Murine Lung Fibroblasts Research Articles

Mechanomemory of pulmonary fibroblasts demonstrates reversibility of transcriptomics and contraction phenotypes

Fibroblasts are cells responsible for producing extracellular matrix (ECM) components, which provides physical support for organs. Although these mesenchymal cells are responsive to mechanical cues in their environment, the permanence of these mechanophenotypes is not well defined. We investigated the mechanomemory of lung fibroblasts and determined how switching culture conditions modulate cell responses and function. Primary murine lung fibroblasts were isolated and cultured on 2D tissue culture plates or within 3D collagen hydrogels and were then passaged within the same or opposite culture condition to assess changes in gene expression, protein production, fibroblast subpopulation, contractile behavior, and traction forces. Compared to fibroblasts isolated on 2D tissue culture plates, fibroblasts within 3D hydrogels exhibited a decreased activation phenotype including reduced contraction profiles, diminished cell traction forces and decreased αSMA gene expression. Cells initially isolated via 2D culture and then cultured in 3D hydrogels exhibited a reversal in activation phenotype as measured by gene expression and contraction profiles. Bulk RNAseq identified groups of genes that exhibit reversible and non-reversable expression patterns. Overall, these findings indicate that lung fibroblasts have a mechanical memory that is altered by culture condition and can be reversible through precondition of cells within a softer 3D microenvironment.

Cell-Type-Specific Effect of Innate Immune Signaling on Stress Granules

Stress granules (SGs) are cytoplasmic membraneless compartments that can form in stressed cells. There is an intricate relationship between SGs and innate immune signaling pathways. A previous study reported that the innate immune signaling mediated by Toll-like receptors (TLRs) can inhibit SGs induced by endoplasmic reticulum stress (ER stress) in bone-marrow-derived macrophages (BMDMs) and the chemotherapy drug oxaliplatin in B16 melanoma cells. We wanted to test if this observation can be generalized to other cell types. First, we recapitulated the results from the previous study showing TLR signaling-mediated inhibition of SGs in BMDMs induced by ER stress. However, SGs formed in response to ER stress were either not inhibited or only very weakly inhibited by TLR4 stimulation in human lung cancer-derived A549 cells, murine immortalized mouse lung fibroblasts (iMLFs) and primary murine mouse lung fibroblasts. This correlated with a weak induction of IKK complex kinase activity by TLR4 stimulation in these cells. SGs formed by sodium arsenite treatment also remained unaffected by TLR4 signaling. Our results indicate that the innate immune signaling-mediated inhibition of SGs is cell-type-dependent, thus opening a new avenue for mechanistic studies of the crosstalk between innate immune and stress signaling pathways.

Selenite selectively kills lung fibroblasts to treat bleomycin-induced pulmonary fibrosis

BackgroundInterstitial lung disease (ILD) treatment is a critical unmet need. Selenium is an essential trace element for human life and an antioxidant that activates glutathione, but the gap between its necessity and its toxicity is small and requires special attention. Whether selenium can be used in the treatment of ILD remains unclear. MethodsWe investigated the prophylactic and therapeutic effects of selenite, a selenium derivative, in ILD using a murine model of bleomycin-induced idiopathic pulmonary fibrosis (IPF). We further elucidated the underlying mechanism using in vitro cell models and examined their relevance in human tissue specimens. The therapeutic effect of selenite in bleomycin-administered mice was assessed by respiratory function and histochemical changes. Selenite-induced apoptosis and reactive oxygen species (ROS) production in murine lung fibroblasts were measured. ResultsSelenite, administered 1 day (inflammation phase) or 8 days (fibrotic phase) after bleomycin, prevented and treated deterioration of lung function and pulmonary fibrosis in mice. Mechanistically, selenite inhibited the proliferation and induced apoptosis of murine lung fibroblasts after bleomycin treatment both in vitro and in vivo. In addition, selenite upregulated glutathione reductase (GR) and thioredoxin reductase (TrxR) in murine lung fibroblasts, but not in lung epithelial cells, upon bleomycin treatment. GR and TrxR inhibition eliminates the therapeutic effects of selenite. Furthermore, we found that GR and TrxR were upregulated in the human lung fibroblasts of IPF patient samples. ConclusionsSelenite induces ROS production and apoptosis in murine lung fibroblasts through GR and TrxR upregulation, thereby providing a therapeutic effect in bleomycin-induced IPF.

