Mouse Model Of Lung Fibrosis Research Articles

Time and phenotype-dependent transcriptome analysis in AAV-TGFβ1 and Bleomycin-induced lung fibrosis models

We have previously established a novel mouse model of lung fibrosis based on Adeno-associated virus (AAV)-mediated pulmonary overexpression of TGFβ1. Here, we provide an in-depth characterization of phenotypic and transcriptomic changes (mRNA and miRNA) in a head-to-head comparison with Bleomycin-induced lung injury over a 4-week disease course. The analyses delineate the temporal state of model-specific and commonly altered pathways, thereby providing detailed insights into the processes underlying disease development. They further guide appropriate model selection as well as interventional study design. Overall, Bleomycin-induced fibrosis resembles a biphasic process of acute inflammation and subsequent transition into fibrosis (with partial resolution), whereas the TGFβ1-driven model is characterized by pronounced and persistent fibrosis with concomitant inflammation and an equally complex disease phenotype as observed upon Bleomycin instillation. Finally, based on an integrative approach combining lung function data, mRNA/miRNA profiles, their correlation and miRNA target predictions, we identify putative drug targets and miRNAs to be explored as therapeutic candidates for fibrotic diseases. Taken together, we provide a comprehensive analysis and rich data resource based on RNA-sequencing, along with a strategy for transcriptome-phenotype coupling. The results will be of value for TGFβ research, drug discovery and biomarker identification in progressive fibrosing interstitial lung diseases.

Pivotal role of micro-CT technology in setting up an optimized lung fibrosis mouse model for drug screening

Idiopathic pulmonary fibrosis (IPF) is a progressive disease with no curative pharmacological treatment. The most used animal model of IPF for anti-fibrotic drug screening is bleomycin (BLM)-induced lung fibrosis. However, several issues have been reported: the balance among disease resolution, an appropriate time window for therapeutic intervention and animal welfare remain critical aspects yet to be fully elucidated. In this study, C57Bl/6 male mice were treated with BLM via oropharyngeal aspiration (OA) following either double or triple administration. The fibrosis progression was longitudinally assessed by micro-CT every 7 days for 4 weeks after BLM administration. Quantitative micro-CT measurements highlighted that triple BLM administration was the ideal dose regimen to provoke sustained lung fibrosis up to 28 days. These results were corroborated with lung histology and Bronchoalveolar Lavage Fluid cells. We have developed a mouse model with prolonged lung fibrosis enabling three weeks of a curative therapeutic window for the screening of putative anti-fibrotic drugs. Moreover, we have demonstrated the pivotal role of longitudinal micro-CT imaging in reducing the number of animals required per experiment in which each animal can be its own control. This approach permits a valuable decrease in costs and time to develop disease animal models.

754-P: Antifibrotic Potential of a Novel Long-Acting Glucagon/GIP/GLP-1 Triple Agonist (HM15211) in Preclinical Models of IPF

