Artem Vorobyev

B Cells, Dendritic Cells, and Macrophages Are Required To Induce an Autoreactive CD4 Helper T Cell Response in Experimental Epidermolysis Bullosa Acquisita

In autoimmune bullous dermatoses (AIBD), autoantibodies induce blisters on skin or mucous membranes, or both. Mechanisms of continued autoantibody production and blistering have been well characterized using AIBD animal models. Mechanisms leading to the initial autoantibody production, however, have not been investigated in detail. Epidermolysis bullosa acquisita (EBA) is an AIBD associated with autoantibodies to type VII collagen (COL7). The majority of EBA patients’ sera recognize the noncollagenous domain 1, including the von Willebrand factor A–like domain 2 (vWFA2). In experimental EBA induced by immunization with GST-COL7, disease manifestation depended on the genetic background, a Th1 polarization, and the GST-tag. In this model, nude mice neither produced autoantibodies nor blisters. It has remained uncertain which APC and T cell subsets are required for EBA induction. We established a novel EBA model by immunization with vWFA2 fused to intein (lacking the GST-tag). All tested mouse strains developed autoantibodies, but blisters were exclusively observed in mice carrying H2s. In immunized mice, CD4 T cells specific for vWFA2 were detected, and their induction required presence of B cells, dendritic cells, and macrophages. Anti-vWFA2 autoantibodies located at the lamina densa bound to the dermal side of salt-split skin and induced blisters when transferred into healthy mice. Absence of CD8 T cells at time of immunization had no effect, whereas depletion of CD4 T cells during the same time period delayed autoantibody production and blisters. Collectively, we demonstrate the pathogenic relevance of Abs targeting the vWFA2 domain of COL7 and show the requirement of APC-induced CD4 T cells to induce experimental EBA.

Serum levels of anti-type VII collagen antibodies detected by enzyme-linked immunosorbent assay in patients with epidermolysis bullosa acquisita are correlated with the severity of skin lesions.

Background  Epidermolysis bullosa acquisita (EBA) is a chronic autoimmune subepidermal bullous disease characterized by circulating autoantibodies against type VII collagen. Detecting these autoantibodies is crucial for the diagnosis of this disease, and is also useful for measuring disease activity. Enzyme‐linked immunosorbent assay (ELISA), a quantitative method to measure anti‐type VII collagen antibody levels, is currently available to diagnose EBA.

Clinical features and diagnosis of epidermolysis bullosa acquisita

ABSTRACT Introduction: Epidermolysis bullosa acquisita (EBA) is a rare autoimmune blistering disease of skin and mucous membranes. EBA is caused by autoantibodies against type VII collagen, which is a major component of anchoring fibrils, attaching epidermis to dermis. Binding of autoantibodies to type VII collagen leads to skin fragility and, finally, blister formation. The clinical picture of EBA is polymorphic, with several distinct phenotypes being described. Despite recent progress in understanding the pathophysiology of EBA, its diagnosis is still challenging. Areas covered: This review provides an update on the clinical manifestations and diagnostic methods of EBA. We searched PubMed using the terms ‘epidermolysis bullosa acquisita’ covering articles in English between 1 January 2005 and 31 May 2016. Relevant older publications were retrieved form cited literature. Expert commentary: While the clinical picture is highly variable, diagnosis relies on direct immunofluorescence (IF) microscopy of a perilesional skin biopsy. Linear deposits of IgG, IgA and/or C3 along the dermal-epidermal junction with an u-serrated pattern are diagnostic for EBA alike the detection of serum autoantibodies against type VII collagen. Several test systems for the serological diagnosis of EBA have recently become widely available. In some patients, sophisticated diagnostic approaches only available in specialized centers are required.

Recombinant soluble CD32 suppresses disease progression in experimental epidermolysis bullosa acquisita.

