Human Fibrotic Lung Research Articles

YAP Alleviates Pulmonary Fibrosis Through Promoting Alveolar Regeneration via Modulating the Stemness of Alveolar Type 2 Cells.

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with no cure except transplantation. Abnormal alveolar epithelial regeneration is a key driver of IPF development. The function of Yes1 Associated Transcriptional Regulator (YAP) in alveolar regeneration and IPF pathogenesis remains elusive. Here, we first revealed the activation of YAP in alveolar epithelium 2 cells (AEC2s) from human IPF lungs and fibrotic mouse lungs. Notably, conditional deletion of YAP in mouse AEC2s exacerbated bleomycin-induced pulmonary fibrosis. Intriguingly, we showed in both conditional knockout mice and alveolar organoids that YAP deficiency impaired AEC2 proliferation and differentiation into alveolar epithelium 1 cells (AEC1s). Mechanistically, YAP regulated expression levels of genes associated with cell cycle progression and AEC1 differentiation. Furthermore, overexpression of YAP in vitro promoted cell proliferation. These results indicate the critical role of YAP in alveolar regeneration and IPF pathogenesis. Our findings provide new insights into the regulation of alveolar regeneration and IPF pathogenesis, paving the road for developing novel treatment strategies.

Gucy1α1 specifically marks kidney, heart, lung and liver fibroblasts.

Fibrosis is a common outcome of numerous pathologies, including chronic kidney disease (CKD), a progressive renal function deterioration. Current approaches to target activated fibroblasts, key effector contributors to fibrotic tissue remodeling, lack specificity. Here, we report Gucy1α1 as a specific kidney fibroblast marker. Gucy1α1 levels significantly increased over the course of two clinically relevant murine CKD models and directly correlated with established fibrosis markers. Immunofluorescent (IF) imaging showed that Gucy1α1 comprehensively labelled cortical and medullary quiescent and activated fibroblasts in the control kidney and throughout injury progression, respectively. Unlike traditionally used markers platelet derived growth factor receptor beta (Pdgfrβ) and vimentin (Vim), Gucy1α1 did not overlap with off-target populations such as podocytes. Notably, Gucy1α1 labelled kidney fibroblasts in both male and female mice. Furthermore, we observed elevated Gucy1α1 expression in the human fibrotic kidney and lung. Studies in the murine models of cardiac and liver fibrosis revealed Gucy1α1 elevation in activated Pdgfrβ-, Vim- and alpha smooth muscle actin (αSma)-expressing fibroblasts paralleling injury progression and resolution. Overall, we demonstrate Gucy1α1 as an exclusive fibroblast marker in both sexes. Due to its multiorgan translational potential, Gucy1α1 might provide a novel promising strategy to specifically target and mechanistically examine fibroblasts.

Lung injury-induced activated endothelial cell states persist in aging-associated progressive fibrosis

Progressive lung fibrosis is associated with poorly understood aging-related endothelial cell dysfunction. To gain insight into endothelial cell alterations in lung fibrosis we performed single cell RNA-sequencing of bleomycin-injured lungs from young and aged mice. Analysis reveals activated cell states enriched for hypoxia, glycolysis and YAP/TAZ activity in ACKR1+ venous and TrkB+ capillary endothelial cells. Endothelial cell activation is prevalent in lungs of aged mice and can also be detected in human fibrotic lungs. Longitudinal single cell RNA-sequencing combined with lineage tracing demonstrate that endothelial activation resolves in young mouse lungs but persists in aged ones, indicating a failure of the aged vasculature to return to quiescence. Genes associated with activated lung endothelial cells states in vivo can be induced in vitro by activating YAP/TAZ. YAP/TAZ also cooperate with BDNF, a TrkB ligand that is reduced in fibrotic lungs, to promote capillary morphogenesis. These findings offer insights into aging-related lung endothelial cell dysfunction that may contribute to defective lung injury repair and persistent fibrosis.

Sanglifehrin A mitigates multiorgan fibrosis by targeting the collagen chaperone cyclophilin B.

