Bullous pemphigoid induced by IgG targeting type XVII collagen non-NC16A/NC15A extracellular domains is driven by Fc gamma receptor- and complement-mediated effector mechanisms and is ameliorated by neonatal Fc receptor blockade.

Bullous pemphigoid (BP) is an autoimmune blistering disease characterized by autoantibodies targeting type XVII collagen (Col17) with the noncollagenous 16A (NC16A) ectodomain representing the immunodominant site. The role of additional extracellular targets of Col17 outside NC16A has not been unequivocally demonstrated. In this study, we showed that Col17 ectodomain-reactive patient sera depleted in NC16A IgG induced dermal-epidermal separation in a cryosection model indicating the pathogenic potential of anti-Col17 non-NC16A extracellular IgG. Moreover, injection of IgG targeting the murine Col17 NC14-1 domains (downstream of NC15A, the murine homologue of human NC16A) into C57BL/6J mice resulted in erythematous skin lesions and erosions. Clinical findings were accompanied by IgG/C3 deposits along the basement membrane and subepidermal blistering with inflammatory infiltrates. Disease development was significantly reduced in either Fc-gamma receptor (FcγR)- or complement-5a receptor-1 (C5aR1)-deficient mice. Inhibition of the neonatal FcR (FcRn), an atypical FcγR regulating IgG homeostasis, with the murine Fc fragment IgG2c-ABDEG, a derivative of efgartigimod, reduced anti-NC14-1 IgG levels, resulting in ameliorated skin inflammation compared with isotype-treated controls. These data demonstrate that the pathogenic effects of IgG targeting the Col17 domain outside human NC16A/murine NC15A are partly attributable to antibody-mediated FcγR- and C5aR1 effector mechanisms while pharmacological inhibition of the FcRn represents a promising treatment for BP. The mouse model of BP will be instrumental in further investigating the role of Col17 non-NC16A/NC15A extracellular epitopes and validating new therapies for this disease. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

C3d-Targeted factor H inhibits tissue complement in disease models and reduces glomerular injury without affecting circulating complement.

Complement-mediated diseases can be treated using systemic inhibitors. However, complement components are abundant in circulation, affecting systemic inhibitors' exposure and efficacy. Furthermore, because of complement's essential role in immunity, systemic treatments raise infection risk in patients. To address these challenges, we developed antibody fusion proteins combining the alternative-pathway complement inhibitor factor H (fH1-5) with an anti-C3d monoclonal antibody (C3d-mAb-2fH). Because C3d is deposited at sites of complement activity, this molecule localizes to tissue complement while minimizing circulating complement engagement. These fusion proteins bind to deposited complement in diseased human skin sections and localize to activated complement in a primate skin injury model. We further explored the pharmacology of C3d-mAb-2fH proteins in rodent models with robust tissue complement activation. Doses of C3d-mAb-2fH >1 mg/kg achieved >75% tissue complement inhibition in mouse and rat injury models while avoiding circulating complement blockade. Glomerular-specific complement inhibition reduced proteinuria and preserved podocyte foot-process architecture in rat membranous nephropathy, indicating disease-modifying efficacy. These data indicate that targeting local tissue complement results in durable and efficacious complement blockade in skin and kidney while avoiding systemic inhibition, suggesting broad applicability of this approach in treating a range of complement-mediated diseases.

Comparing the risk of death, major adverse cardiac events, and relapse in bullous pemphigoid patients treated with systemic or topical corticosteroids.

