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1.
Stem Cell Reports ; 17(7): 1699-1713, 2022 07 12.
Article in English | MEDLINE | ID: covidwho-1907809

ABSTRACT

Conjunctival epithelial cells, which express viral-entry receptors angiotensin-converting enzyme 2 (ACE2) and transmembrane protease serine type 2 (TMPRSS2), constitute the largest exposed epithelium of the ocular surface tissue and may represent a relevant viral-entry route. To address this question, we generated an organotypic air-liquid-interface model of conjunctival epithelium, composed of basal, suprabasal, and superficial epithelial cells, and fibroblasts, which could be maintained successfully up to day 75 of differentiation. Using single-cell RNA sequencing (RNA-seq), with complementary imaging and virological assays, we observed that while all conjunctival cell types were permissive to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome expression, a productive infection did not ensue. The early innate immune response to SARS-CoV-2 infection in conjunctival cells was characterised by a robust autocrine and paracrine NF-κB activity, without activation of antiviral interferon signalling. Collectively, these data enrich our understanding of SARS-CoV-2 infection at the human ocular surface, with potential implications for the design of preventive strategies and conjunctival transplantation.


Subject(s)
COVID-19 , Epithelial Cells/metabolism , Humans , Peptidyl-Dipeptidase A/genetics , Peptidyl-Dipeptidase A/metabolism , Receptors, Virus/metabolism , SARS-CoV-2
2.
Nat Commun ; 12(1): 7092, 2021 12 07.
Article in English | MEDLINE | ID: covidwho-1561304

ABSTRACT

The nasal epithelium is a plausible entry point for SARS-CoV-2, a site of pathogenesis and transmission, and may initiate the host response to SARS-CoV-2. Antiviral interferon (IFN) responses are critical to outcome of SARS-CoV-2. Yet little is known about the interaction between SARS-CoV-2 and innate immunity in this tissue. Here we apply single-cell RNA sequencing and proteomics to a primary cell model of human nasal epithelium differentiated at air-liquid interface. SARS-CoV-2 demonstrates widespread tropism for nasal epithelial cell types. The host response is dominated by type I and III IFNs and interferon-stimulated gene products. This response is notably delayed in onset relative to viral gene expression and compared to other respiratory viruses. Nevertheless, once established, the paracrine IFN response begins to impact on SARS-CoV-2 replication. When provided prior to infection, recombinant IFNß or IFNλ1 induces an efficient antiviral state that potently restricts SARS-CoV-2 viral replication, preserving epithelial barrier integrity. These data imply that the IFN-I/III response to SARS-CoV-2 initiates in the nasal airway and suggest nasal delivery of recombinant IFNs to be a potential chemoprophylactic strategy.


Subject(s)
Epithelial Cells/virology , Interferon Type I/immunology , Interferons/immunology , Nasal Mucosa/virology , SARS-CoV-2/physiology , Antiviral Agents/immunology , Antiviral Agents/pharmacology , COVID-19/immunology , COVID-19/virology , Cells, Cultured , Epithelial Cells/cytology , Epithelial Cells/immunology , Humans , Immunity, Innate , Kinetics , Nasal Mucosa/cytology , Nasal Mucosa/immunology , SARS-CoV-2/drug effects , Signal Transduction/drug effects , Viral Tropism , Virus Replication/drug effects , Interferon Lambda
3.
Ocul Surf ; 19: 190-200, 2021 01.
Article in English | MEDLINE | ID: covidwho-1065414

ABSTRACT

PURPOSE: The high infection rate of SARS-CoV-2 necessitates the need for multiple studies identifying the molecular mechanisms that facilitate the viral entry and propagation. Currently the potential extra-respiratory transmission routes of SARS-CoV-2 remain unclear. METHODS: Using single-cell RNA Seq and ATAC-Seq datasets and immunohistochemical analysis, we investigated SARS-CoV-2 tropism in the embryonic, fetal and adult human ocular surface. RESULTS: The co-expression of ACE2 receptor and entry protease TMPRSS2 was detected in the human adult conjunctival, limbal and corneal epithelium, but not in the embryonic and fetal ocular surface up to 21 post conception weeks. These expression patterns were corroborated by the single cell ATAC-Seq data, which revealed a permissive chromatin in ACE2 and TMPRSS2 loci in the adult conjunctival, limbal and corneal epithelium. Co-expression of ACE2 and TMPRSS2 was strongly detected in the superficial limbal, corneal and conjunctival epithelium, implicating these as target entry cells for SARS-CoV-2 in the ocular surface. Strikingly, we also identified the key pro-inflammatory signals TNF, NFKß and IFNG as upstream regulators of the transcriptional profile of ACE2+TMPRSS2+ cells in the superficial conjunctival epithelium, suggesting that SARS-CoV-2 may utilise inflammatory driven upregulation of ACE2 and TMPRSS2 expression to enhance infection in ocular surface. CONCLUSIONS: Together our data indicate that the human ocular surface epithelium provides an additional entry portal for SARS-CoV-2, which may exploit inflammatory driven upregulation of ACE2 and TMPRSS2 entry factors to enhance infection.


Subject(s)
Angiotensin-Converting Enzyme 2/genetics , COVID-19 , Conjunctiva/metabolism , Epithelium, Corneal/metabolism , Receptors, Virus/genetics , Serine Endopeptidases/genetics , Aged , Aged, 80 and over , Conjunctiva/virology , Epithelium, Corneal/virology , Humans , Middle Aged , SARS-CoV-2
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