary endothelium in postmortem lung samples from individuals who died of COVID-19, demonstrating both heterogeneous ACE2 expression and endothelial damage. In primary endothelial cell cultures, we show that SARS-CoV-2 infection is dependent on the induction of ACE2 protein expression and that this process is facilitated by type 1 interferon-alpha (IFNα) or -beta(ß)-two of the main anti-viral cytokines induced in severe SARS-CoV-2 infection-but not significantly by other cytokines (including interleukin 6 and interferon γ/λ). Our findings suggest that the stereotypical anti-viral interferon response may paradoxically facilitate the propagation of COVID-19 from the respiratory epithelium to the vasculature, raising concerns regarding the use of exogenous IFNα/ß in the treatment of patients with COVID-19.


Subject(s)
COVID-19 , Angiotensin-Converting Enzyme 2 , Cytokines , Endothelial Cells , Humans , Interferon-alpha , SARS-CoV-2
8.
Stem Cell Reports ; 16(4): 940-953, 2021 04 13.
Article in English | MEDLINE | ID: covidwho-1180038

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection leading to coronavirus disease 2019 (COVID-19) usually results in respiratory disease, but extrapulmonary manifestations are of major clinical interest. Intestinal symptoms of COVID-19 are present in a significant number of patients, and include nausea, diarrhea, and viral RNA shedding in feces. Human induced pluripotent stem cell-derived intestinal organoids (HIOs) represent an inexhaustible cellular resource that could serve as a valuable tool to study SARS-CoV-2 as well as other enteric viruses that infect the intestinal epithelium. Here, we report that SARS-CoV-2 productively infects both proximally and distally patterned HIOs, leading to the release of infectious viral particles while stimulating a robust transcriptomic response, including a significant upregulation of interferon-related genes that appeared to be conserved across multiple epithelial cell types. These findings illuminate a potential inflammatory epithelial-specific signature that may contribute to both the multisystemic nature of COVID-19 as well as its highly variable clinical presentation.


Subject(s)
COVID-19/pathology , Colon/pathology , Intestinal Mucosa/pathology , Organoids/pathology , Cell Line , Colon/virology , Epithelial Cells/virology , Humans , Induced Pluripotent Stem Cells/cytology , Inflammation/virology , Intestinal Mucosa/virology , Models, Biological , Organoids/cytology , Organoids/virology , SARS-CoV-2 , Virus Replication/physiology
9.
Mol Cell ; 80(6): 1104-1122.e9, 2020 12 17.
Article in English | MEDLINE | ID: covidwho-933377

ABSTRACT

Human transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causative pathogen of the COVID-19 pandemic, exerts a massive health and socioeconomic crisis. The virus infects alveolar epithelial type 2 cells (AT2s), leading to lung injury and impaired gas exchange, but the mechanisms driving infection and pathology are unclear. We performed a quantitative phosphoproteomic survey of induced pluripotent stem cell-derived AT2s (iAT2s) infected with SARS-CoV-2 at air-liquid interface (ALI). Time course analysis revealed rapid remodeling of diverse host systems, including signaling, RNA processing, translation, metabolism, nuclear integrity, protein trafficking, and cytoskeletal-microtubule organization, leading to cell cycle arrest, genotoxic stress, and innate immunity. Comparison to analogous data from transformed cell lines revealed respiratory-specific processes hijacked by SARS-CoV-2, highlighting potential novel therapeutic avenues that were validated by a high hit rate in a targeted small molecule screen in our iAT2 ALI system.


Subject(s)
Alveolar Epithelial Cells/metabolism , COVID-19/metabolism , Phosphoproteins/metabolism , Proteome/metabolism , SARS-CoV-2/metabolism , Alveolar Epithelial Cells/pathology , Alveolar Epithelial Cells/virology , Animals , Antiviral Agents , COVID-19/drug therapy , COVID-19/genetics , COVID-19/pathology , Chlorocebus aethiops , Cytopathogenic Effect, Viral , Cytoskeleton , Drug Evaluation, Preclinical , Humans , Induced Pluripotent Stem Cells/metabolism , Induced Pluripotent Stem Cells/pathology , Induced Pluripotent Stem Cells/virology , Phosphoproteins/genetics , Protein Transport , Proteome/genetics , SARS-CoV-2/genetics , Signal Transduction , Vero Cells
10.
Cell Stem Cell ; 27(6): 962-973.e7, 2020 12 03.
Article in English | MEDLINE | ID: covidwho-779662

ABSTRACT

A hallmark of severe COVID-19 pneumonia is SARS-CoV-2 infection of the facultative progenitors of lung alveoli, the alveolar epithelial type 2 cells (AT2s). However, inability to access these cells from patients, particularly at early stages of disease, limits an understanding of disease inception. Here, we present an in vitro human model that simulates the initial apical infection of alveolar epithelium with SARS-CoV-2 by using induced pluripotent stem cell-derived AT2s that have been adapted to air-liquid interface culture. We find a rapid transcriptomic change in infected cells, characterized by a shift to an inflammatory phenotype with upregulation of NF-κB signaling and loss of the mature alveolar program. Drug testing confirms the efficacy of remdesivir as well as TMPRSS2 protease inhibition, validating a putative mechanism used for viral entry in alveolar cells. Our model system reveals cell-intrinsic responses of a key lung target cell to SARS-CoV-2 infection and should facilitate drug development.