Inhibition of Galectin-1 attenuates lung fibroblast activation and proliferation in lung fibrosis.

The purpose of this study was to investigate the parenchymal changes in idiopathic pulmonary fibrosis (IPF) caused by massive fibroblastic infiltration and proliferation in lung tissue. Galectin-1 (Gal-1) has been reported to be involved in angiogenesis and fibrosis via modification of TGF-b receptor signaling pathways. However, it remains unknown whether Galectin-1 plays a critical role in IPF. In the current study, we aimed to identify Gal-1 as a crucial fibrotic protein in IPF process. Murine lung fibroblast was pre-treated using Gal-1 inhibitor OTX-008 or overexpression of Gal-1 and then activated using transforming growth factor-beta (TGF-β). Adult male C57BL/6J mice were conducted intratracheal injection of bleomycin (BLM) for lung fibrosis. Mice were conducted OTX-008 administration. Gal-1 expression, fibroblast activation and proliferation, extracellular matrix (ECM), lung fibrosis, lung histology and pulmonary function were investigated respectively. We demonstrated that Gal-1, as a positive pro-fibrotic marker, could promote lung fibroblast activation and proliferation. Inhibition of Gal-1 reduced fibroblast activation and proliferation through negative regulation of TGF-β/Erk1/2 and AKT pathway. In vivo, Gal-1 inhibition ameliorates lung fibroblast accumulation and protects lung histology and function. Gal-1 is verified to be a pro-fibrotic gene in IPF pathogenesis, which promotes fibroblast activation and proliferation via TGF-β/Erk1/2 and AKT pathway. Moreover, inhibition of Gal-1 in lung fibrosis model attenuates lung fibroblast bioactivity and reduces ECM, leading to improved pulmonary histology and function. Hence, knockdown of Gal-1 in IPF may be a promising target therapy.

Paroxetine protects against bleomycin-induced pulmonary fibrosis by blocking GRK2/Smad3 pathway.

G protein-coupled receptor kinase-2 (GRK2) is involved in TGF-β1-induced activation of lung fibroblasts, which could give rise to the pathogenesis of pulmonary fibrosis. Paroxetine (PRXT) serves as a selective GRK2 inhibitor which is widely used to treat anxiety and depression for several decades. However, whether PRXT could inhibit TGF-β1-induced activation of lung fibroblasts and combat bleomycin-induced pulmonary fibrosis remains unclear. Here, we investigated the effects of PRXT on pulmonary fibrosis in C57/BL6 caused by bleomycin as well as on the activation of murine primary lung fibroblasts stimulated with TGF-β1. The results demonstrated that PRXT markedly improved the pulmonary function and 21-day survival in bleomycin-induced mice. Meanwhile, PRXT significantly decreased collagen deposition, inflammation, and oxidative stress in lung tissues from bleomycin-induced mice. Furthermore, we found that PRXT could inhibit the protein and mRNA expression of GRK2 and Smad3 in lung tissues from bleomycin-induced mice. In vitro experiments also PRXT could inhibit cell activation and collagen synthesis in a concentration-dependent manner in TGF-β1-induced lung fibroblasts. In addition, we found that Smad3 overexpression by adenovirus transfection could offset anti-fibrotic and antioxidative effects from PRXT in TGF-β1-induced lung fibroblasts, which showed no effects on the protein expression of GRK2. In conclusion, PRXT mediates the inhibition of GRK2, which further blocks the transcription of Smad3 in TGF-β1-induced lung fibroblasts, providing an attractive therapeutic target for pulmonary fibrosis.

