Idiopathic Pulmonary Fibrosis Tissue Research Articles

Novel AT2 Cell Subpopulations and Diagnostic Biomarkers in IPF: Integrating Machine Learning with Single-Cell Analysis.

Idiopathic pulmonary fibrosis (IPF) is a long-term condition with an unidentified cause, and currently there are no specific treatment options available. Alveolar epithelial type II cells (AT2) constitute a heterogeneous population crucial for secreting and regenerative functions in the alveolus, essential for maintaining lung homeostasis. However, a comprehensive investigation into their cellular diversity, molecular features, and clinical implications is currently lacking. In this study, we conducted a comprehensive examination of single-cell RNA sequencing data from both normal and fibrotic lung tissues. We analyzed alterations in cellular composition between IPF and normal tissue and investigated differentially expressed genes across each cell population. This analysis revealed the presence of two distinct subpopulations of IPF-related alveolar epithelial type II cells (IR_AT2). Subsequently, three unique gene co-expression modules associated with the IR_AT2 subtype were identified through the use of hdWGCNA. Furthermore, we refined and identified IPF-related AT2-related gene (IARG) signatures using various machine learning algorithms. Our analysis demonstrated a significant association between high IARG scores in IPF patients and shorter survival times (p-value < 0.01). Additionally, we observed a negative correlation between the percent predicted diffusing capacity for lung carbon monoxide (% DLCO) and increased IARG scores (cor = -0.44, p-value < 0.05). The cross-validation findings demonstrated a high level of accuracy (AUC > 0.85, p-value < 0.01) in the prognostication of patients with IPF utilizing the identified IARG signatures. Our study has identified distinct molecular and biological features among AT2 subpopulations, specifically highlighting the unique characteristics of IPF-related AT2 cells. Importantly, our findings underscore the prognostic relevance of specific genes associated with IPF-related AT2 cells, offering valuable insights into the advancement of IPF.

Identification of CFH and FHL2 as biomarkers for idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a fatal disease of unknown etiology with a poor prognosis, characterized by a lack of effective diagnostic and therapeutic interventions. The role of immunity in the pathogenesis of IPF is significant, yet remains inadequately understood. This study aimed to identify potential key genes in IPF and their relationship with immune cells by integrated bioinformatics analysis and verify by in vivo and in vitro experiments. Gene microarray data were obtained from the Gene Expression Omnibus (GEO) for differential expression analysis. The differentially expressed genes (DEGs) were identified and subjected to functional enrichment analysis. By utilizing a combination of three machine learning algorithms, specific genes associated with idiopathic pulmonary fibrosis (IPF) were pinpointed. Then their diagnostic significance and potential co-regulators were elucidated. We further analyzed the correlation between key genes and immune infiltrating cells via single-sample gene set enrichment analysis (ssGSEA). Subsequently, a single-cell RNA sequencing data (scRNA-seq) was used to explore which cell types expressed key genes in IPF samples. Finally, a series of in vivo and in vitro experiments were conducted to validate the expression of candidate genes by western blot (WB), quantitative real-time PCR (qRT-PCR), and immunohistochemistry (IHC) analysis. A total of 647 DEGs of IPF were identified based on two datasets, including 225 downregulated genes and 422 upregulated genes. They are closely related to biological functions such as cell migration, structural organization, immune cell chemotaxis, and extracellular matrix. CFH and FHL2 were identified as key genes with diagnostic accuracy for IPF by three machine learning algorithms. Analysis using ssGSEA revealed a significant association of both CFH and FHL2 with diverse immune cells, such as B cells and NK cells. Further scRNA-seq analysis indicated CFH and FHL2 were specifically upregulated in human IPF tissues, which was confirmed by in vitro and in vivo experiments. In this study, CFH and FHL2 have been identified as novel potential biomarkers for IPF, with potential diagnostic utility in future clinical applications. Subsequent investigations into the functions of these genes in IPF and their interactions with immune cells may enhance comprehension of the disease's pathogenesis and facilitate the identification of therapeutic targets.

