Abstract
Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal degenerative lung disease of unknown etiology. Although in its final stages it implicates, in a reactive manner, all lung cell types, the initial damage involves the alveolar epithelial compartment, in particular the alveolar epithelial type 2 cells (AEC2s). AEC2s serve dual progenitor and surfactant secreting functions, both of which are deeply impacted in IPF. Thus, we hypothesize that the size of the surfactant processing compartment, as measured by LysoTracker incorporation, allows the identification of different epithelial states in the IPF lung. Flow cytometry analysis of epithelial LysoTracker incorporation delineates two populations (Lysohigh and Lysolow) of AEC2s that behave in a compensatory manner during bleomycin injury and in the donor/IPF lung. Employing flow cytometry and transcriptomic analysis of cells isolated from donor and IPF lungs, we demonstrate that the Lysohigh population expresses all classical AEC2 markers and is drastically diminished in IPF. The Lysolow population, which is increased in proportion in IPF, co-expressed AEC2 and basal cell markers, resembling the phenotype of the previously identified intermediate AEC2 population in the IPF lung. In that regard, we provide an in-depth flow-cytometry characterization of LysoTracker uptake, HTII-280, proSP-C, mature SP-B, NGFR, KRT5, and CD24 expression in human lung epithelial cells. Combining functional analysis with extracellular and intracellular marker expression and transcriptomic analysis, we advance the current understanding of epithelial cell behavior and fate in lung fibrosis.
Highlights
The human lung is a highly complex organ designed for gas exchange.In idiopathic pulmonary fibrosis (IPF), chronic epithelial injury leads to excessive deposition of rigid extra-cellular matrix and a progressive decrease in lung compliance and gas-exchange surface, causing inevitable and fatal lung failure within 2–5 years after diagnosis [1,2,3]
To identify AEC2s, dead cells (PI+ or DAPI+) and AEC1 cells were first excluded, and the epithelial compartment was further identified by EpCAM expression within the CD45− and CD31− population
At all time-points analyzed, the Lysohigh population was decreased in number, with the greatest decrease registered at days 7 and 14, when AEC2 injury was maximal [43,44]
Summary
The human lung is a highly complex organ designed for gas exchange.In idiopathic pulmonary fibrosis (IPF), chronic epithelial injury leads to excessive deposition of rigid extra-cellular matrix and a progressive decrease in lung compliance and gas-exchange surface, causing inevitable and fatal lung failure within 2–5 years after diagnosis [1,2,3]. New therapies significantly increased the duration and quality of life of IPF patients, a therapeutic regimen that can arrest or, even better, reverse disease progression remains to be discovered [4]. Responsible for this situation is our limited understanding of the cellular states and processes that each of the more than 40 cell types in the lung undergoes, in an active (causative) or reactive manner in homeostatic and injury contexts [5,6]. AEC2s are facultative progenitors in the distal lung, which, in a differentiated state, serve the vital function of surfactant production and secretion, but can act as progenitors for other AEC2s and AEC1s in homeostatic and injury-repair situations [10,11]
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