Idiopathic Pulmonary Fibrosis Tissue Research Articles

Three-Dimensional Idiopathic Pulmonary Fibrosis Model Using a Layer-by-Layer Cell Coating Technique.

Idiopathic pulmonary fibrosis (IPF) is a severe health problem characterized by progressive fibroblast proliferation and aberrant vascular remodeling. However, the lack of a suitable in vitro model that replicates cell-specific changes in IPF tissue is a crucial issue. Three-dimensional (3D) cell cultures allow the mimicking of cell-specific functions, facilitating development of novel antifibrosis drugs. We have established a layer-by-layer (LbL) cell coating technique that enables the construction of 3D tissue and also vascularized 3D tissue. This study evaluated whether this technique is beneficial for constructing an in vitro IPF-3D model using human lung fibroblasts and microvascular endothelial cells. We fabricated an in vitro IPF-3D model to provide IPF-derived fibroblasts-specific function and aberrant microvascular structure using the LbL cell coating technique. We also found that this in vitro IPF-3D model showed drug responsiveness to two antifibrosis drugs that have recently been approved worldwide. This in vitro IPF-3D model constructed by a LbL cell coating technique would help in the understanding of fibroblast function and the microvascular environment in IPF and could also be used to predict the efficacy of novel antifibrosis drugs. Impact statement We established a novel in vitro model mimicking idiopathic pulmonary fibrosis. Three-dimensional culture was constructed by layer-by-layer cell coating technique. This novel model provides a visualization of fibroblast-specific function. This assay allows for the assessment of pulmonary microvascular environment. Our model may be useful for predicting the efficacy of novel antifibrosis drugs.

Reduced SOCS1 Expression in Lung Fibroblasts from Patients with IPF Is Not Mediated by Promoter Methylation or Mir155.

The interleukin (IL)-6 family of cytokines and exaggerated signal transducer and activator of transcription (STAT)3 signaling is implicated in idiopathic pulmonary fibrosis (IPF) pathogenesis, but the mechanisms regulating STAT3 expression and function are unknown. Suppressor of cytokine signaling (SOCS)1 and SOCS3 block STAT3, and low SOCS1 levels have been reported in IPF fibroblasts and shown to facilitate collagen production. Fibroblasts and lung tissue from IPF patients and controls were used to examine the mechanisms underlying SOCS1 down-regulation in IPF. A significant reduction in basal SOCS1 mRNA in IPF fibroblasts was confirmed. However, there was no difference in the kinetics of activation, and methylation of SOCS1 in control and IPF lung fibroblasts was low and unaffected by 5′-aza-2′-deoxycytidine’ treatment. SOCS1 is a target of microRNA-155 and although microRNA-155 levels were increased in IPF tissue, they were reduced in IPF fibroblasts. Therefore, SOCS1 is not regulated by SOCS1 gene methylation or microRNA155 in these cells. In conclusion, we confirmed that IPF fibroblasts had lower levels of SOCS1 mRNA compared with control fibroblasts, but we were unable to determine the mechanism. Furthermore, although SOCS1 may be important in the fibrotic process, we were unable to find a significant role for SOCS1 in regulating fibroblast function.

Disparate Interferon Signaling and Shared Aberrant Basaloid Cells in Single-Cell Profiling of Idiopathic Pulmonary Fibrosis and Systemic Sclerosis-Associated Interstitial Lung Disease.

