Pathogenesis Of Lung Fibrosis Research Articles

A Tale of Two Proteolytic Machines: Matrix Metalloproteinases and the Ubiquitin-Proteasome System in Pulmonary Fibrosis.

Pulmonary fibrosis is a chronic and progressive lung disease characterized by the activation of fibroblasts and the irreversible deposition of connective tissue matrices that leads to altered pulmonary architecture and physiology. Multiple factors have been implicated in the pathogenesis of lung fibrosis, including genetic and environmental factors that cause abnormal activation of alveolar epithelial cells, leading to the development of complex profibrotic cascade activation and extracellular matrix (ECM) deposition. One class of proteinases that is thought to be important in the regulation of the ECM are the matrix metalloproteinases (MMPs). MMPs can be up- and down- regulated in idiopathic pulmonary fibrosis (IPF) lungs and their role depends upon their location and function. Furthermore, alterations in the ubiquitin-proteosome system (UPS), a major intracellular protein degradation complex, have been described in aging and IPF lungs. UPS alterations could potentially lead to the abnormal accumulation and deposition of ECM. A better understanding of the specific roles MMPs and UPS play in the pathophysiology of pulmonary fibrosis could potentially drive to the development of novel biomarkers that can be as diagnostic and therapeutic targets. In this review, we describe how MMPs and UPS alter ECM composition in IPF lungs and mouse models of pulmonary fibrosis, thereby influencing the alveolar epithelial and mesenchymal cell behavior. Finally, we discuss recent findings that associate MMPs and UPS interplay with the development of pulmonary fibrosis.

Non-cell-autonomous role of myofibroblasts in pulmonary fibrosis through lactate induction of M2 macrophage polarization

Abstract Rationale There has been growing evidence that metabolic dysregulations play critical roles in the pathogenesis of lung fibrosis, particularly idiopathic pulmonary fibrosis (IPF). Recent studies from our group as well as others’ showed that lung myofibroblasts displayed augmented glycolysis and secreted more lactate than normal fibroblasts. In this study, we examined the effect of lactate, the end product of myofibroblast glycolysis, on the phenotypic alterations of macrophages that has been frequently implicated in lung fibrosis. Methods Levels of M2 macrophage markers were determined by real-time PCR and Western blotting. Lactate transportation into cells was inhibited by siRNA knockdown of lactate MCT1 or MCT1 inhibitors. Results First, we confirmed that alveolar macrophages (AM) from fibrotic mouse lungs exhibited a M2 macrophage phenotype, as evidenced by increased expression of M2 markers Arginase 1 (Arg1) and vascular endothelial growth factor (VEGF) in these cells. We found that lactate induced M2 polarization of bone marrow derived macrophages (BMDM) and AMs. Furthermore, conditional media from lung myofibroblasts as well as coculture with lung myofibroblasts promoted M2 differentiation of BMDMs and AMs. Lactate induced M2 polarization could be inhibited by incapacitating lactate transporter MCT1 through siRNA knockdown or specific inhibitors. Conclusions Our data suggest that lung myofibroblast derived lactate promotes the pro-fibrotic differentiation of lung macrophages. We provide novel insight into the mechanism by which lung myofibroblasts participate in the pathogenesis of lung fibrosis. Targeting lactate secretion or intake by lung macrophages could be novel strategies to treat lung fibrosis.

Airway epithelial specific deletion of Jun-N-terminal kinase 1 attenuates pulmonary fibrosis in two independent mouse models.

