Tschumperlin, D J

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 Rationale Pulmonary Fibrosis is characterized by excessive accumulation of extracellular matrix (ECM), primarily collagen I, leading to progressive lung scarring and loss of function. Many anti-fibrotic compounds have been found to attenuate collagen deposition. However, identifying compounds that stimulate collagen I degradation, a necessary step in fibrosis clearance, is less common. To identify such compounds, we developed in vitro and ex vivo assays to quantify extracellular collagen I and collagen I degradation products. Methods Primary lung fibroblasts (passage 1-3, N = 5) from patients with idiopathic pulmonary fibrosis (IPF) were cultured in DMEM/F12 with 2% FBS, 2 ng/mL TGF-β, and 50 µg/mL ascorbic acid for 72 hours to generate collagen-rich cell-derived ECM. Cells were then treated with test compounds for 24 hours in the same media. Nuclear labeling with Hoechst 33342 was used to quantify cell numbers by automated microscopy. Decellularization was then performed with a solution containing 20 mM ammonium hydroxide and 0.5% Triton X-100. Cell-derived ECM was then fixed, stained with anti-Collagen I antibody, and quantified by automated microscopy. Collagen I degradation was further assessed by measuring type I collagen C-telopeptide (ICTP) via ELISA in cell culture media. To investigate the role of matrix metalloproteinase-13 (MMP-13) in collagen degradation, we used T26-C, a selective MMP-13 inhibitor, or transfected cells with MMP-13 siRNA. Ex vivo validation of collagen-degrading activity was conducted by measuring type I collagen N-terminal telopeptide (NTX-I) in the culture media of human precision-cut lung slices (PCLS, N = 3) generated from upper and lower lobes of IPF tissue and stimulated with select compounds. Results Thirty-four candidate compounds (metabolic, epigenetic, and cAMP modulators) were tested for their ability to reduce collagen I levels in cell-derived ECM. Six hits and two positive controls were then validated using ICTP ELISAs. In total five compounds were found to stimulate collagen degradation: EGCG (1 μM), Nintedanib (0.25 μM), SB431542 (1 μM), and two dopamine receptor agonists, A68930 (10 μM) and Dihydrexidine (10 µM). MMP-13 inhibition or knockdown prevented collagen degradation by all five compounds. In human IPF PCLS, collagen degradation was confirmed in response to EGCG, Nintedanib and SB431542 but not Dihydrexidine, with much greater responses seen in PCLS from upper lobes than lower lobes. Conclusion Collagen degradation can be stimulated in cultured fibroblasts and IPF PCLS. These systems will assist in uncovering mechanisms and regulators of collagen degradation and may pave the way to better fibrosis-resolving therapies. This abstract is funded by: NIH U01 HL152967

Abstract Rationale Systemic sclerosis (SSc) is a rare, chronic autoimmune disease characterized by vasculopathy and fibrosis of the skin and internal organs. Among its manifestations, interstitial lung disease (ILD) is the leading cause of mortality, yet its clinical course is variable, ranging from stable to progressive pulmonary fibrosis (PPF). The 2022 ATS/ERS/JRS/ALAT guidelines defined progressive PPF as a phenotype of non- Idiopathic Pulmonary Fibrosis (IPF) - ILDs that behave similarly to IPF. Although a subset of SSc-ILD patients develop PPF, biomarkers predicting progression remain lacking. We and others have shown that endothelial dysfunction contributes to the progression of fibrotic lung disease, and that the pulmonary vasculature, continuously exposed to the bloodstream, is influenced by soluble circulating factors, including circulating extracellular vesicles (cEVs), which carry bioactive cargo capable of modulating endothelial behavior. Here, we investigated whether cEVs from SSc patients with distinct fibrotic phenotypes differentially influence human lung endothelial cells (ECs), promoting a pro-fibrotic endothelial phenotype. Methods plasma samples were collected from five groups: 1) Healthy controls (n = 4); 2) SSc-noILD (no high-resolution-CT (HCRT( or functional evidence of ILD at time of enrollment, n = 4); 3) SSc-ILD (HCRT and functional evidence of ILD without meeting the PPF criteria at time of sampling, n = 3); 4) SSc-PPF (meeting the PPF criteria, plasma collected prior to anti-fibrotic therapy, n = 4); 5) IPF (plasma collected prior to anti-fibrotic therapy, n = 3). cEVs were isolated using size-exclusion-chromatography and characterized using Nanoparticle Tracking Analysis (NTA). Human lung ECs were treated with cEVs from the five groups (2x109 particles/ml, 48 hours), followed by qPCR for markers selected for their established involvement in endothelial dysfunction during lung fibrotic progression. Results NTA showed no significant differences in plasma cEV concentrations across the five groups. Human lung ECs exposed to cEVs from SSc or SSc-ILD patients exhibited no significant gene expression changes compared to healthy controls. In contrast, cEVs from SSc-PPF patients markedly downregulated endothelial identity (PECAM1, ERG, TEK), barrier integrity (CDH13, S1PR1), and basement membrane (COL4A1, HSPG2) genes, similarly to changes induced by cEVs from IPF patients. Conclusion cEVs from SSc-PPF patients induce pro-fibrotic transcriptional changes in lung ECs resembling those induced by IPF cEVs and distinct from SSc-ILD cEVs, suggesting that EVs carry cargo that both reflect and influence fibrosis progression. These findings highlight the potential of cEVs as biomarkers for identifying SSC-ILD patients at risk of progression and highlight the clinical relevance of future studies aimed at characterizing their cargo. This abstract is funded by: James Depauw Grant for Pilot Projects

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