Idiopathic pulmonary fibrosis (IPF) is an irreversible and progressive pulmonary disorder in which scarring of lung tissue eventually leads to death. A large of studies have demonstrated that the core mechanisms in fibrosis across various organs are similar. Previously, excellent preclinical efficacy of HM15211, a novel long-acting Glucagon/GIP/GLP-1 triple agonist, on liver fibrosis was confirmed (the major distribution tissues of HM152were liver and lung) . These findings prompted us to hypothesize that HM152might be also a promising drug candidate for IPF. Its effect on pulmonary function and mortality was evaluated in bleomycin (BLM) -induced mouse model of lung fibrosis. BLM was instilled intratracheally to assess the therapeutic benefits of HM152over pirfenidone (PIRF) and nintedanib (NINT) (FDA-approved drugs for IPF) . Briefly, HM152was administered 7 days post BLM instillation, and then saturation of peripheral oxygen (SpO2) and survival rate were monitored until day 14 and day 21, respectively. 1.5 U/kg BLM mice showed markedly impaired lung function at day 7 as indicated by SpO2 (84.0 vs. 95.6% for normal) , which was further exacerbated until day 14 (77.1% vs. 96.0% for normal) . Interestingly, while PIRF (81.9%) and NINT (81.3%) prevent additional lung function impairment without any reversal effects, HM152treatment significantly restored impaired lung function (91.5%) at day 14. In addition, at 21 days after 2.5 U/kg BLM treatment, substantial decline in survival rate (17%) was effectively inhibited by HM15211, NINT, and PIRF. Notably, HM152 (61%) showed more improved survival rate than NINT (33%) , and PIRF (28%) , suggesting potential superiority of HM152over current IPF drugs. In conclusion, HM152may be a novel therapeutic potential for pulmonary fibrosis over current standard of care for IPF. Further evidences from the mechanistic studies will shed light on a direct inhibitory effect of HM152on pulmonary fibrosis. Disclosure S.Lee: None. I.Choi: None. J.A.Kim: None. J.Lee: None. J.Kim: None. Y.Ban: None. J.Lee: None. S.Bae: None. D.Kim: None. S.Lee: n/a.

BI 1015550 is a PDE4B Inhibitor and a Clinical Drug Candidate for the Oral Treatment of Idiopathic Pulmonary Fibrosis.

The anti-inflammatory and immunomodulatory abilities of oral selective phosphodiesterase 4 (PDE4) inhibitors enabled the approval of roflumilast and apremilast for use in chronic obstructive pulmonary disease and psoriasis/psoriatic arthritis, respectively. However, the antifibrotic potential of PDE4 inhibitors has not yet been explored clinically. BI 1015550 is a novel PDE4 inhibitor showing a preferential enzymatic inhibition of PDE4B. In vitro, BI 1015550 inhibits lipopolysaccharide (LPS)-induced tumor necrosis factor-α (TNF-α) and phytohemagglutinin-induced interleukin-2 synthesis in human peripheral blood mononuclear cells, as well as LPS-induced TNF-α synthesis in human and rat whole blood. In vivo, oral BI 1015550 shows potent anti-inflammatory activity in mice by inhibiting LPS-induced TNF-α synthesis ex vivo and in Suncus murinus by inhibiting neutrophil influx into bronchoalveolar lavage fluid stimulated by nebulized LPS. In Suncus murinus, PDE4 inhibitors induce emesis, a well-known gastrointestinal side effect limiting the use of PDE4 inhibitors in humans, and the therapeutic ratio of BI 1015550 appeared to be substantially improved compared with roflumilast. Oral BI 1015550 was also tested in two well-known mouse models of lung fibrosis (induced by either bleomycin or silica) under therapeutic conditions, and appeared to be effective by modulating various model-specific parameters. To better understand the antifibrotic potential of BI 1015550 in vivo, its direct effect on human fibroblasts from patients with idiopathic pulmonary fibrosis (IPF) was investigated in vitro. BI 1015550 inhibited transforming growth factor-β-stimulated myofibroblast transformation and the mRNA expression of various extracellular matrix proteins, as well as basic fibroblast growth factor plus interleukin-1β-induced cell proliferation. Nintedanib overall was unremarkable in these assays, but interestingly, the inhibition of proliferation was synergistic when it was combined with BI 1015550, leading to a roughly 10-fold shift of the concentration–response curve to the left. In summary, the unique preferential inhibition of PDE4B by BI 1015550 and its anticipated improved tolerability in humans, plus its anti-inflammatory and antifibrotic potential, suggest BI 1015550 to be a promising oral clinical candidate for the treatment of IPF and other fibro-proliferative diseases.

