Distinct Groups of Epithelial Cells Identified That May Drive PF Progression

Researchers have discovered the existence of unique subtypes of epithelial cells in the lungs of people with pulmonary fibrosis (PF) that may drive tissue scarring, or fibrosis.

Their findings were published in the study, “Single-cell RNA sequencing reveals profibrotic roles of distinct epithelial and mesenchymal lineages in pulmonary fibrosis,” in the journal Science Advances.

The cells that make up the lung epithelium — the protective lining of the lungs — play a central role in PF progression. However, it is increasingly recognized that these cells are heterogeneous, meaning that it is likely that not all of them participate in disease onset and progression.

Researchers at the Translational Genomics Research Institute (TGen), an affiliate of City of Hope, and their colleagues used a technique called single-cell RNA sequencing to analyze individual cells that make up the lung epithelium of PF patients and controls.

Through this method, they were able to analyze the entire set of gene transcripts — messenger RNA molecules constituting copies of the DNA sequence used to build proteins — in each single cell and assess whether a particular group of cells may drive PF onset and progression.

They examined cells from a total of 20 lungs derived from people with a lung disease — 12 had idiopathic PF (IPF), three had chronic hypersensitivity pneumonitis, two had nonspecific interstitial pneumonia, another two sarcoidosis, and one had unclassifiable interstitial lung disease. As controls, the team analyzed lungs from tobacco smokers who were declined for organ donation.

Diseased lungs were collected at two lung transplant centers, the Vanderbilt University Medical Center and the Norton Thoracic Institute, while control lungs were provided by the Donor Network of Arizona and the Tennessee Donor Services.

After comparing cells from diseased lungs with those from control lungs, the researchers identified 31 distinct cell types/states.

They first noticed that the levels of known biomarkers for fibrosis, such as the MMP7 gene, changed across many cell types. Another marker, called CDKN2A, was more restricted. These findings suggested that different cell types, depending on the stimuli they receive, may participate in the tissue remodeling that leads to IPF.

“Classically, the field used a small number of genes to determine cell types. With the single cell RNA sequencing approach, we find that it is often hard to draw a firm line between different types of cells. Instead of thinking of them as discrete cell types, we should think of them more along a continuum, and given the right stimulus, these cells can change their state,” Nicholas E. Banovich, PhD, an assistant professor in TGen’s Integrated Cancer Genomics Division and co-lead author of the study, said in a press release.

Additionally, the analysis revealed the existence of previously unknown epithelial cell types. A particular cell type — identified by the activity of the KRT17 gene but no activity for KRT5 (KRT5-/KRT17+ cells) — was found exclusively on PF lungs. These cells were actively producing many of the components of the extracellular matrix — the mesh-like scaffold surrounding cells — that drive lung fibrosis, including collagen.

“These cells are incredibly unique as they are clearly epithelial, but are also producing collagen and components of extra-cellular matrix (ECM), which make scar tissue,” Banovich said. “They are directly contributing to fibrosis.”

These cells also share characteristics of both the airway and alveolar epithelium, the respiratory membrane that allows the exchange of gases in the lungs.

Another cell type found almost exclusively in PF lungs was characterized by the activity of the SCGB3A2 gene. These cells seemed to be able to transform into another cell type called type 1 alveolar cells (AT1). AT1 cells are essential for the gas-exchange function of lungs — oxygen gets in and carbon dioxide is expelled — and the transition toward this cell type may indicate an effort to repair damage to the lung.

However, SCGB3A2-positive cells may also become KRT5-/KRT17+ cells and ultimately boost the fibrosis process in PF.

“In addition to becoming AT1 cells, our results suggest the SCGB3A2+ cells can also become the KRT5-/KRT17+ cells. It actually appears that, during the transformation into AT1 cells, the process is being hijacked and instead of helping repair the lungs these cells are pushed toward this weird pro-fibrotic epithelial cell that continues to drive fibrosis,” Banovich said.

Two other cells types found in PF lungs were a subtype of fibroblasts — the cells responsible for the synthesis and buildup of scar tissue — marked by high levels of the genes PLIN2 or HAS1.

Overall, the study identified five unique cells types associated with lung fibrosis and sheds light on the “genes, pathways, and programs that characterize [disease] lung remodeling in PF,” the researchers wrote.

“Future studies investigating the origin, behavior, and function of these cell types, subtypes, states, and pathologic gene expression programs should provide additional insights into the foundational mechanisms regulating … disease in the human lung,” they concluded.