The stress-induced kinase, c-Jun-N-terminal kinase 1 (JNK1) has previously been implicated in the pathogenesis of lung fibrosis. However, the exact cell type(s) wherein JNK1 exerts its pro-fibrotic role(s) remained enigmatic. Herein we demonstrate prominent activation of JNK in bronchial epithelia using the mouse models of bleomycin- or AdTGFβ1-induced fibrosis. Furthermore, in lung tissues of patients with idiopathic pulmonary fibrosis (IPF), active JNK was observed in various regions including type I and type II pneumocytes and fibroblasts. No JNK activity was observed in adjacent normal tissue or in normal control tissue. To address the role of epithelial JNK1, we ablated Jnk1 form bronchiolar and alveolar type II epithelial cells using CCSP-directed Cre recombinase-mediated ablation of LoxP-flanked Jnk1 alleles. Our results demonstrate that ablation of Jnk1 from airway epithelia resulted in a strong protection from bleomycin- or adenovirus expressing active transforming growth factor beta-1 (AdTGFβ1)-induced fibrosis. Ablation of the Jnk1 allele at a time when collagen increases were already present showed a reversal of existing increases in collagen content. Epithelial Jnk1 ablation resulted in attenuation of mesenchymal genes and proteins in lung tissue and preserved expression of epithelial genes. Collectively, these data suggest that epithelial JNK1 contributes to the pathogenesis of pulmonary fibrosis. Given the presence of active JNK in lungs from patients with IPF, targeting JNK1 in airway epithelia may represent a potential treatment strategy to combat this devastating disease.

Dual Inhibition of HDAC and Tyrosine Kinase Signaling Pathways with CUDC-907 Inhibits TGFβ1 Induced Lung and Tumor Fibrosis

Object: TGFβ1 signaling is a critical driver of collagen accumulation in pulmonary fibrotic diseases and a well-characterized regulator of cancer associated fibroblasts (CAF) activation in lung cancer. Myofibroblasts induced by TGFβ1 and other factors are key players in the pathogenesis of lung fibrosis and tumor. Tremendous attention has been gained to targeting myofibroblasts in order to inhibit the progression of fibrosis and myofibroblast-induced tumor progression and metastasis. Our aim was to determine the therapeutic efficacy of simultaneously targeting PI3K and HDAC pathways in lung myofibroblasts and CAF with a single agent and to evaluate biomarkers of treatment response. Method: CUDC-907 is a first-in-class compound, functioning as a dual inhibitor of HDACs and PI3K/AKT pathway. We investigated its effects in counteracting the activity of TGFβ1-induced myofibroblasts/CAF in regard to cell proliferation, migration, invasion, apoptosis in vitro and antifibrosis efficiency in vivo. Result: CUDC-907 inhibited myofibroblasts/CAF cell proliferation, migration and apoptosis in a dose-dependent manner and caused cell cycle arrest at G1-S phase. CUDC-907 not only inhibited myofibroblasts markers expression, but also significantly inhibited the phosphorylation level of AKT, mTOR, Smad2/3, and promoted acetylation of histones. Furthermore, the observed inhibitory effect was also confirmed in bleomycin-induced mice lung fibrosis and nude mouse transplanted tumor model. Conclusion: Dual inhibition of HDAC and the tyrosine kinase signaling pathways with CUDC-907 is a promising treatment strategy for TGFβ1-induced lung and tumor fibrosis. Funding Statement: This study was financially supported by China National Nature Science Foundation (81871882), Science and Technology Commission of Shanghai Municipality Medical Guidance Science &Technology Support Project (16411966100), Shanghai Municipal Education Commission-Gaofeng Clinical Medicine Grant Support (20172005) and Shanghai Municipal Commission of Health and Family Planning Outstanding Academic Leaders Training Program (2017BR055). Declaration of Interests: The authors declare no potential conflicts of interest. Ethics Approval Statement: This protocol was approved by the Ethics Committee of Ruijin Hospital (KY 2018-104). All patients were enrolled after written informed consent.

YY1 mediates TGF-\u03b21-induced EMT and pro-fibrogenesis in alveolar epithelial cells

