Intravenous Ig Ameliorates Disease in a Murine Model of Anti-Laminin 332 Mucous Membrane Pemphigoid.

Intravenous Ig (IVIg) is used to treat mucous membrane pemphigoid, although its therapeutic effectivity is not sufficiently supported by randomized controlled clinical trials, and its mode of action is only insufficiently understood. We have examined the effect of IVIg in a mouse model of anti-laminin 332 mucous membrane pemphigoid and found that IVIg ameliorates both cutaneous and mucosal inflammatory lesions. Our investigation into the modes of action of IVIg in mucous membrane pemphigoid indicated effective anti-inflammatory mechanisms beyond the enhanced degradation of IgG mediated through inhibition of the FcRn. Our results suggest that IVIg curbs the activation of neutrophils at several levels. This includes a direct, immediate inhibitory effect on neutrophil activation by immune complexes but not C5a, which blunts the release of ROS and leukotriene B4 from neutrophils. IVIg also suppresses the formation of neutrophil extracellular traps in response to calcium ion ionophore. In vivo treatment with IVIg altered the transcriptome of blood leukocytes and bone marrow neutrophils toward less proinflammatory phenotypes. Collectively, our results support the effectivity of IVIg in the treatment of mucous membrane pemphigoid and indicate that effects on neutrophils at multiple levels may significantly contribute to its therapeutic effects.

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.

Autoimmunity against laminin 332.

Laminin 332 is a heterotrimeric structural protein of the basal membrane zone (BMZ) of the skin and adjacent mucosal tissues. The importance of laminin 332 for the structural integrity of the BMZ is demonstrated by mutations in any of the three genes encoding for its three chains causing variants of junctional epidermolysis bullosa. Autoimmunity against laminin 332 is observed in mucous membrane pemphigoid (MMP) and in the rare patients with orf-induced pemphigoid. MMP is an autoimmune blistering disease with predominant mucosal manifestations and autoantibodies against the BMZ of the skin and orifice-close mucous membranes. The main autoantigens of MMP are type XVII collagen (BP180) and laminin 332 targeted in about 80% and 10-20% of patients, respectively. An increasing number of studies has highlighted the association of anti-laminin 332 MMP and malignancies that can be revealed in about a quarter of these patients. This data has led to the recommendation of current guidelines to assay for anti-laminin 332 reactivity in all MMP patients. The present review focuses on anti-laminin 332 MMP describing clinical features, its pathophysiology, and detection of serum anti-laminin 332 IgG. In addition, the available data about the occurrence of malignancies in anti-laminin 332 MMP, the underlying tumor entities, and its biology are detailed.

[Immunopathogenesis of mucous membrane pemphigoid]. / Immunpathogenese des Schleimhautpemphigoids.

A detailed understanding of the immunopathogenesis of mucous membrane pemphigoid (MMP) is of particular importance in view of the mostly difficult diagnostics and treatment of this blistering autoimmune dermatosis. A still unknown disturbance of the body's own immune tolerance leads to the formation of autoreactive cells. As the disease progresses these produce autoantibodies which are directed against structural proteins in the basement membrane zone (BMZ). After they bind to the target antigen, complement factors are deposited along the BMZ and inflammatory cells invade the underlying tissue and produce the characteristic subepithelial blistering. This inflammatory response is associated with fibrosis and scarring in many affected tissues. Most phases of MMP pathogenesis are poorly understood; however, the last few years have shed more light on this processes. These advances are mostly the result of animal and cell culture models. Typical clinical and immunopathological characteristics of MMP, such as oral, conjunctival and skin lesions, are reflected, for example, in an antibody transfer-induced mouse model for anti-laminin 332 MMP in adult mice. Dapsone, as first-line treatment for MMP patients, significantly reduced the severity of these symptoms, and fibrosis in the skin and mucous membranes was also found histologically, which makes the model well-suited for testing new therapeutic approaches for MMP patients and might be of help for further elucidation of the immunopathogenesis of MMP.

Corrigendum: Cutaneous kinase activity correlates with treatment outcomes following PI3K delta inhibition in mice with experimental pemphigoid diseases.

[This corrects the article DOI: 10.3389/fimmu.2022.865241.].

Cutaneous kinase activity correlates with treatment outcomes following PI3K delta inhibition in mice with experimental pemphigoid diseases.