64Cu]Cu-PEG-FUD peptide for noninvasive and sensitive detection of murine pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease resulting in irreversible scarring within the lungs. However, the lack of biomarkers that enable real-time assessment of disease activity remains a challenge in providing efficient clinical decision-making and optimal patient care in IPF. Fibronectin (FN) is highly expressed in fibroblastic foci of the IPF lung where active extracellular matrix (ECM) deposition occurs. Functional upstream domain (FUD) tightly binds the N-terminal 70-kilodalton domain of FN that is crucial for FN assembly. In this study, we first demonstrate the capacity of PEGylated FUD (PEG-FUD) to target FN deposition in human IPF tissue ex vivo. We subsequently radiolabeled PEG-FUD with 64Cu and monitored its spatiotemporal biodistribution via μPET/CT imaging in mice using the bleomycin-induced model of pulmonary injury and fibrosis. We demonstrated [64Cu]Cu-PEG-FUD uptake 3 and 11 days following bleomycin treatment, suggesting that radiolabeled PEG-FUD holds promise as an imaging probe in aiding the assessment of fibrotic lung disease activity.

Identification of brominated extracellular matrix proteins in idiopathic pulmonary fibrosis tissues

Identification of brominated extracellular matrix proteins in idiopathic pulmonary fibrosis tissues

Integrated bioinformatics analysis for the identification of idiopathic pulmonary fibrosis–related genes and potential therapeutic drugs

ObjectiveThe pathogenesis of idiopathic pulmonary fibrosis (IPF) remains unclear. We sought to identify IPF-related genes that may participate in the pathogenesis and predict potential targeted traditional Chinese medicines (TCMs).MethodsUsing IPF gene-expression data, Wilcoxon rank-sum tests were performed to identify differentially expressed genes (DEGs). Protein–protein interaction (PPI) networks, hub genes, and competitive endogenous RNA (ceRNA) networks were constructed or identified by Cytoscape. Quantitative polymerase chain reaction (qPCR) experiments in TGF-β1-induced human fetal lung (HFL) fibroblast cells and a pulmonary fibrosis mouse model verified gene reliability. The SymMap database predicted potential TCMs targeting IPF. The reliability of TCMs was verified in TGF-β1-induced MRC-5 cells.MaterialsMultiple gene-expression profile data of normal lung and IPF tissues were downloaded from the Gene Expression Omnibus database. HFL fibroblast cells and MRC-5 cells were purchased from Wuhan Procell Life Science and Technology Co., Ltd. (Wuhan, China). C57BL/12 mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China).ResultsIn datasets GSE134692 and GSE15197, DEGs were identified using Wilcoxon rank-sum tests (both p < 0.05). Among them, 1885 DEGs were commonly identified, and 87% (1640 genes) had identical dysregulation directions (binomial test, p < 1.00E-16). A PPI network with 1623 nodes and 8159 edges was constructed, and 18 hub genes were identified using the Analyze Network plugin in Cytoscape. Of 18 genes, CAV1, PECAM1, BMP4, VEGFA, FYN, SPP1, and COL1A1 were further validated in the GeneCards database and independent dataset GSE24206. ceRNA networks of VEGFA, SPP1, and COL1A1 were constructed. The genes were verified by qPCR in samples of TGF-β1-induced HFL fibroblast cells and pulmonary fibrosis mice. Finally, Sea Buckthorn and Gnaphalium Affine were predicted as potential TCMs for IPF. The TCMs were verified by qPCR in TGF-β1-induced MRC-5 cells.ConclusionThis analysis strategy may be useful for elucidating novel mechanisms underlying IPF at the transcriptome level. The identified hub genes may play key roles in IPF pathogenesis and therapy.

Machine learning-based prediction of candidate gene biomarkers correlated with immune infiltration in patients with idiopathic pulmonary fibrosis.

