EXTRACELLULAR VESICLES AS VEHICLES OF AUTOANTIGENS IN AUTOIMMUNE BULLOUS DISEASES

Background Autoimmune bullous diseases (AIBDs) are organ specific rare autoimmune diseases associated with significantly morbidity and mortality. The most common AIBD is bullous pemphigoid (BP), in which pathogenic autoantibodies target the haemidesmosomal proteins of the dermo-epidermal junction, namely BP180 (collagen 17A) and BP230. Extracellular vesicles (EVs) are small membranous structures, which encapsulate proteins, miRNA, mRNA and lipids, and represent pivotal mediators of intercellular communication. EVs have been demonstrated to carry tissue-specific autoantigens in other autoimmune diseases, such as rheumatoid arthritis and lupus erythematosus, and in transplant organ rejection; this phenomenon was demonstrated to have pathogenic implications in autoimmune diseases and to correlate with transplant rejection severity. Objectives The purpose of this study was to identify the presence targeted autoantigens in EVs derived from the sera and blister fluid of BP patients. Methods We isolated, by size exclusion chromatography, EVs derived from serum and blisters of BP-patients and from serum or suction blisters of healthy donors. EV characterization was performed by flow cytometry analysis and nanoparticle tracking analysis. Flow cytometry analysis was also used to investigated surface expression of skin autoantigen in serum derived EVs. Western blot analysis was used to investigate the presence of autoantigens in blister fluid derived EVs of BP patients and in EVs derived from suction, burn or pressure blisters of healthy controls. Results Flow cytometry analysis confirmed that the retrieved nanoparticle suspension was enriched in EVs and nanoparticle tracking analysis indicated that the EV-enriched fractions were enriched in particles with a size distribution characterizing small-EVs (main peak was present at 94.5nm). BP180 was found, by western blot analysis, in EVs derived from blister fluid of 6 out 11 BP patients and in none of EVs isolated from suction blister fluid of healthy donors. BP230 and Dsg1 were not detectable in EVs of none of samples. No specific clinical characteristics seemed to correlate to the presence of BP180 in EVs. EV-encapsulated autoantigens in BP might represent a direct or indirect route of antigen presentation without cell-to-cell contact; a depletion route of BP180 contributing to the mechanical detachment of keratinocytes during blister formation and a way to spread among adjacent keratinocytes signals of internalization or depletion of BP180; or a prognostic marker of disease severity. Conclusions This is a preliminary study but, the discovery of BP180, the most important autoantigen target in BP, in EVs derived from blister fluid might contribute to the unraveling of BP pathogenesis.