BACKGROUND: According to current guidelines, systemic or topical corticosteroids are recommended as first-line treatments for bullous pemphigoid (BP). There is evidence suggesting that topical application may be associated with a lower risk of mortality. However, there is a lack of comprehensive large-scale data comparing mortality rates, as well as the risk of major adverse cardiac events (MACE), infections, and relapse, between systemic and topical corticosteroid treatments. OBJECTIVES: To evaluate the risk of death, MACE, infections, and relapse in BP patients treated with systemic or topical corticosteroids. METHODS: A population-based retrospective cohort study was performed in the TriNetX US Collaborative Network. As a measure against bias, propensity-score matching for age, sex, ten diseases and six medications, and three sensitivity analyses were conducted. RESULTS: All-time risk of death was increased in US BP patients exposed to any dose of systemic corticosteroids (n=2,917) compared to topical clobetasol propionate treated patients (n=2,932, hazard ratio [HR], 1.43, 95% confidence interval [CI] 1.28-1.58, p<0.0001). This was consistent in time-stratified analysis (1- and 3-year mortality rates), and in analysis contrasting prednisone (equivalent) does of 1-10 mg (low) or 30-100 mg (medium-high) systemic corticosteroid to topical treatment. The increased risk of death in US BP patients exposed to any dose of systemic corticosteroids compared to topical treatment was accompanied by increased risks for MACE (HR 1.33, CI 1.08-1.64, p=0.0075) and infections (HR 1.33, CI 1.15-1.54, p=0.0001). The risk of continued disease or relapse was decreased in patients treated with systemic as opposed to topical corticosteroid (HR 0.85, CI 0.77-0.94, p=0.0016). Results regarding mortality and continued disease or relapse persisted in three of three sensitivity analyses. Potential limitations are the retrospective data collection, bias for treatment selection and miscoding. CONCLUSION: Pending validation in prospective studies, where feasible, and despite the heightened risk of relapse, topical corticosteroid treatment may be advantageous compared to systemic corticosteroid treatment due to its significantly lower risk of death.

Forward genetics and functional analysis highlight Itga11 as a modulator of murine psoriasiform dermatitis.

Psoriasis is a chronic inflammatory skin condition. Repeated epicutaneous application of Aldara® (imiquimod) cream results in psoriasiform dermatitis in mice. The Aldara®-induced psoriasiform dermatitis (AIPD) mouse model has been used to examine the pathogenesis of psoriasis. Here, we used a forward genetics approach in which we compared AIPD that developed in 13 different inbred mouse strains to identify genes and pathways that modulated disease severity. Among our primary results, we found that the severity of AIPD differed substantially between different strains of inbred mice and that these variations were associated with polymorphisms in Itga11. The Itga11 gene encodes the integrin α11 subunit that heterodimerizes with the integrin ß1 subunit to form integrin α11ß1. Less information is available about the function of ITGA11 in skin inflammation; however, a role in the regulation of cutaneous wound healing, specifically the development of dermal fibrosis, has been described. Experiments performed with Itga11 gene-deleted (Itga11-/- ) mice revealed that the integrin α11 subunit contributes substantially to the clinical phenotype as well as the histopathological and molecular findings associated with skin inflammation characteristic of AIPD. Although the skin transcriptomes of Itga11-/- and WT mice do not differ from one another under physiological conditions, distinct transcriptomes emerge in these strains in response to the induction of AIPD. Most of the differentially expressed genes contributed to extracellular matrix organization, immune system, and metabolism of lipids pathways. Consistent with these findings, we detected a reduced number of fibroblasts and inflammatory cells, including macrophages, T cells, and tissue-resident memory T cells in skin samples from Itga11-/- mice in response to AIPD induction. Collectively, our results reveal that Itga11 plays a critical role in promoting skin inflammation in AIPD and thus might be targeted for the development of novel therapeutics for psoriasiform skin conditions. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Evidence for a role of extracellular heat shock protein 70 in epidermolysis bullosa acquisita.

