Search

In response to viral infection, neutrophils release inflammatory mediators as part of the innate immune response, contributing to pathogen clearance through virus internalization and killing. Pre-existing co-morbidities, correlating to incidence of severe COVID-19, are associated with chronic airway neutrophilia and examination of COVID-19 lung tissue revealed a series of epithelial pathologies associated with infiltration and activation of neutrophils. To determine the impact of neutrophil-epithelial interactions on the infectivity and inflammatory response to SARS-CoV-2 infection, we developed a co-culture model of airway neutrophilia. We discovered that SARS-CoV-2 infection of the airway epithelium alone does not result in a notable release of pro-inflammatory cytokines, however in the presence of neutrophils, the inflammatory response is both polarized and significantly augmented, epithelial barrier integrity in impaired and viral load of the airway epithelium increased. This study reveals a key role for neutrophil-epithelial interactions in determining inflammation, infectivity, and outcomes in response to SARS-CoV-2 infection. HighlightsO_LIWe have developed a model to study neutrophil-epithelial interactions which better reflects the in vivo situation than monocultures C_LIO_LINeutrophils significantly augment SARS-CoV-2 mediated, pro-inflammatory cytokine release from the epithelium indicating a key interaction C_LIO_LISARS-CoV-2 infection leads to a polarized inflammatory response in differentiated airway epithelium C_LIO_LIDisruption of the epithelial barrier via addition of neutrophils or cytokines leads to increased infection C_LIO_LIStudy reveals a key role for neutrophil-epithelial interactions in determining outcome/infectivity C_LI

RATIONALE: Novel coronavirus infectious disease (COVID-19) is caused by the severe acute respiratory distress syndrome related coronavirus 2 (SARS-CoV-2). Patients with underlying respiratory disease are considered at increased risk of contracting severe COVID-19. Many respiratory diseases including chronic obstructive pulmonary disease, asthma, and cystic fibrosis, have elevated inflammation driven by recruitment of neutrophils, known as neutrophilia, and contributes to disease pathogenesis. In response to viral infection, neutrophils are the first, and predominant, leukocyte recruited to the respiratory tract. Neutrophils release inflammatory mediators as part of the acute inflammatory response and contribute to pathogen clearance through virus internalization and killing, secretion of antimicrobial peptides and extracellular traps. It remains uncertain how critical the neutrophil response is in SARS-CoV-2 and to what extent this contributes to the cytokine response of the airways in COVID-19. We hypothesized that neutrophils are responsible for elevated inflammatory responses in patients who progress to severe COVID-19. Methods: To investigate, we developed a co-culture model of neutrophilic airways to study SARS-CoV-2 infection, combining primary human neutrophils and airway epithelial cells differentiated at the air-liquid interface. These cultures, with and without neutrophils, were infected with live SARS-CoV-2 and the inflammatory responses analyzed. In addition, extensive analysis of ACE2, neutrophils and inflammatory response, was performed comparing our models and COVID-19 patient and non-infected lung tissues. Results: ACE2 protein, the receptor binding to the S-protein of SARS-CoV-2, was predominantly expressed in submucosal glands, ciliated cells and in alveolar type 2 cells in human lung tissues. Surprisingly, substantial co-localization of ACE2 was also observed with neutrophil elastase and CD15 expressing neutrophils infiltrating the airways. Analysis of mono-and co-cultures of airway epithelial cells and neutrophils in the presence or absence of SARS-CoV-2 infection indicated significant increases in the expression of squamous epithelium associated cytokeratins, damage associated molecular patterns (DAMPS) and a downregulation of interleukin 8 (IL-8) specifically in the co-culture models. Airway supernatants from the basolateral and apical surfaces were profiled. In the neutrophil co-cultures significant increases in IFNγ, IL-1β, IL-6 and TNFα were observed in response to SARS-CoV-2 infection. Conclusions: In conclusion, we demonstrate that co-culture with neutrophils has a significant impact on SARS-CoV-2 infection, changing the inflammatory response and upregulating gene expression patterns associated with tissue remodeling and tissue damage. This study highlights the need to study SARS-CoV-2 infection in more complex, tissue-level models to fully understand the mechanisms driving the severe inflammatory response and the long-term consequences of infection.

Despite drastic improvements in the treatment of cystic fibrosis (CF), there are still no curative options and severe CF remains a terminal diagnosis. With the recent advances in state-of-the-art techniques such as induced pluripotent stem cells (iPSC), stem cell expansion, differentiation, and gene editing technologies, we now have the ability to expand patient-specific cells for therapeutic application. These autologous cells can be readily genetically edited and clonally expanded with the potential for differentiation into the desired mature cell types. Such techniques hold enormous potential for therapeutic application. This chapter aims to provide an overview of the current state of iPSCs, proximal lung stem cells and gene editing techniques focusing on their potential for clinical application in CF.