Chronic blistering at the skin and/or mucous membranes, accompanied by a varying degree of inflammation, is the clinical hallmark of pemphigoid diseases that impose a major medical burden. Pemphigoid diseases are caused by autoantibodies targeting structural proteins of the epithelial basement membrane. One major pathogenic pathway of skin blistering and inflammation is activation of myeloid cells following Fc gamma receptor-dependent binding to the skin-bound immune complexes. This process requires activation of specific kinases, such as PI3Kδ, which have emerged as potential targets for the treatment of pemphigoid diseases. Yet, it is unknown if global cutaneous kinase activity present in lesional pemphigoid disease correlates with therapeutic effects following treatment with a given target-selective kinase inhibitor. To address this, we here first determined the kinase activity in three different mouse models of pemphigoid diseases: Antibody transfer-induced mucous membrane pemphigoid (MMP), antibody transfer-induced epidermolysis bullosa acquisita (EBA) and immunization-induced EBA. Interestingly, the kinome signatures were different among the three models. More specifically, PI3Kδ was within the kinome activation network of antibody transfer-induced MMP and immunization-induced EBA, but not in antibody transfer-induced EBA. Next, the therapeutic impact of the PI3Kδ-selective inhibitor parsaclisib was evaluated in the three model systems. In line with the kinome signatures, parsaclisib had therapeutic effects in antibody transfer-induced MMP and immunization-induced EBA, but not in autoantibody-induced EBA. In conclusion, kinase activation signatures of inflamed skin, herein exemplified by pemphigoid diseases, correlate with the therapeutic outcomes following kinase inhibition, demonstrated here by the PI3Kδ inhibitor parsaclisib.

Mucous membrane pemphigoid.

Mucous membrane pemphigoid (MMP) is a clinically and immunopathologically heterogenous disease with an incidence of about 2/million inhabitants/year in central Europe. Pemphigoid diseases are characterized by autoantibodies against structural proteins of the epidermis and/or surface-close epithelia. MMP has been defined as pemphigoid disease with predominant mucosal lesions. Most frequently, the oral cavity and the conjunctivae are affected. Lesions outside the mouth tend to heal with scarring leading to visual impairment and finally blindness, as well as, more rarely, impairment of breathing and food intake. Autoantibodies target BP180 (collagen type XVII), laminin 332, BP230 (nearly always in conjunction with other antigens), and type VII collagen in about 75%, 10-20%, 10-30%, and <5% of MMP patients, respectively. While the main autoantibody isotype is IgG, additional, and less frequently exclusive, IgA autoantibodies can be detected in the majority of patients. Assaying for anti-laminin 332 reactivity is pivotal, since in about a quarter of patients with anti-laminin 332 MMP, a malignancy, mainly solid cancers, is associated. The pathophysiology of MMP is yet incompletely understood. A recent mouse model of anti-laminin 332 MMP replicating characteristic clinical and immunopathological findings of the human disease may be helpful to close this knowledge gap. Diagnosis is established by the clinical picture with predominant mucosal lesions and visualization of tissue-bound anti-basement membrane zone antibodies by direct immunofluorescence microscopy. In recent S3 guidelines initiated by the European Academy of Dermatology and Venereology, the clinical spectrum and diagnostic strategies are detailed. In addition, treatment regimens for different clinical situations including patients with exclusive oral or ocular involvement are outlined. Future studies are needed to better understand the clinical complexity and associations as well as to establish widely available diagnostic assays and evidence-based therapeutic strategies.

Immunization with desmoglein 3 induces non-pathogenic autoantibodies in mice.

BACKGROUND: Pemphigus vulgaris (PV) is a rare autoimmune blistering disease characterized by the development of autoantibodies targeting desmoglein (Dsg) 3, but also against Dsg1 in mucocutaneous disease. Given that existing PV animal models only recapitulate aspects of the disease, we aimed to establish a more comprehensive disease model based on the immunization of mice with PV autoantigen(s). METHODS: The following immunization strategies were tested: (i) C57Bl/6J, B6.SJL-H2s C3c/1CyJ, DBA2/J, or SJL/J mice were immunized with recombinant murine Dsg3 (mDsg3), (ii) DBA2/J and SJL/J mice were immunized with mDsg3 and additionally injected a single non-blister inducing dose of exfoliative toxin A (ETA), and (iii) DBA2/J and SJL/J mice were immunized with human Dsg (hDsg) 1 and 3. RESULTS: Despite the induction of autoantibodies in each immunization protocol, the mice did not develop a clinical phenotype. Tissue-bound autoantibodies were not detected in the skin or mucosa. Circulating autoantibodies did not bind to the native antigen in indirect immunofluorescence microscopy using monkey esophagus as a substrate. CONCLUSION: Immunization with PV autoantigens induced non-pathogenic Dsg1/3 antibodies, but did not cause skin/mucous membrane disease in mice. These findings, confirmed by failure of binding of the induced autoantibodies to their target in the skin, suggest that the autoantibodies which were formed were unable to bind to the conformational epitope present in vivo.