Heat shock protein 90 (Hsp90) and Hsp70 are chaperones implicated in different inflammatory disorders, given their property to impact innate and adaptive immune responses. Here, we determined the so far unknown role of extracellular Hsp70 in epidermolysis bullosa acquisita (EBA), an anti-type VII collagen autoantibody-mediated blistering dermatosis. The in vivo pathophysiological relevance of extracellular Hsp70 was demonstrated in an anti-type VII collagen antibody transfer-induced EBA mouse model in which elevated blood levels of this chaperone were recorded. We found that Hsp70-treated mice had a more intense clinical disease severity compared to controls that were paralleled by increased levels of cutaneous matrix metalloproteinase 9 and plasma hydrogen peroxide. The latter finding was confirmed in an independent reactive oxygen species release assay using EBA-specific immune complexes combined with recombinant Hsp70. Finally, cell culture experiments using human naive peripheral blood mononuclear cells (PBMC) revealed that extracellular Hsp70 stimulated the secretion of the T cell-derived pro-inflammatory cytokines IL-6 and IL-8. This work extends knowledge about the role of Hsps in autoimmune bullous diseases, suggesting that extracellular Hsp70 represents a pathophysiological factor and potential treatment target in EBA.

DNA chip-based diagnosis of onychomycosis and tinea pedis.

BACKGROUND AND OBJECTIVES: Onychomycosis (OM) and tinea pedis (TP) are common fungal infections. Currently, diagnosis is based on direct microscopy and culture that have a low to moderate sensitivity and/or require up to 3-4 weeks until results are obtained. PCR techniques have emerged for the diagnosis of fungal infections, but little is known about their sensitivity and specificity in diagnosing. Here, we compared the diagnostic value of a DNA-chip technology, that detects 56 fungal pathogens, in a single-center prospective diagnostic study with microscopy and culture in suspected OM/TP. PATIENTS AND METHODS: Microscopy, culture and DNA microarray assays were performed on scraping material from patients with suspected OM (n = 67) or TP (n = 73). To test whether swabs can be used as an alternative for scraping, PCR yields were compared in a further 13 patients with OM and 11 patients with TP. RESULTS: DNA microarrays had the highest sensitivity. Combination of DNA-chip technology with microscopy further increased the sensitivity, and results from this combined laboratory diagnosis can be obtained within 24 hours. Comparison of sampling techniques (scraping, dry or wet swab) for DNA-chip assays showed similar results in suspected OM or TP. CONCLUSIONS: DNA-chip technology shows high sensitivity for OM and TP diagnosis, especially when combined with microscopy.

DNA-Chip-basierte Diagnose der Onychomykose und Tinea pedis.

HINTERGRUND UND ZIELE: Onychomykose (OM) und Tinea pedis (TP) sind häufige Pilzinfektionen der Haut. Aktuell basiert die Diagnose vornehmlich auf mikroskopischem Direktnachweis und/oder Kultur. Beide Methoden haben jedoch eine geringe bis mäßige Sensitivität und benötigen teilweise mehrere Wochen, bis endgültige Laborergebnisse vorliegen. Um die Diagnose kutaner Pilzinfektionen zu verbessern, wurden PCR-basierte Methoden entwickelt. Hier haben wir hier die Sensitivität und Spezifität einer Chip-basierten Multiplex-PCR mit mikroskopischen Direktnachweis und verglichen. PATIENTEN UND METHODIK: In einer monozentrischen, prospektiven Studie wurden bei Patienten mit Verdacht auf OM (n  =  67) oder TP (n  =  73) Schuppenpräparate entnommen und mittels mikroskopischem Direktnachweis, Kultur und DNA-Chip-Technologie der Erregernachweis durchgeführt. In einem weiteren Ansatz wurde überprüft, ob Abstriche als Alternative zur Entnahme eines Schuppenpräparates verwendet werden können. Hierfür wurden 24 weitere OM/TP-Patienten rekrutiert und die Ergebnisse der DNA-Chip-Technologie aus Abstrichen mit denen aus den Schuppenpräparaten verglichen. ERGEBNISSE: Im Vergleich aller Methoden hatte die DNA-Chip-Technologie die höchste Sensitivität, eine Kombination von DNA-Chip-Technologie mit mikroskopischem Direktnachweis erhöhte dies weiter. Ergebnisse dieser kombinierten Labordiagnostik sind innerhalb von 24 Stunden verfügbar. Der Vergleich der Probenentnahmetechniken (Abstrich beziehungsweise Schuppenpräparat) zeigte vergleichbare Ergebnisse. SCHLUSSFOLGERUNGEN: Die molekulare Diagnostik (mittels DNA-Chip-Technologie) hat eine hohe Sensitivität für die OM- und TP-Diagnostik, insbesondere in Kombination mit dem mikroskopischen Direktnachweis.

