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Design of a gene signature to assess the proinflammatory and the profibrotic polarization of murine fibroblasts

ABSTRACT: We provide an original multi-stage approach identifying a gene signature to assess the fibroblast polarization. Prototypic polarizations (inflammatory/fibrotic) were induced by seeded mouse embryonic fibroblasts (MEFs) with TNFα or TGFß1, respectively. The transcriptomic and proteomic profiles were obtained by RNA microarray and LC/MS-MS. Gene Ontology and pathways analysis were performed among the differentially expressed genes (DEGs) and proteins (DEPs). Balb/c mice underwent daily intradermal injections of HOCl (or PBS) as an experimental murine model of inflammation-mediated fibrosis in a time-dependent manner. As results, 1,456 and 2,215 DEGs, and 289 and 233 DEPs were respectively found in MEFs in response to TNFα or TGFß1, respectively. Among the most significant pathways, we combined 26 representative genes to encompass the proinflammatory and profibrotic polarizations of fibroblasts. Based on principal component analysis, this signature deciphered baseline state, proinflammatory polarization, and profibrotic polarization as accurately as did RNA microarray and LC/MS-MS. Then, we assessed the gene signature on dermal fibroblasts isolated from the experimental murine model. We observed a proinflammatory polarization at day 7, and a mixture of a proinflammatory and profibrotic polarizations at day 42 in line with histological findings. Our approach provides a small-size and convenient gene signature to assess murine fibroblast polarization.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Skin, Fibroblast

DISEASE(S): Disease Free

SUBMITTER: BRAY FABRICE

LAB HEAD: Fabrice Brat

PROVIDER: PXD034078 | Pride | 2022-07-12

REPOSITORIES: Pride

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Project description:Langerhans cells (LCs) are antigen presenting cells residing in the epidermis. Due to the difficulties with obtaining sufficient quantities of LCs for functional studies, many controversies exist about their origin and function. To gain insights into the molecular mechanisms underpinning LC biology and to elucidate how similar they are to a classical tissue resident DCs of myeloid origin, we undertook a whole transcriptome analysis of human skin migratory CD1a+ LCs and CD11c+ DDCs, stimulated with an epidermal pro-inflammatory cytokine, TNF-α over a time course of 24h. RNA was extracted from 250,000 human skin migratory CD1a+ LCs and CD11c+ DDCs stimulated in culture with 25ng/ml of TNF-α for 0, 2, 8 and 24h. RNA concentration and integrity was determined with an Agilent Bioanalyser (RIN of 7.0 or above) and gene expression analysis was carried out using the Human Genome U-219 Affymetrix platform by ARK-Genomics Centre, The Roslin Institute, Edinburgh. Expression data were normalised using the Robust Multichip Average (RMA) package. 2,334 transcripts regulated by exposure of skin DCs to TNF-α were identified with Bayesian Estimation of Temporal Regulation, a cut-off threshold 0.05 , for genes showing ≥ 1.5 fold difference between the maximum gene expression level and time 0 control in log2(x) RMA normalised gene expression. The interactive 3D diagram presenting the transcriptomic networks in human skin DCs can be viewed at: http://www.macrophages.com/LC_vs_DC RNA was extracted from 250,000 human skin migratory CD1a+ LCs and DDCs stimulated in culture with 25ng/ml of TNF-α for 0, 2, 8 and 24h. RNA concentration and integrity was determined with an Agilent Bioanalyser (RIN of 7.0 or above) and gene expression analysis was carried out using the Human Genome U-219 Affymetrix platform by ARK-Genomics Centre, The Roslin Institute, Edinburgh. Expression data were normalised using the Robust Multichip Average (RMA) package. 2,334 transcripts regulated by exposure of skin DCs to TNF-α were identified with Bayesian Estimation of Temporal Regulation, a cut-off threshold 0.05 , for genes showing ≥ 1.5 fold difference between the maximum gene expression level and time 0 control in log2(x) RMA normalised gene expression. *submitter cannot locate the CEL files

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Design of a gene signature to assess the proinflammatory and the profibrotic polarization of murine fibroblasts

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