Granzyme B inhibition reduces disease severity in autoimmune blistering diseases

Sho Hiroyasu,Nick J Carr,Daisuke Tsuruta,Megan A Pawluk,Anika Kasprick,Hiroshi Shimizu,Angela Burleigh,Richard I Crawford,Nancy Van Laeken,David J Granville,Peter A Lennox,Hongyan Zhao,Frank Petersen,Christopher T Turner,Wataru Nishie,Matthew R Zeglinski,Chiharu Tateishi,Ralf J Ludwig

doi:10.1038/s41467-020-20604-3

Abstract

Pemphigoid diseases refer to a group of severe autoimmune skin blistering diseases characterized by subepidermal blistering and loss of dermal-epidermal adhesion induced by autoantibody and immune cell infiltrate at the dermal-epidermal junction and upper dermis. Here, we explore the role of the immune cell-secreted serine protease, granzyme B, in pemphigoid disease pathogenesis using three independent murine models. In all models, granzyme B knockout or topical pharmacological inhibition significantly reduces total blistering area compared to controls. In vivo and in vitro studies show that granzyme B contributes to blistering by degrading key anchoring proteins in the dermal-epidermal junction that are necessary for dermal-epidermal adhesion. Further, granzyme B mediates IL-8/macrophage inflammatory protein-2 secretion, lesional neutrophil infiltration, and lesional neutrophil elastase activity. Clinically, granzyme B is elevated and abundant in human pemphigoid disease blister fluids and lesional skin. Collectively, granzyme B is a potential therapeutic target in pemphigoid diseases.

Highlights

Pemphigoid diseases refer to a group of severe autoimmune skin blistering diseases characterized by subepidermal blistering and loss of dermal-epidermal adhesion induced by autoantibody and immune cell infiltrate at the dermal-epidermal junction and upper dermis
Pathogenesis, an inflammatory epidermolysis bullosa acquisita (EBA) murine model was induced through intraperitoneal transfer of pathogenic anti-type VII collagen (COL7) IgG into both wild-type (WT) and Granzyme B (GzmB)−/− mice based on an established method[20]
Double immunostaining of GzmB and mouse mast cell protease-8 (mMCP-8) in mast cell-deficient mice (diphtheria toxin (DT)-treated Mcpt5-Cre iDTR mice) with EBA further confirmed that basophils were a source of GzmB (Supplementary Fig. 1b)

Summary

Introduction

Pemphigoid diseases refer to a group of severe autoimmune skin blistering diseases characterized by subepidermal blistering and loss of dermal-epidermal adhesion induced by autoantibody and immune cell infiltrate at the dermal-epidermal junction and upper dermis. It was proposed that neutrophil elastase induces subepidermal blistering in PDs through proteolytic degradation of hemidesmosomal proteins, which form an anchoring complex between the epidermis and dermis[6] Other proteases such as matrix metalloprotease-9 (MMP-9) and plasmin/plasminogen/plasminogen activators (PAs) have been proposed to contribute to PD pathology by regulating the proteolytic activity of neutrophil elastase[7]. It is recognized that other immune and nonimmune cell types secrete GzmB, independent of perforin, to mediate non-cytotoxic, extracellular functions[13,14,15,16] Unlike other proteases such as neutrophil elastase and MMPs, GzmB has no known endogenous extracellular inhibitor[17]; GzmB accumulates and retains its proteolytic activity in the extracellular space. Our data indicate that genetic deficiency or topical inhibition of GzmB in PDs reduces disease severity, prevents hemidesmosomal protein loss, impedes neutrophil infiltration, and impairs lesional neutrophil elastase activity

Methods

Results

Conclusion