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Idiopathic pulmonary fibrosis (IPF) is marked by the activation of fibroblasts, leading to excessive production and deposition of extracellular matrix (ECM) within the lung parenchyma. Despite the pivotal role of ECM overexpression in IPF, potential negative regulators of ECM production in fibroblasts have yet to be identified. Semaphorin class 3B (SEMA3B), a secreted protein highly expressed in lung tissues, has established roles in axonal guidance and tumor suppression. However, the role of SEMA3B in ECM production by fibroblasts in the pathogenesis of IPF remains unexplored. Here, we show the downregulation of SEMA3B and its cognate binding receptor, neuropilin 1 ( NRP1 ), in IPF lungs compared with healthy controls. Notably, the reduced expression of SEMA3B and NRP1 is associated with a decline in lung function in IPF. The downregulation of SEMA3B and NRP1 transcripts was validated in the lung tissues of patients with IPF, and two alternative mouse models of pulmonary fibrosis. In addition, we show that transforming growth factor-β (TGFβ) functions as a negative regulator of SEMA3B and NRP1 expression in lung fibroblasts. Furthermore, we demonstrate the antifibrotic effects of SEMA3B against TGFβ-induced ECM production in IPF lung fibroblasts. Overall, our findings uncovered a novel role of SEMA3B in the pathogenesis of pulmonary fibrosis and provided novel insights into modulating the SEMA3B-NRP1 axis to attenuate pulmonary fibrosis. NEW & NOTEWORTHY The excessive production and secretion of collagens and other extracellular matrix proteins by fibroblasts lead to the scarring of the lung in severe fibrotic lung diseases. This study unveils an antifibrotic role for semaphorin class 3B (SEMA3B) in the pathogenesis of idiopathic pulmonary fibrosis. SEMA3B functions as an inhibitor of transforming growth factor-β-driven fibroblast activation and reduced levels of SEMA3B and its receptor, neuropilin 1, are associated with decreased lung function in idiopathic pulmonary fibrosis.

The extracellular matrix (ECM) is a dynamic complex protein network that provides structural integrity and plays an active role in shaping fibroblast behavior both in health and disease. Despite its essential functions, the impact of age-associated post-translational modifications on ECM-driven fibroblast activities such as proliferation, survival, fibroblast-to-myofibroblast transformation (FMT), and extracellular matrix production remains largely unknown. Nε-carboxymethyl-lysine (CML) is one of the well-characterized advanced glycation end-products (AGEs) that can occur on lysine residues within ECM proteins through non-enzymatic glycation. In this study, we determined the accumulation and the effects of the CML-modified ECM (CML-ECM) on fibroblast activation. Immunostainings and immunoblot analysis demonstrated significant increases in CML-AGE content in idiopathic pulmonary fibrosis (IPF) compared to age-matched healthy lungs. Gene expression analysis and fibroblast activation assays collectively implicate the ECM as a negative regulator of fibroblast activation. Notably, the CML modification of the ECM resulted in a significant decrease in its anti-fibrotic effects including proliferation, FMT, apoptosis, and ECM production. Together, the results of this study revealed an unexplored pathological role played by the CML-ECM on fibroblast activation, which has wide implications in IPF and other fibrotic diseases.

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease that is often fatal due to the formation of irreversible scar tissue in the distal areas of the lung. Although the pathological and radiological features of IPF lungs are well defined, the lack of insight into the fibrogenic role of fibroblasts that accumulate in distinct anatomical regions of the lungs is a critical knowledge gap. Fibrotic lesions have been shown to originate in the subpleural areas and extend into the lung parenchyma through processes of dysregulated fibroproliferation, migration, fibroblast-to-myofibroblast transformation, and extracellular matrix production. Identifying the molecular targets underlying subpleural thickening at the early and late stages of fibrosis could facilitate the development of new therapies to attenuate fibroblast activation and improve the survival of patients with IPF. Here, we discuss the key cellular and molecular events that contribute to (myo)fibroblast activation and subpleural thickening in IPF. In particular, we highlight the transcriptional programs involved in mesothelial to mesenchymal transformation and fibroblast dysfunction that can be targeted to alter the course of the progressive expansion of fibrotic lesions in the distal areas of IPF lungs.

