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Microarray profiling reveals suppressed interferon stimulated gene program in fibroblasts from scleroderma-associated interstitial lung disease.

Lindahl GE, Stock CJ, Shi-Wen X, Leoni P, Sestini P, Howat SL, Bou-Gharios G, Nicholson AG, Denton CP, Grutters JC, Maher TM, Wells AU, Abraham DJ, Renzoni EA - Respir. Res. (2013)

Bottom Line: Interstitial lung disease is a major cause of morbidity and mortality in systemic sclerosis (SSc), with insufficiently effective treatment options.Genes differentially expressed were identified using two separate analysis programs following a set of pre-determined criteria: only genes significant in both analyses were considered.This study identified a strongly suppressed interferon-stimulated gene program in fibroblasts from fibrotic lung.

View Article: PubMed Central - HTML - PubMed

Affiliation: Interstitial Lung Disease Unit, Royal Brompton Hospital and National Heart and Lung Institute, Imperial College London, Emmanuel Kaye Building, 1B Manresa Road, London SW3 6LR, UK. g.lindahl@imperial.ac.uk

ABSTRACT

Background: Interstitial lung disease is a major cause of morbidity and mortality in systemic sclerosis (SSc), with insufficiently effective treatment options. Progression of pulmonary fibrosis involves expanding populations of fibroblasts, and the accumulation of extracellular matrix proteins. Characterisation of SSc lung fibroblast gene expression profiles underlying the fibrotic cell phenotype could enable a better understanding of the processes leading to the progressive build-up of scar tissue in the lungs. In this study we evaluate the transcriptomes of fibroblasts isolated from SSc lung biopsies at the time of diagnosis, compared with those from control lungs.

Methods: We used Affymetrix oligonucleotide microarrays to compare the gene expression profile of pulmonary fibroblasts cultured from 8 patients with pulmonary fibrosis associated with SSc (SSc-ILD), with those from control lung tissue peripheral to resected cancer (n=10). Fibroblast cultures from 3 patients with idiopathic pulmonary fibrosis (IPF) were included as a further comparison. Genes differentially expressed were identified using two separate analysis programs following a set of pre-determined criteria: only genes significant in both analyses were considered. Microarray expression data was verified by qRT-PCR and/or western blot analysis.

Results: A total of 843 genes were identified as differentially expressed in pulmonary fibroblasts from SSc-ILD and/or IPF compared to control lung, with a large overlap in the expression profiles of both diseases. We observed increased expression of a TGF-β response signature including fibrosis associated genes and myofibroblast markers, with marked heterogeneity across samples. Strongly suppressed expression of interferon stimulated genes, including antiviral, chemokine, and MHC class 1 genes, was uniformly observed in fibrotic fibroblasts. This expression profile includes key regulators and mediators of the interferon response, such as STAT1, and CXCL10, and was also independent of disease group.

Conclusions: This study identified a strongly suppressed interferon-stimulated gene program in fibroblasts from fibrotic lung. The data suggests that the repressed expression of interferon-stimulated genes may underpin critical aspects of the profibrotic fibroblast phenotype, identifying an area in pulmonary fibrosis that requires further investigation.

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Gene expression profiles of control, SSc-ILD, and IPF pulmonary fibroblasts. A) Supervised hierarchical clustering of probes. The gene set used comprised the 843 probes with a fold change ≥2, difference in means ≥100, and p<0.05 (dChip) and FDR<0.01 (SAM), in both the SSc-ILD or IPF samples, compared to controls, which includes also 8 probes differentially expressed between SSc-ILD and IPF samples when compared directly. Each column corresponds to an individual sample (C= control, S=SSc-ILD, U=IPF), and each row corresponds to an individual probe. The coloured bars to the right of the heatmap identify the location of the insets displayed in B-J. Panels B-J were selected to highlight sample heterogeneity and/or genes co-expressed with high scoring differentially expressed genes.
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Figure 4: Gene expression profiles of control, SSc-ILD, and IPF pulmonary fibroblasts. A) Supervised hierarchical clustering of probes. The gene set used comprised the 843 probes with a fold change ≥2, difference in means ≥100, and p<0.05 (dChip) and FDR<0.01 (SAM), in both the SSc-ILD or IPF samples, compared to controls, which includes also 8 probes differentially expressed between SSc-ILD and IPF samples when compared directly. Each column corresponds to an individual sample (C= control, S=SSc-ILD, U=IPF), and each row corresponds to an individual probe. The coloured bars to the right of the heatmap identify the location of the insets displayed in B-J. Panels B-J were selected to highlight sample heterogeneity and/or genes co-expressed with high scoring differentially expressed genes.

