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

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.

Insights Into the Pathogenesis of Bullous Pemphigoid: The Role of Complement-Independent Mechanisms

“…Serum levels of these interleukins have also been correlated with disease activity in BP (73). Overall these observations indicate that keratinocytes, by releasing pro-inflammatory cytokines, also play a previously unrecognized role in the pathogenesis of pemphigoid diseases (67). Keratinocyte dependent complement-independent mechanisms are summarized in Figure 1.…”

“…Noteworthy, IgE appears to be a more potent stimulator of cytokine production compared to IgG ( 44 ). In this context, we recently identified a similar pro-inflammatory response from keratinocytes treated with IgG obtained from patients with laminin-332 pemphigoid, particularly with autoantibodies against the β3 subunit ( 67 ).…”

Gene expression profiling of laminin α3‐blocked keratinocytes reveals an immune‐independent mechanism of blistering

“…We have recently studied the subunit-dependent responses of keratinocytes treated with IgG from a series of patients with antibodies against only the laminin α3,β3, or γ2. 50 Here, we identified a pro-inflammatory effect, particularly in patients with antibodies against laminin β3. Likewise, keratinocyte-treated antibodies from a small cohort of patients with antibodies against laminin α3 did not demonstrate significant differentiation markers.…”

“…Previous studies have provided conflicting evidence regarding complement‐independent blistering mechanisms. We have recently studied the subunit‐dependent responses of keratinocytes treated with IgG from a series of patients with antibodies against only the laminin α3,β3, or γ2 50 . Here, we identified a pro‐inflammatory effect, particularly in patients with antibodies against laminin β3.…”

Einblicke in die Pathogenese des bullösen Pemphigoids: Die Rolle komplementunabhängiger Mechanismen

“…Bemerkenswerterweise scheint IgE im Vergleich zu IgG ein stärkerer Stimulator der Zytokinproduktion zu sein [44]. In diesem Zusammenhang haben wir kürzlich eine ähnliche entzündungsfördernde Reaktion von Keratinozyten identifiziert, die mit IgG behandelt wurden, das von Patienten mit Laminin-332-Pemphigoid erhalten wurde, insbesondere mit Autoantikörpern gegen die 03-Untereinheit [67].…”