Nesfatin-1, a novel energy-regulating peptide, alleviates pulmonary fibrosis by blocking TGF-β1/Smad pathway in an AMPKα-dependent manner

Pulmonary fibrosis is a chronic progressive disease which steadily causes a critical public health concern. Nesfatin-1, a novel energy-regulating peptide discovered in 2006, could increase the level of AMPK phosphorylation. Previous studies have unveiled that Nesfatin-1 possessed many pharmacological effects including anti-inflammation, anti-oxidative stress, anti-fibrosis, and the regulation of lipid metabolism. Here, we investigated the impact of Nesfatin-1 on pulmonary fibrosis. Male C57BL/6J mice were intraperitoneally injected with Nesfatin-1 (10 μg·kg−1·day−1) for 21 days since mice were intratracheally administrated with bleomycin (BLM) (2 U/kg). Primary murine lung fibroblasts were stimulated with TGF-β1 (10 ng/ml) for 48 h. The results showed that Nesfatin-1 treatment significantly improved pulmonary function and decreased the production of collagen in BLM-treated mice. Meantime, Nesfatin-1 treatment also inhibited oxidative stress and inflammation in lung tissues from BLM-treated mice. Mechanically, Nesfatin-1 blocked the activation of TGF-β1/Smad2/3 signaling pathway in lung tissues challenged with BLM. In addition, we found that Nesfatin-1 enhanced the phosphorylation of AMPKα during pulmonary fibrosis. However, pharmacological inhibition or genetic deletion of AMPKα could both offset the pulmonary protection mediated by Nesfatin-1 during pulmonary fibrosis. Our experimental results firstly show Nesfatin-1 might serve as a novel treatment or adjuvant against pulmonary fibrosis by blocking TGF-β1/Smad pathway in an AMPKα-dependent manner.

Thyroid hormone modulates hyperoxic neonatal lung injury and mitochondrial function.

Mitochondrial dysfunction at birth predicts bronchopulmonary dysplasia (BPD) in extremely low-birth weight (ELBW) infants. Recently, nebulized thyroid hormone (TH), given as triiodothyronine (T3), was noted to decrease pulmonary fibrosis in adult animals through improved mitochondrial function. In this study, we tested the hypothesis that TH may have similar effects on hyperoxia-induced neonatal lung injury and mitochondrial dysfunction by testing whether i.n. T3 decreases neonatal hyperoxic lung injury in newborn mice; whether T3 improves mitochondrial function in lung homogenates, neonatal murine lung fibroblasts (NMLFs), and umbilical cord-derived mesenchymal stem cells (UC-MSCs) obtained from ELBW infants; and whether neonatal hypothyroxinemia is associated with BPD in ELBW infants. We found that inhaled T3 (given i.n.) attenuated hyperoxia-induced lung injury and mitochondrial dysfunction in newborn mice. T3 also reduced bioenergetic deficits in UC-MSCs obtained from both infants with no or mild BPD and those with moderate to severe BPD. T3 also increased the content of peroxisome proliferator-activated receptor γ coactivator 1α in lung homogenates of mice exposed to hyperoxia as well as mitochondrial potential in both NMLFs and UC-MSCs. ELBW infants who died or developed moderate to severe BPD had lower total T4 (TT4) compared with survivors with no or mild BPD. In conclusion, TH signaling and function may play a critical role in neonatal lung injury, and inhaled T3 supplementation may be useful as a therapeutic strategy for BPD.

Optimized Protocol for Isolation and Culture of Murine Neonatal Primary Lung Fibroblasts.

During all the stages of lung development, the lung mesoderm (or mesenchyme) is closely related to the endoderm, and their cross-regulation promotes, controls, and drives all lung developmental processes. Generation of 3D organoids in vitro, RNA assays, and mitochondrial respiration studies are used to analyze lung development and regeneration to better understand the interactions between epithelium and mesenchyme, as well as for the study of redox alterations and the metabolic status of the cells. Moreover, to avoid using immortalized cell lines in these in vitro approaches, standardized murine neonatal primary lung fibroblast isolation techniques are essential. Here, we present an optimized method to isolate, culture, and freeze primary lung fibroblasts from neonatal lungs. Our current method includes step-by-step instructions accompanied by graphical explanations and critical steps.

IRAK-M Regulates Proliferative and Invasive Phenotypes of Lung Fibroblasts.