This study aimed to identify candidate gene biomarkers associated with immune infiltration in idiopathic pulmonary fibrosis (IPF) based on machine learning algorithms. Microarray datasets of IPF were extracted from the Gene Expression Omnibus (GEO) database to screen for differentially expressed genes (DEGs). The DEGs were subjected to enrichment analysis, and two machine learning algorithms were used to identify candidate genes associated with IPF. These genes were verified in a validation cohort from the GEO database. Receiver operating characteristic (ROC) curves were plotted to assess the predictive value of the IPF-associated genes. The cell-type identification by estimating relative subsets of RNA transcripts (CIBERSORT) algorithm was used to evaluate the proportion of immune cells in IPF and normal tissues. Additionally, the correlation between the expression of IPF-associated genes and the infiltration levels of immune cells was examined. A total of 302 upregulated and 192 downregulated genes were identified. Functional annotation, pathway enrichment, Disease Ontology and gene set enrichment analyses revealed that the DEGs were related to the extracellular matrix and immune responses. COL3A1, CDH3, CEBPD, and GPIHBP1 were identified as candidate biomarkers using machine learning algorithms, and their predictive value was verified in a validation cohort. Additionally, ROC analysis revealed that the four genes had high predictive accuracy. The infiltration levels of plasma cells, M0 macrophages and resting dendritic cells were higher and those of resting natural killer (NK) cells, M1 macrophages and eosinophils were lower in the lung tissues of patients with IPF than in those of healthy individuals. The expression of the abovementioned genes was correlated with the infiltration levels of plasma cells, M0 macrophages and eosinophils. COL3A1, CDH3, CEBPD, and GPIHBP1 are candidate biomarkers of IPF. Plasma cells, M0 macrophages and eosinophils may be involved in the development of IPF and may serve as immunotherapeutic targets in IPF.

MiRNA Expression in Fibroblastic Foci within Idiopathic Pulmonary Fibrosis Lungs Reveals Novel Disease-Relevant Pathways

miRNAs are 22 nucleotides long and belong to a class of noncoding RNAs that plays an important role in regulating gene expression at a post-transcriptional level. Studies show aberrant levels of miRNAs to be associated with profibrotic processes in idiopathic pulmonary fibrosis (IPF). However, most of these studies used whole IPF tissue or invitro monocultures in which fibrosis was artificially induced. The current study used laser microdissection to collect fibroblastic foci (FF), the key pathologic lesion in IPF, isolated miRNAs, and compared their expression levels with those found in whole IPF lung tissue and/or invitro cultured fibroblast from IPF or normal lungs. Sequencing libraries were generated, and data generated were bioinformatically analyzed. A total of 18 miRNAs were significantly overexpressed in FF tissue when compared with whole IPF tissue. Of those, 15 were unique to FF. Comparison of FF with cultured IPF fibroblasts also revealed differences in miRNA composition that impacted several signaling pathways. The miRNA composition of FF is both overlapping and distinct from that of whole IPF tissue or cultured IPF fibroblasts and highlights the importance of characterizing FF biology as a phenotypically and functionally discrete tissue microenvironment.

Inhibition of MRTF activation as a clinically achievable anti-fibrotic mechanism for pirfenidone.

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic disease characterised by aberrant fibroblast/myofibroblast accumulation and excessive collagen matrix deposition in the alveolar areas of lungs. As the first approved IPF medication, pirfenidone (PFD) significantly decelerates lung function decline while its underlying anti-fibrotic mechanism remains elusive. We performed transcriptomic and immunofluorescence analyses of primary human IPF tissues. We showed that myocardin-related transcription factor (MRTF) signalling is activated in myofibroblasts accumulated in IPF lungs. Furthermore, we showed that PFD inhibits MRTF activation in primary human lung fibroblasts at clinically achievable concentrations (half-maximal inhibitory concentration 50-150 µM, maximal inhibition >90%, maximal concentration of PFD in patients <100 µM). Mechanistically, PFD appears to exert its inhibitory effects by promoting the interaction between MRTF and actin indirectly. Finally, PFD-treated IPF lungs exhibit significantly less MRTF activation in fibroblast foci areas than naïve IPF lungs. Our results suggest MRTF signalling as a direct target for PFD and implicate that some of the anti-fibrotic effects of PFD may be due to MRTF inhibition in lung fibroblasts.