Pulmonary fibrosis is a chronic, progressive lung disease associated with lung damage and scarring. The pathological mechanism causing pulmonary fibrosis remains unknown. Emerging evidence suggests prominent roles of epithelial–mesenchymal transition (EMT) of alveolar epithelial cells (AECs) in myofibroblast formation and progressive pulmonary fibrosis. Our previous work has demonstrated the regulation of YY1 in idiopathic pulmonary fibrosis and pathogenesis of fibroid lung. However, the specific function of YY1 in AECs during the pathogenesis of pulmonary fibrosis is yet to be determined. Herein, we found the higher level of YY1 in primary fibroblasts than that in primary epithelial cells from the lung of mouse. A549 and BEAS-2B cells, serving as models for type II alveolar pulmonary epithelium in vitro, were used to determine the function of YY1 during EMT of AECs. TGF-β-induced activation of the pro-fibrotic program was applied to determine the role YY1 may play in pro-fibrogenesis of type II alveolar epithelial cells. Upregulation of YY1 was associated with EMT and pro-fibrotic phenotype induced by TGF-β treatment. Targeted knockdown of YY1 abrogated the EMT induction by TGF-β treatment. Enforced expression of YY1 can partly mimic the TGF-β-induced pro-fibrotic change in either A549 cell line or primary alveolar epithelial cells, indicating the induction of YY1 expression may mediate the TGF-β-induced EMT and pro-fibrosis. In addition, the translocation of NF-κB p65 from the cytoplasm to the nucleus was demonstrated in A549 cells after TGF-β treatment and/or YY1 overexpression, suggesting that NF-κB-YY1 signaling pathway regulates pulmonary fibrotic progression in lung epithelial cells. These findings will shed light on the better understanding of mechanisms regulating pro-fibrogenesis in AECs and pathogenesis of lung fibrosis.

MiR-34b-5p knockdown attenuates bleomycin-induced pulmonary fibrosis by targeting tissue inhibitor of metalloproteinase 3 (TIMP3).

To examine the effects of microRNA-34b-5p (miR-34b-5p) on bleomycin-induced lung fibrosis in mice and the underlying mechanism. TIMP3-deficient (Timp3-/-) and wild-type mice were administered with bleomycin before to detect the miR-34b-5p expression using quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Luciferase reporter assay was used to identify the target of miR-34b-5p in human lung fibroblast MRC-5. MiR-34b-5p was then silenced in vivo before lung histologic analysis and evaluation of extracellular matrix (ECM) genes as well as myofibroblast marker. The protein and mRNA expression levels were detected by Western blot and qRT-PCR, respectively. We found that miR-34b-5p was significantly increased in lung tissues from bleomycin-stimulated mice. TIMP3 was identified as a direct target of miR-34b-5p by using dual-luciferase reporter assay, and enhanced expression of miR-34b-5p led to a decrease in TIMP3 whereas miR-34b-5p knockdown was responsible for TIMP3 elevation in MRC-5 cells. MiR-34b-5p knockdown in vivo attenuated the bleomycin-induced pulmonary fibrosis in wild-type mice, displayed by a reduced expression of Col1A1, fibronectin (Fn), and α-SMA. Furthermore, histological examination of lung sections also verified a diminishing fibrotic phenotype caused by the miR-34b-5p knockdown. But in Timp3-/- mice, down-regulation of miR-34b-5p did not exert an effect on the severe fibrotic lung injury after bleomycin exposure. MiR-34b-5p knockdown appears to enhance the resistance to bleomycin by regulating its target gene TIMP3 during the pathogenesis of lung fibrosis.

Effect of Thalidomide on Cox-2 expression in bleomycin-induced pulmonary fibrosis in mice

Introduction: Lung fibrosis is a progressive, fatal disease that is characterized by increasing fibroblasts proliferation and extracellular matrix precipitation. Studies have shown that cyclooxygenase- 2 (Cox-2) could play a crucial role in the pathogenesis of lung fibrosis. In the current study, the effect of thalidomide on bleomycin-induced pulmonary fibrosis was qualitatively studied in a laboratory animal model. Methods: Thirty-two adult male C57BL/6 mice were randomly assigned to the following four groups: Group one received 2 mg bleomycin, group two received bleomycin in addition to 4 mg of thalidomide; group three received 4 mg of thalidomide, and Group 4 received 0.1 mg of 0.5% carboxymethyl cellulose (CMC) via intraperitoneal (IP) administration. Finally, the expression of Cox-2 protein and the percentage of contact points of alveolar spaces and pulmonary connective tissue were determined. Results: Our results showed that in the Bleo + Thal group compared to the Bleo group, the percentage of contact points of pulmonary connective tissue decreased significantly (P<0.001), while the percentage of contact points among the alveolar spaces increased significantly (P = 0.01). Also, immunohistochemical studies have demonstrated the number of Cox-2+ cells in the volume unit in the Bleo + Thal group decreased significantly in comparison with the group that received only Bleo (P = 0.012). Conclusion: In conclusion, these results suggest thalidomide could alleviate the bleomycin-induced lung fibrosis and decreases the expression of Cox-2 protein.

