Phenotype Of Lung Fibroblasts Research Articles

NLRP3 inflammasome activation contributes to the development of the pro-fibrotic phenotype of lung fibroblasts

Idiopathic pulmonary fibrosis (IPF) is an irreversible progressive interstitial lung disease of unknown cause. The poorly understood pathophysiology of IPF poses substantial challenges to the development of effective anti-lung fibrotic drugs. The NLRP3 inflammasome, a key component of the innate immune system, has recently been linked to the pathogenesis of lung fibrosis. However, the specific contributions of NLRP3 inflammasomes to determination of the pro-fibrotic phenotype of lung fibroblasts, which play a central role in the production of extracellular matrix protein, remain to be investigated. Therefore, the present study was performed to elucidate the involvement of NLRP3 inflammasome signalling pathways in modulation of lung fibroblast proliferation and differentiation. We found that activation of NLRP3 inflammasomes increased in lung fibroblasts derived from individuals with pulmonary fibrosis and in normal lung fibroblasts stimulated with transforming growth factor β and platelet-derived growth factor. Importantly, blockage of NLRP3 inflammasome signalling, either by gene silencing of NLRP3 or using pharmacological inhibitors of NLRP3, caspase-1, or IL-1 receptor, inhibited the proliferation, differentiation, and extracellular matrix protein synthesis of activated lung fibroblasts. Moreover, induction of the reactive oxygen species/thioredoxin-interacting protein axis, an upstream signalling pathway of NLRP3 inflammasomes, was essential for maintenance of the pro-fibrotic phenotype of lung fibroblasts. Interestingly, treatments with pharmacological inhibitors of NLRP3 inflammasomes prevented the progression of bleomycin-induced pulmonary fibrosis in mice. Collectively, these findings suggest that aberrant activation of NLRP3 inflammasomes is a critical event in the pathogenesis of IPF and that targeting NLRP3 inflammasomes may serve as a therapeutic strategy for IPF.

Endoplasmic reticulum stress-induced senescence in human lung fibroblasts.

Loss of proteostasis and cellular senescence have been previously established as characteristics of aging; however, their interaction in the context of lung aging and potential contributions to aging-associated lung remodeling remains understudied. In this study, we aimed to characterize endoplasmic reticulum (ER) stress response, cellular senescence, and their interaction in relation to extracellular matrix (ECM) production in lung fibroblasts from young (25-45 yr) and old (>60 yr) humans. Fibroblasts from young and old patients without significant preexisting lung disease were exposed to vehicle, MG132, etoposide, or salubrinal. Afterward, cells and cell lysates or supernatants were analyzed for ER stress, cellular senescence, and ECM changes using protein analysis, proliferation assay, and senescence-associated beta-galactosidase (SA-β-Gal) staining. At baseline, fibroblasts from aging individuals showed increased levels of ER stress (ATF6 and PERK), senescence (p21 and McL-1), and ECM marker (COL1A1) compared to those from young individuals. Upon ER stress induction and etoposide exposure, fibroblasts showed an increase in senescence (SA-β-Gal, p21, and Cav-1), ER stress (PERK), and ECM markers (COL1A1 and LUM) compared to vehicle. Additionally, IL-6 and IL-8 levels were increased in the supernatants of MG132- and etoposide-treated fibroblasts, respectively. Finally, the ER stress inhibitor salubrinal decreased the expression of p21 compared to vehicle and MG132 treatments; however, salubrinal inhibited COL1A1 but not p21 expression in MG132-treated fibroblasts. Our study suggests that ER stress response plays an important role in establishment and maintenance of a senescence phenotype in lung fibroblasts and therefore contributes to altered remodeling in the aging lung.NEW & NOTEWORTHY The current study establishes functional links between endoplasmic reticulum (ER) stress and cellular senescence per se in the specific context of aging human lung fibroblasts. Recognizing that the process of aging per se is complex, modulated by the myriad of lifelong and environmental exposures, it is striking to note that chronic ER stress may play a crucial role in the establishment and maintenance of cellular senescence in lung fibroblasts.

