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Differences between fibroblasts derived from normal and IPF lungs

ABSTRACT: Objective: To evaluate the characteristics of IPF lungs in fibroblasts, we performed RNA-Sequencing of fibroblasts derived from normal and IPF lungs Method:　NHLF（Normal human lung fibroblast）　and DHLF-IPF (DIPF; Diseased human lung fibroblasts derived from idiopathic pulmonary fibrosis)　were purchased from Lonza (Walkersville, MD, USA) .Total RNA was extracted from fibroblasts (NHLF and DIPF) using an RNeasy® Mini Kit (#74106; Qiagen, Valencia, CA, USA). Preparation of a next-generation sequencing library was performed using a SMARTer® Stranded Total RNA Sample Prep Kit–Pico Input Mammalian (TaKaRa, Shiga, Japan). Sequencing was performed on an Illumina HiSeq 2500 platform in 75-base single-end mode with Illumina Casava 1.8.2 software for base calling. Sequenced reads were mapped to the human reference genome sequence (hg19) using TopHat v.2.0.13 software, in combination with Bowtie 2 v.2.2.3 and SAMtools v.0.1.19. Result: Of the 26,257 genes analyzed, for NHLF and DHLF-IPF (DIPF), FPKM was required to be greater than or equal to 0.3. Under these conditions, 764 genes were up regulated inDHLF-IPF (DIPF) and 691 genes were down regulated.

ORGANISM(S): Homo sapiens

PROVIDER: GSE185259 | GEO | 2021/10/06

REPOSITORIES: GEO

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Project description:RNA sequencing has been performed to investigate the transcriptomic profile of fibroblasts derived from apical and basal regions of idiopathic pulmonary fibrosis (IPF) and control (CTR) lungs. Lung fibroblasts were cultured onto T25 flasks until full confluence was reached. Fibroblasts were then harvested by trypsinization using 0.05% trypsin-EDTA and washed in Phosphate Buffered Saline (PBS). RNA was extracted using the RNeasy Mini kit protocol. Samples were then sent to the Australian Genome Research Facility (AGRF) where 1μg of RNA was submitted for next-generation sequencing. Once raw data returned, bioinformatics analysis was conducted. Results revealed little difference at the transcriptomic level between apical lung fibroblasts isolated from IPF and CTR donors. In contrast, there was a significant difference in gene expression between IPF and CTR lung basal fibroblasts with 90 differentially expressed genes (DEGs) identified in IPF basal fibroblasts. Gene ontology analysis of these 90 DEGs suggested that the most important functions were associated with receptor ligand activity, cell adhesion molecule binding, and integrin binding. Furthermore, fibroblasts isolated from the basal and apical sites of control lung were not significantly different. Interestingly, the lung basal fibroblasts from IPF patients were significantly different in their transcriptomic profile to that of apical fibroblasts from the same patients. 303 DEGs were identified in IPF basal fibroblasts compared with IPF apical fibroblasts. According to the GO analysis on these 303 DEGs, the most important function of the identified dysregulated genes was associated with the composition and function of the extracellular (ECM). Further pathway analysis identified 4 signaling pathways, namely arrhythmogenic right ventricular cardiomyopathy, calcium signaling pathway, dilated cardiomyopathy, and hematopoietic cell lineage. An interesting finding was that these IPF basal fibroblasts were clustered into two groups (Group 1 and Group 2). Analysis on these two groups found 3594 DEGs in Group 1 compared to Group 2. Go analysis suggested most of the identified dysregulated genes was involved in the composition and function of the ECM. Pathway analysis identified 28 dysregulated signaling pathways, including PI3K-Akt signaling pathway, TGF-β signaling pathway and Wnt signaling pathway. The results of the transcriptome analysis intimate that the IPF is more predominant in the basal lungs and the fibroblast-derived from the basal region of the fibrotic lungs may serve as a central role in IPF. Moreover, the dramatic difference between two groups of IPF basal fibroblasts might be linked to the differential response to the same drug occurred in IPF patients.

Project description:The activated fibroblast is the central effector cell for the progressive fibrotic process that characterizes idiopathic pulmonary fibrosis (IPF). An understanding of the genomic phenotype of this cell in isolation is essential to the understanding of disease pathogenesis and is integral to strategizing therapeutic trials. Employing a unique technique that minimizes cellular phenotypic alterations, we characterized the genomic phenotype of non-cultured pulmonary fibroblasts from the lungs of patients with advanced IPF. This approach revealed several novel genes and pathways previously unreported in IPF fibroblasts. Specifically, we demonstrate altered expression in proteasomal constituents, ubiquitination mediators, the Wnt pathway and several cell cycle regulators suggestive of loss of normal cell cycle controls. The pro-inflammatory cytokine CXCL12 was also up-regulated which may provide a mechanism for fibrocytes’ recruitment, while up-regulated oncogenic KIT may promote fibroblast over proliferation. Paradoxically, pro-apoptotic inducers such as death inducing ligand TRAIL (TNFSF10) and pro-apoptotic Bax were also up-regulated. This comprehensive description of altered gene expression within IPF fibroblasts sheds further light on the complex interactions that characterize IPF. Further studies including therapeutic interventions directed at these pathways hold promise for the treatment of this devastating disease. 58 samples of total RNA isolated from 12 lungs of patients with end-stage idiopathic pulmonary fibrosis and 6 donors of normal lungs (controls) who were designated brain dead, non-diseased donors whose lungs failed criteria for transplantation and who were organ donors for research. RNA extraction followed the Qiagen RNeasy Kit using QIshredder columns for shredding of DNA contiminants. Experimental/control samples were amplified amino-allylated RNA labeled with Cy5 and Stratagene Reference RNA was amplified and amino-allylated and labeled with Cy3. Amplification was one round using Ambion MessageAmp II kit with amino-allylated UTP according to the protocol of the Duke University Institute for Genome Sciences and Policy. Amplification and amino-allylation of the Stratagene Reference RNA and Hybridization of Reference with patient samples and controls was done by the Duke Institute for Genomic Sciences and Policy.

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Differences between fibroblasts derived from normal and IPF lungs

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