We provide an original multi-stage approach identifying a gene signature to assess murine 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, 1456 and 2215 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 RNA microarray and LC-MS/MS did. 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.

Autoantibodies (Aabs) are frequent in systemic sclerosis (SSc). Although recognized as potent biomarkers, their pathogenic role is debated. This study explored the effect of purified immunoglobulin G (IgG) from SSc patients on protein and mRNA expression of dermal fibroblasts (FBs) using an innovative multi-omics approach. Dermal FBs were cultured in the presence of sera or purified IgG from patients with diffuse cutaneous SSc (dcSSc), limited cutaneous SSc or healthy controls (HCs). The FB proteome and transcriptome were explored using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) and microarray assays, respectively. Proteomic analysis identified 3,310 proteins. SSc sera and purified IgG induced singular protein profile patterns. These FB proteome changes depended on the Aab serotype, with a singular effect observed with purified IgG from anti-topoisomerase-I autoantibody (ATA) positive patients compared to HC or other SSc serotypes. IgG from ATA positive SSc patients induced enrichment in proteins involved in focal adhesion, cadherin binding, cytosolic part, or lytic vacuole. Multi-omics analysis was performed in two ways: first by restricting the analysis of the transcriptomic data to differentially expressed proteins; and secondly, by performing a global statistical analysis integrating proteomics and transcriptomics. Transcriptomic analysis distinguished 764 differentially expressed genes and revealed that IgG from dcSSc can induce extracellular matrix (ECM) remodeling changes in gene expression profiles in FB. Global statistical analysis integrating proteomics and transcriptomics confirmed that IgG from SSc can induce ECM remodeling and activate FB profiles. This effect depended on the serotype of the patient, suggesting that SSc Aab might play a pathogenic role in some SSc subsets.

BackgroundFibroblasts (Fb) are key effectors cells in systemic sclerosis (SSc).1 Fb stimulation with TGF-β1 is usually considered as the positive control in studies assessing the fibrogenesis in SSc.2 Yet, the lack of standardisation of TGF-β1 stimulation might be responsible for discrepancies in experiments performed in different conditions. Proteomic approach allows the analysis of differential expression of the whole proteins (proteome) in Fb, and appears an interesting approach to compare different culture conditions.ObjectivesWe designed this study to compare the whole protein expression in Fb stimulated by TGF-β1 in different conditions.MethodsAt fifth passage, primary culture of human Fb from healthy subjects (ATCC; PCS-201–012) were stimulated or not with different concentrations of recombinant human active TGF-β1 (0.04, 1, and 5 ng/mL) (R and D Systems; 240-B-002) during 24, 48 and 72 hours. Proteins were extracted and analysed using an eFASP LC-MS/MS approach on an Orbitrap mass spectrometer (Thermo Scientific; Q Exactive +). Proteins quantitation was performed by Maxquant and statistical analysis by Perseus using ANOVA and principal component analysis (PCA).ResultsA total of 3267 proteins were identified, of which 1957 showed differential expression using ANOVA analysis. PCA revealed several clusters of differential proteins expression (figure 1). There were clear clusters of protein expression related to (i) unstimulated and stimulated conditions, (ii) between the three different times of stimulation and (iii) to TGF-β1 concentrations used. Although the expression of proteins in Fb exposed to 0.04 and 1 ng/mL of TGF-β1 during 72 hour were rather close, there was a unique proteins profile related to the condition with 5 ng/mL of TGF-β1 during 72 hour.Figure 1: PCA representation of differential proteins expression in different conditions. [TGF-β1]=0.04 ng/mL: square; [TGF-β1]=1 ng/mL: circle; [TGF-β1]=5 ng/mL: diamond. The more the points appear distanced, the more different is the protein expression.Abstract AB0195 – Figure 1PCA representation of differential proteins expression in different conditions.ConclusionsThis study highlights a variation of proteins expression depending on both stimulation time and TGF-β1 concentrations in Fb culture. The identification of protein differentially expressed will provide insights in the impact of TGF-β1 on Fb physiology under stimulation conditions. These data underline the need of standardisation of culture conditions to allow inter-data comparisons using in sensitive “omic” approaches.References[1] Garret SM, Frost DB, Feghali-Bostwick C. The mighty fibroblast and its utility in scleroderma research. J Scleroderma Relat Disord2017;2(2):69–134.[2] Verrecchia F, Mauviel A. Transforming growth factor-beta signaling through the Smad pathway: role in extracellular matrix gene expression and regulation. J Invest Dermatol2002Feb;118(2):211–5.Disclosure of InterestNone declared

Fibroblasts (Fb) are key effector cells in systemic sclerosis (SSc). Fb stimulation with transforming growth factor beta 1 (TGF-β1) is considered as a positive control in studies assessing fibrogenesis. The lack of standardization of TGF-β1 stimulation might be responsible for discrepancies in experiments performed in different conditions. Using quantitative proteomics analysis, we evaluated the impact of changes in experimental conditions on proteomic profiles of primary Fb. Principal component analysis (PCA) identified several groups of differentially expressed proteins influenced by cell passage, culture medium, and both concentration and duration of exposure to TGF-β1 stimulation. Bioinformatics analysis revealed that late passages expressed proteins involved in senescence. TGF-β1 concentration and time of stimulation were correlated with the expression of proteins involved in the fibrogenesis and inflammatory processes. These data underline the need for standardization of culture conditions to allow inter-data comparisons in future in vitro studies, especially when using "omics" approaches.