Invadosome Formation by Lung Fibroblasts in Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is characterized by abnormal fibroblast accumulation in the lung leading to extracellular matrix deposition and remodeling that compromise lung function. However, the mechanisms of interstitial invasion and remodeling by lung fibroblasts remain poorly understood. The invadosomes, initially described in cancer cells, consist of actin-based adhesive structures that coordinate with numerous other proteins to form a membrane protrusion capable of degrading the extracellular matrix to promote their invasive phenotype. In this regard, we hypothesized that invadosome formation may be increased in lung fibroblasts from patients with IPF. Public RNAseq datasets from control and IPF lung tissues were used to identify differentially expressed genes associated with invadosomes. Lung fibroblasts isolated from bleomycin-exposed mice and IPF patients were seeded with and without the two approved drugs for treating IPF, nintedanib or pirfenidone on fluorescent gelatin-coated coverslips for invadosome assays. Several matrix and invadosome-associated genes were increased in IPF tissues and in IPF fibroblastic foci. Invadosome formation was significantly increased in lung fibroblasts isolated from bleomycin-exposed mice and IPF patients. The degree of lung fibrosis found in IPF tissues correlated strongly with invadosome production by neighboring cells. Nintedanib suppressed IPF and PDGF-activated lung fibroblast invadosome formation, an event associated with inhibition of the PDGFR/PI3K/Akt pathway and TKS5 expression. Fibroblasts derived from IPF lung tissues express a pro-invadosomal phenotype, which correlates with the severity of fibrosis and is responsive to antifibrotic treatment.

Cell-Specific Response of NSIP- and IPF-Derived Fibroblasts to the Modification of the Elasticity, Biological Properties, and 3D Architecture of the Substrate

The fibrotic fibroblasts derived from idiopathic pulmonary fibrosis (IPF) and nonspecific interstitial pneumonia (NSIP) are surrounded by specific environments, characterized by increased stiffness, aberrant extracellular matrix (ECM) composition, and altered lung architecture. The presented research was aimed at investigating the effect of biological, physical, and topographical modification of the substrate on the properties of IPF- and NSIP-derived fibroblasts, and searching for the parameters enabling their identification. Soft and stiff polydimethylsiloxane (PDMS) was chosen for the basic substrates, the properties of which were subsequently tuned. To obtain the biological modification of the substrates, they were covered with ECM proteins, laminin, fibronectin, and collagen. The substrates that mimicked the 3D structure of the lungs were prepared using two approaches, resulting in porous structures that resemble natural lung architecture and honeycomb patterns, typical of IPF tissue. The growth of cells on soft and stiff PDMS covered with proteins, traced using fluorescence microscopy, confirmed an altered behavior of healthy and IPF- and NSIP-derived fibroblasts in response to the modified substrate properties, enabling their identification. In turn, differences in the mechanical properties of healthy and fibrotic fibroblasts, determined using atomic force microscopy working in force spectroscopy mode, as well as their growth on 3D-patterned substrates were not sufficient to discriminate between cell lines.

Identification of diagnostic gene biomarkers related to immune infiltration in patients with idiopathic pulmonary fibrosis based on bioinformatics strategies.

The study aims to identify potential diagnostic markers of idiopathic pulmonary fibrosis (IPF) and analyze the significance of immune cell infiltration in this pathology. Download two publicly available gene expression profiles (GSE10667 and GSE24206 datasets) from the GEO database including 48 Idiopathic pulmonary fibrosis (IPF) samples and 21 human control samples and select for distinctly expressed genes (DEG) from them. Lasso regression model and support vector machine recursive feature elimination S,V,R,F analysis were used to check candidate biomarkers. The area under the subject's work characteristic curve (AUC) value is used to evaluate its recognition ability. The GSE53845 dataset (40 IPF patients and 8 controls) continue to validate the expression level and diagnostic value of biomarkers in IPF. Comprehensive analysis of immune infiltrated cells of IPF was performed using R software and immune cell infiltration estimation analysis tool- deconvolution algorithm (CIBERSORT). 43 DEGs were identified in total. The identified DEGs mostly involve pneumonia, lung disease, collagen disease, obstructive pulmonary disease and other diseases. The activation of IL-17 signaling pathways, amoebic disease, interaction of viral proteins with cytokines and cytokine receptors, protein digestion and absorption, and flaccid hormone signaling pathways in IPF were different from the control group. The expression degree of CRTAC1, COL10A1, COMP, RPS4Y1, IGFL2, NECAB1, SCG5, SLC6A4, and SPP1 in IPF tissue were prominently higher than the normal group. Immune cell infiltration analysis showed that CRTAC1, COL10A1, COMP, IGFL2, NECAB1, SCG5, SLC6A4, and SPP1 were associated with monocytes, plasma cells, neutrophils, and regulatory (treg) T cells. CRTAC1, COL10A1, COMP, IGFL2, NECAB1, SCG5, SLC6A4, and SPP1 can be used as diagnostic markers for IPF, providing new ideas for the future study of IPF occurrence and molecular mechanisms.

