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Disease-specific transcriptomic changes induced by NFκB inhibition in idiopathic pulmonary fibrosis

ABSTRACT: Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease of unknown etiology and poor prognosis. In IPF, aberrant extracellular matrix production by activated, hyperproliferative fibroblasts drives disease progression but the exact mechanisms by which this occurs remains undefined. We previously showed that inhibition of transcription factor nuclear factor kappa-B (NFκB) with ACT001 reduced pro-fibrotic fibroblast behaviors such as fibroblast-to-myofibroblast transition. The aim of this follow-up study was to characterize the transcriptional profile of primary fibroblasts treated with ACT001 and identify differences between cells from patients with IPF and non-diseased control donors. Primary lung fibroblasts derived from eight patients with IPF and eight age-matched non-diseased controls (NDC) were treated with 0, 3 or 10 µM ACT001 in the presence/absence of pro-fibrotic cytokine transforming growth factor (TGF)-β1 for 24 hours after which whole genome RNA sequencing was performed. Gene set enrichment analysis (GSEA) of the Hallmark gene sets was also completed. Although the majority of genes differentially expressed after ACT001 treatment were the same in NDC and IPF cells, several genes were uniquely changed in IPF. GSEA indicated that ACT001 treatment most upregulates the reactive oxygen species pathway. ACT001 is a water-soluble compound with a stable half-life in plasma, thus making it an attractive candidate for further investigation as a therapeutic in IPF. This study adds to the growing body of literature that demonstrates anti-fibrotic activity of NF-ĸB inhibition in the context of IPF.

ORGANISM(S): Homo sapiens

PROVIDER: GSE188464 | GEO | 2023/11/05

REPOSITORIES: GEO

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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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Disease-specific transcriptomic changes induced by NFκB inhibition in idiopathic pulmonary fibrosis

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