Abbreviations: EBA, epidermolysis bullosa acquisita; DEJ, dermal–epidermal junction; FcγR, Fc gamma receptor; IC, immune complex; PBS, phosphate-buffered saline; ROS, reactive oxygen species

Recombinant Human IgA1 and IgA2 Autoantibodies to Type VII Collagen Induce Subepidermal Blistering Ex Vivo

Subepidermal autoimmune blistering dermatoses (AIBD) are prototypic autoantibody-mediated diseases. In epidermolysis bullosa acquisita (EBA), an autoimmune disease with severe and chronic skin blistering, autoantibodies are directed against type VII collagen. IgG is the predominant autoantibody isotype of EBA, the pathogenicity of which has been demonstrated in a variety of in vivo and ex vivo disease models. In contrast, there is not much evidence for the pathogenicity of IgA, which may appear as the only autoantibody isotype in some EBA patients. To investigate the pathogenic potential of IgA autoantibodies, we generated chimeric V gene–matched human IgA1, IgA2, and control IgG1 autoantibodies directed against type VII collagen. Immobilized immune complexes containing the rIgA1 and rIgA2 autoantibodies induced the dose-dependent release of reactive oxygen species from neutrophil granulocytes, a precondition for blister formation. Moreover, both rIgA1 and rIgA2 induced leukocyte-dependent dermal–epidermal separation in cryosections of human skin. In contrast with rIgG1, neither rIgA1 nor rIgA2 was capable of inducing complement deposition at the dermal–epidermal junction. Because complement activation is a prerequisite for blister induction, this lack of function compared with IgG1 may be compensated for by the stronger activation of neutrophil granulocytes by both IgA1 and IgA2. For IgG-mediated AIBD, immunoadsorption therapy is a convenient treatment modality for the removal of pathogenic autoantibodies, particularly in treatment-resistant cases. The results of this study show the pathogenic potential of IgA autoantibodies and support the development of adsorber matrices for IgA-mediated AIBD.

Dissecting genetics of cutaneous miRNA in a mouse model of an autoimmune blistering disease

BackgroundMicroRNAs (miRNAs) are small endogenous non-coding RNAs that control genes at post-transcriptional level. They are essential for development and tissue differentiation, and such altered miRNA expression patterns are linked to the pathogenesis of inflammation and cancer. There is evidence that miRNA expression is genetically controlled similar to the transcription of protein-coding genes and previous studies identified quantitative trait loci (QTL) for miRNA expression in the liver. So far, little attention has been paid to miRNA expression in the skin. Moreover, epistatic control of miRNA expression remains unknown. In this study, we characterize genetic regulation of cutaneous miRNA and their correlation with skin inflammation using a previously established murine autoimmune-prone advanced intercross line.ResultsWe identified in silico 42 eQTL controlling the expression of 38 cutaneous miRNAs and furthermore found two chromosomal hot-spots on chromosomes 2 and 8 that control the expression of multiple miRNAs. Moreover, for 8 miRNAs an interacting effect from pairs of SNPs was observed. Combining the constraints on genes from the statistical interaction of their loci and further using curated protein interaction networks, the number of candidate genes for association of miRNAs was reduced to a set of several genes. A cluster analysis identified miR-379 and miR-223 to be associated with EBA severity/onset, where miR-379 was observed to be associated to loci on chromosome 6.ConclusionThe murine advanced intercross line allowed us to identify the genetic loci regulating multiple miRNA in skin. The recurrence of trans-eQTL and epistasis suggest that cutaneous miRNAs are regulated by yet an unexplored complex gene networks. Further, using co-expression analysis of miRNA expression levels we showed that multiple miRNA contribute to multiple pathways that might be involved in pathogenesis of autoimmune skin blistering disease. Specifically, we provide evidence that miRNA such as miR-223 and miR-379 may play critical role in disease progression and severity.

