Proteomic analyses of two distinct exosome fractions present in peritoneal exudates from mice with pristane-induced lupus.

Abstract

Background: Intraperitoneal injection of pristane to C57BL6/J WT mice provokes a strong inflammatory reaction with a massive influx of pro-inflammatory Ly6Chi monocytes and neutrophils to the peritoneum that ends up in overt autoimmunity (a murine lupus model of systemic lupus erythematosus (SLE) human disease). The inflammation is milder in CD38-deficient (Cd38-/-) and in double-deficient (Cd38-/- Art2-/-) mice, while it is similar to WT in Art2-deficient (Art2-/-) mice, which suggests an important role of CD38 on the onset of the disease. The aim of this work has been to isolate exosomes from peritoneal exudates (PE-EXO) of mice treated with pristane and to identify/quantify the proteins by mass spectrometry. Functional analyses were performed in silico with the use of FunRich and ClueGO software. Methods: Two types of PE-EXOs (Fr5-10 and Fr11-12) were isolated from pristane-treated mice by qEV size exclusion column methodology. Protein extracts were analyzed by LC-MS/MS. Protein identification was performed with ProteinScape 4.0 (Bruker) and MASCOT data searching using Swiss-Prot database. For relative quantification the emPAI-based method was used. The functional enrichment analysis was based on the latest publicly available data from multiple annotation and ontology resources that can be automatically accessed through FunRich and ClueGO. Results: Gene Ontology (GO) enrichment analyses of Fr5-10 and Fr11-12 PE-EXOs with FunRich showed that 65-75% of the identified proteins clustered in the GO term extracellular vesicular exosomes, while the comparative analyses with CluGO showed that these PE-EXOs were functionally quite distinct. Thus, Fr5-10 PE-EXOs were enriched in terms such as ¿regulation of myeloid leukocyte mediated immunity¿, ¿positive regulation of adaptive immune response¿, while Fr11-12 exosomes were enriched in terms such as ¿complement activation¿, and ¿regulation of humoral immune response¿. Interestingly the predominance of given GO terms in Fr5-10 vs Fr11-12 PE-EXOs seemed to vary with the extent of the inflammatory reaction to pristane, suggesting that these exosomes are representative of the distinct inflammatory cells that are recruited to the peritoneum. Conclusions: Proteomics with the help of appropriate bioinformatics tools such as ClueGO visualizes GO terms and pathways as networks, and groups them based on their biological role. This may contribute to a better diagnosis/prognosis of autoimmune diseases such as lupus. Keywords: exosomes; inflammation; lupus; bioinformatics; gene ontology; omics data analysis; biological networks