Idiopathic pulmonary fibrosis (IPF) is a severe fibrotic lung disease characterized by irreversible scarring of the lung parenchyma leading to dyspnea, progressive decline in lung function, and respiratory failure. We analyzed lung transcriptomic data from independent IPF cohorts using weighted gene co-expression network analysis (WGCNA) to identify gene modules based on their preservation status in these cohorts. The consensus gene modules were characterized by leveraging existing clinical and molecular data such as lung function, biological processes, pathways, and lung cell types. From a total of 32 consensus gene modules identified, two modules were found to be significantly correlated with the disease, lung function, and preserved in other IPF datasets. The upregulated gene module was enriched for extracellular matrix, collagen metabolic process, and BMP signaling while the downregulated module consisted of genes associated with tube morphogenesis, blood vessel development, and cell migration. Using a combination of connectivity-based and trait-based significance measures, we identified and prioritized 103 "hub" genes (including 25 secretory candidate biomarkers) by their similarity to known IPF genetic markers. Our validation studies demonstrate the dysregulated expression of CRABP2 , a retinol-binding protein, in multiple lung cells of IPF, and its correlation with the decline in lung function.

Idiopathic Pulmonary Fibrosis (IPF) is a severe fibrotic lung disease characterized by excessive collagen deposition and progressive decline in lung function. Th2 T cell-derived cytokines including IL-4 and IL-13 have been shown to contribute to inflammation and fibrotic remodeling in multiple tissues. Interleukin-31 (IL-31) is a newly identified cytokine that is predominantly produced by CD4 Th2 T cells, but its signaling receptor IL-31RA is primarily expressed by non-hematopoietic cells. However, the potential role of the IL-31-IL31RA axis in pulmonary inflammation and fibrosis has remained largely unknown. To determine the role of IL-31RA deficiency in pulmonary fibrosis, wildtype, and IL-31RA knockout mice were treated with bleomycin and measured changes in collagen deposition and lung function. Notably, the loss of IL-31 signaling attenuated collagen deposition and lung function decline during bleomycin-induced pulmonary fibrosis. The total lung transcriptome analysis showed a significant reduction in fibrosis-associated gene transcripts including extracellular matrix and epithelial cell-associated gene networks. Furthermore, the lungs of human IPF showed an elevated expression of IL-31 when compared to healthy subjects. In support, the percentage of IL-31 producing CD4 + T cells was greater in the lungs and PBMCs from IPF patients compared to healthy controls. Our findings suggest a pathogenic role for IL-31/IL-31RA signaling during bleomycin-induced pulmonary fibrosis. Thus, therapeutic targeting the IL-31-IL-31RA axis may prevent collagen deposition, improve lung function, and have therapeutic potential in pulmonary fibrosis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Yombo, Odayar, Gupta, Jegga and Madala.)

Fibroblast activation including proliferation, survival, and ECM production is central to initiation and maintenance of fibrotic lesions in idiopathic pulmonary fibrosis (IPF). However, druggable molecules that target fibroblast activation remain limited. In this study, we show that multiple pro-fibrotic growth factors, including TGFα, CTGF, and IGF1, increase aurora kinase B (AURKB) expression and activity in fibroblasts. Mechanistically, we demonstrate that Wilms tumor 1 (WT1) is a key transcription factor that mediates TGFα-driven AURKB upregulation in fibroblasts. Importantly, we found that inhibition of AURKB expression or activity is sufficient to attenuate fibroblast activation. We show that fibrosis induced by TGFα is highly dependent on AURKB expression and treating TGFα mice with barasertib, an AURKB inhibitor, reverses fibroblast activation, and pulmonary fibrosis. Barasertib similarly attenuated fibrosis in the bleomycin model of pulmonary fibrosis. Together, our preclinical studies provide important proof-of-concept that demonstrate barasertib as a possible intervention therapy for IPF., (© 2020 The Authors. Published under the terms of the CC BY 4.0 license.)