Gene Expression Analysis Reveals Novel Shared Gene Signatures and Candidate Molecular Mechanisms between Pemphigus and Systemic Lupus Erythematosus in CD4+ T Cells

Pemphigus and systemic lupus erythematosus (SLE) are severe potentially life-threatening autoimmune diseases. They are classified as B-cell-mediated autoimmune diseases, both depending on autoreactive CD4+ T lymphocytes to modulate the autoimmune B-cell response. Despite the reported association of pemphigus and SLE, the molecular mechanisms underlying their comorbidity remain unknown. Weighted gene co-expression network analysis (WGCNA) of publicly available microarray datasets of CD4+ T cells was performed, to identify shared gene expression signatures and putative overlapping biological molecular mechanisms between pemphigus and SLE. Using WGCNA, we identified 3,280 genes co-expressed genes and 14 co-expressed gene clusters, from which one was significantly upregulated for both diseases. The pathways associated with this module include type-1 interferon gamma and defense response to viruses. Network-based meta-analysis identified RSAD2 to be the most highly ranked hub gene. By associating the modular genes with genome-wide association studies (GWASs) for pemphigus and SLE, we characterized IRF8 and STAT1 as key regulatory genes. Collectively, in this in silico study, we identify novel candidate genetic markers and pathways in CD4+ T cells that are shared between pemphigus and SLE, which in turn may facilitate the identification of novel therapeutic targets in these diseases.

IgE-Selective Immunoadsorption for Severe Atopic Dermatitis

Introduction Recent reports proposed the application of immunoadsorption (IA) for patients with recalcitrant atopic dermatitis (AD) and high-serum IgE levels. However, experience with this novel treatment approach, especially with the newly available IgE-specific adsorber, is limited and recommendation for its use in clinical practice awaits evidence from more studies. Materials and methods Patients with severe AD (SCORAD ≥ 60) and total serum IgE levels ≥750 kU/L were included in this study. The treatment protocol consisted of two cycles of five consecutive treatments with IgE-selective IA 3 weeks apart. Results Ten patients were enrolled and four patients completed the study. The mean SCORAD was significantly improved by up to 43% within a few weeks and until the end of a 6-month follow-up period, with 50% of patients achieving an at least 50% individual reduction of the baseline SCORAD. Each IA cycle induced a temporal average decrement of total serum levels of IgE, IgM, IgA, and IgG by 92, 43, 38, and 35%, respectively. Except for one case of Staphylococcus aureus septicemia, no major adverse events occurred. Conclusion Although limited by a considerable withdrawal rate, our observations strengthen our and other recent results further suggesting that IgE-selective IA is an effective treatment option for patients severely affected by AD with highly elevated IgE levels.

Serration pattern analysis for differentiating epidermolysis bullosa acquisita from other pemphigoid diseases

Background: Direct immunofluorescence (DIF) microscopy of a skin biopsy specimen is the reference standard for the diagnosis of pemphigoid diseases (PDs). Serration pattern analysis enables the differentiation of epidermolysis bullosa acquisita (EBA) from other PDs using DIF microscopy alone. However, practice gaps need to be addressed in order to implement this technique in the routine diagnostic procedure. Objective: We sought to determine and optimize the technical requirements for serration pattern analysis of DIF microscopy and determine interrater conformity of serration pattern analysis. Methods: We compared serration pattern analysis of routine DIF microscopy from laboratories in Groningen, The Netherlands and Lübeck, Germany with 4 blinded observers. Skin biopsy specimens from 20 patients with EBA and other PDs were exchanged and analyzed. Various factors were evaluated, including section thickness, transport medium, and biopsy specimen processing. Results: The interrater conformity of our 4 observers was 95.7%. Recognition of serration patterns was comparable in samples transported in saline and in Michels medium and with section thicknesses of 4, 6, and 8 &mgr;m. Limitations: Limitations include our small sample size and the availability of 20 samples that were compared retrospectively. Conclusion: DIF serration pattern analysis is not restricted by variation in laboratory procedures, transport medium, or experience of observers. This learnable technique can be implemented as a routine diagnostic method as an extension of DIF microscopy for subtyping PD. Graphical abstract: Figure. No caption available.