Mechanisms of gasdermin pore formation in response to viral sensing in human respiratory epithelial cells
Citation:
Guy, Coralie, Mechanisms of gasdermin pore formation in response to viral sensing in human respiratory epithelial cells, Trinity College Dublin, School of Biochemistry & Immunology, Biochemistry, 2023Download Item:
Abstract:
A key component of the innate immune response to infection or cellular stress is the activation of inflammasomes which leads to gasdermin (GSDM) pore formation, interleukin-1β (IL-1β) release and lytic cell death. GSDMs are a family of pore-forming proteins with six members namely GSDMA, GSDMD, GSDMC, GSDMD, GSDME and Pejvakin. Most inflammasome and GSDM studies have been performed in myeloid cells where the NLRP3 inflammasome is often activated after viral infection leading to formation of GSDMD pores. Respiratory epithelial cells are an essential line of defence to initiate a robust immune response against viral pathogens but the mechanism of GSDM activation in epithelial cells upon sensing of double-stranded RNA (dsRNA), a key viral pathogen-associated molecular pattern, and of viral infection, remains unclear. Therefore the aim of this study was to investigate the mechanism of inflammasome activation and GSDM pore formation in response to viruses and dsRNA in epithelial cells. Our results demonstrated that undifferentiated primary normal human bronchial epithelial (NHBE) cells are a better model to study inflammasome and GSDM activation compared to commonly used immortalised and cancerous cell lines such as BEAS-2B and A549 cells. Transfection of poly(I:C), a mimic of viral dsRNA, both triggered caspase-1-dependent GSDMD cleavage into a pore-forming fragment but also led to caspase-3, -8, -9-mediated GSDMD inactivation and GSDME pore formation. Further investigation using pharmacological inhibitors and siRNA technology showed that NLRP1 inflammasome activation, but not NLRP3, resulted in GSDMD pore formation, whereas PKR activation led to GSDMD inactivation and GSDME activation. Additionally, TRIF, the adaptor for the dsRNA receptor TLR3, contributed to early GSDME cleavage, lytic cell death and cytokine secretion following dsRNA delivery. In contrast MAVS, the adaptor for the viral RNA sensors RIG-I and MDA5, wasn't involved in GSDM processing. While GSDME triggered lytic cell death, both GSDME and GSDMD pores were required for robust IL-1β secretion. Preliminary results indicated that dsRNA sensing also triggered GSDMA pore formation at the plasma membrane. Importantly, NLRP1 and PKR were also involved in upregulation of pro-IL-1β, RIG-I and MDA5 expression in dsRNA-stimulated NHBE cells. Infection of NHBE cells with influenza A virus (IAV) mainly led to GSDME pore formation and GSDMD inactivation in a PKR-caspase-3-dependent but inflammasome- and MAVS-independent manner. Interestingly, caspase-3 and caspase-8, but not caspase-1 were required for IAV-mediated lytic cell death and IL-1β secretion. Suppression of GSDMD and GSDME expression using siRNA led to enhanced IAV replication suggesting that GSDM pore formation in response to IAV is intrinsically antiviral in basal respiratory epithelial cells. Preliminary results with respiratory syncytial virus (RSV) suggested that PKR might also contribute to caspase-3-mediated GSDME pore formation during RSV infection. Overall different pathways lead to GSDM cleavage during viral sensing in NHBE cells. While intracellular dsRNA triggers NLRP1 inflammasome-dependent GSDMD pore formation, both dsRNA sensing and IAV infection activate PKR that mediates caspase-3-dependent GSDMD inactivation and GSDME activation.
Sponsor
Grant Number
H2020-MSCA-ITN-2019 (Grant No 813343)
Marie Sklodowska-Curie Actions (MSCA) Innovative Training Networks (ITN)
Description:
APPROVED
Author: Guy, Coralie
Advisor:
Bowie, AndrewPublisher:
Trinity College Dublin. School of Biochemistry & Immunology. Discipline of BiochemistryType of material:
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