A Mitochondrial Polymorphism Alters Immune Cell Metabolism and Protects Mice from Skin Inflammation.

Several genetic variants in the mitochondrial genome (mtDNA), including ancient polymorphisms, are associated with chronic inflammatory conditions, but investigating the functional consequences of such mtDNA polymorphisms in humans is challenging due to the influence of many other polymorphisms in both mtDNA and the nuclear genome (nDNA). Here, using the conplastic mouse strain B6-mtFVB, we show that in mice, a maternally inherited natural mutation (m.7778G > T) in the mitochondrially encoded gene ATP synthase 8 (mt-Atp8) of complex V impacts on the cellular metabolic profile and effector functions of CD4+ T cells and induces mild changes in oxidative phosphorylation (OXPHOS) complex activities. These changes culminated in significantly lower disease susceptibility in two models of inflammatory skin disease. Our findings provide experimental evidence that a natural variation in mtDNA influences chronic inflammatory conditions through alterations in cellular metabolism and the systemic metabolic profile without causing major dysfunction in the OXPHOS system.

Drug Development in Pemphigoid Diseases.

Pemphigoid diseases are organ-specific autoimmune diseases of the skin and/or mucous membranes. They are caused by autoantibodies targeting adhesion molecules located at the dermal-epidermal junction. While the diagnostics of pemphigoid diseases and insights into their pathogenesis have improved significantly, the development of novel treatments that are effective and safe remains an unmet medical need. However, numerous pre-clinical studies and early clinical trials have recently been launched. This review summarizes some pathways leading to drug development in pemphigoid diseases, namely: (i) hypothesis-driven drug development; (ii) omics-based drug development; (iii) drug repurposing; (iv) screening-based drug development; and (v) drug development based on careful clinical observations. Ultimately, it is hoped that this will lead to personalized and curative treatments.

Regulatory T-cell deficiency leads to pathogenic bullous pemphigoid antigen 230 autoantibody and autoimmune bullous disease.

BACKGROUND: Autoimmune bullous diseases/dermatoses (AIBDs) are severe autoantibody-mediated skin diseases. The pathogenic relevance of autoreactive CD4+ T cells for the induction of autoantibody production remains to be fully evaluated. Scurfy mice lack functional regulatory T (Treg) cells, experience spontaneous activation of autoreactive CD4+ T cells, and display severe erosive skin lesions suggestive of AIBDs. OBJECTIVE: We sought to determine whether AIBDs develop in Treg cell-deficient scurfy mice. METHODS: Histology, indirect immunofluorescence (IF) microscopy, direct IF, and ELISA were used to prove the presence of AIBDs in scurfy mice. Monoclonal autoantibodies from sera of scurfy mice were screened by using indirect IF on murine skin, and immunoprecipitation and mass spectrometry were used for target antigen identification, followed by confirmation in modified human embryonic kidney cells and murine keratinocytes. Pathogenicity was determined by injecting the autoantibody into neonatal mice and transferring scurfy CD4+ T cells into nu/nu mice. RESULTS: Autoantibodies against different known autoantigens of AIBDs spontaneously develop in scurfy mice. Histology reveals subepidermal blisters, and direct IF of skin of scurfy mice shows a predominant linear staining pattern. The mAb 20B12 shows a linear staining pattern in indirect IF, recognizes the murine hemidesmosomal protein bullous pemphigoid antigen 230 (BP230) as the target antigen, and cross-reacts with human BP230. Purified mAb 20B12 induces subepidermal blisters in neonatal mice. Transfer of scurfy CD4+ T cells is sufficient to induce antibodies with reactivity to AIBD autoantigens and subepidermal blisters in the skin of recipient T cell-deficient nu/nu mice. CONCLUSION: We show that the absence of Treg cells leads to AIBDs by pathogenic autoantibodies targeting BP230.