Dipeptidyl peptidase 4 expression is not associated with an activated fibroblast phenotype in idiopathic pulmonary fibrosis.

Dipeptidyl peptidase 4 (DPP4) has been proposed as a marker for activated fibroblasts in fibrotic disease. We aimed to investigate whether a profibrotic DPP4 phenotype is present in lung tissue from patients with idiopathic pulmonary fibrosis (IPF). The presence of DPP4+ fibroblasts in normal and IPF lung tissue was investigated using flow cytometry and immunohistology. In addition, the involvement of DPP4 in fibroblast activation was examined in vitro, using CRISPR/Cas9 mediated genetic inactivation to generate primary DPP4 knockout lung fibroblasts. We observed a reduced frequency of primary DPP4+ fibroblasts in IPF tissue using flow cytometry, and an absence of DPP4+ fibroblasts in pathohistological features of IPF. The in vivo observations were supported by results in vitro showing a decreased expression of DPP4 on normal and IPF fibroblasts after profibrotic stimuli (transforming growth factor β) and no effect on the expression of activation markers (α-smooth muscle actin, collagen I and connective tissue growth factor) upon knockout of DPP4 in lung fibroblasts with or without activation with profibrotic stimuli.

Bioinformatic identification and analysis of immune-related chromatin regulatory genes as potential biomarkers in idiopathic pulmonary fibrosis.

BackgroundIdiopathic pulmonary fibrosis (IPF) is a disease with a very poor prognosis. The search for new IPF biomarkers is particularly urgent due to the uncertainty of the mechanisms and treatment. Studies have shown that chromatin regulators (CRs) are involved in the development of IPF and are associated with tumor immunity. However, there are no studies on immune-related CRs in IPF. Therefore, we conducted a systematic study to analyze the expression levels and immune correlation of CRs in IPF tissues and normal tissues and to explore their potential as diagnostic biomarkers.MethodsGSE53845, GSE179781 and GSE24206 datasets from Gene Expression Omnibus (GEO) database were merged into an integrated dataset as the training set; GSE70866 was used as the validation dataset. The cr-related differentially expressed genes (DEGs) between normal and IPF tissues were identified using the “Limma” software package. Weighted gene co-expression network analysis (WGCNA) was performed using the “WGCNA” package to screen eigengenes, which were intersected with DEGs to identify hub genes. The “ggcorrplot” package was used to analyze the correlation between hub genes and immunity, and immune-related hub genes were defined as immHub. A logistic regression model was constructed using immHub as the independent variable and whether the diagnosis was IPF as the dependent variable.ResultsOne hundred and sixty-nine DEGs were identified between IPF and normal tissues. wGCNA identified 3 key modules in brown, green and yellow genes that were present in all 3 modules and met module membership (MM) >0.8 and gene significance (GS) >0.5 were called signature genes (n=390). Four intersecting genes were obtained by intersecting DEGs with signature genes (PADI4, IGFBP7, GADD45A, and SETBP1) all associated with immunity were defined as immHub genes Logistic regression models were constructed based on immHub genes. The area under the curve (AUC) of the ROC curve is used to evaluate the diagnostic accuracy of the logistic regression model for IPF. The AUC in the ROC analysis was 0.771 for the training dataset, and 0.759 for the validation dataset.ConclusionsPADI4, IGFBP7 and GADD45A may be biomarkers for IPF, which will provide assistance in the diagnosis, treatment and prognostic assessment of IPF patients, and provide an important basis for future studies on the relationship between CRs genes and IPF.

Prevalence and lethality of interstitial pulmonary diseases in the Region of Los Ríos, Chile

Idiopathic Pulmonary Fibrosis (IPF) is a subtype of interstitial lung disease (ILD) that significantly affects people's quality of life, whose prevalence and mortality has increased. To determine the prevalence and lethality of ILD in the Los Ríos Region between the years 2018 and 2019. Review of a database of all patients attended at an outpatient clinic of the regional hospital. Those patients with the diagnosis of ILD and seen between 2018 and 2019 were selected. Mortality and its causes were verified with death certificates. In the study period, 339 cases with ILD, aged 71 ± 10 years (64% women) were identified. The calculated ILD prevalence was 84 cases per 100,000 inhabitants, with a higher predominancy in Futrono and Paillaco communes. IPF and Connective Tissue Diseases were the predominant subtypes. Overall lethality was 18%, with more deaths among patients with IPF (n = 31). In the Los Ríos Region, ILDs have a relevant prevalence and lethality.