Lung fibroblasts play an important role in subepithelial fibrosis, one feature for airway remodeling. IL-1 receptor-associated kinase (IRAK)-M was shown to involve fibrosis formation in airways and lung through regulation of inflammatory responses. IRAK-M is expressed by lung fibroblasts, whether IRAK-M has direct impact on lung fibroblasts remains unclear. In this investigation, we evaluated in vitro effect of IRAK-M on phenotypes of lung fibroblasts by silencing or overexpressing IRAK-M. Murine lung fibroblasts (MLg) were stimulated with house dust mite (HDM), IL-33, and transforming growth factor (TGF) β1. Techniques of small interfering RNA or expression plasmid were employed to silence or overexpress IRAK-M in MLg fibroblast cells. Proliferation, migration, invasiveness, and fibrosis-related events were evaluated. Significant upregulation of IRAK-M expression in MLg cells was caused by these stimuli. Silencing IRAK-M significantly increased proliferation, migration, and invasiveness of lung fibroblasts regardless of stimulating conditions. By contrast, IRAK-M overexpression significantly inhibited proliferation and motility of MLg lung fibroblasts. IRAK-M overexpression also significantly decreased the expression of fibronectin, collagen I, and α-SMA in MLg cells. Under stimulation with TGFβ1 or IL-33, IRAK-M silencing reduced MMP9 production, while IRAK-M overexpression increased MMP9 production. Modulation of IRAK-M expression affected cytokines production, either decreased or increased expression of TNFα and CXCL10 by the cells regardless of stimulation. Our in vitro data reveal that IRAK-M directly impacts on lung fibroblasts through modulation of cellular motility, release of inflammatory, and fibrotic cytokines of lung fibroblasts. These might suggest a new target by regulation of IRAK-M in slowing airway remodeling.

Verbascoside and isoverbascoside ameliorate transforming growth factor β1-induced collagen expression by lung fibroblasts through Smad/non-Smad signaling pathways

AimsPulmonary fibrosis (PF) is a chronic, irreversible, and debilitating lung disease that typically leads to respiratory failure, and is a major cause of morbidity and mortality. Few drugs are effective for the treatment of patients with PF or for reducing the rate of disease progression. Main methodsTransforming growth factor-β1 (TGF-β1) is a profibrotic cytokine that signals through Smad and non-Smad pathways. Verbascoside (VB) and isoverbascoside (isoVB) exhibit anti-oxidative and anti-inflammatory activities, however, their anti-fibrotic effects remain unclear. This study evaluated the effects of VB and isoVB on TGF-β1-stimulated murine lung fibroblasts (MLg 2908) and also human lung fibroblasts (confirmed by immunostaining). Key findingsNeither VB nor isoVB had a cytotoxic effect on MLg 2908 fibroblasts. Both compounds (10 μM) reduced intracellular reactive oxygen species and markedly attenuated collagen I expression in TGF-β1 (5 ng/ml)-induced MLg 2908 cells compared to TGF-β1 alone. Both compounds suppressed the TGF-β1-induced phosphorylation of Smad2/3 and ERK/p38 mitogen-activated protein kinases (MAPKs). VB and isoVB, but not pirfenidone and nintedanib, inhibited TGF-β1-induced pSmad2/3, ERK/p38 MAPK, and collagen I expression. VB and isoVB also decreased collagen I deposition in TGF-β1-induced MLg 2908 cells. Only isoVB significantly suppressed collagen I deposition in TGF-β1-induced human pulmonary cells. Our results indicated that VB and isoVB may exert antifibrotic effects by inhibiting TGF-β1-induced collagen I expression via inhibition of oxidative stress and downregulation of the Smad/non-Smad pathway. SignificanceThe present findings suggest that VB or isoVB may be used as a supplement to alleviate PF.