Purinergic signaling promotes premature senescence

Extracellular ATP activates P2 purinergic receptors. Whether purinergic signaling is functionally coupled to cellular senescence is largely unknown. We find that oxidative stress induced release of ATP and caused senescence in human lung fibroblasts. Inhibition of P2 receptors limited oxidative stress-induced senescence, while stimulation with exogenous ATP promoted premature senescence. Pharmacological inhibition of P2Y11 receptor (P2Y11R) inhibited premature senescence induced by either oxidative stress or ATP, while stimulation with a P2Y11R agonist was sufficient to induce cellular senescence. Our data show that both extracellular ATP and a P2Y11R agonist induced calcium (Ca++) release from the endoplasmic reticulum (ER) and that either inhibition of phospholipase C or intracellular Ca++ chelation impaired ATP-induced senescence. We also find that Ca++ that was released from the ER, following ATP-mediated activation of phospholipase C, entered mitochondria in a manner dependent on P2Y11R activation. Once in mitochondria, excessive Ca++ promoted the production of reactive oxygen species in a P2Y11R-dependent fashion, which drove development of premature senescence of lung fibroblasts. Finally, we show that conditioned medium derived from senescent lung fibroblasts, which were induced to senesce through the activation of ATP/P2Y11R-mediated signaling, promoted the proliferation of triple-negative breast cancer cells and their tumorigenic potential by secreting amphiregulin. Our study identifies the existence of a novel purinergic signaling pathway that links extracellular ATP to the development of a protumorigenic premature senescent phenotype in lung fibroblasts that is dependent on P2Y11R activation and ER-to-mitochondria calcium signaling.

Disruption of extracellular redox balance drives persistent lung fibrosis and impairs fibrosis resolution

Lung fibrosis is a devastating outcome of various diffuse parenchymal lung diseases. Despite rigorous research efforts, the mechanisms that propagate its progressive and nonresolving nature remain enigmatic. Oxidative stress has been implicated in the pathogenesis of lung fibrosis. However, the role of extracellular redox state in disease progression and resolution remains largely unexplored. Here, we show that compartmentalized control over extracellular reactive oxygen species (ROS) by aerosolized delivery of recombinant extracellular superoxide dismutase (ECSOD) suppresses an established bleomycin-induced fibrotic process in mice. Further analysis of publicly available microarray, RNA-seq and single-cell RNAseq datasets reveals a significant decrease in ECSOD expression in fibrotic lung tissues that can be spontaneously restored during fibrosis resolution. Therefore, we investigate the effect of siRNA-mediated ECSOD depletion during the established fibrotic phase on the self-limiting nature of the bleomycin mouse model. Our results demonstrate that in vivo knockdown of ECSOD in mouse fibrotic lungs impairs fibrosis resolution. Mechanistically, we demonstrate that transforming growth factor (TGF)-β1 downregulates endogenous ECSOD expression, leading to the accumulation of extracellular superoxide via Smad-mediated signaling and the activation of additional stores of latent TGF-β1. In addition, depletion of endogenous ECSOD during the fibrotic phase in the bleomycin model induces an apoptosis-resistant phenotype in lung fibroblasts through unrestricted Akt signaling. Taken together, our data strongly support the critical role of extracellular redox state in fibrosis persistence and resolution. Based on these findings, we propose that compartment-specific control over extracellular ROS may be a potential therapeutic strategy for managing fibrotic lung disorders.

Involvement of ACACA (acetyl-CoA carboxylase α) in the lung pre-metastatic niche formation in breast cancer by senescence phenotypic conversion in fibroblasts