Dapsone Suppresses Disease in Preclinical Murine Models of Pemphigoid Diseases.

Epidermolysis bullosa acquisita and mucous membrane pemphigoid are autoimmune blistering diseases characterized by mucocutaneous blisters elicited by an autoantibody-mediated immune response against specific proteins of the epidermal basement membrane. The antibiotic dapsone is frequently used to treat both diseases, but its therapeutic effectiveness is uncertain, and its mode of action in these diseases is largely unknown. We evaluated the effect of dapsone in antibody transfer mouse models of epidermolysis bullosa acquisita and mucous membrane pemphigoid, which do not allow the drawing of conclusions on clinical treatment regimens but can be instrumental to partially uncover the mode(s) of action of dapsone in these diseases. Dapsone significantly mitigated inflammation in both models, reducing the recruitment of neutrophils into the skin and disrupting their release of leukotriene B4 (LTB4) and ROS in response to immune complexes. LTB4 has been implicated in numerous diseases, but effective LTB4 inhibitors for clinical use are not available. Our findings indicate that the mode of action of dapsone in these models may be based on the inhibition of LTB4 and ROS release from neutrophils. Moreover, they encourage testing the use of dapsone as an effective, albeit nonspecific, inhibitor of LTB4 biosynthesis in other LTB4-driven diseases.

Increased Fibrosis in a Mouse Model of Anti-Laminin 332 Mucous Membrane Pemphigoid Remains Unaltered by Inhibition of Aldehyde Dehydrogenase.

Mucous membrane pemphigoid (MMP) is an autoimmune blistering disease characterized by autoantibodies against the basal membrane zone of skin and surface-close epithelia and predominant mucosal lesions. The oral cavity and conjunctivae are most frequently affected, albeit clinical manifestations can also occur on the skin. MMP-associated lesions outside the oral cavity typically lead to scarring. Mechanisms underlying scarring are largely unknown in MMP and effective treatment options are limited. Herein, we assessed the collagen architecture in tissue samples of an antibody-transfer mouse model of anti-laminin-332 MMP. In MMP mice, increased collagen fibril density was observed in skin and conjunctival lesions compared to mice injected with normal rabbit IgG. The extracellular matrix of MMP skin samples also showed altered post-translational collagen cross-linking with increased levels of both lysine- and hydroxylysine-derived collagen crosslinks supporting the fibrotic phenotype in experimental MMP compared to control animals. In addition, we evaluated a potential anti-fibrotic therapy in experimental anti-laminin-332 MMP using disulfiram, an inhibitor of the aldehyde dehydrogenase (ALDH), which has been implicated in immune-mediated mucosal scarring. In addition, disulfiram also acts as a copper chelator that was shown to block lysyl oxidase activity, an enzyme involved in formation of collagen crosslinks. Topical use of disulfiram (300 µM in 2% [w/v] methocel) did not improve ocular lesions in experimental MMP over the 12-day treatment period in disulfiram-treated mice compared to vehicle-treated mice (n=8/group). Furthermore, C57BL6/J mice (n=8/group) were treated prophylactically with 200 mg/kg p.o. disulfiram or the solvent once daily over a period of 12 days. Systemic treatment did not show any reduction in the severity of oral and ocular lesions in MMP mice, albeit some improvement in skin lesions was observed in disulfiram- vs. vehicle-treated mice (p=0.052). No reduction in fibrosis was seen, as assessed by immunohistochemistry. Whilst blocking of ALDH failed to significantly ameliorate disease activity, our data provide new insight into fibrotic processes highlighting changes in the collagenous matrix and cross-linking patterns in IgG-mediated MMP.