Granzyme B inhibition reduces autoantibody-induced dermal-epidermal separation in an ex vivo model of epidermolysis bullosa acquisita.

The pemphigoid disease epidermolysis bullosa acquisita (EBA) is an autoimmune blistering skin disease characterized by autoantibodies against type VII collagen (COL7), immune cell infiltrates at the dermal-epidermal junction and subepidermal blistering. Proteases, particularly granzyme B (GzmB), have been established as therapeutic targets for the treatment of EBA and other pemphigoid diseases. We investigated the impact of the novel GzmB inhibitor SNT-6935 on anti-COL7 IgG-induced subepidermal blistering in a well-established EBA ex vivo model. Our findings demonstrate that pharmacological targeting of GzmB with its selective inhibitor SNT-6935 significantly reduced autoantibody-induced dermal-epidermal separation in human skin cryosections. Interestingly, treatment of skin cryosections with recombinant human GzmB alone did not cause dermal-epidermal separation, suggesting that additional mechanisms alongside GzmB are required for tissue damage in EBA. In conclusion, our study highlights the significant contribution of GzmB to the pathogenesis of EBA and supports the notion of GzmB as a therapeutic target in EBA and other pemphigoid diseases.

Inhibition of interferon gamma impairs induction of experimental epidermolysis bullosa acquisita.

Epidermolysis bullosa acquisita (EBA) is a muco-cutaneous autoimmune disease characterized and caused by autoantibodies targeting type VII collagen (COL7). The treatment of EBA is notoriously difficult, with a median time to remission of 9 months. In preclinical EBA models, we previously discovered that depletion of regulatory T cells (Treg) enhances autoantibody-induced, neutrophil-mediated inflammation and blistering. Increased EBA severity in Treg-depleted mice was accompanied by an increased cutaneous expression of interferon gamma (IFN-γ). The functional relevance of IFN-γ in EBA pathogenesis had been unknown. Given that emapalumab, an anti-IFN-γ antibody, is approved for primary hemophagocytic lymphohistiocytosis patients, we sought to assess the therapeutic potential of IFN-γ inhibition in EBA. Specifically, we evaluated if IFN-γ inhibition has modulatory effects on skin inflammation in a pre-clinical EBA model, based on the transfer of COL7 antibodies into mice. Compared to isotype control antibody, anti-IFN-γ treatment significantly reduced clinical disease manifestation in experimental EBA. Clinical improvement was associated with a reduced dermal infiltrate, especially Ly6G+ neutrophils. On the molecular level, we noted few changes. Apart from reduced CXCL1 serum concentrations, which has been demonstrated to promote skin inflammation in EBA, the expression of cytokines was unaltered in the serum and skin following IFN-γ blockade. This validates IFN-γ as a potential therapeutic target in EBA, and possibly other diseases with a similar pathogenesis, such as bullous pemphigoid and mucous membrane pemphigoid.

Clinical Features of Paediatric Inflammatory Epidermolysis Bullosa Acquisita: A Case Series Study.

Epidermolysis bullosa acquisita (EBA) rarely develops in childhood. This study retrospectively recruited paediatric patients with EBA (age ≤ 16 years), diagnosed by clinical and histopathological features and results of immunofluorescence, immunoblotting and enzyme-linked immunosorbent assay (ELISA), and reviews their clinical manifestations, histopathology, immunological features, and responses to various treatments. All 7 included patients presented with inflammatory EBA. Among them, 3 had a bullous pemphigoid-like phenotype. Pathologically, in addition to dermal-epidermal blistering, in all patients, the distribution of neutrophils was superficial perivascular or interstitial, or in the dermal papilla. Mixed neutrophils and eosinophils were detected in 2 of the 3 patients with bullous pemphigoid-like phenotypes. In addition to treatment with glucocorticoids, dapsone was administered in 4 patients, while thalidomide and sulfasalazine were administered in 1 patient. All patients responded to the these therapies. Relapse was mainly due to reduction and cessation of glucocorticoids. In conclusion, EBA in childhood may be unique, and thus distinct from its adult counterpart. Specific treatment and follow-up protocols are required for therapy of this rare autoimmune skin disease in children.

