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Single-cell RNA-sequencing reveals profibrotic roles of distinct epithelial and mesenchymal lineages in pulmonary fibrosis

Arun C. Habermann, Austin J. Gutierrez, Linh T. Bui, Stephanie L. Yahn, Nichelle I. Winters, Carla L. Calvi, Lance Peter, Mei-I Chung, Chase J. Taylor, Christopher Jetter, Latha Raju, Jamie Roberson, Guixiao Ding, Lori Wood, Jennifer MS Sucre, Bradley W. Richmond, Ana P. Serezani, Wyatt J. McDonnell, Simon B. Mallal, Matthew J. Bacchetta, James E. Loyd, Ciara M. Shaver, Lorraine B. Ware, Ross Bremner, Rajat Walia, Timothy S. Blackwell, Nicholas E. Banovich, Jonathan A. Kropski

Preprint posted on September 06, 2019 https://www.biorxiv.org/content/10.1101/753806v1

and

Single Cell RNA-seq reveals ectopic and aberrant lung resident cell populations in Idiopathic Pulmonary Fibrosis

Taylor S. Adams, Jonas C. Schupp, Sergio Poli, Ehab A. Ayaub, Nir Neumark, Farida Ahangari, Sarah G. Chu, Benjamin A. Raby, Giuseppe DeIuliis, Michael Januszyk, Qiaonan Duan, Heather A. Arnett, Asim Siddiqui, George R. Washko, Robert Homer, Xiting Yan, Ivan O. Rosas, Naftali Kaminski

Preprint posted on September 09, 2019 https://www.biorxiv.org/content/10.1101/759902v2

The application of single cell RNA sequencing (scRNAseq) approaches in the context of idiopathic pulmonary fibrosis (IPF) highlights dramatic remodelling of epithelial, stromal and vascular transcriptomes.

Selected by Rob Hynds

Background

Idiopathic pulmonary fibrosis (IPF) is a fatal chronic lung disease characterised by the destruction of the gas exchange unit, the alveolus, and the irregular accumulation of extracellular matrix in the peripheral lungs. There are limitations of in vitro and in vivo models of IPF that limit our understanding of the disease in patients. These pre-prints apply scRNAseq approaches to this problem, comparing human IPF to control lungs. Previous scRNAseq studies in this area identified alterations in the macrophage compartment, revealing alveolar macrophages with a potentially pro-fibrotic phenotype. Abnormalities in IPF epithelial cells have also been described in a study analyzing a smaller number of patients/cells: broad changes were found in gene expression across all epithelial subpopulations as well as an additional ‘indeterminate’ epithelial cell population, which expressed both basal cell (SOX2) and AT2 markers (SOX9) and genes suggestive of ‘epithelial-to-mesenchymal transition’.

Key Findings

More than 400,000 cells were RNA sequenced between the two pre-prints, giving new resolution on IPF pathogenesis. Both pre-prints demonstrate that the transcriptomes of all major airway cell types are altered in IPF lungs, reflecting broad microenvironmental changes in the lungs of these patients.

Figure 1B from Habermann et al.

 

Importantly, the composition of the lungs changed substantially in IPF with the proportion of airway to alveolar cells shifting in favour of more airway-like cells in IPF, reflecting the known proximalization of the distal airways during disease pathogenesis. Further, within specific compartments, transcriptional re-wiring of normal cell types was observed in IPF. For example, Adams et al. identify fibroblast and myofibroblast populations in both control and IPF lungs that are similar to expression profiles described in mice. In IPF, fibroblasts expressed higher levels of HAS1, HAS2 and FBN while myofibroblasts were enriched in collagens and ACTA2. Importantly, lineage reconstruction suggested limited connectivity between fibroblasts and myofibroblasts implying that these two cellular pools are maintained independently, rather than by on-going differentiation of fibroblasts to myofibroblasts. Habermann et al. found four distinct stromal subpopulations: fibroblasts, ACTA2+myofibroblasts, PLIN2lipofibroblasts and a HAS1hi population that was unique to IPF and expressed genes associated with cell stress, IL4/13 signalling and EMT. As expected, myofibroblasts were found near airways and lipofibroblasts were found in the interstitium. HAS1hi fibroblasts were subpleural and co-localised with COL1A1 by RNA in situ hybridization. Analysis of potential interactions between cell types emphasised integrins as key mediators of mesenchymal-to-epithelial interaction while epithelial-to-mesenchymal signalling was more diverse, mediated by a range of growth factors and cytokines.