Pathological deposition and crosslinking of collagen type I by activated myofibroblasts drives progressive tissue fibrosis. Therapies that inhibit collagen synthesis have potential as antifibrotic agents. We identify the collagen chaperone cyclophilin B as a major cellular target of the natural product sanglifehrin A (SfA) using photoaffinity labeling and chemical proteomics. Mechanistically, SfA inhibits and induces the secretion of cyclophilin B from the endoplasmic reticulum (ER) and prevents TGF-β1-activated myofibroblasts from synthesizing and secreting collagen type I in vitro, without inducing ER stress or affecting collagen type I mRNA transcription, myofibroblast migration, contractility, or TGF-β1 signaling. In vivo, SfA induced cyclophilin B secretion in preclinical models of fibrosis, thereby inhibiting collagen synthesis from fibrotic fibroblasts and mitigating the development of lung and skin fibrosis in mice. Ex vivo, SfA induces cyclophilin B secretion and inhibits collagen type I secretion from fibrotic human lung fibroblasts and samples from patients with idiopathic pulmonary fibrosis (IPF). Taken together, we provide chemical, molecular, functional, and translational evidence for demonstrating direct antifibrotic activities of SfA in preclinical and human ex vivo fibrotic models. Our results identify the cellular target of SfA, the collagen chaperone cyclophilin B, as a mechanistic target for the treatment of organ fibrosis.

Osteoprotegerin is an Early Marker of the Fibrotic Process and of Antifibrotic Treatment Responses in Ex Vivo Lung Fibrosis

BackgroundLung fibrosis is a chronic lung disease with a high mortality rate with only two approved drugs (pirfenidone and nintedanib) to attenuate its progression. To date, there are no reliable biomarkers to assess fibrosis development and/or treatment effects for these two drugs. Osteoprotegerin (OPG) is used as a serum marker to diagnose liver fibrosis and we have previously shown it associates with lung fibrosis as well.MethodsHere we used murine and human precision-cut lung slices to investigate the regulation of OPG in lung tissue to elucidate whether it tracks with (early) fibrosis development and responds to antifibrotic treatment to assess its potential use as a biomarker.ResultsOPG mRNA expression in murine lung slices was higher after treatment with profibrotic cytokines TGFβ1 or IL13, and closely correlated with Fn and PAI1 mRNA expression. More OPG protein was released from fibrotic human lung slices than from the control human slices and from TGFβ1 and IL13-stimulated murine lung slices compared to control murine slices. This OPG release was inhibited when murine slices were treated with pirfenidone or nintedanib. OPG release from human fibrotic lung slices was inhibited by pirfenidone treatment.ConclusionOPG can already be detected during the early stages of fibrosis development and responds, both in early- and late-stage fibrosis, to treatment with antifibrotic drugs currently on the market for lung fibrosis. Therefore, OPG should be further investigated as a potential biomarker for lung fibrosis and a potential surrogate marker for treatment effect.

Genome-wide screens identify SEL1L as an intracellular rheostat controlling collagen turnover

Accumulating evidence has implicated impaired extracellular matrix (ECM) clearance as a key factor in fibrotic disease. Despite decades of research elucidating the effectors of ECM clearance, relatively little is understood regarding the upstream regulation of this process. Collagen is the most abundant constituent of normal and fibrotic ECM in mammalian tissues. Its catabolism occurs through extracellular proteolysis and cell-mediated uptake of collagen fragments for intracellular degradation. Given the paucity of information regarding the regulation of this latter process, here we execute unbiased genome-wide screens to understand the molecular underpinnings of cell-mediated collagen clearance. Using this approach, we discover a mechanism through which collagen biosynthesis is sensed by cells internally and directly regulates clearance of extracellular collagen. The sensing mechanism appears to be dependent on endoplasmic reticulum-resident protein SEL1L and occurs via a noncanonical function of this protein. This pathway functions as a homeostatic negative feedback loop that limits collagen accumulation in tissues. In human fibrotic lung disease, the induction of this collagen clearance pathway by collagen synthesis is impaired, thereby contributing to the pathological accumulation of collagen in lung tissue. Thus, we describe cell-autonomous, rheostatic collagen clearance as an important pathway of tissue homeostasis.