Subject(s)
Alveolar Epithelial Cells/virology , Inflammation/virology , SARS-CoV-2/physiology , Adenosine Monophosphate/analogs & derivatives , Adenosine Monophosphate/pharmacology , Alanine/analogs & derivatives , Alanine/pharmacology , Animals , Antiviral Agents/pharmacology , COVID-19/virology , Cells, Cultured , Drug Development , Enzyme Inhibitors/pharmacology , Humans , Models, Biological , Pluripotent Stem Cells/cytology , Pluripotent Stem Cells/virology , RNA-Seq , Serine Endopeptidases/metabolism , Virus Replication
11.
Science ; 370(6513): 241-247, 2020 10 09.
Article in English | MEDLINE | ID: covidwho-733186

ABSTRACT

Recent outbreaks of Ebola virus (EBOV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have exposed our limited therapeutic options for such diseases and our poor understanding of the cellular mechanisms that block viral infections. Using a transposon-mediated gene-activation screen in human cells, we identify that the major histocompatibility complex (MHC) class II transactivator (CIITA) has antiviral activity against EBOV. CIITA induces resistance by activating expression of the p41 isoform of invariant chain CD74, which inhibits viral entry by blocking cathepsin-mediated processing of the Ebola glycoprotein. We further show that CD74 p41 can block the endosomal entry pathway of coronaviruses, including SARS-CoV-2. These data therefore implicate CIITA and CD74 in host defense against a range of viruses, and they identify an additional function of these proteins beyond their canonical roles in antigen presentation.


Subject(s)
Antigens, Differentiation, B-Lymphocyte/physiology , Betacoronavirus/physiology , Coronavirus Infections/immunology , Ebolavirus/physiology , Hemorrhagic Fever, Ebola/immunology , Histocompatibility Antigens Class II/physiology , Host-Pathogen Interactions/immunology , Nuclear Proteins/physiology , Pneumonia, Viral/immunology , Trans-Activators/physiology , Virus Internalization , Antigens, Differentiation, B-Lymphocyte/genetics , COVID-19 , Cell Line, Tumor , Coronavirus Infections/virology , DNA Transposable Elements , Endosomes/virology , Genetic Testing , Hemorrhagic Fever, Ebola/virology , Histocompatibility Antigens Class II/genetics , Host-Pathogen Interactions/genetics , Humans , Nuclear Proteins/genetics , Pandemics , Pneumonia, Viral/virology , SARS-CoV-2 , Trans-Activators/genetics , Transcription, Genetic
12.
bioRxiv ; 2020 Aug 06.
Article in English | MEDLINE | ID: covidwho-637839

ABSTRACT

The most severe and fatal infections with SARS-CoV-2 result in the acute respiratory distress syndrome, a clinical phenotype of coronavirus disease 2019 (COVID-19) that is associated with virions targeting the epithelium of the distal lung, particularly the facultative progenitors of this tissue, alveolar epithelial type 2 cells (AT2s). Little is known about the initial responses of human lung alveoli to SARS-CoV-2 infection due in part to inability to access these cells from patients, particularly at early stages of disease. Here we present an in vitro human model that simulates the initial apical infection of the distal lung epithelium with SARS-CoV-2, using AT2s that have been adapted to air-liquid interface culture after their derivation from induced pluripotent stem cells (iAT2s). We find that SARS-CoV-2 induces a rapid global transcriptomic change in infected iAT2s characterized by a shift to an inflammatory phenotype predominated by the secretion of cytokines encoded by NF-kB target genes, delayed epithelial interferon responses, and rapid loss of the mature lung alveolar epithelial program. Over time, infected iAT2s exhibit cellular toxicity that can result in the death of these key alveolar facultative progenitors, as is observed in vivo in COVID-19 lung autopsies. Importantly, drug testing using iAT2s confirmed an antiviral dose-response to remdesivir and demonstrated the efficacy of TMPRSS2 protease inhibition, validating a putative mechanism used for viral entry in human alveolar cells. Our model system reveals the cell-intrinsic responses of a key lung target cell to infection, providing a physiologically relevant platform for further drug development and facilitating a deeper understanding of COVID-19 pathogenesis.

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