LncRNA DACH1 protects against pulmonary fibrosis by binding to SRSF1 to suppress CTNNB1 accumulation

Idiopathic pulmonary fibrosis (IPF) is a progressive disease with unknown etiology and limited therapeutic options. Activation of fibroblasts is a prominent feature of pulmonary fibrosis. Here we report that lncRNA DACH1 (dachshund homolog 1) is downregulated in the lungs of IPF patients and in an experimental mouse model of lung fibrosis. LncDACH1 knockout mice develop spontaneous pulmonary fibrosis, whereas overexpression of LncDACH1 attenuated TGF-β1-induced aberrant activation, collagen deposition and differentiation of mouse lung fibroblasts. Similarly, forced expression of LncDACH1 not only prevented bleomycin (BLM)-induced lung fibrosis, but also reversed established lung fibrosis in a BLM model. Mechanistically, LncDACH1 binding to the serine/arginine-rich splicing factor 1 (SRSF1) protein decreases its activity and inhibits the accumulation of Ctnnb1. Enhanced expression of SRSF1 blocked the anti-fibrotic effect of LncDACH1 in lung fibroblasts. Furthermore, loss of LncDACH1 promoted proliferation, differentiation, and extracellular matrix (ECM) deposition in mouse lung fibroblasts, whereas such effects were abolished by silencing of Ctnnb1. In addition, a conserved fragment of LncDACH1 alleviated hyperproliferation, ECM deposition and differentiation of MRC-5 cells driven by TGF-β1. Collectively, LncDACH1 inhibits lung fibrosis by interacting with SRSF1 to suppress CTNNB1 accumulation, suggesting that LncDACH1 might be a potential therapeutic target for pulmonary fibrosis.

Intracellular hydroxyproline imprinting following resolution of bleomycin-induced pulmonary fibrosis.

BackgroundIdiopathic pulmonary fibrosis (IPF) is a fatal lung disease with few treatment options. The poor success in developing anti-IPF strategies has impelled researchers to reconsider the importance of the choice of animal model and assessment methodologies. Currently, it is still not settled whether the bleomycin-induced lung fibrosis mouse model finally returns to resolution.MethodsThis study aimed to follow the dynamic fibrotic features of bleomycin-treated mouse lungs over extended durations through a combination of the latest technologies (micro-computed tomography imaging and histological detection of degraded collagens) and traditional methods. In addition, we also applied immunohistochemistry to explore the distribution of all hydroxyproline-containing molecules.ResultsAs determined by classical biochemical methods, total lung hydroxyproline contents reached a peak at 4 weeks after bleomycin injury and maintained a steady high level thereafter until the end of the experiments (16 weeks). This result seemed to partially contradict with the changes of other fibrosis evaluation parameters, which indicated a gradual degradation of collagens and a recovery of lung aeration after the fibrosis peak. This inconsistency was well reconciled by our data from immunostaining against hydroxyproline and fluorescent peptide staining against degraded collagen, together showing large amounts of hydroxyproline-rich degraded collagen fragments detained and enriched within the intracellular regions at 10 or 16 weeks rather than at 4 weeks after bleomycin treatment.ConclusionsOur present data not only offer respiratory researchers a new perspective towards the resolution nature of mouse lung fibrosis, but also remind them to be cautious when using the hydroxyproline content assay to evaluate the severity of fibrosis.

GSK-3 Inhibition Modulates Metalloproteases in a Model of Lung Inflammation and Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is mainly characterized by aberrant extracellular matrix deposition, consequent to epithelial lung injury and myofibroblast activation, and inflammatory response. Glycogen synthase kinase 3 (GSK-3) is a serine–threonine kinase involved in several pathways, and its inhibition has been already suggested as a therapeutic strategy for IPF patients. There is evidence that GSK-3 is able to induce matrix metalloproteinase (MMP) expression and that its inhibition modulates MMP expression in the tissues. The aim of our study was to investigate the role of GSK-3 and its inhibition in the modulation of MMP-9 and -2 in an in vivo mouse model of lung fibrosis and in vitro using different cell lines exposed to pro-inflammatory or pro-fibrotic stimuli. We found that GSK-3 inhibition down-modulates gene expression and protein levels of MMP-9, MMP-2, and their inhibitors TIMP-1 and TIMP-2 in inflammatory cells harvested from bronchoalveolar lavage fluid (BALF) of mice treated with bleomycin as well as in interstitial alveolar macrophages and cuboidalized epithelial alveolar cells. To the same extent, GSK-3 inhibition blunted the increased MMP-9 and MMP-2 activity induced by pro-fibrotic stimuli in a human lung fibroblast cell line. Moreover, the αSMA protein level, a marker of fibroblast-to-myofibroblast transition involved in fibrosis, was decreased in primary fibroblasts treated with TGFβ following GSK-3 inhibition. Our results confirm the implication of GSK-3 in lung inflammation and fibrosis, suggesting that it might play its role by modulating MMP expression and activity but also pushing fibroblasts toward a myofibroblast phenotype and therefore enhancing extracellular matrix deposition. Thus, its inhibition could represent a possible therapeutic strategy.