Endothelial H2S-AMPK dysfunction upregulates the angiocrine factor PAI-1 and contributes to lung fibrosis

Dysfunction of the vascular angiocrine system is critically involved in regenerative defects and fibrosis of injured organs. Previous studies have identified various angiocrine factors and found that risk factors such as aging and metabolic disorders can disturb the vascular angiocrine system in fibrotic organs. One existing key gap is what sense the fibrotic risk to modulate the vascular angiocrine system in organ fibrosis. Here, using human and mouse data, we discovered that the metabolic pathway hydrogen sulfide (H2S)-AMP-activated protein kinase (AMPK) is a sensor of fibrotic stress and serves as a key mechanism upregulating the angiocrine factor plasminogen activator inhibitor-1 (PAI-1) in endothelial cells to participate in lung fibrosis. Activation of the metabolic sensor AMPK was inhibited in endothelial cells of fibrotic lungs, and AMPK inactivation was correlated with enriched fibrotic signature and reduced lung functions in humans. The inactivation of endothelial AMPK accelerated lung fibrosis in mice, while the activation of endothelial AMPK with metformin alleviated lung fibrosis. In fibrotic lungs, endothelial AMPK inactivation led to YAP activation and overexpression of the angiocrine factor PAI-1, which was positively correlated with the fibrotic signature in human fibrotic lungs and inhibition of PAI-1 with Tiplaxtinin mitigated lung fibrosis. Further study identified that the deficiency of the antioxidative gas metabolite H2S accounted for the inactivation of AMPK and activation of YAP-PAI-1 signaling in endothelial cells of fibrotic lungs. H2S deficiency was involved in human lung fibrosis and H2S supplement reversed mouse lung fibrosis in an endothelial AMPK-dependent manner. These findings provide new insight into the mechanism underlying the deregulation of the vascular angiocrine system in fibrotic organs.

Pulmonary siRNA Delivery with Sophisticated Amphiphilic Poly(Spermine Acrylamides) for the Treatment of Lung Fibrosis.

RNA interference (RNAi)is an efficient strategy to post-transcriptionally silence gene expression. While all siRNA drugs on the market target the liver, the lung offers a variety of currently undruggable targets, which can potentially be treated with RNA therapeutics. To achieve this goal, the synthesis of poly(spermine acrylamides) (P(SpAA) is reported herein. Polymers are prepared via polymerization of N-acryloxysuccinimide (NAS) and afterward this active ester is converted into spermine-based pendant groups. Copolymerizations with decylacrylamide are employed to increase the hydrophobicity of the polymers. After deprotection, polymers show excellent siRNA encapsulation to obtain perfectly sized polyplexes at very low polymer/RNA ratios. In vitro 2D and 3D cell culture, ex vivo and in vivo experiments reveal superior properties of amphiphilic spermine-copolymers with respect to delivery of siRNA to lung cells in comparison to commonly used lipid-based transfection agents. In line with the in vitro results, siRNA delivery to human lung explants confirm more efficient gene silencing of protease-activated receptor 2 (PAR2), a G protein-coupled receptor involved in fibrosis. This study reveals the importance of the balance between efficient polyplex formation, cellular uptake, gene knockdown, and toxicity for efficient siRNA delivery in vitro, in vivo, and in fibrotic human lung tissue ex vivo.

Role of Extracellular Vesicles in the Propagation of Lung Fibrosis in Systemic Sclerosis.