A pathologic two-way street: how innate immunity impacts lung fibrosis and fibrosis impacts lung immunity.

Lung fibrosis is characterised by the accumulation of extracellular matrix within the lung and is secondary to both known and unknown aetiologies. This accumulation of scar tissue limits gas exchange causing respiratory insufficiency. The pathogenesis of lung fibrosis is poorly understood, but immunologic‐based treatments have been largely ineffective. Despite this, accumulating evidence suggests that innate immune cells and receptors play important modulatory roles in the initiation and propagation of the disease. Paradoxically, while innate immune signalling may be important for the pathogenesis of fibrosis, there is also evidence to suggest that innate immune function against pathogens may be impaired, leading to dysregulated and/or impaired host defence. This review summarises the evidence for this pathologic two‐way street, highlights new concepts of pathogenesis and recommends future directions for research emphasis.

Reversing Mechanoinductive DSP Expression by CRISPR/dCas9-mediated Epigenome Editing.

DSP (desmoplakin), the most abundant component of desmosomes, which maintain the mechanical integrity of epithelium, is a genome-wide association study-identified genetic risk locus in human idiopathic pulmonary fibrosis (IPF). Subjects with IPF express a significantly higher level of DSP than control subjects. Determine potential mechanisms by which DSP is regulated in lung fibrosis. Matrigel-coated soft and stiff polyacrylamide gels were made to simulate the stiffness of normal and fibrotic lungs. Quantitative chromatin immunoprecipitation and electrophoretic mobility shift assay were used to evaluate transcription factor binding to the DSP promoter. Targeted DNA methylation was achieved by CRISPR (clustered regularly interspaced short palindromic repeats)/dCas9 (deactivated CRISPR-associated protein-9 nuclease)-mediated Dnmt3A (DNA methyltransferase 3A) expression under the guidance of sequence-specific single guide RNAs. Stiff matrix promotes DSP gene expression in both human and rodent lung epithelial cells as compared with soft matrix. A conserved region in the proximal DSP promoter is hypermethylated under soft matrix conditions and becomes hypomethylated/demethylated under stiff matrix conditions. Demethylation of this conserved DSP promoter region is associated with transactivation of transcription factor EGR1 (early growth response protein 1), resulting in EGR1-dependent DSP overexpression. Targeted DNA methylation by CRISPR/dCas9/Dnmt3A-mediated epigenome editing blocks EGR1 binding to the DSP promoter and inhibits stiff matrix-induced DSP overexpression. DSP is a matrix stiffness-regulated mechanosensitive gene. CRISPR/dCas9-Dnmt3A-mediated epigenome editing reverses DSP overexpression by reestablishment of the epigenetic control of DSP under the mechanically homeostatic environment. It provides a useful tool for investigations of the functional role of DSP in the pathogenesis of lung fibrosis.

Current and future perspectives in the management of gastroesophageal reflux disease.

Although the prevalence of gastroesophageal reflux disease (GERD) used to be lower in East Asia as compared to Western countries, it has recently been increasing, most likely due to the socioeconomic development in the East. The prevalence of both GERD and functional gastrointestinal disorders (FGIDs) ranges between 10% and 25% worldwide and there are distinct subgroups of patients with overlapping of GERD and FGIDs. However, the true prevalence of an overlap between GERD and FGIDs can be determined only when a formal pathophysiological evaluation has been performed. Nocturnal reflux symptoms have a significant impact on patients' sleep quality and quality of life. Although proton pump inhibitors (PPIs) can improve both reflux and sleep-related symptoms, the relationship between nocturnal reflux events and sleep disturbance is not fully understood. GERD plays an important role in the pathogenesis of lung fibrosis, and PPIs or fundoplication may decrease the likelihood of pulmonary exacerbation or even improve pulmonary function. Refractory reflux symptoms without esophagitis have become one of the most common presentations of GERD in gastroenterology clinics. There are several new medical therapies, and endoscopic as well as laparoscopic techniques that have been increasingly used in these patients. The selection of options should be tailored and individualized based on the pathophysiology of refractory GERD.

