Neutrophilic inflammation promotes SARS-CoV-2 infectivity and augments the inflammatory responses in airway epithelial cells.

Introduction: 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 to severe COVID-19 are associated with chronic airway neutrophilia. Furthermore, examination of COVID-19 explanted lung tissue revealed a series of epithelial pathologies associated with the infiltration and activation of neutrophils, indicating neutrophil activity in response to SARS-CoV-2 infection. Methods: To determine the impact of neutrophil-epithelial interactions on the infectivity and inflammatory responses to SARS-CoV-2 infection, we developed a co-culture model of airway neutrophilia. This model was infected with live SARS-CoV-2 virus the epithelial response to infection was evaluated. Results: SARS-CoV-2 infection of airway epithelium alone does not result in a notable pro-inflammatory response from the epithelium. The addition of neutrophils induces the release of proinflammatory cytokines and stimulates a significantly augmented proinflammatory response subsequent SARS-CoV-2 infection. The resulting inflammatory responses are polarized with differential release from the apical and basolateral side of the epithelium. Additionally, the integrity of the \epithelial barrier is impaired with notable epithelial damage and infection of basal stem cells. Conclusions: This study reveals a key role for neutrophil-epithelial interactions in determining inflammation and infectivity.

Implications for Extracellular Matrix Interactions With Human Lung Basal Stem Cells in Lung Development, Disease, and Airway Modeling.

The extracellular matrix (ECM) is not simply a quiescent scaffold. This three-dimensional network of extracellular macromolecules provides structural, mechanical, and biochemical support for the cells of the lung. Throughout life, the ECM forms a critical component of the pulmonary stem cell niche. Basal cells (BCs), the primary stem cells of the airways capable of differentiating to all luminal cell types, reside in close proximity to the basolateral ECM. Studying BC-ECM interactions is important for the development of therapies for chronic lung diseases in which ECM alterations are accompanied by an apparent loss of the lung's regenerative capacity. The complexity and importance of the native ECM in the regulation of BCs is highlighted as we have yet to create an in vitro culture model that is capable of supporting the long-term expansion of multipotent BCs. The interactions between the pulmonary ECM and BCs are, therefore, a vital component for understanding the mechanisms regulating BC stemness during health and disease. If we are able to replicate these interactions in airway models, we could significantly improve our ability to maintain basal cell stemness ex vivo for use in in vitro models and with prospects for cellular therapies. Furthermore, successful, and sustained airway regeneration in an aged or diseased lung by small molecules, novel compounds or via cellular therapy will rely upon both manipulation of the airway stem cells and their immediate niche within the lung. This review will focus on the current understanding of how the pulmonary ECM regulates the basal stem cell function, how this relationship changes in chronic disease, and how replicating native conditions poses challenges for ex vivo cell culture.

Transcriptional analysis of cystic fibrosis airways at single-cell resolution reveals altered epithelial cell states and composition.

Cystic fibrosis (CF) is a lethal autosomal recessive disorder that afflicts more than 70,000 people. People with CF experience multi-organ dysfunction resulting from aberrant electrolyte transport across polarized epithelia due to mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CF-related lung disease is by far the most important determinant of morbidity and mortality. Here we report results from a multi-institute consortium in which single-cell transcriptomics were applied to define disease-related changes by comparing the proximal airway of CF donors (n = 19) undergoing transplantation for end-stage lung disease with that of previously healthy lung donors (n = 19). Disease-dependent differences observed include an overabundance of epithelial cells transitioning to specialized ciliated and secretory cell subsets coupled with an unexpected decrease in cycling basal cells. Our study yields a molecular atlas of the proximal airway epithelium that will provide insights for the development of new targeted therapies for CF airway disease.

Derivation of induced pluripotent stem cells from ferret somatic cells.

Ferrets are an attractive mammalian model for several diseases, especially those affecting the lungs, liver, brain, and kidneys. Many chronic human diseases have been difficult to model in rodents due to differences in size and cellular anatomy. This is particularly the case for the lung, where ferrets provide an attractive mammalian model of both acute and chronic lung diseases, such as influenza, cystic fibrosis, A1A emphysema, and obliterative bronchiolitis, closely recapitulating disease pathogenesis, as it occurs in humans. As such, ferrets have the potential to be a valuable preclinical model for the evaluation of cell-based therapies for lung regeneration and, likely, for other tissues. Induced pluripotent stem cells (iPSCs) provide a great option for provision of enough autologous cells to make patient-specific cell therapies a reality. Unfortunately, they have not been successfully created from ferrets. In this study, we demonstrate the generation of ferret iPSCs that reflect the primed pluripotent state of human iPSCs. Ferret fetal fibroblasts were reprogrammed and acquired core features of pluripotency, having the capacity for self-renewal, multilineage differentiation, and a high-level expression of the core pluripotency genes and pathways at both the transcriptional and protein level. In conclusion, we have generated ferret pluripotent stem cells that provide an opportunity for advancing our capacity to evaluate autologous cell engraftment in ferrets.