Systemic sclerosis (SSc) has the highest mortality rate among the rheumatic diseases, with lung fibrosis leading as the cause of death. A characteristic of severe SSc-related lung fibrosis is its progressive nature. Although most research has focused on the pathology of the fibrosis, the mechanism mediating the fibrotic spread remains unclear. We hypothesized that extracellular vesicle (EV) communication drives the propagation of SSc lung fibrosis. EVs were isolated from normal (NL) or SSc-derived human lungs and primary lung fibroblasts (pLFs). EVs were also isolated from human fibrotic lungs and pLFs induced experimentally with transforming growth factor-β (TGFβ). Fibrotic potency of EVs was assessed using functional assays in vitro and in vivo. Transmission electron microscopy, nanoparticle tracking analysis, real-time quantitative polymerase chain reaction(RT-qPCR), immunoblotting, and immunofluorescence were used to analyze EVs, their cargo, extracellular matrix (ECM) fractions, and conditioned media. SSc lungs and pLFs released significantly more EVs than NL lungs, and their EVs showed increased fibrotic content and activity. TGFβ-stimulated NL lung cores and pLFs increased packaging of fibrotic proteins, including fibronectin, collagens, and TGFβ, into released EVs. The EVs induced a fibrotic phenotype in recipient pLFs and in vivo in mouse lungs. Furthermore, EVs interacted with and contributed to the ECM. Finally, suppressing EV release in vivo reduced severity of murine lung fibrosis. Our findings highlight EV communication as a novel mechanism for propagation of SSc lung fibrosis. Identifying therapies that reduce EV release, activity, and/or fibrotic cargo in SSc patient lungs may be a viable therapeutic strategy to improve fibrosis.

Targeted imaging of very late antigen-4 for noninvasive assessment of lung inflammation-fibrosis axis

BackgroundThe lack of noninvasive methods for assessment of dysregulated inflammation as a major driver of fibrosis (i.e., inflammation-fibrosis axis) has been a major challenge to precision management of fibrotic lung diseases. Here, we determined the potential of very late antigen-4 (VLA-4)-targeted positron emission tomography (PET) to detect inflammation in a mouse model of bleomycin-induced fibrotic lung injury.MethodSingle time-point and longitudinal VLA-4-targeted PET was performed using a high-affinity peptidomimetic radiotracer, 64Cu-LLP2A, at weeks 1, 2, and 4 after bleomycin-induced (2.5 units/kg) lung injury in C57BL/6J mice. The severity of fibrosis was determined by measuring the hydroxyproline content of the lungs and expression of markers of extracellular matrix remodeling. Flow cytometry and histology was performed to determine VLA-4 expression across different leukocyte subsets and their spatial distribution.ResultsLung uptake of 64Cu-LLP2A was significantly elevated throughout different stages of the progression of bleomycin-induced injury. High lung uptake of 64Cu-LLP2A at week-1 post-bleomycin was a predictor of poor survival over the 4-week follow up, supporting the prognostic potential of 64Cu-LLP2A PET during the early stage of the disease. Additionally, the progressive increase in 64Cu-LLP2A uptake from week-1 to week-4 post-bleomycin correlated with the ultimate extent of lung fibrosis and ECM remodeling. Flow cytometry revealed that LLP2A binding was restricted to leukocytes. A combination of increased expression of VLA-4 by alveolar macrophages and accumulation of VLA-4-expressing interstitial and monocyte-derived macrophages as well as dendritic cells was noted in bleomycin-injured, compared to control, lungs. Histology confirmed the increased expression of VLA-4 in bleomycin-injured lungs, particularly in inflamed and fibrotic regions.ConclusionsVLA-4-targeted PET allows for assessment of the inflammation-fibrosis axis and prediction of disease progression in a murine model. The potential of 64Cu-LLP2A PET for assessment of the inflammation-fibrosis axis in human fibrotic lung diseases needs to be further investigated.

