Autoimmune skin blistering diseases (AIBD) are characterized by autoantibodies that are directed against structural proteins in the skin and adjacent mucous membranes. Some clinical signs are typical for a specific AIBD, however, correct diagnosis requires the detection of tissue-bound or circulating autoantibodies. The gold standard for diagnosis of AIBD is the detection of autoantibodies or complement component 3 by direct immunofluorescence (DIF) microscopy of a perilesional biopsy. Circulating antibodies can be detected via indirect immunofluorescence (IIF) microscopy of different tissue substrates including human skin, monkey esophagus, and more recently, recombinant forms of the different target antigens. Latter are also employed in various commercial ELISA systems and by immunoblotting in in-house assays available in specialized laboratories. ELISA systems are also particularly valuable for monitoring of the disease activity during the disease course which can be helpful for treatment decisions. Exact diagnosis is essential for both treatment and prognosis, since some AIBD are associated with malign tumors such as paraneoplastic pemphigus and anti-laminin 332 mucous membrane pemphigoid. This review presents clinical and immunopathological features of AIBD for the state-of the art diagnosis of these disorders.

Host defense against pathogens relies on neutrophil activation. Inadequate neutrophil activation is often associated with chronic inflammatory diseases. Neutrophils also constitute a significant portion of infiltrating cells in chronic inflammatory diseases; eg psoriasis and multiple sclerosis. Fumarates improve the latter diseases, which so far has been attributed to effects on lymphocytes and dendritic cells. Here, we focused on effects of dimethyl fumarate (DMF) on neutrophils. In vitro, DMF inhibited neutrophil activation, including changes in surface marker expression, reactive oxygen species production, formation of neutrophil extracellular traps and migration. Phagocytic ability and autoantibody-induced, neutrophil-dependent tissue injury ex vivo was also impaired by DMF. Regarding the mode of action, DMF modulates -in a stimulus-dependent manner- neutrophil activation using the PI3K/Akt-p38 MAPK- and ERK 1/2 pathways. For in vivo validation, mouse models of epidermolysis bullosa acquisita (EBA), an organ-specific autoimmune disease caused by autoantibodies to type VII collagen (COL7), were employed. In the presence of DMF, blistering induced by injection of anti-COL7 antibodies into mice was significantly impaired. DMF treatment of mice with clinically already manifested EBA led to disease improvement. Collectively, we demonstrate a profound inhibitory activity of DMF on neutrophil functions. These findings encourage wider use of DMF in patients with neutrophil-mediated diseases.Journal of Investigative Dermatology accepted article preview online, 05 October 2015. doi:10.1038/jid.2015.361.

T cells are key players in autoimmune diseases by supporting the production of autoantibodies. However, their contribution to the effector phase of antibody-mediated autoimmune dermatoses, i.e., tissue injury and inflammation of the skin, has not been investigated. In this paper, we demonstrate that T cells amplify the development of autoantibody-induced tissue injury in a prototypical, organspecific autoimmune disease, namely epidermolysis bullosa acquisita ( T cells are essential regulators of host defense and exhibit direct cytotoxic as well as regulatory properties. The presence or absence of pro-inflammatory compared with regulatory T cell subsets affects the development and outcome of inflammatory reactions. Misbalance of T cell populations leads to autoimmune disorders, including systemic lupus erythematosus (SLE), different autoimmune bullous dermatoses (AIBDs) and rheumatoid arthritis (RA) [1][2][3] . In these diseases, the contribution of T cells to antibody production and maintenance of the autoimmune response has clearly been demonstrated 4,5 . In recent decades, the understanding of autoantibody-induced tissue injury has greatly improved. However, the role of T cells during the effector phase of autoimmune skin blistering diseases, i.e., tissue injury and inflammation in the targeted organs, is not completely understood. In this study, we investigated the role of T cells during this phase, using a mouse model of epidermolysis bullosa acquisita (EBA), a prototypical organ-specific autoimmune disease 6,7 . EBA is caused by autoantibodies directed against type VII collagen (COL7), an integral component of anchoring fibrils 8 . Animal models, employing antibody transfer into mice 9,10 , have added to a greater understanding of the mechanisms leading to blistering in EBA 9,11,12 . Based on the current understanding of EBA pathogenesis, the effector phase of EBA is predominantly driven by neutrophils -their depletion leading to a complete absence of experimental EBA 13. With regard to T cell involvement during this phase, in vivo and in vitro data have been contradictory. In vivo data indicated a T cell-independent process: Transfer of total IgG isolated from rabbits that had been immunized with COL7 into T cell-deficient mice induced subepidermal blistering 9 . However, in that study, no wild-type control for evaluation of the extent of blistering was included. In other