IL-18 binding protein can be a prognostic biomarker for idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis is a chronic, fibrosing interstitial pneumonia that presents with various clinical courses and progression ranging from rapid to slow. To identify novel biomarkers that can support the diagnosis and/or prognostic prediction of idiopathic pulmonary fibrosis, we performed gene expression analysis, and the mRNA of interleukin-18 binding protein was increasingly expressed in patients with idiopathic pulmonary fibrosis compared with healthy controls. Therefore, we hypothesized that the interleukin-18 binding protein can serve as a diagnostic and/or prognostic biomarker for idiopathic pulmonary fibrosis. We investigated the expression of interleukin-18 binding protein in lung tissue, bronchoalveolar lavage fluid, and serum. Additionally, the correlation between interleukin-18 binding protein expression levels and the extent of fibrosis was investigated using mouse models of lung fibrosis induced by subcutaneous bleomycin injections. Serum interleukin-18 binding protein levels were significantly higher in idiopathic pulmonary fibrosis patients (5.06 ng/mL, interquartile range [IQR]: 4.20-6.35) than in healthy volunteers (3.31 ng/mL, IQR: 2.84-3.99) (p < 0.001). Multivariate logistic regression models revealed that the correlation between serum interleukin-18 binding protein levels and idiopathic pulmonary fibrosis was statistically independent after adjustment for age, sex, and smoking status. Multivariate Cox proportional hazard models revealed that serum interleukin-18 binding protein levels were predictive of idiopathic pulmonary fibrosis disease prognosis independent of other covariate factors (hazard ratio: 1.655, 95% confidence interval: 1.224-2.237, p = 0.001). We also demonstrated a significant positive correlation between lung hydroxyproline expression levels and interleukin-18 binding protein levels in bronchoalveolar lavage fluid from bleomycin-treated mice (Spearman r = 0.509, p = 0.004). These results indicate the utility of interleukin-18 binding protein as a novel prognostic biomarker for idiopathic pulmonary fibrosis.

Club cell-specific role of programmed cell death 5 in pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) causes progressive fibrosis and worsening pulmonary function. Prognosis is poor and no effective therapies exist. We show that programmed cell death 5 (PDCD5) expression is increased in the lungs of patients with IPF and in mouse models of lung fibrosis. Lung fibrosis is significantly diminished by club cell-specific deletion of Pdcd5 gene. PDCD5 mediates β-catenin/Smad3 complex formation, promoting TGF-β-induced transcriptional activation of matricellular genes. Club cell Pdcd5 knockdown reduces matricellular protein secretion, inhibiting fibroblast proliferation and collagen synthesis. Here, we demonstrate the club cell-specific role of PDCD5 as a mediator of lung fibrosis and potential therapeutic target for IPF.