Idiopathic pulmonary fibrosis (IPF) and systemic sclerosis-associated interstitial lung disease (SSc-ILD) differ in the predominant demographics and identified genetic risk alleles of effected patients, however both diseases frequently progress to respiratory failure and death. Contrasting advanced SSc-ILD to IPF provides insight to the role dysregulated immunity may play in pulmonary fibrosis. To analyze cell-type specific transcriptome commonalities and differences between IPF and SSc-ILD, we compared single-cell RNA-sequencing (scRNA-seq) of 21 explanted lung tissue specimens from patients with advanced IPF, SSc-ILD, and organ donor controls. Comparison of IPF and SSc-ILD tissue identified divergent patterns of interferon signaling, with interferon-gamma signaling upregulated in the SPP1 hi and FABP4 hi macrophages, cytotoxic T cells, and natural kill cells of IPF, while type I interferon signaling and production was upregulated in the corresponding SSc-ILD populations. Plasmacytoid dendritic cells were found in diseased lungs only, and exhibited upregulated cellular stress pathways in SSc-ILD compared to IPF. Alveolar type I cells were dramatically decreased in both IPF and SSc-ILD, with a distinct transcriptome signature separating these cells by disease. KRT5-/KRT17+ aberrant basaloid cells exhibiting markers of cellular senescence and epithelial-mesenchymal transition were identified in SSc-ILD for the first time. In summary, our study utilizes the enriched capabilities of scRNA-seq to identify key divergent cell types and pathways between IPF and SSc-ILD, providing new insights into the shared and distinct mechanisms between idiopathic and autoimmune interstitial lung diseases.

Using bioinformatics approach identifies key genes and pathways in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis is a chronic and irreversible respiratory disease with a high incidence worldwide and no specific treatment. Currently, the etiology and pathogenesis of this disease remain largely unknown. In main purpose of this study, bioinformatics analysis was used to uncover key genes and pathways related to idiopathic pulmonary fibrosis (IPF). Gene expression profiles of GSE2052 and GSE35145 were obtained. After combining the 2 chip groups; then, we normalized the data, eliminating batch difference. R software was used to process and to screen differentially expressed genes (DEGs) between the IPF and normal tissues. Then, functional enrichment analysis of these DEGs was carried out, and a protein-protein interaction network (PPI) was also constructed. A total of 276 DEGs (152 up and 134 down-regulated genes) were identified in the IPF lung samples. The PPI network was established with 227 nodes and 763 edges. The top 10 hub genes were CAM1, CDH1, CXCL12, JUN, CTGF, SERPINE1, CXCL1, EDN1, COL1A2, and SPARC. Analyzing the PPI network modules with close interaction, the 3 key modules in the whole PPI network were screened out. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enriched for the module containing DEGs contained the viral protein interaction with cytokine and the cytokine receptor, the TNF signaling pathway, and the chemokine signaling pathway. The identified key genes and pathways may play an important role in the occurrence and development of IPF, and may be expected to be biomarkers or therapeutic targets for the diagnosis of IPF.

Paired genetic analysis by next-generation sequencing of lung cancer and associated idiopathic pulmonary fibrosis.

The pathogenesis of lung cancer associated with idiopathic pulmonary fibrosis (IPF) has remained largely uncharacterized. To provide insight into this condition, we undertook genomic profiling of IPF‐associated lung cancer as well as of adjacent fibrosing lung tissue in surgical specimens. Isolated DNA and RNA from 17 IPF‐associated non‐small cell lung cancer and 15 paired fibrosing lung tissue specimens were analyzed by next‐generation sequencing with a panel that targets 161 cancer‐related genes. Somatic genetic alterations were frequently identified in TP53 (n = 6, 35.3%) and PIK3CA (n = 5, 29.4%) genes in tumor samples as well as in EGFR (n = 7, 46.7%), PIK3CA (n = 5, 33.3%), ERBB3 (n = 4, 26.7%), and KDR (n = 4, 26.7%) in IPF samples. Genes related to the RAS‐RAF signaling pathway were also frequently altered in tumor (n = 7, 41.2%) and IPF (n = 3, 20.0%) samples. The number of somatic alterations identified in IPF samples was almost as large as that detected in paired tumor samples (81 vs 90, respectively). However, only 6 of the 81 somatic alterations detected in IPF samples overlapped with those in paired tumor samples. The accumulation of somatic mutations was thus apparent in IPF tissue of patients with IPF‐associated lung cancer, and the RAS‐RAF pathway was implicated in lung tumorigenesis. The finding that somatic alterations were not frequently shared between tumor and corresponding IPF tissue indicates that IPF‐associated lung cancer does not develop through the stepwise accumulation of somatic alterations in IPF.