Ovatodiolide inhibits SARS-CoV-2 replication and ameliorates pulmonary fibrosis through suppression of the TGF-β/TβRs signaling pathway

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection continues to pose threats to public health. The clinical manifestations of lung pathology in COVID-19 patients include sustained inflammation and pulmonary fibrosis. The macrocyclic diterpenoid ovatodiolide (OVA) has been reported to have anti-inflammatory, anti-cancer, anti-allergic, and analgesic activities. Here, we investigated the pharmacological mechanism of OVA in suppressing SARS-CoV-2 infection and pulmonary fibrosis in vitro and in vivo. Our results revealed that OVA was an effective SARS-CoV-2 3CLpro inhibitor and showed remarkable inhibitory activity against SARS-CoV-2 infection. On the other hand, OVA ameliorated pulmonary fibrosis in bleomycin (BLM)-induced mice, reducing inflammatory cell infiltration and collagen deposition in the lung. OVA decreased the levels of pulmonary hydroxyproline and myeloperoxidase, as well as lung and serum TNF-ɑ, IL-1β, IL-6, and TGF-β in BLM-induced pulmonary fibrotic mice. Meanwhile, OVA reduced the migration and fibroblast-to-myofibroblast conversion of TGF-β1-induced fibrotic human lung fibroblasts. Consistently, OVA downregulated TGF-β/TβRs signaling. In computational analysis, OVA resembles the chemical structures of the kinase inhibitors TβRI and TβRII and was shown to interact with the key pharmacophores and putative ATP-binding domains of TβRI and TβRII, showing the potential of OVA as an inhibitor of TβRI and TβRII kinase. In conclusion, the dual function of OVA highlights its potential for not only fighting SARS-CoV-2 infection but also managing injury-induced pulmonary fibrosis.

Transcriptional factor RUNX1: A potential therapeutic target for fibrotic pulmonary disease

Runt-related transcription factor-1 (RUNX1), also known as the core-binding factor alpha 2 subunit, is closely related to human leukemia. The functions of RUNX1 in modulating cell proliferation, differentiation, and survival in multiple systems have been gradually discovered with the emergence of transgenic mice. RUNX1 is a powerful transcription factor implicated in diverse signaling pathways and cellular mechanisms that participate in lung development and pulmonary diseases. RUNX1 has recently been identified as a target regulator of fibrotic remodeling diseases, particularly in the kidney. However, the role of RUNX1 in pulmonary fibrosis is unclear. Pulmonary fibrosis is characterized by obscure nosogenesis, limited therapy, and poor prognosis. Moreover, the population of patients with pulmonary fibrosis is gradually increasing. Thus, there is an unmet need for therapeutic targets. In this review, we retrospectively discuss the alteration in RUNX1 mRNA expression in the RNA sequencing data of human fibrotic lungs and the protein levels in mouse pulmonary fibrosis. Subsequently, we focused on the interaction between RUNX1 and several signaling pathways involved in pulmonary fibrosis. Finally, this review highlights the therapeutic potential of RUNX1 as a target for slowing the progression of fibrotic lung disease.

Lack of bombesin receptor-activated protein attenuates bleomycin-induced pulmonary fibrosis in mice.

Bombesin receptor-activated protein (BRAP) was found to express in the interstitial cells of human fibrotic lungs with unknown function. Its homologous protein, encoded by BC004004 gene, was also present in mouse lung tissues. We used BC004004 -/- mice which lack BRAP homologous protein expression to establish a bleomycin-induced lung fibrotic model. After bleomycin treatment, BC004004 -/- mice exhibited attenuation of pulmonary injury and less pulmonary fibrosis. Fibroblasts from BC004004 -/- mice proliferated at a lower rate and produced less collagen. Autophagy-related gene 5 (ATG5) was identified as a partner interacting with human BRAP. Lacking BRAP homologous protein led to enhanced autophagy activity in mouse lung tissues as well as in isolated lung fibroblasts, indicating a negative regulatory role of this protein in autophagy via interaction with ATG5. Enhanced autophagy process in fibroblasts due to lack of BRAP homologous protein might contribute to the resistance of BC004004 -/- mice to pulmonary fibrosis.

