Masonou, Tereza; (2024) Investigating neutrophil function during SARS-CoV-2 infection of human airway epithelial cells from elderly and young individuals. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Background: The COVID-19 pandemic caused by SARS-CoV-2 has resulted in over 7 million deaths, disproportionately affecting the elderly. There is little understanding regarding how disease severity increases with age, and the role of neutrophils found in elevated numbers in severe COVID-19 patients.// Aims: My investigation focuses on understanding neutrophil function during SARS-CoV-2 infection and their interaction with nasal airway cells, the primary viral entry point, from children and elderly. The Thesis aims to 1) characterize airway epithelial cells before and after infection, 2) describe neutrophil migration across the epithelium and 3) measure the activation of neutrophils following interaction with the infected epithelium. // Methods: Nasal airway cells obtained from healthy children (<12y) and elderly (>70y) individuals were differentiated at air-liquid interface, infected with SARS-CoV-2, and analysed by single-cell RNA sequencing (scRNA-seq) to identify genes that could influence neutrophil migration. To test this functionally, human neutrophils were added to the basolateral (blood) side so that they migrate to the apical (air) and infected side of the epithelium as like a physiological airway. Neutrophils were then recovered for flow cytometric analyses, to count migrated neutrophils, and measure the expression of markers and the release of enzymes on the apical surface. Additionally, a sub-analysis of an in vivo scRNA-seq dataset was performed to examine neutrophils recovered from COVID-19+ paediatric and adult airways.// Results: Following SARS-CoV-2 infection CD44 was more highly expressed on the elderly, while ICAM-1 was more highly expressed in the paediatric epithelium. I found greater neutrophil adherence to SARS-CoV-2 infected paediatric epithelium compared to elderly. Neutrophils migrating through the elderly airway display increased activation (CD11b+) and an elevated release of neutrophilic myeloperoxidase and LDH, a marker of cell death. Airway neutrophils from COVID-19 patients, also showed a heightened activation in adults characterised by a higher expression of CD44, and its ligands, compared to paediatrics. Conclusion: These findings suggest an inflammatory neutrophil phenotype influenced by an elderly epithelium and highlight the importance in understanding age-related immune interactions with the airway during SARS-CoV-2 infection.

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