Monocyte-derived multipotent cell delivered programmed therapeutics to reverse idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a highly heterogeneous and fatal disease. However, IPF treatment has been limited by the low drug delivery efficiency to lungs and dysfunctional "injured" type II alveolar epithelial cell (AEC II). Here, we present surface-engineered nanoparticles (PER NPs) loading astaxanthin (AST) and trametinib (TRA) adhered to monocyte-derived multipotent cell (MOMC) forming programmed therapeutics (MOMC/PER). Specifically, the cell surface is designed to backpack plenty of PER NPs that reach directly to the lungs due to the homing characteristic of the MOMC and released PER NPs retarget injured AEC II after responding to the matrix metalloproteinase-2 (MMP-2) in IPF tissues. Then, released AST can enhance synergetic effect of TRA for inhibiting myofibroblast activation, and MOMC can also repair injured AEC II to promote damaged lung regeneration. Our findings provide proof of concept for developing a strategy for cell-mediated lung-targeted delivery platform carrying dual combined therapies to reverse IPF.

Comprehensive analysis of lncRNA-associated competing endogenous RNA network and immune infiltration in idiopathic pulmonary fibrosis.

BackgroundIdiopathic pulmonary fibrosis (IPF) is a life-threatening lung disorder with an unknown aetiology. The roles of long non-coding RNAs (lncRNAs) and its related competing endogenous RNAs (ceRNA) network in IPF remains poorly understood. In this study, we aimed to build a lncRNA-miRNA-mRNA network and explore the pathogenesis of IPF.MethodsWe screened differentially expressed lncRNAs (DElncRNAs) and mRNAs (DEmRNAs) between IPF and control lung tissues from two datasets. The ceRNA network was built according to the interactions between DElncRNA, miRNA, and DEmRNA. Functional enrichment analysis of DemRNAs was performed using Metascape. CIBERSORT (Cell type Identification by Estimating Relative Subsets Of known RNA Transcripts) was applied to estimate the fraction of 22 immune cells in IPF and controls lung tissue samples. Then we investigated the correlation between immune cells and clinical traits.ResultsWe constructed a lncRNA-miRNA-mRNA network, which was composed of two DElncRNAs, 18 miRNAs, 66 DemRNAs. Functional enrichment analysis showed that the DEmRNAs mainly participated in MicroRNAs in cancer. By applying CIBERSORT, we found that IPF tissue samples had a higher proportion of plasma cells, resting mast cells and a lower proportion of resting NK cells, monocytes, neutrophils compared with control tissue samples. Also, our results indicated that immune cells were associated with the severity of IPF.ConclusionsIn summary, this is the first study to build lncRNA-miRNA-mRNA ceRNA network of IPF, which may improve our understanding of IPF pathogenesis. Our study indicates that immune cells in lung tissues may predict disease severity and participate in the development of IPF. Future prospective studies are required to confirm the findings of the current study.

Small airways pathology in idiopathic pulmonary fibrosis: a retrospective cohort study