Disruption of Proteostasis Causes IRE1 Mediated Reprogramming of Alveolar Epithelial Cells in Lung Fibrosis

Pulmonary Fibrosis (PF), a devastating lung disease with rising prevalence, is defined by a physiologic defect in gas exchange owing to two cardinal pathological features: intrusion of myofibroblasts into the distal lung parenchyma and an alteration of normal alveolar epithelial composition most clearly defined by a loss of the squamous alveolar epithelial type 1 cell (AEC1) and the presence of a recently identified aberrant alveolar epithelial “transitional state” enriched in profibrotic mediators. While this transitional cell state has been identified transiently in the epithelium of murine lung injury models, the mechanism by which it arises in human fibrotic lung disease is unknown.We previously reported the in vivo modeling of PF utilizing inducible, knock‐in expression of a clinical Surfactant Protein C (SP‐C) mutation in alveolar type 2 cells (AEC2). Expression of the SP‐C mutation in the adult mouse AEC2s led to activation of Unfolded Protein Response (UPR) signaling pathways, endoplasmic reticulum (ER) stress and spontaneous fibrosis providing proof of concept for disruption to proteostasis as a proximal driver of PF. We hypothesized that disruption of AEC2 protein quality control and specifically UPR signaling causes cell autonomous AEC2 reprogramming to the transitional state in the absence of an exogenous lung injury.Using two clinical SP‐C mutation models we discovered that AEC2s experiencing significant ER stress lose quintessential AEC2 features and develop the recently identified transitional cell state. Using single cell RNA sequencing of the epithelium in our murine models we identify that UPR activated AEC2s develop the transitional cell phenotype in the absence of an exogenous lung injury. Through organoid based modeling we validated that this state arises de novo from intrinsic AEC2 dysfunction. The cell autonomous AEC2 reprogramming is mediated through IRE1 signaling as use of a novel IRE1 inhibitor attenuated the development of the transitional cell state and diminished AEC2 driven recruitment of granulocytes, alveolitis, and lung injury arising from the loss of proteostasis.We further show that this transitional state persists into the fibrotic phase of our murine models and is enriched in pro‐fibrotic mediators. These findings identify AEC2 proteostasis, and specifically IRE1 signaling, as a driver of a key AEC2 phenotypic change that has been identified in lung fibrosis.

Atorvastatin attenuates pulmonary fibrosis in mice and human lung fibroblasts, by the regulation of myofibroblast differentiation and apoptosis.

Statins have anti-inflammatory and antifibrotic effects in addition to cholesterol-lowering effect. We aimed to investigate the effect of atorvastatin (ATR) in fibrotic mouse lung and human lung fibroblasts (MRC5s). Pulmonary fibrosis was induced by a single dose of bleomycin by intratracheal instillation in adult mice. ATR was administered (20 mg/kgip) to mice with healthy and pulmonary fibrosis for 10 days from Day 7 of the experiment. Mice were dissected on the 21st day. The levels of alpha-smooth muscle actin (α-SMA), pSMAD2/3, LOXL2, and p-Src were determined by Western blot analysis in the lungs. Furthermore, a group of MRC5 was differentiated into myofibroblasts by transforming growth factor-beta (TGF-β). Another group of MRC5s was treated with 10 µM ATR at 24 h after TGF-β stimulation. Cells were collected at 0, 24, 48, and 72 h. The effects of ATR on myofibroblast differentiation, apoptosis, and TGF-β and Wnt/β-catenin signaling activations were examined by Western blot analysis and flow cytometry in MRC5s. ATR attenuated pulmonary fibrosis by regulating myofibroblast differentiation and interstitial accumulation of collagen, by acting on LOXL2, p-Src, and pSMAD2/3 in mice lungs. Additionally, it blocked myofibroblast differentiation via reduced TGF-β and Wnt/β-catenin signaling and decreased α-SMA in MRC5s stimulated with TGF-β. Moreover, ATR caused myofibroblast apoptosis via caspase-3 activation. ATR treatment attenuates pulmonary fibrosis in mice treated with bleomycin. It also inhibits fibroblast/myofibroblast activation, by both reducing myofibroblasts differentiation and inducing myofibroblast apoptosis.