Fibroblast-derived conditioned media promotes lung cancer progression

Lung cancer is the leading cause of cancer death in men and women in the United States. Recent studies have implicated the tumor microenvironment as a new chemotherapeutic target by demonstrating the importance of tumor cell-stromal interactions in cancer progression. However, the exact mechanisms by which tumor cell-stromal interactions drive lung cancer progression remain undefined, particularly in the lung. We suspect host fibroblasts represent an important component of the tumor microenvironment that drives tumor progression. We found that human non-small cell lung carcinoma cell lines show alterations in cell morphology, proliferation, migration, and colony formation on soft agar when exposed to fibroblast-conditioned media (FCM). Interestingly, FCM also promoted tumor cell resistance to cisplatin-induced apoptosis. These effects varied depending on the cancer cell line used. Similar observations were made when exposing murine Lewis Lung Carcinoma cells to conditioned media harvested from primary murine lung fibroblasts. Certain effects of FCM, but not all, could be prevented by using a cMET inhibitor. In vivo, we observed enhanced growth of the primary tumors when treated with FCM, but no changes in metastatic behavior. Although the identity of the stimulating agent(s) in the fibroblast-conditioned media was not unveiled, further studies revealed that the activity is more than one factor with a high-molecular weight (over 100 kDa). These studies implicate lung fibroblast-derived factors in lung cancer progression. These data suggest that targeting the lung tumor stroma alone, or in combination with other interventions, is a promising concept that warrants further study in the setting of lung cancer.

Piceatannol-mediated JAK2/STAT3 signaling pathway inhibition contributes to the alleviation of oxidative injury and collagen synthesis during pulmonary fibrosis

Pulmonary fibrosis (PF) is characterized by oxidative injury and excessive collagen synthesis in lung fibroblasts, causing impaired pulmonary function and chronic lung injury. Piceatannol, a dietary polyphenol, possesses vital pharmacological effects in metabolic disorders, cancers, cardiovascular disease and infectious disease; however, its role in PF is still not completely elucidated. Mice (8 to 10 weeks old) were administered bleomycin (BLM) intratracheally (2 U/kg) to establish an in vivo PF model. Murine primary lung fibroblasts were isolated and stimulated with TGF-β (10 ng/mL) for 48 h to induce its activation. Meanwhile, mice or primary lung fibroblasts were treated with different doses of piceatannol to observe its protective roles. Pulmonary function and arterial blood gas were detected to assess pulmonary physiological status. Collagen deposition and the mRNA levels of profibrotic genes were determined by H&E staining and RT-PCR. Meanwhile, the protein and mRNA markers, as well as end-product of oxidative stress were detected in vivo and in vitro. The results showed that pulmonary function was significantly impaired in BLM-induced mice, accompanied by elevated oxidative stress and excessive collagen synthesis. Piceatannol significantly improved pulmonary function and decreased oxidative injury as well as collagen synthesis in mice with PF. Mechanically, piceatannol treatment significantly inhibited the activation of JAK2/STAT3 signaling pathway in BLM-induced mice and TGF-β-induced lung fibroblasts. Additional findings also demonstrated that coumermycin A1 (C-A1), an agonist of JAK2, could abolish the effects of piceatannol on TGF-β-induced lung fibroblasts and reactivated the phosphorylation STAT3. Taken together, our study demonstrated that piceatannol could protect against oxidative injury and collagen synthesis during PF in a JAK2/STAT3 signaling pathway-dependent manner.

Loss of p19Arf promotes fibroblast survival during leucine deprivation

ABSTRACTFibroblasts are quiescent and tumor suppressive in nature but become activated in wound healing and cancer. The response of fibroblasts to cellular stress has not been extensively investigated, however the p53 tumor suppressor has been shown to be activated in fibroblasts during nutrient deprivation. Since the p19 Alternative reading frame (p19Arf) tumor suppressor is a key regulator of p53 activation during oncogenic stress, we investigated the role of p19Arf in fibroblasts during nutrient deprivation. Here, we show that prolonged leucine deprivation results in increased expression and nuclear localization of p19Arf, triggering apoptosis in primary murine adult lung fibroblasts (ALFs). In contrast, the absence of p19Arf during long-term leucine deprivation resulted in increased ALF proliferation, migration and survival through upregulation of the Integrated Stress Response pathway and increased autophagic flux. Our data implicates a new role for p19Arf in response to nutrient deprivation. This article has an associated First Person interview with the first author of the paper.