BackgroundReprogramming of metabolism is strongly associated with the development of cancer. However, the role of metabolic reprogramming in the remodeling of pre-metastatic niche (PMN), a key step in metastasis, is still unknown. We aimed to investigate the metabolic alternation during lung PMN formation in breast cancer.MethodsWe assessed the transcriptomes and lipidomics of lung of MMTV-PyVT mice by microarray and liquid chromatography-tandem mass mass spectrometry before lung metastasis. The validation of gene or protein expressions was performed by quantitative real-time polymerase chain reaction or immunoblot and immunohistochemistry respectively. The lung fibroblasts were isolated from mice and then co-cultured with breast cancer to identify the influence of cancer on the change of lung fibroblasts in PMN.ResultsWe demonstrated changes in the lipid profile and several lipid metabolism genes in the lungs of breast cancer-bearing MMTV-PyVT mice before cancer spreading. The expression of ACACA (acetyl-CoA carboxylase α) was downregulated in the lung fibroblasts, which contributed to changes in acetylation of protein’s lysine residues and the synthesis of fatty acid. The downregulation of ACACA in lung fibroblasts triggered a senescent and inflammatory phenotypic shift of lung fibroblasts in both in vivo and in vitro models. The senescence-associated secretory phenotype of lung fibroblasts enabled the recruitment of immunosuppressive granulocytic myeloid-derived suppressor cells into the lungs through the production of CXCL1 in the lungs. Knock-in of ACACA prevented lung metastasis in the MMTV-PyVT mouse model, further supporting that ACACA was involved in the remodeling of the lung PMN.ConclusionsTaken together, these data revealed a mechanism by which ACACA downregulation directed the formation of an immunosuppressive lung PMN in breast cancer.

Tobacco smoke activated fibrogenic MARCKS/AXL complex promotes macrophage reprogramming and pulmonary fibrosis

Abstract Macrophages and tobacco smoke (TS) exposure have been demonstrated to play significant roles in modulating pulmonary fibrosis (PF). However, the mechanisms of how TS exposure modulates pro-fibrotic macrophage polarization and drives lung fibrosis is unclear. In our study, we investigated how TS modulates macrophage polarization and the functional consequences of this polarization. Multicolor flow cytometric data indicated that markers of M2 macrophage polarization were elevated in both human and mouse macrophage cells and tissues upon TS exposure. In addition, multiple primary lung fibroblast cells demonstrated elevated pro-fibrotic markers and aggressive phenotypes upon interacting with TS-exposed macrophage cells in a co-culture system. Elucidation of the signaling pathways activated by TS exposure through a receptor tyrosine kinase array screen revealed AXL receptor as a novel smoke-responsive molecule in macrophage cells. We noted elevated secretion of AXL ligand, Gas6, and AXL activity in TS exposed cells and tissues. Prior work had demonstrated an interaction between MARCKS, a smoke-responsive protein, and AXL in promoting a pro-fibrotic phenotype in lung fibroblasts. Similarly, we observed AXL activity positively correlated with MARCKS phosphorylation in macrophage cells. Pharmacologic and genetic targeting of the MARCKS/AXL signaling complex reduced M2 markers and profibrotic cytokine production in macrophages and reduced fibrotic changes in the co-culture model and in an animal model of smoke-mediated lung fibrosis. In all, our work suggests that the MARCKS/AXL fibrogenic complex is a potential target in attenuating macrophage activity in TS-mediated fibrosis. Supported by grants from NIH/NHLBI (R01HL146802), DOD DCMRP/PRMRP (PR202411), and UCOP TRDRP (28IR-0061 and T31DT1849).

Dedicator of cytokinesis 2 (DOCK2) mediates chronic high‐fat and high‐fructose diet induced lung injury

Obesity is involved in the pathogenesis of multiple lung diseases. However, little is known about the effects of chronic high‐fat and high‐fructose (HFHF) diet‐induced obesity on lung inflammatory/injury. We have reported that dedicator of cytokinesis 2 (DOCK2) is important for high‐fat diet (HFD)‐induced obesity and adipose tissue inflammation. DOCK2 deficient mice were protected from HFD induced body weight gain, insulin resistance, and increased proinflammatory cytokines in the adipose tissue and peripheral circulation. However, it remains elusive whether DOCK2 plays a role in lung injury associated with chronic HFHF diet‐induced obesity. In this study, we showed that chronic HFHF diet (20 weeks) induced lung inflammatory infiltration and collagen expression in the wild‐type (WT) C57BL/6 mice. Macrophage marker CD68 and monocyte chemoattractant protein‐1 (MCP‐1) expression were notably increased in the lungs of WT mice fed a HFHF diet. Further, HFHF diet increased lung DOCK2 expression that co‐localized with fibroblast‐specific protein 1, suggesting a potential role of DOCK2 in regulating proinflammatory phenotype of lung fibroblasts. Conversely, DOCK2 deficiency attenuated lung inflammation and fibrosis induced by chronic HFHF diet. In primary human lung fibroblast, TNF‐α and IL‐1β induced DOCK2 expression concurrent with MCP‐1, IL‐6, and matrix metalloproteinase‐2. DOCK2 knockdown also suppressed TNF‐α induced increase of these inflammatory mediators. Taken together, these findings suggest a previously unrecognized role of DOCK2 in mediating lung inflammation and fibrosis in chronic HFHF diet caused obesity.

