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.
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.
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.
This study reveals a key role for neutrophil-epithelial interactions in determining inflammation and infectivity.
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.
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.
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 (<1% of total lung cells) ...and a lack of established protocols for their isolation. We used induced pluripotent stem cells (iPSCs) to generate induced PNECs (iPNECs), which express core PNEC markers, including ROBO receptors, and secrete major neuropeptides, recapitulating known functions of primary PNECs. Furthermore, we demonstrate that differentiation efficiency is increased in the presence of an air-liquid interface and inhibition of Notch signaling. Single-cell RNA sequencing (scRNA-seq) revealed a PNEC-associated gene expression profile that is concordant between iPNECs and human fetal PNECs. In addition, pseudotime analysis of scRNA-seq results suggests a basal cell origin of human iPNECs. In conclusion, our model has the potential to provide an unlimited source of human iPNECs to explore PNEC pathophysiology associated with several lung diseases.
Display omitted
•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
Introduction
Gene‐corrected autologous cells provide one avenue for treating respiratory diseases, such as Cystic Fibrosis and Primary Ciliary Dyskinesia that harbor an underlying genetic defect. ...Basal cells are critical stem cells in the airways with the capacity to generate all cells of the respiratory epithelium. Use of these cells is limited due to inherent difficulties in gene editing and expansion in vitro. Efficient generation of basal cells from iPSCs has the potential to generate enough cells for both autologous cell therapy and for in vitro disease modelling. Building on our airway differentiation protocol, we have derived a strategy for the specification, isolation and expansion of iPSC‐derived basal cells (iBasal) improving the efficiency of generating respiratory epithelium from iPSC.
Methods
iPSC are cultured using a modified differentiation protocol pushing the formation of lung progenitor cells sorted on day 17 of differentiation for CD47hiCD26lo cells. These cells express transcription factor NKx2.1 and can be cultured in defined serum‐free media in the absence of wnt signaling. Over 4 passages we evaluated; 1) expression of basal cell markers, 2) clonality by a limited dilution clonogenic assay and by spheroid formation and 3) stem cell potential in spheroid and air‐liquid interface differentiation assays. Gene expression was compared to that of primary endogenous basal cells by bulk and single cell RNAseq, and validated by qPCR.
Results
Under our defined culture conditions, iBasal can be derived from NKx2.1 expressing lung progenitor cells. They are clonogenic forming colonies expressing cytokeratin 5 (krt5), p63 and integrin alpha 6 (IGTA6), acquiring more mature basal cell marker, nerve growth factor receptor (NGFR), expression after passaging when cultured on NIH3T3‐J2 feeders or in defined media. iBasal have the capacity for differentiation into a functional, tight junction forming epithelium containing both mucus secreting and motile multiciliated cells at the air liquid interface. In addition, they can form and maintain as spheroids which can also differentiate to generate airway epithelial cells. iBasal can be passaged and maintain a basal cell phenotype similar to that of paired passaged human airway basal cells. Single cell sequence data on iBasal and paired primary and cultured basal cells will provide the ultimate validation of iBasal.
Conclusion
We have generated iBasal from four independent iPSC lines with functional properties akin to that of primary basal cells. iBasal, therefore, provide the opportunity to generate sufficient functional and gene‐corrected autologous basal cells to develop patient specific, high throughput screening platforms overcoming the current limitations.
Support or Funding Information
Cystic Fibrosis Foundation FIRTH17XX0, FIRTH15XX1, NIH:NHLBI R01 HL139828‐01, Hastings Foundation
This is from the Experimental Biology 2019 Meeting. There is no full text article associated with this published in The FASEB Journal.
Introduction
Patients with chronic lung diseases, such as cystic fibrosis (CF), are often characterized with chronic inflammation and impaired bacterial clearance. This suggests that their ...respiratory innate immune response is inadequate. Macrophages are the sentinel cells of the respiratory system acting as the first line of defense against invading pathogens, orchestrating wound healing, and maintaining tissue homeostasis. The phenotype and function of macrophages are governed by signals they receive from their microenvironment. Despite this they are often studied in isolation in vitro. We hypothesized that changes in the innate immune response in CF result from pathological signaling between macrophages and airway epithelium.
Methods
Primary lung macrophages, isolated from lung explants from CF patient and patients with no prior evidence of chronic lung disease, were co‐cultured with human bronchial epithelial cell lines with or without mutations in the cystic fibrosis transmembrane regulator (CFTR), NuLi (control) and CuFi (CF). Cell lines were used for the initial experiments to reduce patient to patient variability due to factors such as treatment and bacterial colonization, allowing to focus on the specific role of CFTR mutations. A co‐culture model at the air‐liquid interface was developed to determine whether epithelial interactions with CFTR‐mutant macrophages influenced basal stem cell differentiation. Tumor necrosis factor alpha (TNFα) and interleukins 6 and 8 (IL‐6/8) were profiled by stimulating cultures with lipopolysaccharide (LPS) for 24 hours and analyzing the culture supernatants.
Results
Substantial differences in theinflammatory profile were observed in the presence of CFTR mutations in the co‐culture model. A 2‐fold increase in TNFα after LPS, was observed comparing CF to WT macrophage monocultures; this suggests they may maintain a hyperinflammatory physiological memory of the CF lung. The presence of epithelial cells suppresses TNFα release when compared to monoculture controls. In contrast, IL‐6 is increased in the co‐cultures. Additionally, CFTR mutant epithelial cells significantly increase IL‐8 (3‐fold increase, P<0.05), even in the absence of LPS, this response further exacerbated after LPS stimulation (1.5‐fold increase, P<0.05), compared to WT controls. Finally, our data supports a role for macrophages in stimulating ciliogenesis in WT epithelium which is suppressed by the presence of CFTR‐mutant macrophages (P<0.05).
Conclusion
This study highlights the importance of studying macrophages in a physiological microenvironment and supports a pathogenic role for CFTR‐mutant epithelium in stimulating lung macrophages to a hyper‐inflammatory phenotype. In addition, CF macrophages lose a capacity to promote efficient epithelial regeneration and homeostasis. This highlights the importance of understanding the cellular mechanisms regulated by macrophage‐epithelial interactions, regulated by CFTR, to identify new therapeutic targets in CF.
PDF