A scalable 3D tissue culture pipeline to enable functional therapeutic screening for pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a lethal lung disease targeting the alveolar gas exchange apparatus, leading to death by asphyxiation. IPF progresses on a tissue scale through aberrant matrix remodeling, enhanced cell contraction, and subsequent microenvironment densification. Although two pharmaceuticals modestly slow progression, IPF patient survival averages less than 5 years. A major impediment to therapeutic development is the lack of high-fidelity models that account for the fibrotic microenvironment. Our goal is to create a three-dimensional (3D) platform to enable lung fibrosis studies and recapitulate IPF tissue features. We demonstrate that normal lung fibroblasts encapsulated in collagen microspheres can be pushed toward an activated phenotype, treated with FDA-approved therapies, and their fibrotic function quantified using imaging assays (extracellular matrix deposition, contractile protein expression, and microenvironment compaction). Highlighting the system's utility, we further show that fibroblasts isolated from IPF patient lungs maintain fibrotic phenotypes and manifest reduced fibrotic function when treated with epigenetic modifiers. Our system enables enhanced screening due to improved predictability and fidelity compared to 2D systems combined with superior tractability and throughput compared to 3D systems.

Analysis of immune-related genes in idiopathic pulmonary fibrosis based on bioinformatics and experimental verification.

Idiopathic pulmonary fibrosis (IPF) is a lung disease involving chronic progressive fibrosis, with unclear pathogenesis. In recent years, people have paid increasing attention to the role of immune mechanism. In this study, bioinformatics analysis was used to determine the potential immune-related biomarkers for the diagnosis of IPF, and further analyze the role of immune cell infiltration in the pathogenesis of IPF. The IPF data set (GSE150910) was downloaded from the Gene Expression Omnibus (GEO) database. We used R software to screen differential immune-related genes (IRGs). Least absolute shrinkage and selection operator (LASSO) regression, random forest algorithm, and support vector machine (SVM) were used to screen and determine IPF IRGs to be diagnostic biomarkers. The GSE32537 and GSE10667 data sets were combined into 1 data set to verify the diagnostic efficacy of biomarkers. Cell-type Identification by Estimating Relative Subsets of RNA Transcripts (CIBERSORT) was used to evaluate the infiltration of immune cells in IPF tissues, and analyze the relationship between diagnostic markers and immune cell infiltration. Meanwhile, clinical specimens were used to verify the diagnostic efficacy of biomarkers and their relationship with immune cell infiltration. In this study, 408 participants were involved in the screening to find that PLXNA4 and SLIT2 can be used as diagnostic biomarkers of IPF, and the results were verified by clinical samples. Immune cell infiltration analysis found that regulatory T cells (Tregs), memory B cells, plasma cells, and eosinophils might be involved in the process of IPF. In addition, Tregs were most closely related to PLXNA4 and SLIT2. In clinical samples, forkhead box p3 (FOXP3), a specific marker of Tregs, was positively correlated with PLXNA4 and negatively correlated with SLIT2, which is consistent with the results of bioinformatics analysis. The genes PLXNA4 and SLIT2 can be used as diagnostic markers of IPF, and immune cell infiltration plays an important role in the occurrence and development of IPF.

Small Airway Reduction and Fibrosis Is an Early Pathologic Feature of Idiopathic Pulmonary Fibrosis.