BMP1 is not required for lung fibrosis in mice

Bone morphogenetic protein 1 (BMP1) belongs to the astacin/BMP1/tolloid-like family of zinc metalloproteinases, which play a fundamental role in the development and formation of extracellular matrix (ECM). BMP1 mediates the cleavage of carboxyl terminal (C-term) propeptides from procollagens, a crucial step in fibrillar collagen fiber formation. Blocking BMP1 by small molecule or antibody inhibitors has been linked to anti-fibrotic activity in the preclinical models of skin, kidney and liver fibrosis. Therefore, we reason that BMP1 may be important for the pathogenesis of lung fibrosis and BMP1 could be a potential therapeutic target for progressive fibrotic disease such as idiopathic pulmonary fibrosis (IPF). Here, we observed the increased expression of BMP1 in both human IPF lungs and mouse fibrotic lungs induced by bleomycin. Furthermore, we developed an inducible Bmp1 conditional knockout (cKO) mouse strain. We found that Bmp1 deletion does not protect mice from lung fibrosis triggered by bleomycin. Moreover, we found no significant impact of BMP1 deficiency upon C-term propeptide of type I procollagen (CICP) production in the fibrotic mouse lungs. Based on these results, we propose that BMP1 is not required for lung fibrosis in mice and BMP1 may not be considered a candidate therapeutic target for IPF.

Airway secretory cell fate conversion via YAP‐mTORC1‐dependent essential amino acid metabolism

Tissue homeostasis requires lineage fidelity of stem cells. Dysregulation of cell fate specification and differentiation leads to various diseases, yet the cellular and molecular mechanisms governing these processes remain elusive. We demonstrate that YAP/TAZ activation reprograms airway secretory cells, which subsequently lose their cellular identity and acquire squamous alveolar type 1 (AT1) fate in the lung. This cell fate conversion is mediated via distinctive transitional cell states of damage‐associated transient progenitors (DATPs), recently shown to emerge during injury repair in mouse and human lungs. We further describe a YAP/TAZ signaling cascade to be integral for the fate conversion of secretory cells into AT1 fate, by modulating mTORC1/ATF4‐mediated amino acid metabolism in vivo. Importantly, we observed aberrant activation of the YAP/TAZ‐mTORC1‐ATF4 axis in the altered airway epithelium of bronchiolitis obliterans syndrome, including substantial emergence of DATPs and AT1 cells with severe pulmonary fibrosis. Genetic and pharmacologic inhibition of mTORC1 activity suppresses lineage alteration and subepithelial fibrosis driven by YAP/TAZ activation, proposing a potential therapeutic target for human fibrotic lung diseases.

IL10 trains macrophage profibrotic function after lung injury.

Cx3cr1+ monocyte-derived macrophages (moMacs) are recruited to tissues after injury and are known to have profibrotic effects, but the cell-cell interactions and specific pathways that regulate this polarization and function are incompletely understood. Here we investigate the role of moMac-derived Pdgfa in bleomycin-induced lung fibrosis in mice. Deletion of Pdgfa with Cx3cr1-CreERT2 decreased bleomycin-induced lung fibrosis. Among a panel of in vitro macrophage polarizing stimuli, robust induction of Pdgfa was noted with IL10 in both mouse and human moMacs. Likewise, analysis of single-cell data revealed high expression of the receptor IL10RA in moMacs from human fibrotic lungs. Studies with IL10-GFP mice revealed that IL10-expressing cells were increased after injury in mice and colocalized with moMacs. Notably, deletion of IL10ra with Csf1r-Cre: IL10ra fl/fl mice decreased both Pdgfa expression in moMacs and lung fibrosis. Taken together, these findings reveal a novel, IL10-dependent mechanism of macrophage polarization leading to fibroblast activation after injury.