Dedicator of Cytokinesis 2 (DOCK2) Deficiency Attenuates Lung Injury Associated with Chronic High-Fat and High-Fructose Diet–Induced Obesity

Obesity is a major risk factor for lung disease development. However, little is known about the impact of chronic high-fat and high-fructose (HFHF) diet-induced obesity on lung inflammation and subsequent pulmonary fibrosis. Herein we hypothesized that dedicator of cytokinesis 2 (DOCK2) promotes a proinflammatory phenotype of lung fibroblasts (LFs) to elicit lung injury and fibrosis in chronic HFHF diet-induced obesity. An HFHF diet for 20 weeks induced lung inflammation and profibrotic changes in wild-type C57BL/6 mice. CD68 and monocyte chemoattractant protein-1 (MCP-1) expression were notably increased in the lungs of wild-type mice fed an HFHF diet. An HFHF diet further increased lung DOCK2 expression that co-localized with fibroblast-specific protein 1, suggesting a role of DOCK2 in regulating proinflammatory phenotype of LFs. Importantly, DOCK2 knockout protected mice from lung inflammation and fibrosis induced by a HFHF diet. In primary human LFs, tumor necrosis factor-α (TNF-α) and IL-1β induced DOCK2 expression concurrent with MCP-1, IL-6, and matrix metallopeptidase 2. DOCK2 knockdown suppressed TNF-α-induced expression of these molecules and activation of phosphatidylinositol 3-kinase/AKT and NF-κB signaling pathways, suggesting a mechanism of DOCK2-mediated proinflammatory and profibrotic changes in human LFs. Taken together, these findings reveal a previously unrecognized role of DOCK2 in regulating proinflammatory phenotype of LFs, potentiation of lung inflammation, and pulmonary fibrosis in chronic HFHF diet-caused obesity.

Investigating the Role of NHE1 in the Regulation of Changes in Cellular Functions Related to the Development of Idiopathic Pulmonary Fibrosis

Idiopathic pulmonary fibrosis is a chronic lung disorder characterized by the thickening, stiffening, and scaring of tissue within the lungs. Affected individuals develop shortness of breath and the progressive reduction of respiratory function. Fibrosis ultimately results in low blood flow and hypoxia in the tissue, a microenvironment which is closely associated with the upregulation of the sodium-hydrogen exchanger isoform 1 (NHE1). NHE1 is a ubiquitously expressed protein that exchanges one extracellular Na+ for one intracellular H+. This regulates intracellular pH and cell progression through the cell cycle. When NHE1 is upregulated it supports increased cell proliferation and migration, indicating that it may play a vital role in the behavior of fibroblasts in the development of idiopathic pulmonary fibrosis. We hypothesized that inhibition of NHE1 activation would decrease cell progression towards a fibrotic phenotype in lung fibroblasts. For this assessment, we compared differences in cellular behavior in three cell lines to evaluate the role of NHE1 in these processes. LL29 cells are human fibroblasts derived from a patient diagnosed with idiopathic pulmonary fibrosis; WI-38 cells which are derived from healthy lung tissue; and PSN cells, which are derived from Chinese hamster lung fibroblasts and express only human NHE1.Transforming growth factor beta 1 (TGF-b1), Lysophosphatidic Acid (LPA) and Serotonin (5-hydroxytryptamine) were evaluated for their ability to alter cell proliferation, stress fiber formation, and myofibroblast differentiation in all three cell lines. TGF-b1 is a cytokine that is a growth factor implicated in the pathogenesis of pulmonary fibrosis and is involved in differentiation, proliferation, and matrix production of fibroblasts. Serotonin (5-HT) is a signaling molecule that binds to G-protein coupled receptors supporting the increase in TGF-b1 and its effects in cells. This hormone is involved in wound healing and stimulating inflammation. LPA is a lipid signaling molecule that plays a role in mediating inflammation and activating TGF-b1 signaling. All experiments were performed in the presence and absence of Ethylisopropylamiloride (EIPA), a potent inhibitor of NHE1, to evaluate the role of NHE1 in fibrotic transformation. In PSN cells, stimulation with 10 µM LPA increased stress fiber formation over 2-fold and this stimulation was absent in cells treated with 10 µM EIPA indicating a role for NHE1 in the regulation of stress fiber formation and related cellular processes.