Tetrandrine Modulates Rheb-mTOR Signaling-Mediated Selective Autophagy and Protects Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis is a progressive fatal disease characterized by interstitial remodeling, with high lethality and a lack of effective medical therapies. Tetrandrine has been proposed to present anti-fibrotic effects, but the efficacy and mechanisms have not been systematically evaluated. We sought to study the potential therapeutic effects and mechanisms of tetrandrine against lung fibrosis. The anti-fibrotic effects of tetrandrine were evaluated in bleomycin-induced mouse models and TGF-β1-stimulated murine lung fibroblasts. We performed Chromatin Immunoprecipitation (ChIP), Immunoprecipitation (IP), and mRFP-GFP-MAP1LC3B adenovirus construct to investigate the novel mechanisms of tetrandrine-induced autophagy. Tetrandrine decreased TGF-β1-induced expression of α-smooth muscle actin, fibronectin, vimentin, and type 1 collagen and proliferation in fibroblasts. Tetrandrine restored TGF-β1-induced impaired autophagy flux, accompanied by enhanced interaction of SQSTM1 and MAP1LC3-Ⅱ. ChIP studies revealed that tetrandrine induced autophagy via increasing binding of NRF2 and SQSTM1 promoter. Furthermore, tetrandrine inhibited TGF-β1-induced phosphorylation of mTOR by reducing activation of Rheb. In vivo tetrandrine suppressed the bleomycin-induced expression of fibrotic markers and improved pulmonary function. Our data suggest that protective effect of tetrandrine against lung fibrosis might be through promoting Rheb-mTOR and NRF2-SQSTM1 mediated autophagy. Tetrandrine may thus be potentially employed as a novel therapeutic medicine against IPF.

Dynamic Regulation of GH-IGF1 Signaling in Injury and Recovery in Hyperoxia-Induced Neonatal Lung Injury.

Prematurely born infants often require supplemental oxygen that impairs lung growth and results in arrest of alveolarization and bronchopulmonary dysplasia (BPD). The growth hormone (GH)- and insulin-like growth factor (IGF)1 systems regulate cell homeostasis and organ development. Since IGF1 is decreased in preterm infants, we investigated the GH- and IGF1 signaling (1) in newborn mice with acute and prolonged exposure to hyperoxia as well as after recovery in room air; and (2) in cultured murine lung epithelial cells (MLE-12) and primary neonatal lung fibroblasts (pLFs) after treatment with GH, IGF1, and IGF1-receptor (IGF1-R) inhibitor or silencing of GH-receptor (Ghr) and Igf1r using the siRNA technique. We found that (1) early postnatal hyperoxia caused an arrest of alveolarization that persisted until adulthood. Both short-term and prolonged hyperoxia reduced GH-receptor expression and STAT5 signaling, whereas Igf1 mRNA and pAKT signaling were increased. These findings were related to a loss of epithelial cell markers (SFTPC, AQP5) and proliferation of myofibroblasts (αSMA+ cells). After recovery, GH-R-expression and STAT5 signaling were activated, Igf1r mRNA reduced, and SFTPC protein significantly increased. Cell culture studies showed that IGF1 induced expression of mesenchymal (e.g., Col1a1, Col4a4) and alveolar epithelial cell type I (Hopx, Igfbp2) markers, whereas inhibition of IGF1 increased SFTPC and reduced AQP5 in MLE-12. GH increased Il6 mRNA and reduced proliferation of pLFs, whereas IGF1 exhibited the opposite effect. In summary, our data demonstrate an opposite regulation of GH- and IGF1- signaling during short-term/prolonged hyperoxia-induced lung injury and recovery, affecting alveolar epithelial cell differentiation, inflammatory activation of fibroblasts, and a possible uncoupling of the GH-IGF1 axis in lungs after hyperoxia.

CG223, a novel BET inhibitor, exerts TGF-β1-mediated antifibrotic effects in a murine model of bleomycin-induced pulmonary fibrosis