Mentions: To visualise the differential expression across samples and to identify co-expressed genes, average linkage cluster analysis was performed using expression data for the 843 probes sets which displayed differential expression in at least one of the two comparisons: SSc-ILD vs. control, and IPF vs. control (Figure 4A). Within this set are also the eight probes differentially expressed when SSc-ILD and IPF samples were compared directly. Parts of identified gene clusters were selected to illustrate co-expression among upregulated (Figure 4, Panels B-F) and downregulated (Panels H-J) genes, and also to highlight different patterns of sample heterogeneity. Overall, a heterogeneous expression pattern was observed in the upregulated genes, whereas downregulated genes had more uniform expression patterns across samples for the majority of genes in both disease groups. Among upregulated genes, a TGF-β response signature [31,32] including genes encoding for fibrosis mediators and myofibroblast markers, such as SERPINE1 (PAI1), connective tissue growth factor, smoothelin, and transgelin (SM22) is prominent (Panel B). Co-expressed with these are strongly upregulated genes: growth arrest and DNA-damage inducible β (GADD45), xylosyltransferase 1 (XYLT1), N-cadherin, and elastin, with potential roles in the fibrotic disease process. Groups B, C and D all contain genes involved in contraction and migration, however, the degree of heterogeneity between samples differ between these groups: in group B, the majority of fibrotic samples have elevated expression of smoothelin and transgelin compared with controls; in group C, fewer fibrotic samples, 7 out of 11, have enhanced levels of α2 smooth muscle actin (ACTA2) expression; and in group D, all three IPF samples, but only 3 out of 8 SSc-ILD samples, have elevated expression of calponin 1 and actin gamma 2 smooth muscle (ACTG2). This may indicate different degrees of contractile/migratory phenotypes among these fibrotic cell preparations. Panel E contains ID1 and ID3, which are in the top 20 differentially regulated genes in both disease groups, and are upregulated in most of the fibrotic samples. Group F depicts a cluster of co-expressed cell-cycle associated genes, including cyclins and TOPO2, which exhibit heterogeneous expression in both disease groups. Panel G illustrates an area with less clustering, which however includes possible disease specific genes, e.g. Secreted protein, acidic, cysteine-rich (SPARC) (IPF, Table 2B) and desmoplakin (SSc-ILD) (Additional file 4). Desmoplakin is among the top 20 most upregulated genes in SSc-ILD with an elevated expression in 7 out of the 8 SSc-ILD fibroblast lines, but with low expression in the three IPF cell preparations and in all controls. Desmoplakin is part of the desmosome complex which forms tight cell-cell contacts [33], and its enhanced expression in SSc-ILD fibroblasts may define a different pathogenesis and cell origin. Panels H-J shows the marked suppressed expression of interferon stimulated genes (ISGs), such as antiviral genes (Group H), chemokine (Group I) and MHC class I genes (Group J). This cluster also includes key regulators of the interferon gene program, STAT1, interferon regulatory factor 1 (IRF1) and interferon regulatory factor 7 (IRF7), as well as chemokine (C-X-C motif) ligand 10 (CXCL10/IP10), one of the most strongly suppressed genes in the study, and the most strongly repressed chemokine in both disease groups (Panel H and I).


