27005 Proteomics analysis of blister fluid from patients with bullous pemphigoid

Abstract

Bullous pemphigoid (BP) is an autoimmune disorder characterized by the development of pruritic lesions and/or blisters. Previous research analyzing the blister fluid from BP patients shows proinflammatory cytokines/chemokines, typically at a higher concentration than in serum. We explored the blister fluid proteome of 8 patients with BP to assess the intraepidermal microenvironment. TMT-labeled proteomics identified a total of 339 proteins. Using gene names for these proteins, Gene Ontology was used for analysis, demonstrating marked enrichment of exocytosis pathways, immune response, and platelet degranulation. In light of an abundance of exosomal proteins, we depleted exosomes using ultracentrifugation, to determine which proteins were exosome-bound and increase sensitivity for soluble proteins. Differential protein analysis was performed between these samples. Notable findings in BP blister fluid proteome include eosinophil degranulation proteins (EPX, ECP, EDN, MBP) at greater abundance than neutrophil-related proteins (neutrophil gelatinase-associated lipocalin, myeloperoxidase, neutrophil defensin 3). We identified IgM/IgA/IgG/IgE, 16 SERPIN-family protease inhibitors, heat shock proteins (HSP90AA1, HSP90AB1, HSP90B1, HSPA1b, HSPA5, HSPA8, HSPB1), neutrophil inflammasome mediators (lipopolysaccharide-binding protein, CD14), clotting factors (factors 2,5,9,10-13 (A1,B), VWF, tissue factor, fibrinogen), and complement mediators (C1-9). Neutrophil elastase, MMP9, ECP, and eosinophil derived neurotoxin appear to be bound or contained in fluid exosomes. The results demonstrate the players that may be responsible for the inflammatory/proteolytic environment in BP. Exosomal proteins appear to be derived from eosinophils, keratinocytes, and neutrophils, suggesting that exosomes likely contribute to the propagation of the inflammatory response. The extent of clotting factor presence in fluid confirms prior studies identifying coagulation activation in BP.