Preclinical MRI to quantify pulmonary disease severity and trajectories in poorly characterized mouse models: A pedagogical example using data from novel transgenic models of lung fibrosis

Structural remodeling in lung disease is progressive and heterogeneous, making temporally and spatially explicit information necessary to understand disease initiation and progression. While mouse models are essential to elucidate mechanistic pathways underlying disease, the experimental tools commonly available to quantify lung disease burden are typically invasive (e.g., histology). This necessitates large cross-sectional studies with terminal endpoints, which increases experimental complexity and expense. Alternatively, magnetic resonance imaging (MRI) provides information noninvasively, thus permitting robust, repeated-measures statistics. Although lung MRI is challenging due to low tissue density and rapid apparent transverse relaxation (T2* <1 ms), various imaging methods have been proposed to quantify disease burden. However, there are no widely accepted strategies for preclinical lung MRI. As such, it can be difficult for researchers who lack lung imaging expertise to design experimental protocols—particularly for novel mouse models. Here, we build upon prior work from several research groups to describe a widely applicable acquisition and analysis pipeline that can be implemented without prior preclinical pulmonary MRI experience. Our approach utilizes 3D radial ultrashort echo time (UTE) MRI with retrospective gating and lung segmentation is facilitated with a deep-learning algorithm. This pipeline was deployed to assess disease dynamics over 255 days in novel, transgenic mouse models of lung fibrosis based on disease-associated, loss-of-function mutations in Surfactant Protein-C. Previously identified imaging biomarkers (tidal volume, signal coefficient of variation, etc.) were calculated semi-automatically from these data, with an objectively-defined high signal volume identified as the most robust metric. Beyond quantifying disease dynamics, we discuss common pitfalls encountered in preclinical lung MRI and present systematic approaches to identify and mitigate these challenges. While the experimental results and specific pedagogical examples are confined to lung fibrosis, the tools and approaches presented should be broadly useful to quantify structural lung disease in a wide range of mouse models.

Restoration of SIRT3 gene expression by airway delivery resolves age-associated persistent lung fibrosis in mice.

Aging is a risk factor for progressive fibrotic disorders involving diverse organ systems, including the lung. Idiopathic pulmonary fibrosis, an age-associated degenerative lung disorder, is characterized by persistence of apoptosis-resistant myofibroblasts. In this report, we demonstrate that sirtuin-3 (SIRT3), a mitochondrial deacetylase, is downregulated in lungs of IPF human subjects and in mice subjected to lung injury. Over-expression of the SIRT3 cDNA via airway delivery restored capacity for fibrosis resolution in aged mice, in association with activation of the forkhead box transcription factor, FoxO3a, in fibroblasts, upregulation of pro-apoptotic members of the Bcl-2 family, and recovery of apoptosis susceptibility. While transforming growth factor-β1 reduced levels of SIRT3 and FoxO3a in lung fibroblasts, cell non-autonomous effects involving macrophage secreted products were necessary for SIRT3-mediated activation of FoxO3a. Together, these findings reveal a novel role of SIRT3 in pro-resolution macrophage functions that restore susceptibility to apoptosis in fibroblasts via a FoxO3a-dependent mechanism.

Aucuparin Suppresses Bleomycin-Induced Pulmonary Fibrosis Via Anti-Inflammatory Activity.

Idiopathic pulmonary fibrosis (IPF) is a lung disease that results in scarring of the lungs for an unknown reason. Although many studies have been conducted on IPF, precise mechanisms and treatments have not yet been identified. In this study, we found that aucuparin, a natural product isolated from Sorbus aucuparia, inhibited pulmonary fibrosis in a bleomycin (BLM)-induced lung fibrosis mouse model. In the lung samples of mice treated with aucuparin, the gene expression of inflammation and macrophage activation markers was reduced compared to those treated with BLM alone. Moreover, aucuparin decreased the expression of profibrotic marker genes and increased the expression of antifibrotic marker genes. Finally, we observed that aucuparin significantly suppressed transforming growth factor-β-induced activation of inflammatory cytokine production and collagen synthesis from macrophages and fibroblasts, respectively. Taken together, these data demonstrate that aucuparin inhibits lung fibrosis via its anti-inflammatory action and support its potential to be a therapeutic drug for IPF treatment.

Stearic acid attenuates profibrotic signalling in idiopathic pulmonary fibrosis.