The observation that patients with idiopathic pulmonary fibrosis (IPF) can have higher than normal expiratory flow rates at low lung volumes led to the conclusion that the airways are spared in IPF. This study aimed to re-examine the hypothesis that airways are spared in IPF using a multiresolution imaging protocol that combines multidetector CT (MDCT), with micro-CT and histology. This was a retrospective cohort study comparing explanted lungs from patients with severe IPF treated by lung transplantation with a cohort of unused donor (control) lungs. The donor control lungs had no known lung disease, comorbidities, or structural lung injury, and were deemed appropriate for transplantation on review of the clinical files. The diagnosis of IPF in the lungs from patients was established by a multidisciplinary consensus committee according to existing guidelines, and was confirmed by video-assisted thoracic surgical biopsy or by pathological examination of the contralateral lung. The control and IPF groups were matched for age, sex, height, and bodyweight. Samples of lung tissue were compared using the multiresolution imaging approach: a cascade of clinical MDCT, micro-CT, and histological imaging. We did two experiments: in experiment 1, all the lungs were randomly sampled; in experiment 2, samples were selected from regions of minimal and established fibrosis. The patients and donors were recruited from the Katholieke Universiteit Leuven (Leuven, Belgium) and the University of Pennsylvania Hospital (Philadelphia, PA, USA). The study took place at the Katholieke Universiteit Leuven, and the University of British Columbia (Vancouver, BC, Canada). Between Oct 5, 2009, and July 22, 2016, explanted lungs from patients with severe IPF (n=11), were compared with a cohort of unused donor (control) lungs (n=10), providing 240 samples of lung tissue for comparison using the multiresolution imaging approach. The MDCT specimen scans show that the number of visible airways located between the ninth generation (control 69 [SD 22] versus patients with IPF 105 [33], p=0·0023) and 14th generation (control 9 [6] versus patients with IPF 49 [28], p<0·0001) of airway branching are increased in patients with IPF, which we show by micro-CT is due to thickening of their walls and distortion of their lumens. The micro-CT analysis showed that compared with healthy (control) lung anatomy (mean 5·6 terminal bronchioles per mL [SD 1·6]), minimal fibrosis in IPF tissue was associated with a 57% loss of the terminal bronchioles (mean 2·4 terminal bronchioles per mL [SD 1·0]; p<0·0001), the appearance of fibroblastic foci, and infiltration of the tissue by inflammatory immune cells capable of forming lymphoid follicles. Established fibrosis in IPF tissue had a similar reduction (66%) in the number of terminal bronchioles (mean 1·9 terminal bronchioles per mL [SD 1·4]; p<0·0001) and was dominated by increased airspace size, Ashcroft fibrosis score, and volume fractions of tissue and collagen. Small airways disease is a feature of IPF, with significant loss of terminal bronchioles occuring within regions of minimal fibrosis. On the basis of these findings, we postulate that the small airways could become a potential therapeutic target in IPF. Katholieke Universiteit Leuven, US National Institutes of Health, BC Lung Association, and Genentech.

Human lung extracellular matrix hydrogels resemble the stiffness and viscoelasticity of native lung tissue.

Chronic lung diseases such as idiopathic pulmonary fibrosis (IPF) and chronic obstructive pulmonary disease (COPD) are associated with changes in extracellular matrix (ECM) composition and abundance affecting the mechanical properties of the lung. This study aimed to generate ECM hydrogels from control, severe COPD [Global Initiative for Chronic Obstructive Lung Disease (GOLD) IV], and fibrotic human lung tissue and evaluate whether their stiffness and viscoelastic properties were reflective of native tissue. For hydrogel generation, control, COPD GOLD IV, and fibrotic human lung tissues were decellularized, lyophilized, ground into powder, porcine pepsin solubilized, buffered with PBS, and gelled at 37°C. Rheological properties from tissues and hydrogels were assessed with a low-load compression tester measuring the stiffness and viscoelastic properties in terms of a generalized Maxwell model representing phases of viscoelastic relaxation. The ECM hydrogels had a greater stress relaxation than tissues. ECM hydrogels required three Maxwell elements with slightly faster relaxation times (τ) than that of native tissue, which required four elements. The relative importance (Ri) of the first Maxwell element contributed the most in ECM hydrogels, whereas for tissue the contribution was spread over all four elements. IPF tissue had a longer-lasting fourth element with a higher Ri than the other tissues, and IPF ECM hydrogels did require a fourth Maxwell element, in contrast to all other ECM hydrogels. This study shows that hydrogels composed of native human lung ECM can be generated. Stiffness of ECM hydrogels resembled that of whole tissue, while viscoelasticity differed.

Galectin-3 levels are elevated following nintedanib treatment

Background and Aims:Idiopathic pulmonary fibrosis (IPF) is a common and severe form of pulmonary fibrosis. Nintedanib, a triple angiokinase inhibitor, is approved for treating IPF. Galectin 3 (Gal-3) activates a variety of profibrotic processes. Currently, the Gal-3 inhibitor TD139 is being tested in phase II clinical trials. Since this treatment is given ‘on top’ of nintedanib, it is important to estimate its effect on Gal-3 levels. Therefore, we evaluated the impact of nintedanib on Gal-3 expression using both in vitro and in vivo models, in addition to serum samples from patients with IPF.Methods:Gal-3 levels were evaluated in IPF and control tissue samples, primary human lung fibroblasts (HLFs) following nintedanib treatment (10–100 nM, quantitative polymerase chain reaction), and in a silica-induced fibrosis mouse model with/without nintedanib (0.021–0.21 mg/kg) by immunohistochemistry. In addition, Gal-3 levels were analyzed in serum samples from 41 patients with interstitial lung disease patients with/without nintedanib treatment by ELISA.Results:Nintedanib addition to HLFs resulted in significant elevations in Gal-3, phospho-signal transducer and activator of transcription 3 (pSTAT3), as well as IL-8 mRNA levels (p < 0.05). Gal-3 expression was higher in samples from IPF patients compared with non-IPF controls at the protein and mRNA levels (p < 0.05). In the in vivo mouse model, Gal-3 levels were increased following fibrosis induction and even further increased with the addition of nintedanib, mostly in macrophages (p < 0.05). Patients receiving nintedanib presented with higher Gal-3 serum levels compared with those who did not receive nintedanib (p < 0.05).Conclusion:Nintedanib elevates Gal-3 levels in both experimental models, along with patient samples. These findings highlight the possibility of using combined inhibition therapy for patients with IPF.