In Vivo Expansion of Endogenous Regulatory T Cell Populations Induces Long-Term Suppression of Contact Hypersensitivity.

Contact hypersensitivity (CHS) of murine skin serves as a model of allergic contact dermatitis. Hapten-specific CD8 T cells and neutrophils represent the major effector cells driving this inflammatory reaction whereas Foxp3(+) regulatory T cells (Tregs) control the severity of inflammation. However, whether in vivo expansion of endogenous Tregs can downregulate CHS-mediated inflammation remains to be elucidated. In this study, we addressed this issue by using injection of an IL-2/anti-IL-2 mAb JES6-1 complex (IL-2/JES6-1) as a means of Treg induction in 2,4,6-trinitrochlorobenzene-induced CHS. IL-2/JES6-1 injection before or after hapten sensitization led to a considerable reduction of skin inflammation, even when rechallenged up to 3 wk after the last treatment. Conversely, Treg depletion re-established the CHS response in IL-2/JES6-1-treated mice. IL-2/JES6-1 injection resulted in increased frequencies of natural and peripheral Tregs in spleen and draining lymph nodes (LNs), elevated IL-10 and TGF-ß production by CD4 T cells, reduced CD86 expression by dendritic cells, and led to lower numbers of hapten-specific IFN-γ-producing CD8 T effector cells in LNs. Neutrophil and CD8 T cell infiltration was reduced in inflamed ear tissue, whereas CTLA-4(+)Foxp3(+) Treg frequencies were augmented. Adoptive transfer of LN cells of sensitized mice into recipients treated with IL-2/JES6-1 showed impaired CHS. Our results show that in vivo Treg expansion results in a prolonged CHS suppression, a sustained reduction of hapten-specific CD8 T cells, and a decrease in effector cell influx in inflamed tissue.

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 an increased concentration of tumor necrosis factor alpha (TNF) in the serum and blister fluid of patients with subepidermal AIBDs. Furthermore, successful anti-TNF treatment has been reported for individual patients with AIBDs. 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 PCR and immunohistochemistry. To investigate if 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 receiving either of these two 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 to controls. Furthermore, etanercept treatment significantly reduced the disease progression in immunization-induced EBA. In conclusion, the 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 the 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.

In vitro and in vivo models to investigate the pathomechanisms and novel treatments for pemphigoid diseases.

Pemphigoid diseases (PD) are a subgroup of rare acute or chronic autoimmune skin disorders characterized and caused by autoantibodies directed against distinct structural components of the dermal-epidermal junction. Binding of autoantibodies to their targets leads to the formation of blisters and erosions in patients. PDs comprise eight disorders for which the molecular target antigens have been identified. First, we review the available in vitro and ex vivo models for analysis of distinct aspects of the pathogenesis of PDs. This includes the binding of autoantibodies to skin sections, the analysis of blister formation capability and skin complement activation as well as investigation of neutrophil and keratinocyte activation. In addition to this, several animal models of PD have been developed during the last decades. These animal models have greatly contributed to our current understanding of the pathogenesis of PDs. We summarize spontaneously arising PD in animals and the induction of PD by transfer of (auto)antibodies, transfer of (auto)-antigen-specific lymphocytes and by immunization. In combined use, these models allow dissecting all aspects of PD pathogenesis, for example loss of tolerance, autoantibody production and inflammatory skin processes that lead to blister formation. Overall, we aimed to foster translational biomedical research, to deepen our understanding of PD pathogenesis and to develop novel treatments for patients suffering from these life-threatening and difficult-to-treat autoimmune diseases.

Evidence for a contributory role of a xenogeneic immune response in experimental epidermolysis bullosa acquisita.