HMGB1 induces lung fibroblast to myofibroblast differentiation through NF‑κB‑mediated TGF‑β1 release.

The proinflammatory factor high-mobility group box protein 1 (HMGB1) has been implicated in the pathogenesis of lung fibrosis; however, the role of HMGB1 in lung fibrosis remains unclear. It has previously been reported that nuclear factor (NF)-κB and transforming growth factor (TGF)-β1 may be involved in lung fibrosis. Therefore, the present study aimed to examine the potential molecular mechanisms that underlie HMGB1-induced lung fibrosis via the regulation of NF-κB and TGF-β1. The results demonstrated that HMGB1 stimulation increased the activation of NF-κB and the release of TGF-β1, as well as the expression of α-smooth muscle actin (α-SMA) and collagen I in human lung fibroblasts in vitro. In addition, inhibition of NF-κB activation blocked HMGB1-induced TGF-β1 release, as well as α-SMA and collagen I expression in lung fibroblasts. Preventing the release of TGF-β1 inhibited HMGB1-induced α-SMA and collagen I expression; however, it had no effect on NF-κB activation. Collectively, these findings indicate that HMGB1 induces fibroblast to myofibroblast differentiation of lung fibroblasts via NF-κB-mediated TGF-β1 release.

Potential Mechanisms Underlying TGF-β-mediated Complement Activation in Lung Fibrosis.

While our previous studies suggest that limiting bleomycin-induced complement activation suppresses TGF-β signaling, the specific hierarchical interactions between TGF-β and complement in lung fibrosis are unclear. Herein, we investigated the mechanisms underlying TGF-β-induced complement activation in the pathogenesis of lung fibrosis. C57-BL6 mice were given intratracheal instillations of adenoviral vectors overexpressing TGF-β (Ad-TGFβ) or the firefly gene-luciferase (Ad-Luc; control). Two weeks later, mice with fibrotic lungs were instilled RNAi specific to receptors for C3a or C5a-C3ar or C5ar, and sacrificed at day 28. Histopathological analyses revealed that genetic silencing of C3ar or C5ar arrested the progression of TGF-β-induced lung fibrosis, collagen deposition and content (hydroxyproline, col1a1/2); and significantly suppressed local complement activation. With genetic silencing of either C3ar or C5ar, in Ad-TGFβ-injured lungs: we detected the recovery of Smad7 (TGF-β inhibitor) and diminished local release of DAF (membrane-bound complement inhibitor); in vitro: TGF-β-mediated loss of DAF was prevented. Conversely, blockade of the TGF-β receptor prevented C3a-mediated loss of DAF in both normal primary human alveolar and small airway epithelial cells. Of the 52 miRNAs analyzed as part of the Affymetrix array, normal primary human SAECs exposed to C3a, C5a or TGF-β caused discrete and overlapping miRNA regulation related to epithelial proliferation or apoptosis (miR-891A, miR-4442, miR-548, miR-4633), cellular contractility (miR-1197) and lung fibrosis (miR-21, miR-200C, miR-31HG, miR-503). Our studies present potential mechanisms by which TGF-β activates complement and promotes lung fibrosis.

MiR-34a Inhibits Lung Fibrosis by Inducing Lung Fibroblast Senescence.

Cellular senescence has been implicated in diverse pathologies. However, there is conflicting evidence regarding the role of this process in tissue fibrosis. Although dysregulation of microRNAs is a key mechanism in the pathogenesis of lung fibrosis, it is unclear whether microRNAs function by regulating cellular senescence in the disease. In this study, we found that miR-34a demonstrated greater expression in the lungs of patients with idiopathic pulmonary fibrosis and in mice with experimental pulmonary fibrosis, with its primary localization in lung fibroblasts. More importantly, miR-34a was up-regulated significantly in both human and mouse lung myofibroblasts. We found that mice with miR-34a ablation developed more severe pulmonary fibrosis than did wild-type animals after fibrotic lung injury. Mechanistically, we found that miR-34a induced a senescent phenotype in lung fibroblasts because this microRNA increased senescence-associated β-galactosidase activity, enhanced expression of senescence markers, and decreased cell proliferative capacities. Consistently, we found that primary lung fibroblasts from fibrotic lungs of miR-34a-deficient mice had a diminished senescent phenotype and enhanced resistance to apoptosis as compared with those from wild-type animals. We also identified multiple miR-34a targets that likely mediated its activities in inducing senescence in lung fibroblasts. In conclusion, our data suggest that miR-34a functions through a negative feedback mechanism to restrain fibrotic response in the lungs by promoting senescence of pulmonary fibroblasts.