Monocyte populations are involved in the pathogenesis of experimental epidermolysis bullosa acquisita.

Monocytes play a significant role in the pathogenesis of most inflammatory diseases, including autoimmune diseases. Herein, different subpopulations of monocytes often play differential, partially antagonistic roles, in the regulation of tissue populations. Pemphigoid diseases constitute a group of autoimmune blistering skin diseases featuring a marked infiltration of the dermis with immune cells, including monocytes. The monocyte subsets infiltrating the skin, however, have largely remained elusive. Monocyte adhesion and recruitment into the inflamed tissues are regulated by chemokine receptors, most prominently by CCR2 and CX3CR1. To delineate the involvement of monocyte populations in autoimmune blistering skin diseases, we spatiotemporally monitored the dynamic spectrum of monocyte populations that infiltrate the inflamed skin using multiphoton intravital imaging and reporter mice for chemokine receptors. Experimental epidermolysis bullosa acquisita (EBA) was induced by injection of anti-murine type VII collagen (amCOLVII) IgG into the Csf1rEGFP-reporter mice, where circulating myeloid cells, such as monocytes and neutrophils, express an EGFP. EGFP+ cells, including neutrophils and monocytes, were present in the skin, immediately after the deposition of the amCOLVII antibody at the dermal-epidermal junction. To investigate the recruitment and involvement of different monocyte-derived cell populations in the disease course further, EBA was induced in CCR2RFP/+-reporter and CX3CR1GFP/+-reporter mice. A comparable distribution of red fluorescent protein (RFP)+ or green fluorescent protein (GFP)+ was found in both diseased mice and their respective controls over time, indicating the similar recruitment of monocytes into the skin following the binding of autoantibodies. Experiments were extended to the CCR2RFP/RFP-deficient and CX3CR1GFP/GFP-deficient mice to determine whether monocyte recruitment and disease severity are compromised in the absence of the receptor. A comparable pattern was seen in the recruitment of monocytes into the skin in both reporter and deficient mice. However, in contrast to similar disease severity between CX3CR1-deficient and reporter mice, CCR2-deficient mice developed significantly less disease than CCR2-reporter mice, as indicated by the percentage of affected area of ears. Collectively, our observations indicate that while CCR2 and CX3CR1 receptors are not involved in the recruitment of monocytes into the skin, CCR2 deficiency is associated with improved disease outcomes in experimental EBA in mice.

Involvement of palms and soles in patients with autoimmune bullous diseases: a comparative analysis of a diagnostically relevant localization.

Introduction: The involvement of palms and soles is variable among disease entities belonging to autoimmune bullous diseases (AIBD). We present our own clinical-laboratory experience concerning presentations of skin lesions on palms and soles in the pemphigus diseases group, pemphigoid diseases group, epidermolysis bullosa acquisita (EBA), and lichen planus pemphigoides (LPP) and discuss the pertinent literature. Methods: Lesions on palms and soles were assessed retrospectively on the basis of just photographic archives from the beginning of 2014 to March 2023. We comparatively evaluated 462 Slavic patients with AIBD. Results: Palmoplantar involvement was observed in only 21 patients with AIBD (12 females and 9 males). There was no statistically significant difference between palmoplantar involvement in the pemphigus diseases group compared to the pemphigoid diseases group and no statistically significant difference between the pemphigus diseases group compared to the subepithelial AIBD. Discussion: Nevertheless, particularly in LPP and EBA, and occasionally in pemphigus diseases and pemphigoid diseases groups of AIBD, localization on palms and soles may be diagnostically important at the clinical level.

C-type lectin receptor expression is a hallmark of neutrophils infiltrating the skin in epidermolysis bullosa acquisita.

