OP0096 CAN IMMUNOGLOBULINS G FROM SYSTEMIC SCLEROSIS PATIENTS INFLUENCE THE SECRETOME OF DERMAL FIBROBLASTS?

Abstract

BackgroundAutoantibodies (Aab) are frequent in systemic sclerosis (SSc) [1]. Recently, it has been showed that immunoglobulins G (IgG) from SSc promoted proinflammatory and profibrotic phenotype in monocyte secretome [2]. Fibroblasts (FB) are key effectors cells in SSc and data on FB proteins secretion in the presence of IgG from SSc patients are lacking. While recognized as potent biomarkers, the pathogenic role of Aab is much more debated.ObjectivesTo explore the FB secretome in the presence of purified IgG from SSc patientsMethodsNormal dermal FB were cultured in the presence of purified IgG from patients with diffuse cutaneous SSc (dcSSc), limited cutaneous SSc (lcSSc) or healthy controls (HC). After 72 h of culture, the cell supernatants were collected, centrifuged and passed through a filter to remove the cells. After proteins digestion, secretome was explored using mass spectrometry coupled with liquid chromatography (LC-MS/MS). Analysis of variance (ANOVA) and hierarchical clustering were used to identify proteins responses patterns.ResultsProteomic identified and quantified 1268 proteins, among them 377 were significant after ANOVA. SSc and HC secretome appeared distinct. Hierarchical clustering on significant proteins after ANOVA showed 3 distinct groups of patients secretome: a first group including mostly dcSSc anti-topoisomerase-I (ATA) positive (dcSSc ATA+) patients, a second group including mostly dcSSc ATA negative (dcSSc ATA-) patients, a third group more heterogeneous including the majority of HC, lcSSc anti-centromere positive (lcSSc ACA+) patients and dcSSc ATA- patients (Figure 1.A). The comparison of FB secretome in the presence of purified IgG from dcSSc ATA+ vs IgG HC revealed 203 differentially expressed proteins (DEP) (Figure 1.B). The enriched Gene Ontology (GO) terms upregulated in IgG dcSSc ATA+ were involved in endocytic vesicle lumen, vesicle lumen, extracellular matrix or glycosaminoglycan binding. The comparisons of IgG dcSSc ATA- vs IgG HC and IgG lcSSc ACA+ vs IgG HC revealed 85 and 15 DEP, respectively. Follistatin, amyloid beta A4 protein, myosin-9 and calreticulin were commonly overexpressed in the 3 subtypes of SSc. 40 proteins were exclusively overexpressed in the condition dcSSc ATA+. Among them, collagen alpha-1(VII) chain, galectin-3-binding protein, desintegrin and metalloproteinase domain-containing 10, low affinity immunoglobulin gamma Fc region receptor III-A, CD59 glycoprotein, growth arrest-arrest specific protein and clusterin were overexpressed in the condition dcSSc ATA+ (Figure 1.C).Figure 1.Heatmap representing the 377 differentially expressed proteins after ANOVA in all samples. Cluster analysis identified 3 group of patients and 2 different clusters of protein expression (A). Volcanoplot represented the comparison IgG dcSSc ATA+ vs IgG HC (B). Venn diagram representing the proteins in common and exclusive of the following comparison: IgG dcSSc ATA+ vs HC, IgG dcSSc ATA- vs IgG HC and IgG dcSSc ATA- vs IgG HC (C). IgG HC: purified IgG from healthy controls; IgG dcSSc ATA+: purified IgG from diffuse systemic sclerosis anti-topoisomerase-I positive patients; IgG dcSSc ATA-: purified IgG from diffuse systemic sclerosis anti-topoisomerase-I negative patients; IgG lcSSc ACA+: purified IgG from limited systemic sclerosis anti-centromere positive patients. ANOVA: analysis of variance.ConclusionUsing sensitive proteomic approach, we identified that purified IgG from SSc patients modified FB secretome in a serotype dependent manner. The data support the pathogenic role of Aab in SSc.