PD-L1 on invasive fibroblasts drives fibrosis in a humanized model of idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive disease with unremitting extracellular matrix deposition, leading to a distortion of pulmonary architecture and impaired gas exchange. Fibroblasts from IPF patients acquire an invasive phenotype that is essential for progressive fibrosis. Here, we performed RNA sequencing analysis on invasive and noninvasive fibroblasts and found that the immune checkpoint ligand CD274 (also known as PD-L1) was upregulated on invasive lung fibroblasts and was required for the invasive phenotype of lung fibroblasts, is regulated by p53 and FAK, and drives lung fibrosis in a humanized IPF model in mice. Activating CD274 in IPF fibroblasts promoted invasion in vitro and pulmonary fibrosis in vivo. CD274 knockout in IPF fibroblasts and targeting CD274 by FAK inhibition or CD274-neutralizing antibodies blunted invasion and attenuated fibrosis, suggesting that CD274 may be a novel therapeutic target in IPF.

Mitochondrial dysfunction contributes to the senescent phenotype of IPF lung fibroblasts.

Increasing evidence highlights that senescence plays an important role in idiopathic pulmonary fibrosis (IPF). This study delineates the specific contribution of mitochondria and the superoxide they form to the senescent phenotype of lung fibroblasts from IPF patients (IPF‐LFs). Primary cultures of IPF‐LFs exhibited an intensified DNA damage response (DDR) and were more senescent than age‐matched fibroblasts from control donors (Ctrl‐LFs). Furthermore, IPF‐LFs exhibited mitochondrial dysfunction, exemplified by increases in mitochondrial superoxide, DNA, stress and activation of mTORC1. The DNA damaging agent etoposide elicited a DDR and augmented senescence in Ctrl‐LFs, which were accompanied by disturbances in mitochondrial homoeostasis including heightened superoxide production. However, etoposide had no effect on IPF‐LFs. Mitochondrial perturbation by rotenone involving sharp increases in superoxide production also evoked a DDR and senescence in Ctrl‐LFs, but not IPF‐LFs. Inhibition of mTORC1, antioxidant treatment and a mitochondrial targeting antioxidant decelerated IPF‐LF senescence and/or attenuated pharmacologically induced Ctrl‐LF senescence. In conclusion, increased superoxide production by dysfunctional mitochondria reinforces lung fibroblast senescence via prolongation of the DDR. As part of an auto‐amplifying loop, mTORC1 is activated, altering mitochondrial homoeostasis and increasing superoxide production. Deeper understanding the mechanisms by which mitochondria contribute to fibroblast senescence in IPF has potentially important therapeutic implications.

Sirtuin 7 is decreased in pulmonary fibrosis and regulates the fibrotic phenotype of lung fibroblasts.

Pulmonary fibrosis is a severe condition with no cure and limited therapeutic options. A better understanding of its pathophysiology is needed. Recent studies have suggested that pulmonary fibrosis may be driven by accelerated aging-related mechanisms. Sirtuins (SIRTs), particularly SIRT1, SIRT3, and SIRT6, are well-known mediators of aging; however, limited data exist on the contribution of sirtuins to lung fibrosis. We assessed the mRNA and protein levels of all seven known sirtuins in primary lung fibroblasts from patients with idiopathic pulmonary fibrosis (IPF) and systemic sclerosis-associated interstitial lung disease (SSc-ILD) in comparison with lung fibroblasts from healthy controls. These unbiased tests revealed a tendency for all sirtuins to be expressed at lower levels in fibroblasts from patients compared with controls, but the greatest decrease was observed with SIRT7. Similarly, SIRT7 was decreased in lung tissues of bleomycin-challenged mice. Inhibition of SIRT7 with siRNA in cultured lung fibroblasts resulted in an increase in collagen and α-smooth muscle actin (α-SMA). Reciprocally, overexpression of SIRT7 resulted in lower basal and TGF-β-induced levels of COL1A1, COL1A2, COL3A1, and α-SMA mRNAs, as well as collagen and α-SMA proteins. Induced changes in SIRT7 had no effect on endogenous TGF-β mRNA levels or latent TGF-β activation, but overexpression of SIRT7 reduced the levels of Smad3 mRNA and protein. In conclusion, the decline in SIRT7 in lung fibroblasts has a profibrotic effect, which is mediated by changes in Smad3 levels.