Introduction: Inflammatory epidermolysis bullosa acquisita (EBA) is characterized by a neutrophilic response to anti-type VII collagen (COL7) antibodies resulting in the development of skin inflammation and blistering. The antibody transfer model of EBA closely mirrors this EBA phenotype. Methods: To better understand the changes induced in neutrophils upon recruitment from peripheral blood into lesional skin in EBA, we performed single-cell RNA-sequencing of whole blood and skin dissociate to capture minimally perturbed neutrophils and characterize their transcriptome. Results: Through this approach, we identified clear distinctions between circulating activated neutrophils and intradermal neutrophils. Most strikingly, the gene expression of multiple C-type lectin receptors, which have previously been reported to orchestrate host defense against fungi and select bacteria, were markedly dysregulated. After confirming the upregulation of Clec4n, Clec4d, and Clec4e in experimental EBA as well as in lesional skin from patients with inflammatory EBA, we performed functional studies in globally deficient Clec4e-/- and Clec4d-/- mice as well as in neutrophil-specific Clec4n-/- mice. Deficiency in these genes did not reduce disease in the EBA model. Discussion: Collectively, our results suggest that while the upregulation of Clec4n, Clec4d, and Clec4e is a hallmark of activated dermal neutrophil populations, their individual contribution to the pathogenesis of EBA is dispensable.

Retrospective analysis of autoimmune bullous diseases in Middle Franconia.

Introduction: Autoimmune bullous diseases (AIBDs) are a group of rare cutaneous disorders affecting cornified skin and mucous membranes. They are characterized by tense or flaccid blistering and erosions due to autoantibodies against desmosomal and hemidesmosomal structural proteins of the skin. This group of disorders can be divided into those of pemphigoid and those of pemphigus diseases. If left untreated, these autoimmune diseases can cause serious or even life-threatening complications such as loss of fluid, superinfections or impaired food intake. Due to modern standardized serological assays, the diagnosis of AIBDs can usually be confirmed in combination with their clinical appearance. Whereas for a long time corticosteroids were the major players in the treatment of these diseases, with the approval of rituximab and other immunosuppressive agents, the therapy has increasingly improved. Methods: In this study, we aimed to investigate epidemiologic and clinical features as well as diagnostics and therapy of bullous autoimmune diseases in Middle Franconia, a governorate within the German federal state of Bavaria. Patients diagnosed or treated because of a AIBDs between 01.04.2013 and 31.03.2019 at the dermatological department of the university hospital Erlangen were included in this retrospective study (n = 242). Patients were either diagnosed for the first time (n=176) or the diagnosis has been confirmed (n=66) at the department. The respective incidence was calculated among the 176 subjects who had been diagnosed at the center in this period. Data was taken from patient records and analyzed with Microsoft® Excel. The evaluation included the diagnoses of pemphigus vulgaris (PV), pemphigus foliaceus (PF), bullous pemphigoid (BP), mucous membrane pemphigoid (MMP), linear IgA dermatosis (LAD), epidermolysis bullosa acquisita (EBA), and dermatitis herpetiformis (DH). Results: This study shows that the incidence of each AIBDs in Middle Franconia is low and comparable (PV, PF, LAD, EBA) or lower (BP, MMP, DH) than in other studies and regions. BP is the most common newly diagnosed AIBD in Middle Franconia. Discussion: Due to the chronic and sometimes severe course of AIBDs, repeated in-house treatments are often necessary. To date, mainly topically and systemically applied corticosteroids in combination with immunomodulators are used as first-line therapy.

Luteolin peracetate and gossypolone inhibit immune complex-mediated neutrophil activation in vitro and dermal-epidermal separation in an ex vivo model of epidermolysis bullosa acquisita.