Pulmonary fibrosis is a progressive disease with poor prognosis and limited therapeutic options. In this study, we evaluated the potential therapeutic effects of CG223, a novel inhibitor of bromodomain and extra-terminal motif (BET) proteins, on pulmonary fibrosis by focusing on the transforming growth factor-β1 (TGF-β1) pathway. In a murine model of bleomycin-induced pulmonary fibrosis, CG223 attenuated fibrosis while reducing the infiltration of inflammatory cells into the lungs. Fibroblasts expressing BRD4, a member of the BET protein family, were enriched in the tissue regions corresponding to bleomycin-induced fibrotic lesions. Additionally, pulmonary fibroblasts isolated from bleomycin-instilled mice showed a significantly increased association of BRD4 with the promoters of two pro-fibrotic genes linked to the entry into the TGF-β1 autocrine/paracrine loop, thrombospondin 1 (Thbs1) and integrin β3 (Itgb3), as well as with the promoter of a myofibroblast marker gene, actin alpha 2 (Acta2). Subsequent in vitro studies with murine primary lung fibroblasts showed that the mRNA induction of Thbs1, Itgb3, and Acta2 by TGF-β1 can be inhibited by CG223 in a dose-dependent manner. Taken together, CG223-induced BRD4 inhibition suppressed lung fibrogenesis by affecting multiple genes, including those involved in the triggering of the TGF-β1 autocrine/paracrine loop.

Differential Engagement Of The Integrin Binding Domain Of Fibronectin Impacts Fibroblast Behavior

The switch between α5β1 and αvβ3 integrin engagement due to unfolding of the integrin binding domain (IBD) of fibronectin (Fn) has been theorized for a long time but was observed in vitro and murine models only recently. Fn is largely present in the extra cellular matrix (ECM) during wound healing and fibroproliferative diseases like idiopathic pulmonary fibrosis or cancer. Thus, further understanding the downstream effects of this differential integrin binding could elucidate several diseases’ mechanisms. Specifically, integrin α5β1 requires the regular, closed conformation of Fn's IBD, while αvβ3 can also bind the unfolded, open conformation. Moreover, cells can generate forces sufficient to cause such unfolding. We hypothesized that this change in Fn conformation affects lung fibroblasts by guiding integrin enrichment, pushing them towards a secretory and disease-relevant phenotype. We tested this premise by engineering Fn fragments containing the IBD site that mimicked either the open or closed conformation. Additionally, these fragments were designed to be covalently bound via thiol chemistry to a variety of substrates in order to preserve their structural integrity. Glass dishes coated with either Fn fragment were used as substrates for Single Cell Force Spectroscopy. By using fibroblasts expressing only α5β1, only αvβ3, and naïve murine lung fibroblasts we observed the need of proper α5β1 engagement to generate high adhesion forces even at short time points (<120s). Plating naïve fibroblasts on similarly functionalized dishes for up to 30 minutes enabled Mass Spec analysis of the adhesome and of the phosphorylation pathways emerging from the differential integrin engagement. Particularly, collagen XIIa and tubulin beta3 appeared to be enriched upon increased αvβ3 binding. Even on soft substrates (4kPa representing healthy lung tissue), lung fibroblasts plated on the open IBD Fn fragment engaged αvβ3 and display increased nuclear translocation of transcription factors associated with a secretory and contractile phenotype, after a 24h incubation. Fn (with Extra Domain A), Tropomyosin, Vimentin, Serpine1, and α integrins were among the genes upregulated on the open Fn fragment, compared to the closed fragment. These results were confirmed by qPCR and then bulk RNAseq assays. In conclusion, these data support that differential integrin engagement due to Fn IBD unfolding appears to affect cell phenotype in both the short and long run. Current efforts are focused on evaluating the efficacy of a novel αvβ3 inhibiting antibody on the progression of murine lung fibrosis. This work contributes to defining the link between changes in the ECM and cell behavior in the context of fibroproliferative diseases.

Fibroblast Expression of Thy-1 Protects Grafts from Chronic Lung Allograft Dysfunction