Dual inhibition of \u03b1v\u03b26 and \u03b1v\u03b21 reduces fibrogenesis in lung tissue explants from patients with IPF

Rationaleαv integrins, key regulators of transforming growth factor-β activation and fibrogenesis in in vivo models of pulmonary fibrosis, are expressed on abnormal epithelial cells (αvβ6) and fibroblasts (αvβ1) in fibrotic lungs.ObjectivesWe evaluated multiple αv integrin inhibition strategies to assess which most effectively reduced fibrogenesis in explanted lung tissue from patients with idiopathic pulmonary fibrosis.MethodsSelective αvβ6 and αvβ1, dual αvβ6/αvβ1, and multi-αv integrin inhibitors were characterized for potency, selectivity, and functional activity by ligand binding, cell adhesion, and transforming growth factor-β cell activation assays. Precision-cut lung slices generated from lung explants from patients with idiopathic pulmonary fibrosis or bleomycin-challenged mouse lungs were treated with integrin inhibitors or standard-of-care drugs (nintedanib or pirfenidone) and analyzed for changes in fibrotic gene expression or TGF-β signaling. Bleomycin-challenged mice treated with dual αvβ6/αvβ1 integrin inhibitor, PLN-74809, were assessed for changes in pulmonary collagen deposition and Smad3 phosphorylation.Measurements and main resultsInhibition of integrins αvβ6 and αvβ1 was additive in reducing type I collagen gene expression in explanted lung tissue slices from patients with idiopathic pulmonary fibrosis. These data were replicated in fibrotic mouse lung tissue, with no added benefit observed from inhibition of additional αv integrins. Antifibrotic efficacy of dual αvβ6/αvβ1 integrin inhibitor PLN-74809 was confirmed in vivo, where dose-dependent inhibition of pulmonary Smad3 phosphorylation and collagen deposition was observed. PLN-74809 also, more potently, reduced collagen gene expression in fibrotic human and mouse lung slices than clinically relevant concentrations of nintedanib or pirfenidone.ConclusionsIn the fibrotic lung, dual inhibition of integrins αvβ6 and αvβ1 offers the optimal approach for blocking fibrogenesis resulting from integrin-mediated activation of transforming growth factor-β.

Thymosin β4 Suppresses LPS-Induced Murine Lung Fibrosis by Attenuating Oxidative Injury and Alleviating Inflammation.

Inflammation plays a critical role in the progression of pulmonary fibrosis. Thymosin β4 (Tβ4) has antioxidant, anti-inflammatory, and antifibrotic effects. Although the potent protective role of Tβ4 in bleomycin-induced pulmonary fibrosis has been validated, the underlying mechanism is not clear; moreover, the influence of Tβ4 on lipopolysaccharide (LPS)-induced lung injury/fibrosis has not been reported. Expression of Tβ4 in fibrotic lung tissues was assessed by real-time quantitative reverse-transcription PCR (rt-PCR), immunohistochemistry (IHC), and western blotting. The effects of intraperitoneal adeno-associated virus-Tβ4 (AAV-Tβ4) on LPS-induced lung injury and fibrosis were observed through the evaluation of collagen deposition and α-smooth muscle actin (SMA) expression. In vitro tests with HPAEpiC and HLF-1 cells were performed to confirm the effects of Tβ4. In this study, we evaluated the role of Tβ4 in pulmonary fibrosis and explored the possible underlying mechanisms. Tβ4 was markedly upregulated in human or mouse fibrotic lung tissues. AAV-Tβ4 markedly alleviated LPS-induced oxidative damage, lung injury, inflammation, and fibrosis in mice. Our in vitro experiments also showed that LPS inhibited mitophagy and promoted inflammation via oxidative stress in HPAEpiC, and Tβ4 significantly attenuated LPS-induced mitophagy inhibition, inflammasome activation, and transforming growth factor-β (TGF)-β1-induced epithelial-mesenchymal transition (EMT) in HPAEpiC. Moreover, Tβ4 suppressed the proliferation and attenuated the TGF-β1-induced activation of HLF-1 cells. In conclusion, Tβ4 alleviates LPS-induced lung injury, inflammation, and subsequent fibrosis in mice, suggesting that Tβ4 has a protective role in the pathogenesis of pulmonary fibrosis. Tβ4 is involved in attenuating oxidative injury, promoting mitophagy, and alleviating inflammation and fibrosis. Modulation of Tβ4 might be a novel strategy for treating pulmonary fibrosis.