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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. Single-cell RNA profiling of human cystic fibrosis proximal airway tissue reveals an overabundance of epithelial cells transitioning to specialized ciliated and secretory cells coupled with a decrease in cycling basal cells.

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

Respiratory disease is one of the leading causes of morbidity and mortality world-wide with an increasing incidence as the aged population prevails. Many lung diseases are treated for symptomatic relief, with no cure available, indicating a critical need for novel therapeutic strategies. Such advances are hampered by a lack of understanding of how human lung pathologies initiate and progress. Research on human lung disease relies on the isolation of primary cells from explanted lungs or the use of immortalized cells, both are limited in their capacity to represent the genomic and phenotypic variability among the population. In an era where we are progressing toward precision medicine the use of patient specific induced pluripotent cells (iPSC) to generate models, where sufficient primary cells and tissues are scarce, has increased our capacity to understand human lung pathophysiology. Directed differentiation of iPSC toward lung presented the initial challenge to overcome in generating iPSC-derived lung epithelial cells. Since then major advances have been made in defining protocols to specify and isolate specific lung lineages, with the generation of airway spheroids and multi cellular organoids now possible. This technological advance has opened up our capacity for human lung research and prospects for autologous cell therapy. This chapter will focus on the application of iPSC to studying human lung disease.

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.

Introduction/AbstractCystic fibrosis (CF) is a lethal autosomal recessive disorder that afflicts in excess of 70,000 people globally. 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 significant determinant of morbidity and mortality. In this study we report results from a multi-institute consortium in which single cell transcriptomics were applied to define disease-related changes to the proximal airway of CF donors (n=19) undergoing transplantation for end-stage lung disease compared to the proximal airway of previously healthy lung donors (n=19). We found that all major airway epithelial cell types were conserved between control and CF donors. Disease-dependent differences were observed, including an overabundance of epithelial cells transitioning to specialized ciliated and secretory cell subtypes coupled with an unexpected decrease in cycling basal cells. This study developed a molecular atlas of the proximal airway epithelium that will provide insights for the development of new targeted therapies for CF airway disease.

Summary Expansion of pulmonary neuroendocrine cells (PNECs) is a pathological feature of many human lung diseases. Human PNECs are inherently difficult to study due to their rarity (, Graphical Abstract, Highlights • PNECs can be efficiently generated from induced pluripotent stem cells (iPSCs) • Induced PNECs express key PNEC markers and express and secrete all major neuropeptides • Induced PNECs resemble the transcriptomic profile of human primary fetal PNECs • Basal cells are able to differentiate into PNECs, Cell Biology; Stem Cells Research; Transcriptomics

Respiratory disease is one of the leading causes of morbidity and mortality world-wide with an increasing incidence as the aged population prevails. Many lung diseases are treated for symptomatic relief, with no cure available, indicating a critical need for novel therapeutic strategies. Such advances are hampered by a lack of understanding of how human lung pathologies initiate and progress. Research on human lung disease relies on the isolation of primary cells from explanted lungs or the use of immortalized cells, both are limited in their capacity to represent the genomic and phenotypic variability among the population. In an era where we are progressing toward precision medicine the use of patient specific induced pluripotent cells (iPSC) to generate models, where sufficient primary cells and tissues are scarce, has increased our capacity to understand human lung pathophysiology. Directed differentiation of iPSC toward lung presented the initial challenge to overcome in generating iPSC-derived lung epithelial cells. Since then major advances have been made in defining protocols to specify and isolate specific lung lineages, with the generation of airway spheroids and multi cellular organoids now possible. This technological advance has opened up our capacity for human lung research and prospects for autologous cell therapy. This chapter will focus on the application of iPSC to studying human lung disease.

Respiratory disease is one of the leading causes of morbidity and mortality world-wide with an increasing incidence as the aged population prevails. Many lung diseases are treated for symptomatic relief, with no cure available, indicating a critical need for novel therapeutic strategies. Such advances are hampered by a lack of understanding of how human lung pathologies initiate and progress. Research on human lung disease relies on the isolation of primary cells from explanted lungs or the use of immortalized cells, both are limited in their capacity to represent the genomic and phenotypic variability among the population. In an era where we are progressing toward precision medicine the use of patient specific induced pluripotent cells (iPSC) to generate models, where sufficient primary cells and tissues are scarce, has increased our capacity to understand human lung pathophysiology. Directed differentiation of iPSC toward lung presented the initial challenge to overcome in generating iPSC-derived lung epithelial cells. Since then major advances have been made in defining protocols to specify and isolate specific lung lineages, with the generation of airway spheroids and multi cellular organoids now possible. This technological advance has opened up our capacity for human lung research and prospects for autologous cell therapy. This chapter will focus on the application of iPSC to studying human lung disease.