Subunit-Specific Reactivity of Autoantibodies Against Laminin-332 Reveals Direct Inflammatory Mechanisms on Keratinocytes

Lei Bao,Xiaoguang Li,Payal M Patel,Kyle T Amber,Takashi Hashimoto,Farzan Solimani,Jing Li,Hua Qian,Norito Ishii,Michael Hertl,Dario Didona

doi:10.3389/fimmu.2021.775412

Abstract

Laminin-332 pemphigoid is a rare and severe autoimmune blistering disease, caused by IgG autoantibodies targeting laminin-332 in the dermal-epidermal basement zone. Laminin-332 pemphigoid is characterized by variable inflammatory infiltrate and the predominance of non-complement-fixing antibodies. Given these findings, we hypothesized that IgG autoantibodies to laminin-332 directly resulted in keratinocyte expression of inflammatory factors. We performed RNA-seq on primary human keratinocytes treated with IgG from patients with laminin-332 pemphigoid. Genes for numerous cytokines and chemokines were upregulated, including CSF2, CSF3, CXCL1, CXCL5, CXCL3, CXCL8, CXCL10, CXCL1, IL6, IL7, IL15, IL23, IL32, IL37, TGFB2 as well as metalloproteases. Considering the pro-inflammatory and proteolytic effect of autoantibodies from patients with laminin-332 pemphigoid identified in our initial experiment, we next questioned whether the reactivity against specific laminin subunits dictates the inflammatory and proteolytic keratinocyte response. Then, we treated keratinocytes with IgG from a separate cohort of patients with reactivity against individual subunits of laminin-332. We identified upregulation of IL-1α, IL-6, IL-8, CXCL1, MMP9, TSLP, and GM-CSF at the protein level, most notably in keratinocytes treated with IgG from laminin β3-reactive patients. We for the first time demonstrated a pro-inflammatory response, similar to that described in keratinocytes treated with IgG autoantibodies from patients with bullous pemphigoid, providing novel insight into the pathogenesis of laminin-332 pemphigoid and laminin-332 biology.

Highlights

Laminin-332 pemphigoid, a subtype of mucous membrane pemphigoid (MMP), is a rare and severe autoimmune blistering disease, caused by IgG autoantibodies targeting laminin-332 in the dermal-epidermal basement zone (BMZ) [1,2,3]
Diagnosis of laminin-332 mucous membrane pemphigoid in the first cohort of patients was based on clinical presentation of MMP, direct immunofluorescence (DIF) demonstrating linear IgG deposition at the cutaneous BMZ, and with indirect immunofluorescence (IIF) against the dermal side of salt-split skin
Considering the pro-inflammatory and proteolytic effect of IgG autoantibodies from patients with laminin-332 pemphigoid on keratinocytes identified in our initial experiment, we looked to validate several of the key findings in a larger cohort of patients with laminin-332 pemphigoid

Summary

Introduction

Laminin-332 pemphigoid, a subtype of mucous membrane pemphigoid (MMP), is a rare and severe autoimmune blistering disease, caused by IgG autoantibodies targeting laminin-332 in the dermal-epidermal basement zone (BMZ) [1,2,3]. The role of complement in inducing local inflammatory response and subsequent blistering remains unclear. Passive transfer experiments in neonatal mice demonstrate anti-laminin-332 IgG induced blistering, but a lack of local inflammation and significant mucosal involvement. In an adult mouse model of laminin-332 using anti-a3 antibodies, a more characteristic clinical phenotype with a local inflammatory response was appreciated in an Fc-dependent manner [14]. It has been reported that non-complement-fixing IgG antibodies against laminin 332 are the predominant class of autoantibodies deposited at the cutaneous BMZ in patients with laminin-332 pemphigoid [16]. It is likely that complement-independent inflammatory pathways exist in human disease

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