Influences of innate immunity, autophagy, and fibroblast activation in the pathogenesis of lung fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease characterized by accumulation of extracellular matrix (ECM) and impaired gas exchange. The pathobiological mechanisms that account for disease progression are poorly understood but likely involve alterations in innate inflammatory cells, epithelial cells, and fibroblasts. Thus we seek to review the most recent literature highlighting the complex roles of neutrophils and macrophages as both promoters of fibrosis and defenders against infection. With respect to epithelial cells and fibroblasts, we review the data suggesting that defective autophagy promotes the fibrogenic potential of both cell types and discuss new evidence related to matrix metalloproteinases, growth factors, and cellular metabolism in the form of lactic acid generation that may have consequences for promoting fibrogenesis. We discuss potential cross talk between innate and structural cell types and also highlight literature that may help explain the limitations of current IPF therapies.

Targeting Inhibitor of Apoptosis Proteins Protects from Bleomycin-Induced Lung Fibrosis.

Accumulation of apoptosis-resistant fibroblasts is a hallmark of pulmonary fibrosis. We hypothesized that disruption of inhibitor of apoptosis protein (IAP) family proteins would limit lung fibrosis. We first show that transforming growth factor-β1 and bleomycin increase X-linked IAP (XIAP) and cellular IAP (cIAP)-1 and -2 in murine lungs and mesenchymal cells. Functional blockade of XIAP and the cIAPs with AT-406, an orally bioavailable second mitochondria-derived activator of caspases (Smac) mimetic, abrogated bleomycin-induced lung fibrosis when given both prophylactically and therapeutically. To determine whether the reduction in fibrosis was predominantly due to AT-406-mediated inhibition of XIAP, we compared the fibrotic response of XIAP-deficient mice (XIAP(-/y)) with littermate controls and found no difference. We found no alterations in total inflammatory cells of either wild-type mice treated with AT-406 or XIAP(-/y) mice. AT-406 treatment limited CCL12 and IFN-γ production, whereas XIAP(-/y) mice exhibited increased IL-1β expression. Surprisingly, XIAP(-/y) mesenchymal cells had increased resistance to Fas-mediated apoptosis. Functional blockade of cIAPs with AT-406 restored sensitivity to Fas-mediated apoptosis in XIAP(-/y) mesenchymal cells in vitro and increased apoptosis of mesenchymal cells in vivo, indicating that the increased apoptosis resistance in XIAP(-/y) mesenchymal cells was the result of increased cIAP expression. Collectively, these results indicate that: (1) IAPs have a role in the pathogenesis of lung fibrosis; (2) a congenital deficiency of XIAP may be overcome by compensatory mechanisms of other IAPs; and (3) broad functional inhibition of IAPs may be an effective strategy for the treatment of lung fibrosis by promoting mesenchymal cell apoptosis.

Glycolytic Reprogramming in Myofibroblast Differentiation and Lung Fibrosis.