Inhibiting eicosanoid degradation exerts antifibrotic effects in a pulmonary fibrosis mouse model and human tissue

Idiopathic pulmonary fibrosis (IPF) is a disease with high 5-year mortality and few therapeutic options. Prostaglandin (PG) E2 exhibits antifibrotic properties and is reduced in bronchoalveolar lavage from patients with IPF. 15-Prostaglandin dehydrogenase (15-PGDH) is the key enzyme in PGE2 metabolism under the control of TGF-β and microRNA 218. We sought to investigate the expression of 15-PGDH in IPF and the therapeutic potential of a specific inhibitor of this enzyme in a mouse model and human tissue. Invitro studies, including fibrocyte differentiation, regulation of 15-PGDH, RT-PCR, and Western blot, were performed using peripheral blood from healthy donors and patients with IPF and A549 cells. Immunohistochemistry, immunofluorescence, 15-PGDH activity assays, and in situ hybridization as well as exvivo IPF tissue culture experiments were done using healthy donor and IPF lungs. Therapeutic effects of 15-PGDH inhibition were studied in the bleomycin mouse model of pulmonary fibrosis. We demonstrate that 15-PGDH shows areas of increased expression in patients with IPF. Inhibition of this enzyme increases PGE2 levels and reduces collagen production in IPF precision cut lung slices and in the bleomycin model. Inhibitor-treated mice show amelioration of lung function, decreased alveolar epithelial cell apoptosis, and fibroblast proliferation. Pulmonary fibrocyte accumulation is also decreased by inhibitor treatment in mice, similar to PGE2 that inhibits fibrocyte differentiation from blood of healthy donors and patients with IPF. Finally, microRNA 218-5p, which is downregulated in patients with IPF, suppressed 15-PGDH expression invivo and invitro. These findings highlight the role of 15-PGDH in IPF and suggest 15-PGDH inhibition as a promising therapeutic approach.

The Oncogene ECT2 Contributes to a Hyperplastic, Proliferative Lung Epithelial Cell Phenotype in Idiopathic Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) and lung cancer are progressive lung diseases with a poor prognosis. IPF is a risk factor for the development of lung cancer, and the incidence of lung cancer is increased in patients with IPF. The disease pathogenesis of IPF and lung cancer involves common genetic alterations, dysregulated pathways, and the emergence of hyperplastic and metaplastic epithelial cells. Here, we aimed to identify novel, common mediators that might contribute to epithelial cell reprogramming in IPF. Gene set enrichment analysis of publicly available non-small cell lung cancer and IPF datasets revealed a common pattern of misregulated genes linked to cell proliferation and transformation. The oncogene ECT2 (epithelial cell transforming sequence 2), a guanine nucleotide exchange factor for Rho GTPases, was highly enriched in both IPF and non-small cell lung cancer compared with nondiseased controls. Increased expression of ECT2 was verified by qPCR and Western blotting in bleomycin-induced lung fibrosis and human IPF tissue. Immunohistochemistry demonstrated strong expression of ECT2 staining in hyperplastic alveolar epithelial type II (ATII) cells in IPF, as well as its colocalization with proliferating cell nuclear antigen, a well-known proliferation marker. Increased ECT2 expression coincided with enhanced proliferation of primary mouse ATII cells as analyzed by flow cytometry. ECT2 knockdown in ATII cells resulted in decreased proliferation and collagen I expression in vitro. These data suggest that the oncogene ECT2 contributes to epithelial cell reprogramming in IPF, and further emphasize the hyperplastic, proliferative ATII cell as a potential target in patients with IPF and lung cancer.