Microarray profiling reveals suppressed interferon stimulated gene program in fibroblasts from scleroderma-associated interstitial lung disease.

Lindahl GE, Stock CJ, Shi-Wen X, Leoni P, Sestini P, Howat SL, Bou-Gharios G, Nicholson AG, Denton CP, Grutters JC, Maher TM, Wells AU, Abraham DJ, Renzoni EA - Respir. Res. (2013)

Gene expression profiles of control, SSc-ILD, and IPF pulmonary fibroblasts. A) Supervised hierarchical clustering of probes. The gene set used comprised the 843 probes with a fold change ≥2, difference in means ≥100, and p<0.05 (dChip) and FDR<0.01 (SAM), in both the SSc-ILD or IPF samples, compared to controls, which includes also 8 probes differentially expressed between SSc-ILD and IPF samples when compared directly. Each column corresponds to an individual sample (C= control, S=SSc-ILD, U=IPF), and each row corresponds to an individual probe. The coloured bars to the right of the heatmap identify the location of the insets displayed in B-J. Panels B-J were selected to highlight sample heterogeneity and/or genes co-expressed with high scoring differentially expressed genes.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 4: Gene expression profiles of control, SSc-ILD, and IPF pulmonary fibroblasts. A) Supervised hierarchical clustering of probes. The gene set used comprised the 843 probes with a fold change ≥2, difference in means ≥100, and p<0.05 (dChip) and FDR<0.01 (SAM), in both the SSc-ILD or IPF samples, compared to controls, which includes also 8 probes differentially expressed between SSc-ILD and IPF samples when compared directly. Each column corresponds to an individual sample (C= control, S=SSc-ILD, U=IPF), and each row corresponds to an individual probe. The coloured bars to the right of the heatmap identify the location of the insets displayed in B-J. Panels B-J were selected to highlight sample heterogeneity and/or genes co-expressed with high scoring differentially expressed genes.
Mentions: To visualise the differential expression across samples and to identify co-expressed genes, average linkage cluster analysis was performed using expression data for the 843 probes sets which displayed differential expression in at least one of the two comparisons: SSc-ILD vs. control, and IPF vs. control (Figure 4A). Within this set are also the eight probes differentially expressed when SSc-ILD and IPF samples were compared directly. Parts of identified gene clusters were selected to illustrate co-expression among upregulated (Figure 4, Panels B-F) and downregulated (Panels H-J) genes, and also to highlight different patterns of sample heterogeneity. Overall, a heterogeneous expression pattern was observed in the upregulated genes, whereas downregulated genes had more uniform expression patterns across samples for the majority of genes in both disease groups. Among upregulated genes, a TGF-β response signature [31,32] including genes encoding for fibrosis mediators and myofibroblast markers, such as SERPINE1 (PAI1), connective tissue growth factor, smoothelin, and transgelin (SM22) is prominent (Panel B). Co-expressed with these are strongly upregulated genes: growth arrest and DNA-damage inducible β (GADD45), xylosyltransferase 1 (XYLT1), N-cadherin, and elastin, with potential roles in the fibrotic disease process. Groups B, C and D all contain genes involved in contraction and migration, however, the degree of heterogeneity between samples differ between these groups: in group B, the majority of fibrotic samples have elevated expression of smoothelin and transgelin compared with controls; in group C, fewer fibrotic samples, 7 out of 11, have enhanced levels of α2 smooth muscle actin (ACTA2) expression; and in group D, all three IPF samples, but only 3 out of 8 SSc-ILD samples, have elevated expression of calponin 1 and actin gamma 2 smooth muscle (ACTG2). This may indicate different degrees of contractile/migratory phenotypes among these fibrotic cell preparations. Panel E contains ID1 and ID3, which are in the top 20 differentially regulated genes in both disease groups, and are upregulated in most of the fibrotic samples. Group F depicts a cluster of co-expressed cell-cycle associated genes, including cyclins and TOPO2, which exhibit heterogeneous expression in both disease groups. Panel G illustrates an area with less clustering, which however includes possible disease specific genes, e.g. Secreted protein, acidic, cysteine-rich (SPARC) (IPF, Table 2B) and desmoplakin (SSc-ILD) (Additional file 4). Desmoplakin is among the top 20 most upregulated genes in SSc-ILD with an elevated expression in 7 out of the 8 SSc-ILD fibroblast lines, but with low expression in the three IPF cell preparations and in all controls. Desmoplakin is part of the desmosome complex which forms tight cell-cell contacts [33], and its enhanced expression in SSc-ILD fibroblasts may define a different pathogenesis and cell origin. Panels H-J shows the marked suppressed expression of interferon stimulated genes (ISGs), such as antiviral genes (Group H), chemokine (Group I) and MHC class I genes (Group J). This cluster also includes key regulators of the interferon gene program, STAT1, interferon regulatory factor 1 (IRF1) and interferon regulatory factor 7 (IRF7), as well as chemokine (C-X-C motif) ligand 10 (CXCL10/IP10), one of the most strongly suppressed genes in the study, and the most strongly repressed chemokine in both disease groups (Panel H and I).