Dysregulation of cellular metabolism has been shown to participate in several pathologic processes. However, the role of metabolic reprogramming is not well appreciated in the pathogenesis of organ fibrosis. To determine if glycolytic reprogramming participates in the pathogenesis of lung fibrosis and assess the therapeutic potential of glycolytic inhibition in treating lung fibrosis. A cell metabolism assay was performed to determine glycolytic flux and mitochondrial respiration. Lactate levels were measured to assess glycolysis in fibroblasts and lungs. Glycolytic inhibition by genetic and pharmacologic approaches was used to demonstrate the critical role of glycolysis in lung fibrosis. Augmentation of glycolysis is an early and sustained event during myofibroblast differentiation, which is dependent on the increased expression of critical glycolytic enzymes, in particular, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3). Augmented glycolysis contributes to the stabilization of hypoxia-inducible factor 1-α, a master regulator of glycolytic enzymes implicated in organ fibrosis, by increasing cellular levels of tricarboxylic acid cycle intermediate succinate in lung myofibroblasts. Inhibition of glycolysis by the PFKFB3 inhibitor 3PO or genomic disruption of the PFKFB3 gene blunted the differentiation of lung fibroblasts into myofibroblasts, and attenuated profibrotic phenotypes in myofibroblasts isolated from the lungs of patients with idiopathic pulmonary fibrosis. Inhibition of glycolysis by 3PO demonstrates therapeutic benefit in bleomycin-induced and transforming growth factor-β1-induced lung fibrosis in mice. Our data support the novel concept of glycolytic reprogramming in the pathogenesis of lung fibrosis and provide proof-of-concept that targeting this pathway may be efficacious in treating fibrotic disorders, such as idiopathic pulmonary fibrosis.

Bidirectional role of IL-6 signal in pathogenesis of lung fibrosis.

BackgroundVarious signals are known to participate in the pathogenesis of lung fibrosis. Our aim was to determine which signal is predominantly mobilized in the early inflammatory phase and thereafter modulates the development of lung fibrosis.MethodsMice received a single dose of 3 mg/kg body weight of bleomycin (BLM) and were sacrificed at designated days post-instillation (dpi). Lung homogenates and sections from mice in the early inflammatory phase were subjected to phospho-protein array analysis and immunofluorescence studies, respectively. Bronchoalveolar lavage fluid (BALF) from mice was subjected to an enzyme-linked immunosorbent assay (EIA) for interleukin (IL)-6 and evaluation of infiltrated cell populations. The effects of endogenous and exogenous IL-6 on the BLM-induced apoptotic signal in A549 cells and type 2 pneumocytes were elucidated. In addition, the effect of IL-6-neutralizing antibody on BLM-induced lung injury was evaluated.ResultsPhospho-protein array revealed that BLM induced phosphorylation of molecules downstream of the IL-6 receptor such as Stat3 and Akt in the lung at 3 dpi. At 3 dpi, immunofluorescence studies showed that signals of phospho-Stat3 and -Akt were localized in type 2 pneumocytes, and that BLM-induced IL-6-like immunoreactivity was predominantly observed in type 2 pneumocytes. Activation of caspases in BLM-treated A549 cells and type 2 pneumocytes was augmented by application of IL-6-neutralizing antibody, a PI3K inhibitor or a Stat3 inhibitor. EIA revealed that BLM-induced IL-6 in BALF was biphasic, with the first increase from 0.5 to 3 dpi followed by the second increase from 8 to 10 dpi. Blockade of the first increase of IL-6 by IL-6-neutralizing antibody enhanced apoptosis of type 2 pneumocytes and neutrophilic infiltration and markedly accelerated fibrosis in the lung. In contrast, blockade of the second increase of IL-6 by IL-6-neutralizing antibody ameliorated lung fibrosis.ConclusionsThe present study demonstrated that IL-6 could play a bidirectional role in the pathogenesis of lung fibrosis. In particular, upregulation of IL-6 at the early inflammatory stage of BLM-injured lung has antifibrotic activity through regulating the cell fate of type 2 pneumocytes in an autocrine/paracrine manner.Electronic supplementary materialThe online version of this article (doi:10.1186/s12931-015-0261-z) contains supplementary material, which is available to authorized users.

Circulating fibrocytes as biomarker of prognosis in Hermansky-Pudlak syndrome.

