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Transcriptional regulatory model of fibrosis progression in the human lung
John E. McDonough, … , Wim A. Wuyts, Naftali Kaminski
John E. McDonough, … , Wim A. Wuyts, Naftali Kaminski
Published October 10, 2019
Citation Information: JCI Insight. 2019;4(22):e131597. https://doi.org/10.1172/jci.insight.131597.
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Research Article Pulmonology

Transcriptional regulatory model of fibrosis progression in the human lung

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Abstract

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.

Authors

John E. McDonough, Farida Ahangari, Qin Li, Siddhartha Jain, Stijn E. Verleden, Jose Herazo-Maya, Milica Vukmirovic, Giuseppe DeIuliis, Argyrios Tzouvelekis, Naoya Tanabe, Fanny Chu, Xiting Yan, Johny Verschakelen, Robert J. Homer, Dimitris V. Manatakis, Junke Zhang, Jun Ding, Karen Maes, Laurens De Sadeleer, Robin Vos, Arne Neyrinck, Panayiotis V. Benos, Ziv Bar-Joseph, Dean Tantin, James C. Hogg, Bart M. Vanaudenaerde, Wim A. Wuyts, Naftali Kaminski

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Figure 4

Pou2af1–/– mice show less fibrotic severity in bleomycin-induced lung fibrosis.

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Pou2af1–/– mice show less fibrotic severity in bleomycin-induced lung f...
(A) qRT-PCR analysis of relative change in collagen type I, α1 (Col1α1) mRNA levels in wild-type (Pou2af1+/+) and Pou2af1–/– mice on day 14 after treatment with saline or bleomycin. Data are presented as box plots showing median and interquartile ranges for each group, with groups compared using linear mixed-effects models. Wild-type saline (WTS) n = 26; wild-type bleomycin (WTB) n = 23; Pou2af1-knockout saline (KOS) n = 23; and Pou2af1-knockout bleomycin (KOB) n = 25. (B) Relative collagen deposition assessed by hydroxyproline content per right lung in wild-type (Pou2af1+/+) and Pou2af1–/– mice as indicated by treatment groups. Data are shown as box plots with median and interquartile ranges and compared using linear mixed-effect models. WTS n = 20; WTB n = 26; KOS n = 22; and KOB n = 29. (C) H&E staining (upper panels), α-SMA (middle panels), and Masson’s trichrome (lower panels) staining of representative lung sections (n = 2 per group). Scale bars: 200 μm. (D) POU2AF1 immunostaining (red) in human IPF lung tissue showed no staining in bronchial epithelium (a) but lymphocytic aggregates (b) were positive. Scale bars: 400 μm (left) or 50 μm (right 2 images). (E) Violin plots from single-cell RNA-sequencing data of control (orange) and IPF (green) lung tissues showing specific cell types expressing POU2AF1. Upper panel shows CD79 expression identifying the B cell population, middle panel shows FOXJ1 expression identifying the ciliated epithelial cell population. POU2AF1 (bottom panel) was highly expressed in CD79 expressing B cells with some expression in ciliated epithelium (FOXJ1). In IPF, POU2AF1 showed increased expression in B cells and a slight reduction of expression in the ciliated cells. *P < 0.05, **P < 0.001, ***P < 0.0001, ns = no significance.

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