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.

Interleukin-1α drives the dysfunctional cross-talk of the airway epithelium and lung fibroblasts in COPD.

Chronic obstructive pulmonary disease (COPD) has been associated with aberrant epithelial-mesenchymal interactions resulting in inflammatory and remodelling processes. We developed a co-culture model using COPD and control-derived airway epithelial cells (AECs) and lung fibroblasts to understand the mediators that are involved in remodelling and inflammation in COPD.AECs and fibroblasts obtained from COPD and control lung tissue were grown in co-culture with fetal lung fibroblast or human bronchial epithelial cell lines. mRNA and protein expression of inflammatory mediators, pro-fibrotic molecules and extracellular matrix (ECM) proteins were assessed.Co-culture resulted in the release of pro-inflammatory mediators interleukin (IL)-8/CXCL8 and heat shock protein (Hsp70) from lung fibroblasts, and decreased expression of ECM molecules (e.g. collagen, decorin) that was not different between control and COPD-derived primary cells. This pro-inflammatory effect was mediated by epithelial-derived IL-1α and increased upon epithelial exposure to cigarette smoke extract (CSE). When exposed to CSE, COPD-derived AECs elicited a stronger IL-1α response compared with control-derived airway epithelium and this corresponded with a significantly enhanced IL-8 release from lung fibroblasts.We demonstrate that, through IL-1α production, AECs induce a pro-inflammatory lung fibroblast phenotype that is further enhanced with CSE exposure in COPD, suggesting an aberrant epithelial-fibroblast interaction in COPD.

Extracellular Matrix Metalloproteinase Inducer (EMMPRIN) promotes lung fibroblast proliferation, survival and differentiation to myofibroblasts

BackgroundIdiopathic pulmonary fibrosis (IPF) is a chronic progressively fatal disease. Extracellular Matrix Metalloproteinase Inducer (EMMPRIN) is a glycosylated transmembrane protein that induces the expression of some matrix metalloproteinase (MMP) in neighboring stromal cells through direct epithelial–stromal interactions. EMMPRIN is highly expressed in type II alveolar epithelial cells at the edges of the fibrotic areas in IPF lung sections. However, the exact role of EMMPRIN in IPF is unknown.MethodsTo determine if EMMPRIN contributes to lung fibroblast proliferation, resistance to apoptosis, and differentiation to myofibroblasts, normal Human lung fibroblasts (NHLF) transiently transfected with either EMMPRIN/GFP or GFP were treated with TGF- β1 from 0 to 10 ng/ml for 48 h and examined for cell proliferation (thymidine incorporation), apoptosis (FACS analysis and Cell Death Detection ELISA assay), cell migration (Modified Boyden chamber) and differentiation to myofibroblasts using Western blot for α–smooth actin of cell lysates. The effect of EMMPRIN inhibition on NHLF proliferation, apoptosis, migration and differentiation to myofibroblasts after TGF- β1 treatment was examined using EMMPRIN blocking antibody. We examined the mechanism by which EMMPRIN induces its effects on fibroblasts by studying the β-catenin/canonical Wnt signaling pathway using Wnt luciferase reporter assays and Western blot for total and phosphorylated β-catenin.ResultsHuman lung fibroblasts overexpressing EMMPRIN had a significant increase in cell proliferation and migration compared to control fibroblasts. Furthermore, EMMPRIN promoted lung fibroblasts resistance to apoptosis. Lung fibroblasts overexpressing EMMPRIN showed a significantly increased expression of α- smooth muscle actin, a marker of differentiation to myofibroblasts compared to control cells. TGF-β1 increased the expression of EMMPRIN in lung fibroblasts in a dose-dependent manner. Attenuation of EMMPRIN expression with the use of an EMMPRIN blocking antibody markedly inhibited TGF-β1 induced proliferation, migration, and differentiation of fibroblasts to myofibroblasts. EMMPRIN overexpression in lung fibroblasts was found to induce an increase in TOPFLASH luciferase reporter activity when compared with control fibroblasts.ConclusionThese findings indicate that TGF-β1 induces the release of EMMPRIN that activates β-catenin/canonical Wnt signaling pathway. EMMPRIN overexpression induces an anti-apoptotic and pro-fibrotic phenotype in lung fibroblasts that may contribute to the persistent fibro-proliferative state seen in IPF.