The rate of progression of most interstitial lung diseases (ILD) is unpredictable. Fibrocytes are circulating bone marrow-derived cells that have been implicated in the pathogenesis of lung fibrosis. Hermansky-Pudlak syndrome (HPS), a genetic cause of ILD in early adulthood, allows for study of biomarkers of ILD in a homogeneous population at near-certain risk of developing fibrotic lung disease. To test the hypothesis that, in subjects with HPS, the number or phenotype of circulating fibrocytes predicts progression and outcome of ILD. We measured circulating fibrocyte counts and chemokine levels in a cohort of subjects with HPS and healthy control subjects and correlated the results to disease outcome. In a cross-sectional analysis, peripheral blood fibrocyte concentrations were markedly elevated in a subset of subjects with HPS who had ILD but not subjects without lung disease or normal control subjects. The blood concentration of fibrocytes expressing the chemokine receptor CXCR4 correlated significantly with the plasma concentration of the CXCR4 ligand, CXCL12. In a longitudinal study, we found marked episodic elevations in circulating fibrocyte counts over a median follow-up period of 614 days. Elevations in both maximal values and final values of peripheral blood CXCR4(+) fibrocyte concentration were strongly associated with death from ILD. CXCR4(+) fibrocyte concentration may be useful as a biomarker for outcome of ILD in subjects with HPS.

Deletion of ADORA2B from myeloid cells dampens lung fibrosis and pulmonary hypertension.

Idiopathic pulmonary fibrosis (IPF) is a lethal, fibroproliferative disease. Pulmonary hypertension (PH) can develop secondary to IPF and increase mortality. Alternatively, activated macrophages (AAMs) contribute to the pathogenesis of both IPF and PH. Here we hypothesized that adenosine signaling through the ADORA2B on AAMs impacts the progression of these disorders and that conditional deletion of ADORA2B on myeloid cells would have a beneficial effect in a model of these diseases. Conditional knockout mice lacking ADORA2B on myeloid cells (Adora2B(f/f)-LysM(Cre)) were exposed to the fibrotic agent bleomycin (BLM; 0.035 U/g body weight, i.p.). At 14, 17, 21, 25, or 33 d after exposure, SpO2, bronchoalveolar lavage fluid (BALF), and histologic analyses were performed. On day 33, lung function and cardiovascular analyses were determined. Markers for AAM and mediators of fibrosis and PH were assessed. Adora2B(f/f)-LysM(Cre) mice presented with attenuated fibrosis, improved lung function, and no evidence of PH compared with control mice exposed to BLM. These findings were accompanied by reduced expression of CD206 and arginase-1, markers for AAMs. A 10-fold reduction in IL-6 and a 5-fold decrease in hyaluronan, both linked to lung fibrosis and PH, were also observed. These data suggest that activation of the ADORA2B on macrophages plays an active role in the pathogenesis of lung fibrosis and PH.

A critical role for the mTORC2 pathway in lung fibrosis.

A characteristic of dysregulated wound healing in IPF is fibroblastic-mediated damage to lung epithelial cells within fibroblastic foci. In these foci, TGF-β and other growth factors activate fibroblasts that secrete growth factors and matrix regulatory proteins, which activate a fibrotic cascade. Our studies and those of others have revealed that Akt is activated in IPF fibroblasts and it mediates the activation by TGF-β of pro-fibrotic pathways. Recent studies show that mTORC2, a component of the mTOR pathway, mediates the activation of Akt. In this study we set out to determine if blocking mTORC2 with MLN0128, an active site dual mTOR inhibitor, which blocks both mTORC1 and mTORC2, inhibits lung fibrosis. We examined the effect of MLN0128 on TGF-β-mediated induction of stromal proteins in IPF lung fibroblasts; also, we looked at its effect on TGF-β-mediated epithelial injury using a Transwell co-culture system. Additionally, we assessed MLN0128 in the murine bleomycin lung model. We found that TGF-β induces the Rictor component of mTORC2 in IPF lung fibroblasts, which led to Akt activation, and that MLN0128 exhibited potent anti-fibrotic activity in vitro and in vivo. Also, we observed that Rictor induction is Akt-mediated. MLN0128 displays multiple anti-fibrotic and lung epithelial-protective activities; it (1) inhibited the expression of pro-fibrotic matrix-regulatory proteins in TGF-β-stimulated IPF fibroblasts; (2) inhibited fibrosis in a murine bleomycin lung model; and (3) protected lung epithelial cells from injury caused by TGF-β-stimulated IPF fibroblasts. Our findings support a role for mTORC2 in the pathogenesis of lung fibrosis and for the potential of active site mTOR inhibitors in the treatment of IPF and other fibrotic lung diseases.