In this study, we explored the regulation and the role of up-regulated microRNAs in idiopathic pulmonary fibrosis (IPF), a progressive interstitial lung disease of unknown origin. We analyzed the expression of microRNAs in IPF lungs and identified 43 significantly upregulated microRNAs. Twenty-four of the 43 increased microRNAs were localized to the chromosome 14q32 microRNA cluster. We validated the increased expression of miR-154, miR-134, miR-299-5p, miR-410, miR-382, miR-409-3p, miR-487b, miR-31, and miR-127 by quantitative RT-PCR and determined that they were similarly expressed in embryonic lungs. We did not find evidence for differential methylation in this region, but analysis of transcription factor binding sites identified multiple SMAD3-binding elements in the 14q32 microRNA cluster. TGF-b1 stimulation of normal human lung fibroblasts (NHLF) caused up-regulation of microRNAs on chr14q32 that were also increased in IPF lungs. Chromatin immunoprecipitation confirmed binding of SMAD3 to the putative promoter of miR-154. Mir-154 was increased in IPF fibroblasts, and transfection of NHLF with miR-154 caused significant increases in cell proliferation and migration. The increase in proliferation induced by TGF-b was not observed when NHLF or IPF fibroblasts were transfected with a mir-154 inhibitor. Transfection with miR-154 caused activation of the WNT pathway in NHLF. ICG-001 and XAV939, inhibitors of the WNT/b-catenin pathway, reduced the proliferative effect of miR-154. The potential role of miR-154, one of multiple chr14q32 micro-RNA cluster members up-regulated in IPF and a regulator of fibroblast migration and proliferation, should be further explored in IPF.

BackgroundIdiopathic Pulmonary Fibrosis (IPF) is characterized by profound changes in the lung phenotype including excessive extracellular matrix deposition, myofibroblast foci, alveolar epithelial cell hyperplasia and extensive remodeling. The role of epigenetic changes in determining the lung phenotype in IPF is unknown. In this study we determine whether IPF lungs exhibit an altered global methylation profile.Methodology/Principal FindingsImmunoprecipitated methylated DNA from 12 IPF lungs, 10 lung adenocarcinomas and 10 normal histology lungs was hybridized to Agilent human CpG Islands Microarrays and data analysis was performed using BRB-Array Tools and DAVID Bioinformatics Resources software packages. Array results were validated using the EpiTYPER MassARRAY platform for 3 CpG islands. 625 CpG islands were differentially methylated between IPF and control lungs with an estimated False Discovery Rate less than 5%. The genes associated with the differentially methylated CpG islands are involved in regulation of apoptosis, morphogenesis and cellular biosynthetic processes. The expression of three genes (STK17B, STK3 and HIST1H2AH) with hypomethylated promoters was increased in IPF lungs. Comparison of IPF methylation patterns to lung cancer or control samples, revealed that IPF lungs display an intermediate methylation profile, partly similar to lung cancer and partly similar to control with 402 differentially methylated CpG islands overlapping between IPF and cancer. Despite their similarity to cancer, IPF lungs did not exhibit hypomethylation of long interspersed nuclear element 1 (LINE-1) retrotransposon while lung cancer samples did, suggesting that the global hypomethylation observed in cancer was not typical of IPF.Conclusions/SignificanceOur results provide evidence that epigenetic changes in IPF are widespread and potentially important. The partial similarity to cancer may signify similar pathogenetic mechanisms while the differences constitute IPF or cancer specific changes. Elucidating the role of these specific changes will potentially allow better understanding of the pathogenesis of IPF.

