Reduced Skin Blistering in Experimental Epidermolysis Bullosa Acquisita After Anti-TNF Treatment

Epidermolysis bullosa acquisita (EBA) is a difficult-to-treat subepidermal autoimmune blistering skin disease (AIBD) with circulating and tissue-bound anti-type VII collagen antibodies. Different reports have indicated increased concentration of tumor necrosis factor a (TNF) in the serum and blister fluid of patients with subepidermal AIBD. Furthermore, successful anti-TNF treatment has been reported for individual patients with AIBD. Here we show that in mice, induction of experimental EBA by repeated injections of rabbit anti-mouse type VII collagen antibodies led to increased expression of TNF in skin, as determined by real-time polymerase chain reaction (PCR) and immunohistochemistry. To investigate whether the increased TNF expression is of functional relevance in experimental EBA, we inhibited TNF function using the soluble TNF receptor fusion protein etanercept (Enbrel) or a monoclonal antibody to murine TNF. Interestingly, mice that received either of these treatments showed significantly milder disease progression than controls. In addition, immunohistochemical staining demonstrated reduced numbers of macrophages in lesional skin in mice treated with TNF inhibitors compared with controls. Furthermore, etanercept treatment significantly reduced disease progression in immunization-induced EBA. In conclusion, increased expression of TNF in experimental EBA is of functional relevance, as both the prophylactic blockade of TNF and the therapeutic use of etanercept impaired induction and progression of experimental EBA. Thus, TNF is likely to serve as a new therapeutic target for EBA and AIBDs with a similar pathogenesis

Recombinant IL-6 treatment protects mice from organ specific autoimmune disease by IL-6 classical signalling-dependent IL-1ra induction

Cytokines are key regulators of physiological inflammatory responses, while aberrant cytokine expression contributes to pathogenesis of autoimmune diseases. We noted increased IL-6 levels in human and murine epidermolysis bullosa acquisita (EBA), a prototypic organ-specific autoimmune bullous dermatoses (AIBD) induced by autoantibodies to type VII collagen (COL7). In contrast to rheumatoid arthritis, blockade of IL-6 led to strikingly enhanced experimental EBA, while treatment with recombinant IL-6 was protective. This was due to classical IL-6 signalling and independent of IL-6 trans-signalling, as treatment of mice with sgp130Fc had no impact on EBA manifestation. Induction of EBA in mice led to increased IL-1ra levels in skin and serum, while blockade of IL-6 completely inhibited IL-1ra expression induced by autoantibodies to COL7. In line, treatment of mice with EBA with recombinant IL-6 induced IL-1ra concentrations exceeding those of untreated animals with EBA, and IL-1ra (anakinra) administration significantly impaired experimental EBA induction. We here identified a novel anti-inflammatory pathway in an organ-specific autoimmune disease. Modulation of this IL-1ra pathway by classical IL-6 signalling demonstrates anti-inflammatory and protective activities of IL-6 in vivo. (C) 2012 Elsevier Ltd. All rights reserved

Methylprednisolone Blocks Autoantibody-Induced Tissue Damage in Experimental Models of Bullous Pemphigoid and Epidermolysis Bullosa Acquisita through Inhibition of Neutrophil Activation

Corticosteroids are regularly used to treat autoimmune diseases, such as bullous pemphigoid (BP). In BP, autoantibodies bind to type XVII collagen (COL17), located at the dermal-epidermal junction. A crucial role of neutrophils in experimental BP has been established. Specifically, reactive oxygen species and proteolytic granule enzymes mediate tissue injury. Therefore, we investigated the effects of methylprednisolone (MP) on neutrophils, which are likely to be affected by topical treatment. First, MP inhibited dermal-epidermal separation ex vivo in cryosections of the human skin induced by co-incubation of BP autoantibodies with neutrophils from healthy volunteers. Next, MP inhibited neutrophil activation in vitro induced by immune complexes (ICs) of COL17 and autoantibodies. This neutrophil activation was associated with phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), p38 mitogen-activated protein kinase (MAPK), and Akt. In turn, inhibition of ERK1/2, p38 MAPK, or Akt phosphorylation inhibited neutrophil activation by IC in vitro and dermal-epidermal separation ex vivo. In addition, we observed an increase of p38 MAPK phosphorylation in dermal infiltrates of BP patients. Treatment of mice with either MP or inhibitors of p38-MAPK or ERK1/2 phosphorylation impaired induction of autoantibody- or irritant-induced neutrophil-dependent inflammation. We here identify the inhibition of Akt, ERK1/2, and p38 MAPK phosphorylation as molecular mechanisms to promote MP's therapeutic effects.Journal of Investigative Dermatology advance online publication, 4 April 2013; doi:10.1038/jid.2013.91

Caspase-1-Independent IL-1 Release Mediates Blister Formation in Autoantibody-Induced Tissue Injury through Modulation of Endothelial Adhesion Molecules