Background: There are two US Food and Drug Administration (FDA)-approved drugs, pirfenidone and nintedanib, for treatment of patients with idiopathic pulmonary fibrosis (IPF). However, neither of these drugs provide a cure. In addition, both are associated with several drug-related adverse events. Hence, the pursuit for newer IPF therapeutics continues. Recent studies show that joint analysis of systems-biology-level information with drug-disease connectivity are effective in discovery of biologically relevant candidate therapeutics., Methods: Publicly available gene expression signatures from patients with IPF were used to query a large-scale perturbagen signature library to discover compounds that can potentially reverse dysregulated gene expression in IPF. Two methods were used to calculate IPF-compound connectivity: gene expression-based connectivity and feature-based connectivity. Identified compounds were further prioritized if their shared mechanism(s) of action were IPF-related., Results: We found 77 compounds as potential candidate therapeutics for IPF. Of these, 39 compounds are either FDA-approved for other diseases or are currently in phase II/III clinical trials suggesting their repurposing potential for IPF. Among these compounds are multiple receptor kinase inhibitors (e.g. nintedanib, currently approved for IPF, and sunitinib), aurora kinase inhibitor (barasertib), epidermal growth factor receptor inhibitors (erlotinib, gefitinib), calcium channel blocker (verapamil), phosphodiesterase inhibitors (roflumilast, sildenafil), PPAR agonists (pioglitazone), histone deacetylase inhibitors (entinostat), and opioid receptor antagonists (nalbuphine). As a proof of concept, we performed in vitro validations with verapamil using lung fibroblasts from IPF and show its potential benefits in pulmonary fibrosis., Conclusions: As about half of the candidates discovered in this study are either FDA-approved or are currently in clinical trials for other diseases, rapid translation of these compounds as potential IPF therapeutics is possible. Further, the integrative connectivity analysis framework in this study can be adapted in early phase drug discovery for other common and rare diseases with transcriptomic profiles. The reviews of this paper are available via the supplemental material section .

Introduction: Fibrosis is an irreversible pathological endpoint in many chronic diseases, including pulmonary fibrosis. Idiopathic pulmonary fibrosis (IPF) is a progressive and often fatal condition characterized by (myo)fibroblast proliferation and transformation in the lung, expansion of the extracellular matrix, and extensive remodeling of the lung parenchyma. Recent evidence indicates that IPF prevalence and mortality rates are growing in the United States and elsewhere. Despite decades of research on the pathogenic mechanisms of pulmonary fibrosis, few therapeutics have succeeded in the clinic, and they have failed to improve IPF patient survival. Areas covered: Based on a literature search and our own results, we discuss the key cellular and molecular responses that contribute to (myo)fibroblast actions and pulmonary fibrosis pathogenesis; this includes signaling pathways in various cells that aberrantly and persistently activate (myo)fibroblasts in fibrotic lesions and promote scar tissue formation in the lung. Expert opinion: Lessons learned from recent failures and successes with new therapeutics point toward approaches that can target multiple pro-fibrotic processes in IPF. Advances in preclinical modeling and single-cell genomics will also accelerate novel discoveries for effective treatment of IPF.

Wilms' tumor 1 (WT1) is a critical transcriptional regulator of mesothelial cells during lung development but is downregulated in postnatal stages and adult lungs. We recently showed that WT1 is upregulated in both mesothelial cells and mesenchymal cells in the pathogenesis of idiopathic pulmonary fibrosis (IPF), a fatal fibrotic lung disease. Although WT1-positive cell accumulation leading to severe fibrotic lung disease has been studied, the role of WT1 in fibroblast activation and pulmonary fibrosis remains elusive. Here, we show that WT1 functions as a positive regulator of fibroblast activation, including fibroproliferation, myofibroblast transformation, and extracellular matrix (ECM) production. Chromatin immunoprecipitation experiments indicate that WT1 binds directly to the promoter DNA sequence of α-smooth muscle actin (αSMA) to induce myofibroblast transformation. In support, the genetic lineage tracing identifies WT1 as a key driver of mesothelial-to-myofibroblast and fibroblast-to-myofibroblast transformation. Importantly, the partial loss of WT1 was sufficient to attenuate myofibroblast accumulation and pulmonary fibrosis in vivo. Further, our coculture studies show that WT1 upregulation leads to non-cell autonomous effects on neighboring cells. Thus, our data uncovered a pathogenic role of WT1 in IPF by promoting fibroblast activation in the peripheral areas of the lung and as a target for therapeutic intervention.