Lipid metabolism dysregulation has been implicated in the pathogenesis of IPF; however, the roles of most lipid metabolites in lung fibrosis remain unexplored. Therefore, we aimed to identify changes in lipid metabolites in the lung tissues of IPF patients and determine their roles in pulmonary fibrosis. Free fatty acids in the lung tissues of IPF patients and controls were quantified using a metabolomic approach. The roles of free fatty acids in fibroblasts or epithelial cells treated with TGF-β1 were evaluated using fibrotic markers. The antifibrotic role of stearic acid was also assessed in a bleomycin-induced lung fibrosis mouse model. Protein levels in cell lysates or tissues were measured by western blotting. The levels of stearic acid were lower in IPF lung tissues than in control lung tissues. Stearic acid significantly reduced TGF-β1-induced α-SMA and collagen type 1 expression in MRC-5 cells. Furthermore, stearic acid decreased the levels of p-Smad2/3 and ROS in MRC-5 cells treated with TGF-β1 and disrupted TGF-β1-induced EMT in Beas-2B cells. Stearic acid reduced the levels of bleomycin-induced hydroxyproline in a mouse model. Changes in the free fatty acid profile, including low levels of stearic acid, were observed in IPF patients. Stearic acid may exert antifibrotic activity by regulating profibrotic signalling.

The p53/RMRP/miR122 signaling loop promotes epithelial-mesenchymal transition during the development of silica-induced lung fibrosis by activating the notch pathway

BackgroundUnderstanding the roles of long noncoding RNAs (lncRNAs) in EMT would help with establishing novel avenues for further uncovering the mechanisms of lung fibrosis and identifying preventative and therapeutic targets. This study aimed to identify silica-induced specific lncRNAs and investigate the feedback loop regulation among their upstream and downstream genes. Methods and materialsA microarray assay, quantitative real-time polymerase chain reaction and Western blot analysis dual-luciferase reporter gene activity and chromatin immunoprecipitation assays were used. Moreover, a silica-induced lung fibrosis mouse model was used to verify the roles of the lncRNAs. ResultsFollowing silica exposure, both RNA component of mitochondrial RNA processing endoribonuclease (RMRP) and p53 were significantly upregulated during the EMT. The upregulation of p53 upon silica exposure activated RMRP expression, which promoted the EMT. When RMRP is overexpressed, additional RMRP acts as a sponge to bind to miR122, thus decreasing miR122 levels. Using microarrays, miR122 was identified as a potential upstream regulator of p53. This relationship was also verified using the dual-luciferase reporter gene. Hence, decreased miR122 levels result in an increase in p53 activity. More importantly, RMRP promotes the transcription of Notch 1, which, in turn, results in Notch pathway activation. We show that the p53/RMRP/miR122 pathway creates a positive feedback loop that promotes EMT progress by activating the Notch signaling pathway. ConclusionOur data indicated that p53/RMRP/miR122 feedback loop might contribute to the EMT development by activating Notch pathway, which provides new sight into understanding of the complex network regulating silica-induced lung fibrosis.

NADPH oxidase DUOX1 sustains TGF-β1 signalling and promotes lung fibrosis.

