Central lung gene expression associates with myofibroblast features in idiopathic pulmonary fibrosis

Abstract

Rationale: Contribution of central lung tissues to pathogenesis of idiopathic pulmonary fibrosis (IPF) remains unknown.

Objective: To ascertain the relationship between cell types of IPF-central and IPF-peripheral lung explants using RNA sequencing (RNA-seq) transcriptome.

Methods: Biopsies of paired IPF-central and IPF-peripheral along with non-IPF lungs were selected by reviewing H&E data. Criteria for differentially expressed genes (DEG) were set at false discovery rate <5% and fold change >2. Computational cell composition deconvolution was performed. Signature scores were computed for each cell type.

Findings: Comparison of central IPF versus non-IPF identified 1723 DEG (1522 upregulated and 201 downregulated). Sixty-two per cent (938/1522) of the mutually upregulated genes in central IPF genes were also upregulated in peripheral IPF versus non-IPF. Moreover, 85 IPF central-associated genes (CAG) were upregulated in central IPF versus both peripheral IPF and central non-IPF. IPF single-cell RNA-seq analysis revealed the highest CAG signature score in myofibroblasts and significantly correlated with a previously published activated fibroblasts signature (r=0.88, p=1.6×10^-4). CAG signature scores were significantly higher in IPF than in non-IPF myofibroblasts (p=0.013). Network analysis of central-IPF genes identified a module significantly correlated with the deconvoluted proportion of myofibroblasts in central IPF and anti-correlated with inflammation foci trait in peripheral IPF. The module genes were over-represented in idiopathic pulmonary fibrosis signalling pathways.

Interpretation: Gene expression in central IPF lung regions demonstrates active myofibroblast features that contributes to disease progression. Further elucidation of pathological transcriptomic state of cells in the central regions of the IPF lung that are relatively spared from morphological rearrangements may provide insights into molecular changes in the IPF progression.

Keywords: interstitial fibrosis.

Figure 1

Spatial differences and features of IPF lung biopsies. (A) Representative CT image of IPF lungs illustrating the locations of the central (C) and peripheral (P) tissues, respectively. (B–E) H&E staining of central and peripheral biopsies derived from non-IPF (both with normal lung architecture) and IPF lungs (central section with mild or no focal fibrosis and peripheral with rearrangement scarring, microscopic honeycombing and fibroblastic foci). IPF, idiopathic pulmonary fibrosis.

Figure 2

Volcano plot of differentially expressed genes (DEGs) by two-group comparison. (A) IPF central (IPF.C) versus non-IPF central (CTR.C); 1522 genes were upregulated and 201 were downregulated; (B) IPF peripheral (IPF.P) versus non-IPF peripheral (CTR.P); 1634 gene were upregulated and 686 were downregulated); (C) IPF peripheral (IPF.P) versus IPF central (IPF.C); 464 gene were upregulated and 379 were downregulated; (D) non-IPF peripheral (CTR.P) versus non-IPF central (CTR.C); only three upregulated genes were identified. Red and green dots represent DEGs. The horizontal dash lines represent FDR <1% in A–C and FDR <5% in D. The vertical dash lines represent FC >8 in A and B; FC >4 in C, and FC >2 in D. The full gene lists of upregulated (red font) and downregulated (green font) and statistical details of two-group comparison (A–C). Detailed DEGs lists can be found in online supplemental table E1 and DEGs passed the vertical lines defined above were in bold. CTR, non-IPF control; FC, fold change; FDR, false discovery rate; IPF, idiopathic pulmonary fibrosis.