<h3>Purpose</h3> Multiple immunologic and physiologic insults can contribute to chronic lung allograft dysfunction (CLAD). Thy-1 is a surface glycoprotein that can downregulate fibroblast differentiation to myofibroblasts and fibroblast activation. The role of Thy-1 in CLAD has not been explored. <h3>Methods</h3> We utilized the minor antigen mismatched C57BL/6 (B6)→C57BL/10 (B10) model of murine lung transplantation. To mimic bacterial infection some mice received LPS (5μg in 50μl PBS/dose) intratracheally every 4 days until sacrifice on postoperative day 30 while some mice were treated with saline. We transplanted either wild type B6 or Thy-1 deficient grafts to B10 recipient mice (B6^wt→B10 vs. B6^Thy1-/−→B10). Grafts were assessed based on histological grading for obliterative bronchiolitis (OB) and parenchymal fibrosis. To assess the role of LPS on fibroblast-specific Thy-1 expression, we utilized primary cell cultures of murine lung fibroblasts isolated from B6 mice, using media containing 1μg/ml of LPS. <h3>Results</h3> LPS treatment resulted in CLAD which was more severe in B6^Thy1-/- donors(Figure A).More importantly immunohistochemical evaluation demonstrated that that all OB and fibrotic lesion, even in wild-type donors consisted of Thy-1 negative fibroblast. In vitro administration of LPS significantly downregulated Thy-1 in wild-type fibroblast through the downregulation of protein-specific mRNA (<i>P</i><0.05) (Figure B). <h3>Conclusion</h3> We for the first time demonstrate the link between the downregulation of Thy-1 and CLAD. We also demonstrate that LPS results in loss of Thy-1 on stromal cells thus contributing to proliferative airway lesions and parenchymal fibrosis. Our findings suggest that Thy-1 replacement may serve as a therapeutic option to prevent chronic rejection.

Extracellular BMP1 is the major proteinase for COOH-terminal proteolysis of type I procollagen in lung fibroblasts.

Proteolytic processing of procollagens is a central step during collagen fibril formation. Bone morphogenic protein 1 (BMP1) is a metalloprotease that plays an important role in the cleavage of carboxy-terminal (COOH-terminal) propeptides from procollagens. Although the removal of propeptides is required to generate mature collagen fibrils, the contribution of BMP1 to this proteolytic process and its action site remain to be fully determined. In this study, using postnatal lung fibroblasts as a model system, we showed that genetic ablation of Bmp1 in primary murine lung fibroblasts abrogated COOH-terminal cleavage from type I procollagen as measured by COOH-terminal propeptide of type I procollagen (CICP) production. We also showed that inhibition of BMP1 by siRNA-mediated knockdown or small-molecule inhibitor reduced the vast majority of CICP production and collagen deposition in primary human lung fibroblasts. Furthermore, we discovered and characterized two antibody inhibitors for BMP1. In both postnatal lung fibroblast and organoid cultures, BMP1 blockade prevented CICP production. Together, these findings reveal a nonredundant role of extracellular BMP1 to process CICP in lung fibroblasts and suggest that development of antibody inhibitors is a viable pharmacological approach to target BMP1 proteinase activity in fibrotic diseases.

Neuropilin-1 directs PDGFRα-entry into lung fibroblasts and signaling from very early endosomes.

Platelet-derived growth factor receptor-α (PDGFRα) is absolutely required for the development of secondary pulmonary alveolar septa. Our earlier observations indicated that PDGFRα resides intracellularly as well as on the plasma membrane of murine lung fibroblasts (LF). We have examined how neuropilin-1 (Nrp1), a surface receptor without kinase activity, regulates the intracellular trafficking of PDGFRα in LF obtained from mice, some bearing a targeted deletion of Nrp1 in myofibroblasts. Using the proximity ligation assay, we observed that PDGFRα and Nrp1 colocalized in both early antigen-1 (EEA1) containing sorting endosomes and with adaptor protein containing a pleckstrin homology domain and a phosphotyrosine-binding domain-1 (APPL1) in very early endosomes (VEE). These findings were confirmed using live-cell imaging, which demonstrated that recently internalized PDGFRα was observed in Rab5-containing vesicles residing within 100 nm of the plasma membrane. Nrp1 deletion reduced the phosphorylation of Akt (protein kinase B), the major downstream target of PDGFRα, and limited accumulation of inositol-3 phosphates in APPL1-containing endosomes after exposure to PDGFA. PDGFRα co-immunoprecipitated with APPL1, indicating that PDGFRα enters VEE. Targeted deletion of Nrp1 or APPL1-depletion in control LF reduced the activity of an Akt1 biosensor following stimulation with PDGFA. Our findings demonstrate that Nrp1 enhances the entry of PDGFRα into APPL1 containing VEE and that APPL1 enhances PDGFRα signaling. Therefore, Nrp1 promotes endosomal signaling by PDGFRα offering a potential mechanism to explain our prior observation that Nrp1 supports the formation of alveolar ducts and alveoli during secondary septation in mice.