Background: Idiopathic Pulmonary Fibrosis (IPF) is a fatal fibrotic lung disease occurring predominantly in middle-aged and older adults. The traditional diagnostic classification of IPF is based on clinical, radiological, and histopathological features. However, the considerable heterogeneity in IPF presentation suggests that differences in gene expression profiles can help to characterize and distinguish disease severity., Methods: We used data-driven unsupervised clustering analysis, combined with a knowledge-based approach to identify and characterize IPF subgroups., Results: Using transcriptional profiles on lung tissue from 131 patients with IPF/UIP and 12 non-diseased controls, we identified six subgroups of IPF that generally correlated with the disease severity and lung function decline. Network-informed clustering identified the most severe subgroup of IPF that was enriched with genes regulating inflammatory processes, blood pressure and branching morphogenesis of the lung. The differentially expressed genes in six subgroups of IPF compared to healthy control include transcripts of extracellular matrix, epithelial-mesenchymal cell cross-talk, calcium ion homeostasis, and oxygen transport. Further, we compiled differentially expressed gene signatures to identify unique gene clusters that can segregate IPF from normal, and severe from mild IPF. Additional validations of these signatures were carried out in three independent cohorts of IPF/UIP. Finally, using knowledge-based approaches, we identified several novel candidate genes which may also serve as potential biomarkers of IPF., Conclusions: Discovery of unique and redundant gene signatures for subgroups in IPF can be greatly facilitated through unsupervised clustering. Findings derived from such gene signatures may provide insights into pathogenesis of IPF and facilitate the development of clinically useful biomarkers.

Idiopathic pulmonary fibrosis (IPF) is a severe fibrotic lung disease associated with fibroblast activation that includes excessive proliferation, tissue invasiveness, myofibroblast transformation, and extracellular matrix (ECM) production. To identify inhibitors that can attenuate fibroblast activation, we queried IPF gene signatures against a library of small-molecule-induced gene-expression profiles and identified Hsp90 inhibitors as potential therapeutic agents that can suppress fibroblast activation in IPF. Although Hsp90 is a molecular chaperone that regulates multiple processes involved in fibroblast activation, it has not been previously proposed as a molecular target in IPF. Here, we found elevated Hsp90 staining in lung biopsies of patients with IPF. Notably, fibroblasts isolated from fibrotic lesions showed heightened Hsp90 ATPase activity compared with normal fibroblasts. 17- N -allylamino-17-demethoxygeldanamycin (17-AAG), a small-molecule inhibitor of Hsp90 ATPase activity, attenuated fibroblast activation and also TGF-β-driven effects on fibroblast to myofibroblast transformation. The loss of the Hsp90AB, but not the Hsp90AA isoform, resulted in reduced fibroblast proliferation, myofibroblast transformation, and ECM production. Finally, in vivo therapy with 17-AAG attenuated progression of established and ongoing fibrosis in a mouse model of pulmonary fibrosis, suggesting that targeting Hsp90 represents an effective strategy for the treatment of fibrotic lung disease., Competing Interests: Conflict of interest: The authors have declared that no conflict of interest exists.

Collagen-producing myofibroblast transdifferentiation is considered a crucial determinant in the formation of scar tissue in the lungs of patients with idiopathic pulmonary fibrosis. Multiple resident pulmonary cell types and bone marrow-derived fibrocytes have been implicated as contributors to fibrotic lesions because of the transdifferentiation potential of these cells into myofibroblasts. In this study, we assessed the expression of Wilms tumor 1 (WT1), a known marker of mesothelial cells, in various cell types in normal and fibrotic lungs. We demonstrate that WT1 is expressed by both mesothelial and mesenchymal cells in idiopathic pulmonary fibrosis lungs but has limited or no expression in normal human lungs. We also demonstrate that WT1(+) cells accumulate in fibrotic lung lesions, using two different mouse models of pulmonary fibrosis and WT1 promoter-driven fluorescent reporter mice. Reconstitution of bone marrow cells into a TGF-α transgenic mouse model demonstrated that fibrocytes do not transform into WT1(+) mesenchymal cells, but they do augment accumulation of WT1(+) cells in severe fibrotic lung disease. Importantly, the number of WT1(+) cells in fibrotic lesions was correlated with severity of lung disease as assessed by changes in lung function, histology, and hydroxyproline levels in mice. Finally, inhibition of WT1 expression was sufficient to attenuate collagen and other extracellular matrix gene production by mesenchymal cells from both murine and human fibrotic lungs. Thus, the results of this study demonstrate a novel association between fibrocyte-driven WT1(+) cell accumulation and severe fibrotic lung disease., (Copyright © 2015 by The American Association of Immunologists, Inc.)