Figure 3

Regional specific and consensus upregulated genes. (A) Principle component analysis plot separates all samples into three dimensions: IPF-peripheral (IPF.P, red) group, IPF-central (IPF.C, green) group, non-IPF control peripheral (CTR.P, blue) and non-IPF control-central (CTR.C, black) group. (B) Venn diagram illustrates 938 mutually upregulated genes (MUG, top panel) increased in both central and peripheral IPF lungs compared with non-IPF control lungs; 414 peripheral-associated genes (PAG, middle panel) upregulated in peripheral IPF compared with peripheral non-IPF control and central IPF; 85 central-associated genes (CAG, bottom panel) upregulated in central IPF compared with central non-IPF control and peripheral IPF. IPF.C/CTR.C: genes upregulated in IPF central compared with non-IPF control central tissues; IPF.P/CTR.P: genes upregulated in IPF peripheral compared with non-IPF control peripheral tissues; IPF.C/IPF.P: genes upregulated in IPF central compared with IPF peripheral tissues; IPF.P/IPF.C: genes upregulated in IPF peripheral compared with IPF central tissues. (C) Ingenuity Pathway Analysis of MUG with adjusted p value<0.0001. Detailed lists of MUG, PAG, CAG, and pathways enriched from MUG can be found in online supplemental table E2. CTR, non-IPF control; IPF, idiopathic pulmonary fibrosis.

Figure 4

Quantitative reverse transcription-PCR validation of dysregulated genes in IPF lungs. (A) PDGFRB displays more significant upregulation in IPF central (IPF.C) than in IPF peripheral (IPF.P) when compared with their corresponding non-IPF regions. (B) TNC is mutually upregulated in both IPF central and peripheral regions compared with the non-IPF regions. (C) ACTA2 is only upregulated in IPF central (IPF.C) compared with non-IPF control central (CTR.C). (D) COL1A1 is mutually upregulated in IPF central and peripheral regions compared with non-IPF regions. (E and F) PDGFB and CTGF are upregulated in IPF central (IPF.C) compared with both IPF peripheral (IPF.P) and non-IPF control central (CTR.C). *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001 are compared between groups as specified. ACTA2, alpha (α)−2 actin; COL1A1, collagen type I alpha 1; CTGF, connective tissue growth factor; CTR, non-IPF control; IPF, idiopathic pulmonary fibrosis; PDGFB, platelet derived growth factor subunit B; PDGFRB, platelet derived growth factor receptor beta; TNC, tenascin C gene; IPF, idiopatheic pulmonary fibrosis.

Figure 5

Central-associated genes (CAG) signature characterises myofibroblasts in IPF. CAG and activated fibroblasts signature genes were mapped to patients with IPF and non-IPF donors of single-cell RNA sequencing data GSE135893. (A) Z-scores of the 85 CAG were summed in each cell and then averaged in each cell type of patient with IPF as a signature score. (B) Correlation of CAG and activated fibroblasts signature scores in myofibroblasts of 12 patients with IPF. (C) Comparison of CAG signature scores between IPF and donor’s myofibroblasts. (D) GO biological process significantly enriched with CAG. FB, fibroblasts; GO, Gene Ontology; IPF, idiopathic pulmonary fibrosis.

Figure 6

Correlation of IPF central gene modules with cell type compositions in central lungs and pathological changes in peripheral lungs. (A) Correlation matrix of gene modules with cell type abundance in IPF central lung and traits of peripheral lungs pathological alterations. Construction of gene modules and correlation analysis were performed using the R/Bioconductor package ‘WGCNA’. Cell type deconvolution of IPF central lung tissues was performed using the R package ‘MuSiC’. Co-expressed gene modules were depicted in unique colour bars on the left y-axis. Correlation coefficient values from −1 (green) to 1 (red) were depicted on the right y-axis. Coloured squares in the correlation matrix plot represent positive (red) or negative (green) correlation between module eigengene with pathological traits or cell type compositions. (B) Ingenuity Pathway Analysis of Green module genes anti-correlated with the trait of peripheral lung inflammation foci. The red line indicates the significance criterion at p<0.01. IPF, idiopathic pulmonary fibrosis; WGCNA, weighted gene co-expression network analysis.