Schussler, N A

Abstract Rationale The governing mechanisms of lung regeneration are still poorly understood. Studies have observed the lung’s regenerative capacity using the pneumonectomy (PNX) model, where removal of the left lung stimulates compensatory growth and neoalveolarization. However, this process is inhibited if a physical object is used to replace the extracted tissue, demonstrating that the lung can sense the available space and regenerate in response. The mechanism of this mechanosensing is unknown. As outermost tissue layer of the lung, the pulmonary mesothelium is well situated to sense the surrounding environment. We and others have hypothesized that the mesothelium can become activated in cases of injury or regeneration, but this has not been fully explored. Understanding the mechanisms controlling neoalveolarizaiton may be key to unlocking the lung’s natural regenerative capacity in humans. Methods & Results In this study, we examined the location and fate of the mesothelium during regeneration using fluorescent lineage tracing and the PNX model. Specifically, we used the mesothelial-specific Wt1CreERT2 crossed with a lox-stop-lox-RFP reporter (Wt1(CreERT2/+);LSL-RFP(+/-)). Following PNX, we collected the lineage-labeled cells along with the CD326+ and CD326-/CD45-/CD31- populations and performed scRNA-sequencing. As quantified by flow-cytometry, the number of RFP+ cells increased following PNX, indicating proliferation of the mesothelium. Clustering analysis elucidated the emergence of 3 activated clusters of mesothelium following PNX, representing 12.9% of the RFP+ population in Sham and 66% in PNX. These activated cells exhibited decreased expression of mesothelial hallmarks and increased expression of extracellular matrix and epithelial-to-mesenchymal transition genes. Using immunofluorescence and confocal microscopy, we observed lineage-labeled mesothelium deep within the parenchyma after PNX. We observed none or singular invaded mesothelium in no-procedure control animals and only minimal occurrence in animals exposed to Sham surgery. Compared to Sham controls, PNX post-surgical (D)ay 3, D7, and D14 had 8.5, 32.7, and 9.2-fold more cells, respectively, in the parenchyma. Moreover, in aged (18 month) mice, where regeneration is known to be significantly reduced, we likewise observed minimal mesothelial invasion following PNX. These cells were often found near or directly wrapping around progenitors of the alveolar epithelium, AT2 cells, whose proliferation is essential to neoalveolarization. Using time-lapse microscopy of precision cut lung slices, we observed the invading mesothelial cells to be highly dynamic, sending out multiple transient protrusions and interacting with neighboring cells. Conclusion Together these data support strong and potentially functional interactions between activated and invading mesothelial cells and lung regeneration following PNX. This abstract is funded by: NIH

Abstract A population of pathologic fibroblasts has been identified in several human fibrotic lung diseases, including IPF. These cells, marked by high expression of collagen triple-helix repeat containing 1 (CTHRC1), display an invasive phenotype and aggressively deposit extracellular matrix. The goal of this study was to identify transcriptional regulators of the CTHRC1+ pathologic fibroblast state. We analyzed published single-cell RNA sequencing data of IPF and control lungs to predict transcription factor activity in CTHRC1+ fibroblasts, which led us to hypothesize that transcription factors in the nuclear factor of activated T-cells (NFAT) family may contribute to the pathologic fibroblast state. To test whether NFAT family members promote and/or maintain pathologic fibroblasts, we silenced each cytosol-localized NFAT (NFATc1-4) in lung fibroblasts from IPF donors (passage 3-6, N = 5 donors) and measured expression of key NFAT target genes related to fibrosis using qPCR. NFATC1 silencing significantly reduced expression of CTHRC1, SERPINE1, FN1, HAS2, and TNC, whereas other computationally predicted regulators showed little effect. Our computational analysis also predicted high activator protein 1 (AP-1) activity in pathologic fibroblasts, which interestingly has been found to act in concert with NFATs to promote an effector response in T-cells. Thus, we hypothesized that NFAT:AP-1 signaling may be involved in lung fibroblast activation. To test this hypothesis, we employed a small molecule inhibitor of NFAT:AP-1 complex formation on DNA (“Compound 10”, 25-50 µM). NFAT:AP-1 inhibition significantly reduced expression of fibrillar collagens COL1A1 and COL3A1, as well as CTHRC1 in primary IPF lung fibroblasts. Additionally, NFAT:AP-1 inhibition prevented TGF-β-mediated increase of several fibrosis-related NFATc1 targets. Aside from CTHRC1, NFAT:AP-1 inhibition led to decrease in a different set of NFATc1 targets than NFATc1 silencing, suggesting a potentially complex relationship between NFATc1 and AP-1 in fibroblast gene regulation. Finally, in mouse precision cut lung slices (PCLS) stimulated with a fibrosis-inducing cocktail ex vivo, NFAT:AP-1 inhibition prevented increased expression of Col1a1 and Cthrc1. Taken together, these studies suggest that NFATc1, together with AP-1, is involved in fibrogenic activation of pathological fibroblasts. Ongoing and future experiments will determine the roles of these signaling mechanisms in matrix deposition and contraction, as well as fibroblast invasion using human fibroblasts and PCLS models. Furthermore, we aim to determine how mechanical forces and soluble factors encountered in IPF lungs promote NFAT signaling in fibroblasts. This abstract is funded by: NHLBI, Pulmonary Fibrosis Foundation