Transcriptional regulatory model of fibrosis progression in the human lung.

To develop a systems biology model of fibrosis progression within the human lung we performed RNA sequencing and microRNA analysis on 95 samples obtained from 10 idiopathic pulmonary fibrosis (IPF) and 6 control lungs. Extent of fibrosis in each sample was assessed by microCT-measured alveolar surface density (ASD) and confirmed by histology. Regulatory gene expression networks were identified using linear mixed-effect models and dynamic regulatory events miner (DREM). Differential gene expression analysis identified a core set of genes increased or decreased before fibrosis was histologically evident that continued to change with advanced fibrosis. DREM generated a systems biology model (www.sb.cs.cmu.edu/IPFReg) that identified progressively divergent gene expression tracks with microRNAs and transcription factors that specifically regulate mild or advanced fibrosis. We confirmed model predictions by demonstrating that expression of POU2AF1, previously unassociated with lung fibrosis but proposed by the model as regulator, is increased in B lymphocytes in IPF lungs and that POU2AF1-knockout mice were protected from bleomycin-induced lung fibrosis. Our results reveal distinct regulation of gene expression changes in IPF tissue that remained structurally normal compared with moderate or advanced fibrosis and suggest distinct regulatory mechanisms for each stage.

MicroRNA-448 overexpression inhibits fibroblast proliferation and collagen synthesis and promotes cell apoptosis via targeting ABCC3 through the JNK signaling pathway.

Idiopathic pulmonary fibrosis (IPF) is a condition that results in the progressive deterioration of lung function with poor prognosis. The current study is aimed at exploring how microRNA-448 (miR-448) targeting ABCC3 affects fibroblast proliferation, apoptosis, and collagen synthesis of mice with IPF via the Jun N-terminal kinase (JNK) signaling pathway. Bioinformatics and dual-luciferase polymerase chain reaction were used to predict the relationship of miR-448 and ABCC3. The expression of miR-448 and ABCC3 was detected in IPF tissues. Using IPF mouse models, lung fibroblasts for the experiments were treated with miR-448 mimic, miR-448 inhibitor, si-ABCC3, or SP600125 (inhibitor of JNK) to evaluate the cell proliferation and apoptosis in response to miR-448. Reverse transcription quantitative polymerase chain reaction and western blot analysis were used to identify the expression of miR-448, ABCC3, and the activation of the JNK signaling pathway. ABCC3 was targeted and downregulated by miR-448 based on bioinformatics prediction and dual-luciferase reporter gene assay. Additionally, miR-448 was found to be highly expressed in IPF lung tissues with low expression levels of ABCC3. In response to the treatment of miR-448 mimic or si-ABCC3, lung fibroblasts exhibited decreased cell proliferation and increased apoptotic rates, whereas the miR-448 inhibitor reversed the conditions. Notably, we also found that miR-448 mimic inhibited the JNK signaling pathway. In conclusion, by using miR-448 to target and downregulate ABCC3 to block the JNK signaling pathway in mice with IPF, we found an increase in fibroblast apoptosis, inhibited cell proliferation, and decreased collagen synthesis of fibroblasts.

The Long Noncoding RNA DNM3OS Is a Reservoir of FibromiRs with Major Functions in Lung Fibroblast Response to TGF-β and Pulmonary Fibrosis.