Interstitial lung fibroblast activation coupled with extracellular matrix production is a pathological signature of pulmonary fibrosis, and is governed by transforming growth factor (TGF)-β1/Smad signalling. TGF-β1 and oxidative stress cooperate to drive fibrosis. Cells can produce reactive oxygen species through activation and/or induction of NADPH oxidases, such as dual oxidase (DUOX1/2). Since DUOX enzymes, as extracellular hydrogen peroxide (H2O2--)-generating systems, are involved in extracellular matrix formation and in wound healing in different experimental models, we hypothesised that DUOX-based NADPH oxidase plays a role in the pathophysiology of pulmonary fibrosis.Our in vivo data (idiopathic pulmonary fibrosis patients and mouse models of lung fibrosis) showed that the NADPH oxidase DUOX1 is induced in response to lung injury. DUOX1-deficient mice (DUOX1+/- and DUOX1-/-) had an attenuated fibrotic phenotype. In addition to being highly expressed at the epithelial surface of airways, DUOX1 appears to be well expressed in the fibroblastic foci of remodelled lungs. By using primary human and mouse lung fibroblasts, we showed that TGF-β1 upregulates DUOX1 and its maturation factor DUOXA1 and that DUOX1-derived H2O2 promoted the duration of TGF-β1-activated Smad3 phosphorylation by preventing phospho-Smad3 degradation. Analysis of the mechanism revealed that DUOX1 inhibited the interaction between phospho-Smad3 and the ubiquitin ligase NEDD4L, preventing NEDD4L-mediated ubiquitination of phospho-Smad3 and its targeting for degradation.These findings highlight a role for DUOX1-derived H2O2 in a positive feedback that amplifies the signalling output of the TGF-β1 pathway and identify DUOX1 as a new therapeutic target in pulmonary fibrosis.

MiR-301a Suppression within Fibroblasts Limits the Progression of Fibrosis through the TSC1/mTOR Pathway

Pulmonary fibrosis has been characterized by abnormal proliferation of fibroblasts and massive deposition of the extracellular matrix, which results from a complex interplay of chronic injury and inflammatory responses. MicroRNA-301a (miR-301a) is activated by multiple inflammatory stimulators, contributing to multiple tumorigenesis and autoimmune diseases. This study showed that miR-301a was overexpressed in a bleomycin-induced murine model of pulmonary fibrosis and patients with idiopathic pulmonary fibrosis (IPF). In addition, miR-301a was activated by transforming growth factor β (TGF-β) and interleukin 6 (IL-6) in normal and IPF fibroblasts, which was markedly reversed by the signal transducer and activator of transcription 3 (STAT3) inhibitor. The genetic ablation of miR-301a in mice reduced bleomycin-induced lung fibrosis, and the downregulation of miR-301a restrained proliferation and activation of fibroblasts. Furthermore, this study demonstrated that TSC1 was a functional target of miR-301a in fibroblasts, and the negative regulation of TSC1 by miR-301a promoted the severity of pulmonary fibrosis through the mammalian target of rapamycin (mTOR) signaling pathway. The blocking of miR-301a by the intravenous injection of antagomiR-301a inhibited the proliferation of fibroblasts and the structural destruction of lung tissues in the bleomycin-induced lung fibrosis mouse model. The findings revealed the crucial role of the miR-301a/TSC1/mTOR axis in the pathogenesis of pulmonary fibrosis, suggesting that miR-301a might serve as a potential therapeutic target.

A single injection of bleomycin reduces glycosaminoglycan sulfation up to 30 days in the C57BL/6 mouse model of lung fibrosis

Bleomycin is a clinically used anticancer drug, but it induces lung fibrosis in certain cancer patients with unknown mechanism. Glycosaminoglycans (GAGs) are required for lung morphogenesis during animal development. In current study, GAG disaccharides including heparan sulfate (HS) and chondroitin sulfate (CS) from bleomycin-induced and control lung tissues in lung fibrosis mouse model were tagged with 1-phenyl-3-methyl-5-pyrazolone (PMP) and deuterated PMP, respectively. The differentially isotope-tagged disaccharides were quantitatively compared by LC-MS. At day 10, the amount of CS disaccharides (U0a0, U0a6, and U0a4) and non-sulfated HS disaccharide (U0A0) were increased by 1.3-, 1.6-, 11.7-, and 2.2-fold, respectively, whereas the amount of CS disaccharide (U0a2), hyaluranan disaccharide (UβA0), and six HS disaccharides (U0A6, U2A0, U0H6, U0S0, U2S0, and U2S6) were decreased from1.1- to 14.3-fold compared to that of the controls. At day 15, under-sulfation of both HS and CS disaccharides was persisted. At day 30, the CS disaccharide compositions were recovered to that of the control levels whereas the HS were still remarkably under-sulfated. In conclusion, GAGs, especially HS, from fibrotic lungs induced by a single injection of bleomycin were significantly under-sulfated up to 30 days, suggesting GAGs might be another class of defective signaling molecules involved in bleomycin-induced lung fibrosis.