Introduction: Natural products have been shown to an important source of therapeutics for human disease. In this study, we aimed to identify natural compounds as potential therapeutics for epidermolysis bullosa acquisita (EBA), an autoimmune disease caused by autoantibodies to type VII collagen (COL7). Methods: Utilizing an in vitro experimental system, we screened a natural product library composed of 800 pure compounds for their inhibitory effect on COL7-anti-COL7 IgG immune complex (IC)-mediated neutrophil activation and on neutrophil-mediated tissue damage. Results: Three natural compounds, namely luteolin peracetate, gossypol, and gossypolone were capable in inhibiting the IC-induced neutrophil adhesion and oxygen burst in vitro. Furthermore, luteolin peracetate and gossypolone were able to inhibit the anti-COL7 IgG induced dermal-epidermal separation in an ex vivo model for EBA. Discussion: In summary, this study demonstrates that luteolin peracetate and gossypolone are potential therapeutics for experimental EBA, which deserves further investigation.

Case Report: Biological treatment of epidermolysis bullosa acquisita: report on four cases and literature review.

Epidermolysis bullosa acquisita (EBA) is a chronic, recurrent autoimmune subepidermal bullous disease characterized by the presence of autoantibodies targeting type VII collagen -- basement membrane zone antigen. Standard therapy for EBA includes a combination of systemic corticosteroids and dapsone; however, severe cases may require advanced treatment. The current article reports on four EBA cases in which biologics: infliximab, rituximab (Rtx), and intravenous immunoglobulin (IVIG) were applied. All patients fulfilled the clinical and immunological criteria of EBA: they presented tense blisters healing with atrophic scars on the skin on traumatized areas and in mucous membranes. The diagnosis of EBA was established using numerous techniques: direct and indirect immunofluorescence, salt split skin, ELISA, Fluorescence Overlay Antigen Mapping using Laser Scanning Confocal Microscopy. Since all the patients did not achieve long-term remission on standard treatment (prednisone, dapsone) due to ineffectiveness or side effects of drugs, they eventually were treated with biologics leading to extraordinary skin improvement and stopping the disease for 1-3 years. Biologics in all patients were tolerated very well. No side effects were observed during application as well as multi-month follow-up. The presented cases provide a premise that biological drugs can be a valuable component of EBA therapy.

Intravenous Immunoglobulin for Autoimmune Bullous Diseases: A Case Series from a Central European Referral Center.

Background and Objectives: Autoimmune bullous diseases (AIBDs) may be treated with intravenous immunoglobulin (IVIG) infusions. This study aimed to evaluate the benefits and safety profiles of high-dose IVIG therapy in AIBD patients, as determined by clinical remission, the glucocorticosteroid-sparing effect, and adverse events at 12 months follow-up in a Central European university dermatology department setting. Materials and Methods: Our case series included 10 patients: five patients with pemphigus vulgaris, one with pemphigus herpetiformis, one with pemphigus foliaceus, one with bullous pemphigoid, two with epidermolysis bullosa acquisita. They underwent 4-12 monthly cycles of IVIG therapy at a dose of 2 g/kg per cycle. Results: The prednisone dosage reduction after 2, 6, and 12 months following the final IVIG course was 65.45%, 70.91%, and 76.37%, respectively. During the 12-month observation period, disease relapse was observed in 20% of patients, while others achieved complete or partial remission without or with minimal therapy. Side effects were seen in 80% of patients; they were transient and did not necessitate discontinuation of IVIG. Conclusions: IVIG demonstrates effectiveness as a treatment with a favorable safety profile. Nevertheless, its high cost remains a significant drawback, particularly in low-income countries. IVIG should be considered, especially in patients opposed to standard therapies or with contraindications to their use.

Acquired epidermolysis bullosa in a pediatric patient.