Autoimmune diseases affect a large fraction of the population in Western countries. To elucidate the underlying causes, autoantibody transfer-induced mouse models have been established that greatly contributed to the understanding of the pathophysiology of these diseases. However, the role of a potentially co-occurring murine xenogeneic immune response to commonly utilized rabbit anti-mouse IgG remains poorly understood. Using the established rabbit anti-mouse type VII collagen (COL7) IgG-induced mouse model of the mucocutaneous blistering disorder epidermolysis bullosa acquisita (EBA), we found in this study a profound T- and B-cell response along with an altered cytokine expression profile in draining lymph nodes of mice injected with the xenogeneic IgG. This was associated with the formation of circulating and skin-bound mouse anti-rabbit IgG in wild-type but not CD154-deficient or B-cell-deficient JHT mice challenged with pathogenic rabbit IgG. Development of EBA skin lesions was attenuated in the two mouse strains lacking a B-cell response at later observation time points, but was not affected in mice treated with the T-cell trafficking blocker FTY720. Collectively, our results implicate a host's xenoreactive immune response to rabbit anti-mouse COL7 IgG, a confounding effect that may contribute to immune complex-driven inflammation and tissue damage in this antibody transfer-induced EBA mouse model, especially at later time points. In this regard, it may be recommended to finish the evaluation of results obtained by experiments employing antibody-transferred mouse models within the first 2 weeks after the pathogenic antibody injection.

Analysis of serum markers of cellular immune activation in patients with bullous pemphigoid.

Experimental models of bullous pemphigoid (BP), the most frequent subepidermal autoimmune bullous disease, revealed that the immune response leading to blister formation represents an incompletely understood complex process involving different inflammatory cells. In contrast to previous reports commonly focusing on limited molecular and cellular phenotypes of the disease, the aim of this study was to investigate a broad spectrum of markers of cellular immune activation in patients with BP. We found that serum levels of soluble CD4, myeloperoxidase, S100A12, eosinophil cationic protein and soluble P-selectin were significantly elevated in patients with active BP compared with normal controls. Mast cell tryptase and neopterin serum levels significantly decreased at the time of clinical remission of the patients. Additionally, serum concentrations of soluble IL-2 receptor, mast cell tryptase and soluble P-selectin were significantly associated with levels of circulating anti-BP180 autoantibodies. Our findings confirm and extend previous reports suggesting some concomitant involvement of a panel of molecules representative for a wide spectrum of cellular players (T cells, mast cells, neutrophils, eosinophils, macrophages and platelets) orchestrating the inflammatory reaction in BP. These data may favour the employment of broad-spectrum or combined immunosuppressants, potentially together with an anticoagulant treatment, over cell- or molecule-specific targeted therapy in patients with this disorder.

Skin microbiota-associated inflammation precedes autoantibody induced tissue damage in experimental epidermolysis bullosa acquisita.

Epidermolysis bullosa acquisita (EBA) is a chronic autoimmune blistering skin disease characterized by autoantibodies against type VII collagen (COL7). Immunization of SJL/J mice with recombinant murine COL7 results in break of tolerance and skin blisters. Strikingly, despite circulating autoantibodies, the same genetic background and identical environmental conditions, 20% of mice remain healthy. To elucidate the regulation of the transition from the presence of autoantibodies to overt autoimmune disease, we characterized the innate and adaptive immune response of mice that remain healthy after immunization and compared it to mice that developed skin disease. Both clinically healthy and diseased SJL/J mice showed circulating autoantibodies and deposition of complement-fixing IgG2c autoantibodies and C3 at the dermal-epidermal junction. However, only in diseased animals significant neutrophil infiltration and increase in FcgRIV expression were observed in the skin. In contrast, the expression of T cell signature cytokines in the T cell zone of the draining lymph node was comparable between clinically healthy and diseased animals after immunization. Surprisingly, health was associated with a decreased expression of CD11c, TNFA and KC (CXCL1) in the skin prior to immunization and could be predicted with a negative predictive value of >80%. Furthermore, mice that did not develop clinical disease showed a significantly higher richness and distinctly clustered diversity of their skin microbiota before immunization. Our data indicate that the decision whether blisters develop in the presence of autoantibodies is governed in the skin rather than in the lymph node, and that a greater richness of cutaneous bacterial species appears to be protective.