Rationale: Idiopathic pulmonary fibrosis (IPF) is an untreatable and often fatal lung disease that is increasing in prevalence and is caused by complex interactions between genetic and environmental factors. Epigenetic mechanisms control gene expression and are likely to regulate the IPF transcriptome.Objectives: To identify methylation marks that modify gene expression in IPF lung. Methods:We assessed DNA methylation (comprehensive highthroughput arrays for relative methylation arrays [CHARM]) and gene expression (Agilent gene expression arrays) in 94 patients with IPF and 67 control subjects, and performed integrative genomic analyses to define methylation-gene expression relationships in IPF lung. We validated methylation changes by a targeted analysis (Epityper), and performed functional validation of one of the genes identified by our analysis.Measurements and Main Results: We identified 2,130 differentially methylated regions (DMRs; ,5% false discovery rate), of which 738 are associated with significant changes in gene expression and enriched for expected inverse relationship between methylation and expression (P , 2.2 3 10 216 ). We validated 13/15 DMRs by targeted analysis of methylation. Methylation-expression quantitative trait loci (methyl-eQTL) identified methylation marks that control cis and trans gene expression, with an enrichment for cis relationships (P , 2.2 3 10 216 ). We found five trans methyl-eQTLs where a methylation change at a single DMR is associated with transcriptional changes in a substantial number of genes; four of these DMRs are near transcription factors (castor zinc finger 1 [CASZ1], FOXC1, MXD4, and ZDHHC4). We studied the in vitro effects of change in CASZ1 expression and validated its role in regulation of target genes in the methyl-eQTL.Conclusions: These results suggest that DNA methylation may be involved in the pathogenesis of IPF.Keywords: DNA methylation; gene expression; pulmonary fibrosis; quantitative trait; mapping Idiopathic pulmonary fibrosis (IPF) appears to result from reprogramming of injured alveolar epithelial cells, which undergo early apoptosis, epithelial-mesenchymal transition, and produce mediators that lead to proliferation of resident fibroblasts and recruitment of fibrocytes. As the extracellular matrix (ECM) expands, myofibroblastic foci develop, resulting in further fibroproliferation in the ECM and the more extensive lung remodeling (1).

In this study, using sLC and CD11b-DTR techniques, monocytes were depleted from both peripheral blood by 70-95% and spleen by 50-70%. Administration of MC21 mAb removed 45% of the PBMs. Whether or not the three techniques have an influence on monocytes and neutrophils in the bone marrow was not reported. It is crucial to cautiously choose depletion strategies because monocytes are able to migrate among different pools (the peripheral blood, spleen, and bone marrow) during inflammation. Monocytes also play a requisite role in regulating the resolution of inflammation by different mechanisms. Otherwise, it is very difficult to interpret the experimental findings. This notion is supported by the following findings: (1) bone marrow can release Ly6C high CCR2 1 monocytes to the blood stream in response to the LPS challenge (12); (2) the spleen is a storage site that contains 50% of monocytes (Ly6C high ) in the body, and splenic monocytes can be rapidly mobilized toward the peripheral blood and inflamed site during inflammation and injury (13); (3) the splenic monocytes are indispensable elements forming immune reflex, which controls production of systemic proinflammatory cytokines (for example, TNF-a) (14) through the cholinergic-a7nAChR pathway; (4) alternatively activated monocytes (M2) in the peripheral blood and spleen might be involved in the resolution of inflammation (15); and (5) activation of CD11b in monocytes is able to limit TLR4dependent proinflammatory cytokine production (16).Alveolar flooding is a hallmark of ALI and ARDS. It should be mentioned that the magnitude of lung injury in this study is not equivalent to the clinical conditions because the dosages of LPS (0.4 mg/kg) and bacteria (10 25 cfu) used in the experiments were relatively low. In the meantime, a clear link among PBM depletion, alveolar neutrophilia, and pulmonary edema has not been established. Alveolar neutrophilia and the slight increase of lung vascular permeability are not parallel to the formation of pulmonary edema. Therefore, the conclusion drawn from the current study should be verified in more severe conditions and the other ALI animal models that are neutrophil dependent, for example, acid aspiration, transfusion-related acute lung injury, and ventilator-induced lung injury. Nevertheless, this study contributes importantly to ongoing research on how neutrophils are guided to the airspaces of the lung to mediate lung inflammation and injury.