Targeting of Proteoglycan Synthesis Pathway: A New Strategy to Counteract Excessive Matrix Proteoglycan Deposition and Transforming Growth Factor-β1-Induced Fibrotic Phenotype in Lung Fibroblasts.

Stimulation of proteoglycan (PG) synthesis and deposition plays an important role in the pathophysiology of fibrosis and is an early and dominant feature of pulmonary fibrosis. Transforming growth factor-β1 (TGF-β1) is a major cytokine associated with fibrosis that induces excessive synthesis of matrix proteins, particularly PGs. Owing to the importance of PGs in matrix assembly and in mediating cytokine and growth factor signaling, a strategy based on the inhibition of PG synthesis may prevent excessive matrix PG deposition and attenuates profibrotic effects of TGF-β1 in lung fibroblasts. Here, we showed that 4-MU4-deoxy-β-D-xylopyranoside, a competitive inhibitor of β4-galactosyltransferase7, inhibited PG synthesis and secretion in a dose-dependent manner by decreasing the level of both chondroitin/dermatan- and heparin-sulfate PG in primary lung fibroblasts. Importantly, 4-MU4-deoxy-xyloside was able to counteract TGF-β1-induced synthesis of PGs, activation of fibroblast proliferation and fibroblast-myofibroblast differentiation. Mechanistically, 4-MU4-deoxy-xyloside treatment inhibited TGF-β1-induced activation of canonical Smads2/3 signaling pathway in lung primary fibroblasts. The knockdown of β4-galactosyltransferase7 mimicked 4-MU4-deoxy-xyloside effects, indicating selective inhibition of β4-galactosyltransferase7 by this compound. Collectively, this study reveals the anti-fibrotic activity of 4-MU4-deoxy-xyloside and indicates that inhibition of PG synthesis represents a novel strategy for the treatment of lung fibrosis.

TGF-β1 epigenetically modifies Thy-1 expression in primary lung fibroblasts.

Idiopathic pulmonary fibrosis is a progressive lung disease that increases in incidence with age. We identified a profibrotic lung phenotype in aging mice characterized by an increase in the number of fibroblasts lacking the expression of thymocyte differentiation antigen 1 (Thy-1) and an increase in transforming growth factor (TGF)-β1 expression. It has been shown that Thy-1 expression can be epigenetically modified. Lung fibroblasts (PLFs) were treated with TGF-β1 ± DNA methyltransferase (DNMT) inhibitor 5-aza-2'-deoxycytidine (5-AZA) and analyzed for Thy-1 gene and protein expression, DNMT protein expression, and activity. α-Smooth muscle actin (α-SMA) and collagen type 1 (Col1A1) gene and protein expression was assessed. PLFs were transfected with DNMT1 silencing RNA ± TGF-β1. TGF-β1 inhibited Thy-1 gene and protein expression in PLFs, and cotreatment with 5-AZA ameliorated this effect and appeared to inhibit DNMT1 activation. TGF-β1 induced Thy-1 promoter methylation as assessed by quantitative methyl PCR. Treatment with 5-AZA attenuated TGF-β1-induced Col1A1 gene and protein expression and α-SMA gene expression (but not α-SMA protein expression). Inhibiting DNMT1 with silencing RNA attenuated TGF-β1-induced DNMT activity and its downstream suppression of Thy-1 mRNA and protein expression as well as inhibited α-SMA mRNA and Col1A1 mRNA and protein expression, and showed a decreased trend in Thy-1 promoter methylation. Immunofluorescence for α-SMA suggested that 5-AZA inhibited stress fiber formation. These findings suggest that TGF-β1 epigenetically regulates lung fibroblast phenotype through methylation of the Thy-1 promoter. Targeted inhibition of DNMT in the right clinical context might prevent fibroblast to myofibroblast transdifferentiation and collagen deposition, which in turn could prevent fibrogenesis in the lung and other organs.