In addition to cell populations that are phenotypically altered in disease, Adams et al. identified a vascular endothelial cell population – characterized by COL15A1 expression – that is ordinarily found underlying major airways, in the distal IPF lung near to fibrotic foci and in areas of bronchialization. These findings mirror the epithelial cell compartment which is similarly proximalized in IPF lungs.

Populations of cells that were unique to IPF lungs are also described. Common to both studies are a population of SOX2/SOX9+/TP63+/KRT5/KRT17epithelial cells, dubbed “aberrant basaloid cells” by Adams et al. and “KRT5/KRT17cells” by Habermann et al., that are implicated in IPF pathogenesis both by their proximity to fibrotic foci and expression of genes such as MMP7, integrin αVβ6 and senescence-associated genes, all of which have been linked to IPF pathogenesis previously. Intriguingly, this population also expresses some genes associated with ‘epithelial-to-mesenchymal transition’, although neither study finds intermediate cells between any epithelial and mesenchymal populations, arguing against a pathogenic cell type arising by transdifferentiation between these compartments. However, the upregulation of gene programmes typically associated with mesenchymal cells, such as invasion, migration and reduced dependence on neighbours for survival signals suggests that these processes – sometimes referred to as ‘partial EMT’ – might be important in IPF pathogenesis.

Figure 1B from Adams et al.

 

Interestingly, Habermann et al. performed pseudotime- and RNA processing-based trajectory analyses that show that these cells might be derived from a transitional AT2 cell population rather than airway basal cells, as had been widely thought previously due to phenotypic similarities. Transitional AT2 cells in turn could be derived either from AT2 cells or from SCGB3A2+secretory cells, which are substantially more abundant in IPF lungs and spatially associated with remodelling. The data suggest that multiple epithelial cell types might converge on a common transitional state to generate AT1 cells in human lungs. Mechanistically, the authors suggest that upregulation of SOX4 and SOX9 concurrent with downregulation of NR1D1, a transcriptional repressor, might be involved in generating the IPF-specific epithelial cell phenotype, making these targets for future functional validation.

Conclusions
These pre-prints highlight the transcriptomic complexity of IPF and describe the behaviour of cell types involved the disease’s pathogenesis in new detail, describing multiple cell lineages that contribute to collagen production and provide a more comprehensive assessment of the epithelial population overlying fibrotic foci. Both pre-prints describe strong spatial influences on cell phenotype, suggesting the value of applying spatial transcriptomics techniques in IPF or specifically microdissecting out fibrotic foci to better understand cellular cross-talk in IPF.

Questions for the authors

Q1. How much consideration was paid to disease heterogeneity during sampling? Would the spared regions of IPF patients’ lungs resemble control or is there a general effect of the disease on transcription?

Q2. Do the authors have any indication of which aspects of proximalization (epithelial, endothelial) occur earliest during disease pathogenesis? Might one be a consequence of the other?

Q3. How do the previously described ‘indeterminate’ cells fit into the picture described here? Are they equivalent to the ‘aberrant basaloid cell’?

Tags: chronic lung disease, epithelium, scrnaseq, stroma

Posted on: 20th September 2019

(2 votes)




  • Author's response

    Dr. Nicholas Banovich and Dr. Jon Kropski shared about Single-cell RNA-sequencing reveals profibrotic roles of distinct epithelial and mesenchymal lineages in pulmonary fibrosis

    R1. These analyses were performed agnostic to the heterogeneity within the lung. However, the degree to which the spared regions of IPF lungs resemble controls is an important question that we are actively following up on.

    R2. We agree that this and related questions are central to bettering our understanding of this process but admittedly are ones that our current study cannot directly address.  Our hope is that by making these data publicly available, researchers around the world can use their expertise to pursue these sorts of questions.

    R3. The previously described “indeterminate” cells likely included multiple different cell phenotypes that did not fit into classical categories, including KRT5/KRT17+/aberrant basaloid cells, but also SCGB3A2+cells and at least a subset of transitional AT2 cells. With greater cell numbers, we were able to more granularly resolve these discrete cell states/phenotypes.

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