Rationale: To improve disease outcomes in idiopathic pulmonary fibrosis (IPF), it is essential to understand its early pathophysiology so that it can be targeted therapeutically. Objectives: Perform three-dimensional assessment of the IPF lung microstructure using stereology and multiresolution computed tomography (CT) imaging. Methods: Explanted lungs from patients with IPF (n = 8) and donor control subjects (n = 8) were inflated with air and frozen. CT scans were used to assess large airways. Unbiased, systematic uniform random samples (n = 8/lung) were scanned with microCT for stereological assessment of small airways (count number, and measure airway wall and lumen area) and parenchymal fibrosis (volume fraction of tissue, alveolar surface area, and septal wall thickness). Measurements and Main Results: The total number of airways on clinical CT was greater in IPF lungs than control lungs (P < 0.01), owing to an increase in the wall (P < 0.05) and lumen area (P < 0.05) resulting in more visible airways with a lumen larger than 2 mm. In IPF tissue samples without microscopic fibrosis, assessed by the volume fraction of tissue using microCT, there was a reduction in the number of the terminal (P < 0.01) and transitional (P < 0.001) bronchioles, and an increase in terminal bronchiole wall area (P < 0.001) compared with control lungs. In IPF tissue samples with microscopic parenchymal fibrosis, terminal bronchioles had increased airway wall thickness (P < 0.05) and dilated airway lumens (P < 0.001) leading to honeycomb cyst formations. Conclusions: This study has important implications for the current thinking on how the lung tissue is remodeled in IPF and highlights small airways as a potential target to modify IPF outcomes.

Adipose Stromal Cell-Secretome Counteracts Profibrotic Signals From IPF Lung Matrices.

Introduction: Idiopathic pulmonary fibrosis (IPF) is a fibrotic lung disease characterized by excess deposition and altered structure of extracellular matrix (ECM) in the lungs. The fibrotic ECM is paramount in directing resident cells toward a profibrotic phenotype. Collagens, an important part of the fibrotic ECM, have been shown to be structurally different in IPF. To further understand the disease to develop better treatments, the signals from the ECM that drive fibrosis need to be identified. Adipose tissue-derived stromal cell conditioned medium (ASC-CM) has demonstrated antifibrotic effects in animal studies but has not been tested in human samples yet. In this study, the collagen structural integrity in (fibrotic) lung tissue, its interactions with fibroblasts and effects of ASC-CM treatment hereon were studied.Methods: Native and decellularized lung tissue from patients with IPF and controls were stained for denatured collagen using a collagen hybridizing peptide. Primary lung fibroblasts were seeded into decellularized matrices from IPF and control subjects and cultured for 7 days in the presence or absence of ASC-CM. Reseeded matrices were fixed, stained and analyzed for total tissue deposition and specific protein expression.Results: In both native and decellularized lung tissue, more denatured collagen was observed in IPF tissue compared to control tissue. Upon recellularization with fibroblasts, the presence of denatured collagen was equalized in IPF and control matrices, whereas total ECM was higher in IPF matrices than in the control. Treatment with ASC-CM resulted in less ECM deposition, but did not alter the levels of denatured collagen.Discussion: Our data showed that ASC-CM can inhibit fibrotic ECM-induced profibrotic behavior of fibroblasts. This process was independent of collagen structural integrity. Our findings open up new avenues for ASC-CM to be explored as treatment for IPF.

Paired analysis of tumor mutation burden for lung adenocarcinoma and associated idiopathic pulmonary fibrosis

Genetic alterations underlying the development of lung cancer in individuals with idiopathic pulmonary fibrosis (IPF) have remained unclear. To explore whether genetic alterations in IPF tissue contribute to the development of IPF-associated lung cancer, we here evaluated tumor mutation burden (TMB) and somatic variants in 14 paired IPF and tumor samples from patients with IPF-associated lung adenocarcinoma. We also determined TMB for 22 samples of lung adenocarcinoma from patients without IPF. TMB for IPF-associated lung adenocarcinoma was significantly higher than that for matched IPF tissue (median of 2.94 vs. 1.26 mutations/Mb, P = 0.002). Three and 102 somatic variants were detected in IPF and matched lung adenocarcinoma samples, respectively, with only one pair of specimens sharing one somatic variant. TMB for IPF-associated lung adenocarcinoma was similar to that for lung adenocarcinoma samples with driver mutations (median of 2.94 vs. 2.51 mutations/Mb) and lower than that for lung adenocarcinoma samples without known driver mutations (median of 2.94 vs. 5.03 mutations/Mb, P = 0.130) from patients without IPF. Our findings suggest that not only the accumulation of somatic mutations but other factors such as inflammation and oxidative stress might contribute to the development and progression of lung cancer in patients with IPF.

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.