An in vivo study of the importance of the innate immune system in the pathogenesis of epidermolysis bullosa acquisita

Resumen

Background: Epidermolysis bullosa acqusita (EBA) is a chronic autoimmune disorder characterized by the presence of circulating and tissue-bound autoantibodies against collagen VII (CVII), a protein localized at the dermal-epidermal basement membrane zone (BMZ). It is clinically manifested with tense blisters and erosions involving mucocutaneous tissues. Parts of the innate immune system have been demonstrated to be involved in tissue damage, particularly components of the complement system, antibodies’ receptors and neutrophils. Specifically, Rac2 protein is a GTPase involved in chemotaxis of neutrophils and reactive oxygen species synthesis through NADPH oxidase activation. Acute lung injury (ALI) is a syndrome induced by the immune response where the recruitment of neutrophils favors tissue damage evolving into an acute respiratory distress syndrome. In vivo studies performed in Rac2 knock-out (KO) mice and the pharmacological inhibition of Rac2 (NSC23766) in ALI have demonstrated a decrease in lung tissue injury. Objectives: To investigate the role of Rac2 protein in tissue injury developed in an in vivo experimental mouse model of EBA. Materials and Methods: EBA phenotype was induced by passive transfer of antibodies against a fragment of murine CVII (mCVIICr). Anti-mCVIICr IgG was obtained after rabbit immunization. IgG from immunized and non-immunized rabbits (used as negative controls) were purified and concentrated to inject mice. Two sets of experiments were performed with Rac2 KO mice (total n=10) and wild type mice as positive controls (n=3) by injecting rabbit anti-mCVIICr IgG. At the same time, a set of Rac2 KO mice (n=4) were injected with unspecific rabbit IgG as negative controls. A third set of experiments consisted in injecting rabbit anti-mCVIICr IgG in mice pre-treated with dexamethasone (n=3), dapsone (n=5) and NSC23766 (n=3), an experimental soluble inhibitor of Rac2. Clinical assessments, histology of skin biopsies, direct immunofluorescence (DIF), and enzyme-linked immunosorbent assays (ELISA) were performed. Results: WT mice injected with anti-mCVIICr IgG successfully developed the EBA phenotype, with significantly increased disease severity with higher doses of IgG (p=.05). In contrast, none of the Rac2 KO mice injected with anti-mCVIICr IgG developed the EBA phenotype, even though IgG and C3 were deposited at the BMZ, and circulating anti-mCVIICr IgG was detected by ELISA. Pre-treated WT mice with NSC23766, dexamethasone and dapsone developed disease initially with a trend of milder disease severity, yet we found no statistically difference in body surface area involvement at the end of the observation period when compared with positive controls (p=0.08). Conclusions: The fact that EBA lesions were not developed in Rac2 KO mice after the passive transfer of anti-mCVIICr IgG clearly suggests the involvement of Rac2 protein in EBA pathogenesis. Therefore, we propose Rac2 as a potential target for designing therapies specific for EBA and other IgG-mediated disease. However, an effective Rac2 inhibitor to be used as therapy is not yet available.