Regulatory T cells (Tregs) are well known for their modulatory functions in adaptive immunity. Through regulation of T cell functions, Tregs have also been demonstrated to indirectly curb myeloid cell-driven inflammation. However, direct effects of Tregs on myeloid cell functions are insufficiently characterized, especially in the context of myeloid cell-mediated diseases, such as pemphigoid diseases (PDs). PDs are caused by autoantibodies targeting structural proteins of the skin. Autoantibody binding triggers myeloid cell activation through specific activation of Fc gamma receptors, leading to skin inflammation and subepidermal blistering. Here, we used mouse models to address the potential contribution of Tregs to PD pathogenesis in vivo. Depletion of Tregs induced excessive inflammation and blistering both clinically and histologically in two different PD mouse models. Of note, in the skin of Treg-depleted mice with PD, we detected increased expression of different cytokines, including Th2-specific IL-4, IL-10, and IL-13 as well as pro-inflammatory Th1 cytokine IFN-γ and the T cell chemoattractant CXCL-9. We next aimed to determine whether Tregs alter the migratory behavior of myeloid cells, dampen immune complex (IC)-induced myeloid cell activation, or both. In vitro experiments demonstrated that co-incubation of IC-activated myeloid cells with Tregs had no impact on the release of reactive oxygen species (ROS) but downregulated β2 integrin expression. Hence, Tregs mitigate PD by altering the migratory capabilities of myeloid cells rather than their release of ROS. Modulating cytokine expression by administering an excess of IL-10 or blocking IFN-γ may be used in clinical translation of these findings.

Animal models have enhanced our understanding of the pathogenesis of autoimmune diseases. For these models, genetically identical, inbred mice have commonly been used. Different inbred mouse strains, however, show a high variability in disease manifestation. Identifying the factors that influence this disease variability could provide unrecognized insights into pathogenesis. We established a novel Ab transfer-induced model of epidermolysis bullosa acquisita (EBA), an autoimmune disease characterized by (muco)-cutaneous blistering caused by anti-type VII collagen (COL7) autoantibodies. Blistering after anti-COL7 IgG (directed against the von Willebrand factor A–like domain 2) transfer showed clear variability among inbred mouse strains, that is, severe cutaneous blistering and inflammation in C57BL/6J and absence of skin lesions in MRL/MpJ mice. The transfer of anti-COL7 IgG into irradiated, EBA-resistant MRL/MpJ mice, rescued by transplantation with bone marrow from EBA-susceptible B6.AK-H2k mice, induced blistering. To the contrary, irradiated EBA-susceptible B6.AK-H2k mice that were rescued using MRL/MpJ bone marrow were devoid of blistering. In vitro, immune complex activation of neutrophils from C57BL/6J or MRL/MpJ mice showed an impaired reactive oxygen species release from the latter, whereas no differences were observed after PMA activation. This finding was paralleled by divergent expression profiles of immune complex–activated neutrophils from either C57BL/6J or MRL/MpJ mice. Collectively, we demonstrate that radiosensitive cells determine the varying extent of skin inflammation and blistering in the end-stage effector phase of EBA.

A link between hypovitaminosis D and development of autoimmune bullous disorders has been suggested recently, but this association has not been elaborated experimentally. Here, the role of vitamin D was investigated in epidermolysis bullosa acquisita (EBA), an anti-type VII collagen autoantibody-induced blistering skin disease. Oral administration of the hormonally active vitamin D metabolite calcitriol ameliorated clinical disease severity and dermal neutrophil infiltration in both an antibody transfer- and immunization-induced EBA mouse model. Mechanistically, calcitriol hindered immune effector cell activation as evidenced by increased L-selectin expression on Gr-1 cells in calcitriol-treated mice with antibody transfer-induced EBA, as well as suppressed in vitro immune complex-induced reactive oxygen species production in calcitriol-treated murine neutrophils. Additionally, calcitriol administration was associated with an increase of regulatory T (CD4FoxP3) and B (CD19IL10) cells as well as reduction of pro-inflammatory T helper 17 (CD4IL-17) cells in mice with immunization-induced EBA. In line, levels of circulating anti-type VII collagen autoantibodies were lower in mice that received calcitriol compared to solvent-treated animals. Together with the observed state of hypovitaminosis D in most cases of an analyzed EBA patient cohort, the results of this study support the use of vitamin D derivatives or analogs for patients with EBA and related diseases.

Cell stress-inducible Hsp90 has been recognized as key player in mediating inflammatory responses. Although its systemic blockade was successfully used to treat autoimmune diseases in preclinical models, efficacy of a topical route of Hsp90 inhibitor administration has so far not been evaluated in chronic inflammatory and autoimmune-mediated dermatoses. Here, effects of the Hsp90 blocker 17-allylamino-demethoxygeldanamycin (17AAG) applied topically to the skin were determined in experimental inflammatory epidermolysis bullosa acquisita (EBA), an anti-type VII collagen autoantibody-induced blistering skin disease. Topical 17AAG ameliorated clinical disease severity when given before or during the occurrence of skin lesions without causing cutaneous or systemic toxicity in mice with antibody transfer- and immunization-induced EBA. In both EBA models and in the setting of locally induced inflammation, topical 17AAG treatment was associated with (i) reduced neutrophilic infiltrates, (ii) decreased NF-κB activation, (iii) lowered expression of matrix metalloproteinases and Flii, and (iv) induction of anti-inflammatory Hsp70 in the skin. Our results suggest that topical delivery of Hsp90 antagonists, offering the benefit of a reduced risk of systemic adverse effects of Hsp90 inhibition, may be useful for the control of EBA and possibly other related inflammatory skin disorders.

Our findings demonstrate that immunohistochemistry for C3d or C4d is a helpful screening procedure for cases of suspected MMP where frozen tissue is not readily available. Negative findings, however, do not exclude a possible diagnosis of MMP and should prompt an additional biopsy for direct immunofluorescence studies. Immunohistochemical detection of IgG or IgA cannot yet be used for the diagnosis of MMP.