Although reports documented aberrant cytokine expression in autoimmune bullous dermatoses (AIBDs), cytokine-targeting therapies have not been established in these disorders. We showed previously that IL-6 treatment protected against tissue destruction in experimental epidermolysis bullosa acquisita (EBA), an AIBD caused by autoantibodies to type VII collagen (COL7). The anti-inflammatory effects of IL-6 were mediated by induction of IL-1ra, and prophylactic IL-1ra administration prevented blistering. In this article, we demonstrate elevated serum concentrations of IL-1β in both mice with experimental EBA induced by injection of anti-COL7 IgG and in EBA patients. Increased IL-1α and IL-1β expression also was observed in the skin of anti-COL7 IgG-injected wild-type mice compared with the significantly less diseased IL-1R-deficient or wild-type mice treated with the IL-1R antagonist anakinra or anti-IL-1β. These findings suggested that IL-1 contributed to recruitment of inflammatory cells into the skin. Accordingly, the expression of ICAM-1 was decreased in IL-1R-deficient and anakinra-treated mice injected with anti-COL7. This effect appeared to be specifically attributable to IL-1 because anakinra blocked the upregulation of different endothelial adhesion molecules on IL-1-stimulated, but not on TNF-α-stimulated, cultured endothelial cells. Interestingly, injection of caspase-1/11-deficient mice with anti-COL7 IgG led to the same extent of skin lesions as in wild-type mice. Collectively, our data suggest that IL-1, independently of caspase-1, contributes to the pathogenesis of EBA. Because anti-IL-1β in a prophylactic setting and anakinra in a quasi-therapeutic setting (i.e., when skin lesions had already developed) improved experimental EBA, IL-1 appears to be a potential therapeutic target for EBA and related AIBDs

Table_2.XLS

<p>Because of the morbidity and limited therapeutic options of autoimmune diseases, there is a high, and thus far, unmet medical need for development of novel treatments. Pemphigoid diseases, such as epidermolysis bullosa acquisita (EBA), are prototypical autoimmune diseases that are caused by autoantibodies targeting structural proteins of the skin, leading to inflammation, mediated by myeloid cells. To identify novel treatment targets, we performed cutaneous genome-wide mRNA expression profiling in 190 outbred mice after EBA induction. Comparison of genome-wide mRNA expression profiles in diseased and healthy mice, and construction of a co-expression network identified Sykb (spleen tyrosine kinase, SYK) as a major hub gene. Aligned, pharmacological SYK inhibition protected mice from experimental EBA. Using lineage-specific SYK-deficient mice, we identified SYK expression on myeloid cells to be required to induce EBA. Within the predicted co-expression network, interactions of Sykb with several partners (e.g., Tlr13, Jdp2, and Nfkbid) were validated by curated databases. Additionally, novel gene interaction partners of SYK were experimentally validated. Collectively, our results identify SYK expression in myeloid cells as a requirement to promote inflammation in autoantibody-driven pathologies. This should encourage exploitation of SYK and SYK-regulated genes as potential therapeutic targets for EBA and potentially other autoantibody-mediated diseases.</p

Table_1.PDF

<p>Because of the morbidity and limited therapeutic options of autoimmune diseases, there is a high, and thus far, unmet medical need for development of novel treatments. Pemphigoid diseases, such as epidermolysis bullosa acquisita (EBA), are prototypical autoimmune diseases that are caused by autoantibodies targeting structural proteins of the skin, leading to inflammation, mediated by myeloid cells. To identify novel treatment targets, we performed cutaneous genome-wide mRNA expression profiling in 190 outbred mice after EBA induction. Comparison of genome-wide mRNA expression profiles in diseased and healthy mice, and construction of a co-expression network identified Sykb (spleen tyrosine kinase, SYK) as a major hub gene. Aligned, pharmacological SYK inhibition protected mice from experimental EBA. Using lineage-specific SYK-deficient mice, we identified SYK expression on myeloid cells to be required to induce EBA. Within the predicted co-expression network, interactions of Sykb with several partners (e.g., Tlr13, Jdp2, and Nfkbid) were validated by curated databases. Additionally, novel gene interaction partners of SYK were experimentally validated. Collectively, our results identify SYK expression in myeloid cells as a requirement to promote inflammation in autoantibody-driven pathologies. This should encourage exploitation of SYK and SYK-regulated genes as potential therapeutic targets for EBA and potentially other autoantibody-mediated diseases.</p

Autoantibodies to Multiple Epitopes on the Non-Collagenous-1 Domain of Type VII Collagen Induce Blisters

Epidermolysis bullosa acquisita (EBA) is an autoimmune blistering disease of the skin and mucous membranes, characterized by autoantibodies against type VII collagen (COL7), a major component of anchoring fibrils. Different clinical EBA phenotypes are described, including mechanobullous and inflammatory variants. Most EBA patients’ sera react with epitopes located within the non-collagenous 1 (NC1) domain of human COL7. However, it has remained unclear whether antibody binding to these different epitopes is pathogenically relevant. To address this issue, we generated recombinant proteins covering the entire NC1 domain. IgG reactivity with these proteins was analyzed in sera of 69 EBA patients. Most recognized clusters of epitopes throughout the NC1 domain. No correlation was detected between antibody specificity and clinical phenotype. To study the pathogenicity of antibodies specific to different NC1 subdomains, rabbit antibodies were generated. All these antibodies caused dermal–epidermal separation ex vivo. Antibodies against two of these subdomains were injected into mice carrying null mutations of mouse COL7 and the human COL7 transgene and induced subepidermal blisters. We here document that autoantibodies to COL7, independent of the targeted epitopes, induce blisters both ex vivo and in vivo. In addition, using COL7-humanized mice, we provide in vivo evidence of pathogenicity of autoantibodies binding to human COL7