Sustained Activation of Toll-Like Receptor 9 Induces an Invasive Phenotype in Lung Fibroblasts: Possible Implications in Idiopathic Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is characterized by excessive scarring of the lung parenchyma, resulting in a steady decline of lung function and ultimately respiratory failure. The disease course of IPF is extremely variable, with some patients exhibiting stability of symptoms for prolonged periods of time, whereas others exhibit rapid progression and loss of lung function. Viral infections have been implicated in IPF and linked to disease severity; however, whether they directly contribute to progression is unclear. We previously classified patients as rapid and slow progressors on the basis of clinical features and expression of the pathogen recognition receptor, Toll-like receptor 9 (TLR9). Activation of TLR9 in vivo exacerbated IPF in mice and induced differentiation of myofibroblasts in vitro, but the mechanism of TLR9 up-regulation and progression of fibrosis are unknown. Herein, we investigate whether transforming growth factor (TGF)-β, a pleiotropic cytokine central to IPF pathogenesis, regulates TLR9 in lung myofibroblasts. Results showed induction of TLR9 expression by TGF-β in lung myofibroblasts and a distinct profibrotic myofibroblast phenotype driven by stimulation with the TLR9 agonist, CpG-DNA. Chronic TLR9 stimulation resulted in stably differentiated α-smooth muscle actin(+)/platelet-derived growth factor receptor α(+)/CD44(+)/matrix metalloproteinase-14(+)/matrix metalloproteinase-2(+) myofibroblasts, which secrete inflammatory cytokines, invade Matrigel toward platelet-derived growth factor, and resist hypoxia-induced apoptosis. These results suggest a mechanism by which TGF-β and TLR9 responses in myofibroblasts collaborate to drive rapid progression of IPF.

HMGB1 contributes to allergen-induced airway remodeling in a murine model of chronic asthma by modulating airway inflammation and activating lung fibroblasts.

The pro-inflammation factor high-mobility group box protein 1 (HMGB1) has been implicated in the pathogenesis of asthma. In this study, we used a murine model of chronic asthma to evaluate the effects of HMGB1 on airway remodeling. Female BALB/c mice were randomly divided into four groups: control, ovalbumin (OVA) asthmatic, OVA+isotype antibody and OVA+anti-HMGB1 antibody. Anti-HMGB1 antibody therapy was started on day 21 and was administered three times per week for 6 weeks before intranasal challenge with OVA. In this mouse model, HMGB1 expression is significantly elevated. The anti-HMGB1 antibody group exhibited decreased levels of immunoglobulin E (IgE) and inflammatory mediators and reduced inflammatory cell accumulation, airway hyperresponsiveness (AHR), mucus synthesis, smooth muscle thickness and lung collagen content compared with the OVA groups. Treatment with HMGB1 increased proliferation, migration, collagen secretion and α-smooth muscle actin (SMA) expression in MRC-5 cells. Treatment with the HMGB1/IL-1β complex significantly increased the expression and secretion of transforming growth factor (TGF-β1), matrix metalloproteinase (MMP)-9 and vascular endothelial growth factor (VEGF). Altogether, these results suggest that blocking HMGB1 activity may reverse airway remodeling by suppressing airway inflammation and modulating lung fibroblast phenotype and activation.

Immunopatogeneza Samoistnego Włóknienia Płuc

The paper presents the state of the art in the pathogenesis of idiopathic pulmonary fibrosis (IPF). Both, etiology and pathogenesis of IPF are unclear. The key elements in the pathogenesis of the disease are epithelial destruction and dysregulation of the phenotype of lung fibroblasts. Currently accepted hypothesis claims that IPF is not related to underlying inflammatory state but it is rather a result of pathological interaction between pulmonary epithelium and mesenchyme followed by disturbed healing of damaged alveolar epithelial cells. The function of lung progenitor cells residing in distant lung structures is also impaired. Some scientists claim that genetic defect causes fast shortening of telomeres reducing lung properties of regeneration. According to the current knowledge, IFP is a multifactorial disease resulting from a repeated cycle of injuries followed by pathological regeneration.