Bottom Line: Interstitial lung disease is a major cause of morbidity and mortality in systemic sclerosis (SSc), with insufficiently effective treatment options.Genes differentially expressed were identified using two separate analysis programs following a set of pre-determined criteria: only genes significant in both analyses were considered.This study identified a strongly suppressed interferon-stimulated gene program in fibroblasts from fibrotic lung.

View Article: PubMed Central - HTML - PubMed

Affiliation: Interstitial Lung Disease Unit, Royal Brompton Hospital and National Heart and Lung Institute, Imperial College London, Emmanuel Kaye Building, 1B Manresa Road, London SW3 6LR, UK. g.lindahl@imperial.ac.uk

ABSTRACT

Background: Interstitial lung disease is a major cause of morbidity and mortality in systemic sclerosis (SSc), with insufficiently effective treatment options. Progression of pulmonary fibrosis involves expanding populations of fibroblasts, and the accumulation of extracellular matrix proteins. Characterisation of SSc lung fibroblast gene expression profiles underlying the fibrotic cell phenotype could enable a better understanding of the processes leading to the progressive build-up of scar tissue in the lungs. In this study we evaluate the transcriptomes of fibroblasts isolated from SSc lung biopsies at the time of diagnosis, compared with those from control lungs.

Methods: We used Affymetrix oligonucleotide microarrays to compare the gene expression profile of pulmonary fibroblasts cultured from 8 patients with pulmonary fibrosis associated with SSc (SSc-ILD), with those from control lung tissue peripheral to resected cancer (n=10). Fibroblast cultures from 3 patients with idiopathic pulmonary fibrosis (IPF) were included as a further comparison. Genes differentially expressed were identified using two separate analysis programs following a set of pre-determined criteria: only genes significant in both analyses were considered. Microarray expression data was verified by qRT-PCR and/or western blot analysis.

Results: A total of 843 genes were identified as differentially expressed in pulmonary fibroblasts from SSc-ILD and/or IPF compared to control lung, with a large overlap in the expression profiles of both diseases. We observed increased expression of a TGF-β response signature including fibrosis associated genes and myofibroblast markers, with marked heterogeneity across samples. Strongly suppressed expression of interferon stimulated genes, including antiviral, chemokine, and MHC class 1 genes, was uniformly observed in fibrotic fibroblasts. This expression profile includes key regulators and mediators of the interferon response, such as STAT1, and CXCL10, and was also independent of disease group.

Conclusions: This study identified a strongly suppressed interferon-stimulated gene program in fibroblasts from fibrotic lung. The data suggests that the repressed expression of interferon-stimulated genes may underpin critical aspects of the profibrotic fibroblast phenotype, identifying an area in pulmonary fibrosis that requires further investigation.

Show MeSH
Related in: MedlinePlus