Rationale: Given the paucity of effective treatments for idiopathic pulmonary fibrosis (IPF), new insights into the deleterious mechanisms controlling lung fibroblast activation, the key cell type driving the fibrogenic process, are essential to develop new therapeutic strategies. TGF-β (transforming growth factor-β) is the main profibrotic factor, but its inhibition is associated with severe side effects because of its pleiotropic role. Objectives: To determine if downstream noncoding effectors of TGF-β in fibroblasts may represent new effective therapeutic targets whose modulation may be well tolerated. Methods: We investigated the whole noncoding fraction of TGF-β-stimulated lung fibroblast transcriptome to identify new genomic determinants of lung fibroblast differentiation into myofibroblasts. Differential expression of the long noncoding RNA (lncRNA) DNM3OS (dynamin 3 opposite strand) and its associated microRNAs (miRNAs) was validated in a murine model of pulmonary fibrosis and in IPF tissue samples. Distinct and complementary antisense oligonucleotide-based strategies aiming at interfering with DNM3OS were used to elucidate the role of DNM3OS and its associated miRNAs in IPF pathogenesis. Measurements and Main Results: We identified DNM3OS as a fibroblast-specific critical downstream effector of TGF-β-induced lung myofibroblast activation. Mechanistically, DNM3OS regulates this process in trans by giving rise to three distinct profibrotic mature miRNAs (i.e., miR-199a-5p/3p and miR-214-3p), which influence SMAD and non-SMAD components of TGF-β signaling in a multifaceted way. In vivo, we showed that interfering with DNM3OS function not only prevents lung fibrosis but also improves established pulmonary fibrosis. Conclusions: Pharmacological approaches aiming at interfering with the lncRNA DNM3OS may represent new effective therapeutic strategies in IPF.

A high content, phenotypic \u2018scar-in-a-jar\u2019 assay for rapid quantification of collagen fibrillogenesis using disease-derived pulmonary fibroblasts

BackgroundExcessive extracellular matrix (ECM) deposition is a hallmark feature in fibrosis and tissue remodelling diseases. Typically, mesenchymal cells will produce collagens under standard 2D cell culture conditions, however these do not assemble into fibrils. Existing assays for measuring ECM production are often low throughput and not disease relevant. Here we describe a robust, high content, pseudo-3D phenotypic assay to quantify mature fibrillar collagen deposition which is both physiologically relevant and amenable to high throughput compound screening. Using pulmonary fibroblasts derived from patients with idiopathic pulmonary fibrosis (IPF), we developed the ‘scar-in-a-jar’ assay into a medium-throughput phenotypic assay to robustly quantify collagen type I deposition and other extracellular matrix (ECM) proteins over 72 h.ResultsThis assay utilises macromolecular crowding to induce an excluded volume effect and enhance enzyme activity, which in combination with TGF-β1 stimulation significantly accelerates ECM production. Collagen type I is upregulated approximately 5-fold with a negligible effect on cell number. We demonstrate the robustness of the assay achieving a Z prime of approximately 0.5, and % coefficient of variance (CV) of < 5 for the assay controls SB-525334 (ALK5 inhibitor) and CZ415 (mTOR inhibitor). This assay has been used to confirm the potency of a number of potential anti-fibrotic agents. Active compounds from the ‘scar-in-a-jar’ assay can be further validated for other markers of ECM deposition and fibroblast activation such as collagen type IV and α-smooth muscle actin exhibiting a 4-fold and 3-fold assay window respectively.ConclusionIn conclusion, we have developed ‘scar -in-a-jar is’ into a robust disease-relevant medium-throughput in vitro assay to accurately quantify ECM deposition. This assay may enable iterative compound profiling for IPF and other fibroproliferative and tissue remodelling diseases.

Pharmacometabolic response to pirfenidone in pulmonary fibrosis detected by MALDI-FTICR-MSI.

Idiopathic pulmonary fibrosis (IPF) is a fatal condition that reduces life expectancy and shows a limited response to available therapies. Pirfenidone has been approved for treatment of IPF, but little is known about the distinct metabolic changes that occur in the lung upon pirfenidone administration.Here, we performed a proof-of-concept study using high-resolution quantitative matrix-assisted laser desorption/ionisation Fourier-transform ion cyclotron resonance mass spectrometry imaging (MALDI-FTICR-MSI) to simultaneously detect, visualise and quantify in situ endogenous and exogenous metabolites in lungs of mice subjected to experimental fibrosis and human patients with IPF, and to assess the effect of pirfenidone treatment on metabolite levels.Metabolic pathway analysis and endogenous metabolite quantification revealed that pirfenidone treatment restores redox imbalance and glycolysis in IPF tissues, and downregulates ascorbate and aldarate metabolism, thereby likely contributing to in situ modulation of collagen processing. As such, we detected specific alterations in metabolite pathways in fibrosis and, importantly, metabolic recalibration following pirfenidone treatment.Together, these results highlight the suitability of high-resolution MALDI-FTICR-MSI for deciphering the therapeutic effects of pirfenidone and provide a preliminary analysis of the metabolic changes that occur during pirfenidone treatment in vivo These data may therefore contribute to improvement of currently available therapies for IPF.

