Biology subjects

Garnett, J. P.

Publications and source records attributed to Garnett, J. P..

4 recordsLinked to original sources

bioRxiv2021

Delayed induction of type I and III interferons and nasal epithelial cell permissiveness to SARS-CoV-2

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 applied single-cell RNA sequencing and proteomics to a primary cell model of human nasal epithelium differentiated at air-liquid interface. SARS-CoV-2 demonstrated widespread tropism for nasal epithelial cell types. The host response was dominated by type I and III IFNs and interferon-stimulated gene products. This response was notably delayed in onset relative to viral gene expression and compared to other respiratory viruses. Nevertheless, once established, the paracrine IFN response began to impact on SARS-CoV-2 replication. When provided prior to infection, recombinant IFN{beta} or IFN{lambda}1 induced an efficient antiviral state that potently restricted SARS-CoV-2 viral replication, preserving epithelial barrier integrity. These data suggest 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.

immunology↗

bioRxiv2020

Differentiation of human induced pluripotent stem cells into functional airway epithelium

RationaleHighly reproducible in vitro generation of human bronchial epithelium from pluripotent stem cells is an unmet key goal for drug screening to treat lung diseases. The possibility of using induced pluripotent stem cells (hiPSC) to model normal and diseased tissue in vitro from a simple blood sample will reshape drug discovery for chronic lung, monogenic and infectious diseases. MethodsWe devised a simple and reliable method that drives a blood sample reprogrammed into hiPSC subsequently differentiated within 45 days into air-liquid interface bronchial epithelium (iALI), through key developmental stages, definitive-endoderm (DE) and Ventralized-Anterior-Foregut-Endoderm (vAFE) cells. ResultsReprogramming blood cells from one healthy and 3 COPD patients, and from skin-derived fibroblasts obtained in one PCD patient, succeeded in 100% of samples using Sendai viruses. Mean cell purity at DE and vAFE stages was greater than 80%, assessed by expression of CXCR4 and NKX2.1, avoiding the need of cell sorting. When transferred to ALI conditions, vAFE cells reliably differentiated within 4 weeks into bronchial epithelium with large zones covered by beating ciliated, basal, goblets, club cells and neuroendocrine cells as found in vivo. Benchmarking all culture conditions including hiPSCs adaptation to single-cell passaging, cell density and differentiation induction timing allowed for consistently producing iALI bronchial epithelium from the five hiPSC lines. ConclusionsReliable reprogramming and differentiation of blood-derived hiPSCs into mature and functional iALI bronchial epithelium is ready for wider use and this will allow better understanding lung disease pathogenesis and accelerating the development of novel gene therapies and drug discovery.

bioengineering↗

bioRxiv2020

A polycomb-independent role of EZH2 in TGFβ1-damaged epithelium triggers a fibrotic cascade with mesenchymal cells

To restore organ homeostasis, a myriad of cell types need to activate rapid and transient programs to adjust cell fate decisions and elicit a collective behaviour. Characterisation of such programs are imperative to elucidate an organs regenerative capacity and its aberrant repair in disease. By modelling epithelial-mesenchymal crosstalk, we provide direct evidence for transforming growth factor {beta}1 (TGF{beta}1)-damaged epithelium initiating a bi-directional fibrotic cascade with the mesenchyme. Strikingly, TGF{beta}1-damaged epithelia facilitates the release of Enhancer of Zester Homolog 2 (EZH2) from Polycomb Repressive Complex 2 (PRC2) to establish a novel fibrotic transcriptional complex of EZH2, RNA-polymerase II (POL2) and nuclear actin. Perturbing this complex by disrupting epithelial EZH2 or actomyosin remodelling abrogates the fibrotic crosstalk. The liberation of EZH2 from PRC2 is accompanied by an EZH2-EZH1 switch to preserve global H3K27me3 occupancy. Our results reveal an important non-canonical function of EZH2, paving the way for therapeutic interventions in fibrotic disease.

cell biology↗

bioRxiv2019

Recapitulating idiopathic pulmonary fibrosis related alveolar epithelial dysfunction in an iPSC-derived air-liquid interface model

An abnormal emergence of airway epithelial-like cells within the alveolar compartments of the lung, herein termed bronchiolization, is a process often observed in patients suffering from idiopathic pulmonary fibrosis (IPF), a fatal disease characterized by progressive fibrotic lung remodeling. However, the origin of this dysfunctional epithelium remains unknown.\n\nIn this study, we aimed to investigate the effects of a pro-fibrotic milieu, similar to that found in an IPF lung, on human alveolar epithelial progenitor cell differentiation. We developed an induced pluripotent stem cell (iPSC)-derived air-liquid interface (ALI) model of alveolar type II (ATII)-like cell differentiation and stimulated it with an IPF-relevant cocktail (IPF-RC), composed of cytokines previously reported to be elevated in IPF lungs. iPSC-derived cultures express ATII markers and contain lamellar body-like structures. Stimulation with IPF-RC during the last two weeks of differentiation increases secretion of IPF biomarkers. Transcriptome analysis of IPF-RC treated cultures reveals significant overlap with human IPF data and enrichment of transcripts associated with extracellular matrix organization. IPF-RC stimulation further impairs ATII differentiation by driving a shift towards an airway epithelial-like expression signature.\n\nIn conclusion, we show for the first time, the establishment of a human model system that recapitulates aspects of IPF-associated bronchiolization in vitro. Our findings reveal how aberrant alveolar epithelial progenitor cell differentiation in a pro-fibrotic environment could contribute to alveolar bronchiolization in the distal IPF lung.\n\nSOURCE OF SUPPORTThe research was funded by Boehringer Ingelheim Pharma GmbH & Co. KG.

cell biology↗