BACKGROUND: Acquired epidermolysis bullosa is a rare and chronic autoimmune subepidermal bullous disease characterized by the formation of autoantibodies against type VII collagen. Presentation in childhood is rare and with several manifestations. CASE REPORT: We report the case of a 12-year-old female patient who presented bullous and polymorphic lesions on the chest and extremities of several months of evolution. Due to the characteristics of the skin lesions, a histopathological and direct immunofluorescence study was conducted, confirming the diagnosis of acquired epidermolysis bullosa. Subsequently, corticosteroid and dapsone treatment was administered, with favorable clinical response during follow-up. CONCLUSIONS: Acquired epidermolysis bullosa is unusual in pediatric age, so it should be considered in the differential diagnosis of other congenital and acquired bullous diseases of childhood. The definitive diagnosis is performed through an immunofluorescence, study, which allows for rapid and effective treatment to control the disease and avoid permanent sequelae.

INTRODUCCIÓN: La epidermólisis bullosa adquirida es una enfermedad ampollar subepidérmica autoinmune, rara y crónica caracterizada por la formación de autoanticuerpos contra colágeno tipo VII. La presentación en la infancia es poco frecuente y con manifestaciones variables. CASO CLÍNICO: Se describe el caso de una paciente de sexo femenino de 12 años de edad que presentó lesiones ampollares y polimórficas en tórax y extremidades de varios meses de evolución. Por las características de las lesiones cutáneas, se realizó un estudio histopatológico y de inmunofluorescencia directa que confirmó el diagnóstico de epidermólisis bullosa adquirida, por lo que se administró tratamiento con corticoide y dapsona, con una respuesta clínica favorable durante el seguimiento. CONCLUSIONES: La epidermólisis bullosa adquirida es inusual en la edad pediátrica. Por tanto, debe considerarse en el diagnóstico diferencial de otras enfermedades ampollares congénitas y adquiridas de la infancia. El diagnóstico definitivo se realiza a través del estudio de inmunofluorescencia, lo que permite instaurar rápidamente un tratamiento rápido y eficaz para controlar la enfermedad y evitar secuelas permanentes.

The complement receptor C5aR2 regulates neutrophil activation and function contributing to neutrophil-driven epidermolysis bullosa acquisita.

Introduction: The function of the second receptor for the complement cleavage product C5a, C5aR2, is poorly understood and often neglected in the immunological context. Using mice with a global deficiency of C5aR2, we have previously reported an important role of this receptor in the pathogenesis of the neutrophil-driven autoimmune disease epidermolysis bullosa acquisita (EBA). Based on in vitro analyses, we hypothesized that the absence of C5aR2 specifically on neutrophils is the cause of the observed differences. Here, we report the generation of a new mouse line with a LysM-specific deficiency of C5aR2. Methods: LysM-specific deletion of C5aR2 was achieved by crossing LysMcre mice with tdTomato-C5ar2fl/fl mice in which the tdTomato-C5ar2 gene is flanked by loxP sites. Passive EBA was induced by subcutaneous injection of rabbit anti-mouse collagen type VII IgG. The effects of targeted deletion of C5ar2 on C5a-induced effector functions of neutrophils were examined in in vitro assays. Results: We confirm the successful deletion of C5aR2 at both the genetic and protein levels in neutrophils. The mice appeared healthy and the expression of C5aR1 in bone marrow and blood neutrophils was not negatively affected by LysM-specific deletion of C5aR2. Using the antibody transfer mouse model of EBA, we found that the absence of C5aR2 in LysM-positive cells resulted in an overall amelioration of disease progression, similar to what we had previously found in mice with global deficiency of C5aR2. Neutrophils lacking C5aR2 showed decreased activation after C5a stimulation and increased expression of the inhibitory Fcγ receptor FcγRIIb. Discussion: Overall, with the data presented here, we confirm and extend our previous findings and show that C5aR2 in neutrophils regulates their activation and function in response to C5a by potentially affecting the expression of Fcγ receptors and CD11b. Thus, C5aR2 regulates the finely tuned interaction network between immune complexes, Fcγ receptors, CD11b, and C5aR1 that is important for neutrophil recruitment and sustained activation. This underscores the importance of C5aR2 in the pathogenesis of neutrophil-mediated autoimmune diseases.