Increased Primary Cilia in Idiopathic Pulmonary Fibrosis.

Primary cilia are solitary, non-motile, axonemal microtubule-based antenna-like organelles that project from the plasma membrane of most mammalian cells and are implicated in transducing hedgehog signals during development. It was recently proposed that aberrant SHH signaling may be implicated in the progression of idiopathic pulmonary fibrosis (IPF). However, the distribution and role of primary cilia in IPF remains unclear. Here, we clearly observed the primary cilia in alveolar epithelial cells, fibroblasts, and endothelial cells of human normal lung tissue. Then, we investigated the distribution of primary cilia in human IPF tissue samples using immunofluorescence. Tissues from six IPF cases showed an increase in the number of primary cilia in alveolar cells and fibroblasts. In addition, we observed an increase in ciliogenesis related genes such as IFT20 and IFT88 in IPF. Since major components of the SHH signaling pathway are known to be localized in primary cilia, we quantified the mRNA expression of the SHH signaling components using qRT-PCR in both IPF and control lung. mRNA levels of SHH, the coreceptor SMO, and the transcription factors GLI1 and GLI2 were upregulated in IPF compared with control. Furthermore, the nuclear localization of GLI1 was observed mainly in alveolar epithelia and fibroblasts. In addition, we showed that defective KIF3A-mediated ciliary loss in human type II alveolar epithelial cell lines leads to disruption of SHH signaling. These results indicate that a significant increase in the number of primary cilia in IPF contributes to the upregulation of SHH signals.

Transcriptomic evidence of immune activation in macroscopically normal-appearing and scarred lung tissues in idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease manifested by overtly scarred peripheral and basilar regions and more normal-appearing central lung areas. Lung tissues from macroscopically normal-appearing (IPFn) and scarred (IPFs) areas of explanted IPF lungs were analyzed by RNASeq and compared with healthy control (HC) lung tissues. There were profound transcriptomic changes in IPFn compared with HC tissues, which included elevated expression of numerous immune-, inflammation-, and extracellular matrix-related mRNAs, and these changes were similar to those observed with IPFs compared to HC. Comparing IPFn directly to IPFs, elevated expression of epithelial mucociliary mRNAs was observed in the IPFs tissues. Thus, despite the known geographic tissue heterogeneity in IPF, the entire lung is actively involved in the disease process, and demonstrates pronounced elevated expression of numerous immune-related genes. Differences between normal-appearing and scarred tissues may thus be driven by deranged epithelial homeostasis or possibly non-transcriptomic factors.

CELLULAR SENESCENCE DRIVES FIBROTIC PULMONARY DISEASE

Idiopathic pulmonary fibrosis (IPF) is a progressive, fatal disease characterized by interstitial remodeling, leading to severely compromised lung structure and function. Cellular senescence, a stress-induced state of replicative arrest, is causally implicated in numerous age-related diseases. Markers of senescence are detectable within parenchyma of chronologically aged and IPF lung tissue. Whether and how senescent cells mechanistically regulate IPF or if their removal may be an efficacious intervention strategy is unknown. Through examination of IPF tissue and transcriptome datasets, we discovered elevated abundance of senescence effectors and senescence associated secretory phenotype (SASP) factors, with select markers increasing concordantly with disease severity. Leveraging intratracheal administration of bleomycin to replicate aspects of IPF, we demonstrate that suicide gene-mediated ablation of p16Ink4a-expressing senescent cells in Ink-Attac mice improves pulmonary function, body composition, and physical performance. Senolytic DQ treatment replicates several benefits of transgenic clearance. Thus, our findings demonstrate for the first time that fibrotic pulmonary disease is mediated in part by senescent cells, which can be pharmacologically targeted to improve physical health and function.