An experimental mouse model of lung fibrosis

Pulmonary fibrosis is a pathologic chronic lung disorder characterized by the deposition of extracellular matrix and damage to lung architecture. Models of pulmonary fibrosis were developed in several animal species. However, murine models are more common due to their low costs, availability and well characterized immune systems. Indeed, these murine models play an important role in understanding the biology of the diseases, deciphering the cellular and molecular mechanisms mediating pulmonary pathobiology and to explore novel therapeutic approaches. Although the majority of these models mimic certain characteristics of human idiopathic pulmonary fibrosis, most of them do not mimic other features especially those related to the progressive and irreversible nature of this disease. The present protocol describes the induction of a pulmonary fibrosis model in mice with bleomycin, which is a risk factor for human lung fibrosis. Specifically, we described the methodology of intratracheal instillation of bleomycin as well as the assessment methods of pulmonary fibrosis either histopathologically based on Ashcroft grading score or by quantitatively based on biochemical biomarkers. Moreover, we proposed a detailed readout that can determine the degree of lung injury in bronchoalveolar lavage fluids. We described different cellular and molecular markers such as by total protein concentration, total cell count, immune cell differential count, and the expression of pro-inflammatory mediators. We present this model as a reference for studies on the pulmonary fibrosis.

Keratinocyte transglutaminase 2 promotes CCR6+ \u03b3\u03b4T-cell recruitment by upregulating CCL20 in psoriatic inflammation

Keratinocyte-derived cytokines and chemokines amplify psoriatic inflammation by recruiting IL-17-producing CCR6+ γδT-cells and neutrophils. The expression of these cytokines and chemokines mainly depends on NF-κB activity; however, the pathway that activates NF-κB in response to triggering factors is poorly defined. Here, we show that transglutaminase 2 (TG2), previously reported to elicit a TH17 response by increasing IL-6 expression in a mouse model of lung fibrosis, mediates the upregulation of cytokines and chemokines by activating NF-κB in imiquimod (IMQ)-treated keratinocytes. TG2-deficient mice exhibited reduced psoriatic inflammation in skin treated with IMQ but showed systemic immune responses similar to wild-type mice. Experiments in bone marrow (BM) chimeric mice revealed that TG2 is responsible for promoting psoriatic inflammation in non-BM-derived cells. In keratinocytes, IMQ treatment activated TG2, which in turn activated NF-κB signaling, leading to the upregulation of IL-6, CCL20, and CXCL8 and increased leukocyte migration, in vitro. Consequently, TG2-deficient mice showed markedly decreased CCR6+ γδT-cell and neutrophil infiltration in IMQ-treated skin. Moreover, TG2 levels were higher in psoriatic skin than in normal skin and correlated with IL-6, CXCL8, and CCL20 levels. Therefore, these results indicate that keratinocyte TG2 acts as a critical mediator in the amplification of psoriatic inflammation.

Metabolic dysregulation into fibrosis

Fibrosis Excessive fibrosis around alveoli, tiny air sacs that promote gas exchange, prevents the lungs from expanding properly. Yin et al. found that the glycolytic enzyme hexokinase 2 (HK2) was abundant in lung fibroblasts from patients with idiopathic pulmonary fibrosis. The profibrotic cytokine transforming growth factor–β (TGF-β) induced HK2 accumulation in mouse and human lung fibroblasts and thus increased glycolysis in these cells. Inhibition of HK2 with the cancer drug lonidamine attenuated the profibrotic actions of TGF-β in fibroblasts and improved lung function in a mouse model of lung fibrosis. These results support a model of HK2-dependent metabolic dysregulation that contributes to lung fibrosis and demonstrates that HK2 is a potential therapeutic target. Sci. Signal. 12 , eaax4067 (2019).