Detection of a natural antibody targeting the shed ectodomain of BP180 in mice.

BACKGROUND: Pemphigoid diseases are characterized by subepidermal blister formation accompanied by autoantibodies targeting skin component molecules, such as BP180. It is suggested that an epitope-phenotype correlation exists among autoantibodies recognizing BP180. However, it is unclear which regions of BP180 are likely targets for autoantibodies. OBJECTIVE: To elucidate the portions of BP180 where antibodies tend to react under the breakdown of immune tolerance. METHODS: We immunized mice with full-length mouse BP180 (mBP180) to produce anti-mBP180 antibodies. Using the immunized mice, hybridoma cells were established to produce anti-mBP180 antibodies. We analyzed the characteristics of the anti-mBP180 antibodies that were produced in terms of epitopes, immunoglobulin subclasses, and somatic hypermutations. RESULTS: Hybridoma cells derived from immunized mice with full-length mBP180 produced antibodies targeting the intracellular domain (IC) and the shed ectodomain (EC) of mBP180. Using the domain-deleted mBP180 recombinant protein, we revealed that monoclonal anti-mBP180 EC antibodies react to neoepitopes on the 13th collagenous region of cleaved mBP180, which corresponds to the epitopes of linear IgA bullous dermatosis antibodies in human BP180. Furthermore, the subclasses of these antibodies could be distinguished by epitope: The subclass of the anti-mBP180 IC monoclonal antibodies was IgG, whereas that of the anti-mBP180 EC antibodies was IgM. Of note, a clone of these IgM mBP180 EC antibodies was a germline antibody without somatic hypermutation, which is also known as a natural antibody. CONCLUSION: These data suggest that mice potentially have natural antibodies targeting the neoepitopes of cleaved mBP180 EC.

Protein kinase Cß is involved in cigarette smoke gas phase-induced ferroptosis in J774 macrophages.

Cigarette smoking is a risk factor for respiratory infection caused by immune cell dysfunction. Cigarette smoke is divided into tar and gas phases. Although the gas phase induces cell death in various cell types, the mechanism for gas phase-induced cell death remains to be clarified. In this study, we have examined the effects of cigarette smoke gas phase on J774 macrophages. Cigarette smoke gas phase and cytotoxic factors in the gas phase induced protein kinase C (PKC)-dependent ferroptosis. Pharmacological studies using isoform-specific PKC inhibitors have revealed that PKCß is involved in cigarette smoke gas phase-induced ferroptosis in J774 macrophages.

Native Autoantigen Complex Detects Pemphigoid Autoantibodies.

Pemphigoid diseases are a group of autoimmune disorders characterized by subepidermal blistering in the skin and mucosa. Among them, mucous membrane pemphigoid (MMP) autoantibodies are characterized by targeting multiple molecules in the hemidesmosomes, including collagen XVII, laminin-332, and integrin a6/ß4. Traditionally, recombinant proteins of the autoantigens have been employed to identify circulating autoantibodies by immune assays. However, developing an efficient detection system for MMP autoantibodies has been challenging because the autoantibodies have heterogeneous profiles and the antibody titers are typically low. In this study, we introduce an ELISA that takes advantage of a native autoantigen complex rather than simple recombinant proteins. We generated HaCaT keratinocytes with a DDDDK-tag knocked in at the COL17A1 locus by CRISPR/Cas9-mediated gene editing. Immunoprecipitation using the DDDDK-tag isolated a native complex that contained full-length and processed collagen XVII and integrin α6/ß4. Then, we used the complex proteins to prepare an ELISA system and enrolled 55 MMP cases to validate its diagnostic performance. The sensitivity and specificity of the ELISA for detecting MMP autoantibodies were 70.9% and 86.7%, respectively, far superior to those of conventional assays. In autoimmune diseases such as MMP, in which autoantibodies target various molecules, isolating the antigen-protein complexes can help establish a diagnostic system.

Case report: Difference in outcomes between two cases of Hailey-Hailey disease treated with apremilast.

Hailey-Hailey disease (HHD) is a rare autosomal dominant acantholytic dermatosis clinically characterized by recurrent erythematous plaques and erosions mainly on the intertriginous regions. Although HHD seriously affects quality of life, conventional treatments often fail to provide long-term relief for most patients. The effectiveness of apremilast, a phosphodiesterase-4 inhibitor, against severe HHD was first reported in 2018, and after further testing, this agent is currently expected to be established as an efficacious and safe therapeutic option. Here we report two cases of HHD treated with apremilast which showed opposite outcomes. Although the case with extremely severe symptoms showed remarkable and long-lasting improvement with apremilast used after acute treatment with oral corticosteroid, the other case, with milder symptoms treated only with apremilast, showed no improvement. Our transcriptome analysis using skin samples collected prior to apremilast administration revealed the involvement of the NF-κB signaling pathway, which is related to the responses to bacteria and other organisms. However, this pathway was more strongly activated in case 2 than in case 1, suggesting that the steroid treatment preceding apremilast may have been effective and supportive in the apremilast-responding case. One of the two cases highlights the potential of apremilast as a treatment option for HHD, but the other underlines the difficulties in managing HHD and the complexity of the disease background. The accumulation of cases and larger clinical studies are expected to precisely evaluate the safety and efficacy of apremilast, and the potential for therapies in combination with conventional treatments.

Hemidesmosomal Reactivity and Treatment Recommendations in Immune Checkpoint Inhibitor-Induced Bullous Pemphigoid-A Retrospective, Monocentric Study.

Immune checkpoint inhibitors (ICI) induce T-cell-mediated antitumour responses. While ICI were initially successfully applied in metastasized melanoma, they are now approved for several tumour entities. Numerous autoimmune disorders have been reported to occur as adverse events of the treatment, among them bullous pemphigoid (BP), with less than 1% of the patients experiencing ICI-induced BP. This number is higher than the estimated prevalence of autoimmune bullous diseases in the general population of Germany, which lies around 0.05%. We here describe our cohort of eight patients, who developed a bullous pemphigoid under or shortly after ICI treatment. Half of them had a severe subtype (as shown by BPDAI >57) and showed a median onset of ICI-BP after 10 months of ICI initiation. Six patients had a palmar and/or plantar involvement, while oral involvement occurred in one case. All patients had linear epidermal IgG depositions in split skin in the indirect immunofluorescence. In four out of five biopsies available for direct immunofluorescence, linear IgG and C3 depositions were detected at the basement membrane, while one patient showed linear IgM staining. Moderate to high levels of FLBP180 autoantibodies were found in seven of eight cases. The disease can still be active after ICI discontinuation, while rituximab might be required for remission. Finally, four tumour samples were stained histochemically for collagen XVII (BP180), but no enhanced expression was found.

The significance of preclinical anti-BP180 autoantibodies.

Bullous pemphigoid (BP) is the most common autoimmune subepidermal blistering disease. Although the pathomechanism of BP onset has yet to be elucidated in detail, BP autoantibodies targeting two hemidesmosomal components, BP180 and BP230, are known to play a pivotal role in BP pathogenesis. Thus, the detection and measurement of BP autoantibodies are necessary for diagnosing BP and monitoring the disease activity. Immune assays such as immunofluorescence microscopy, immunoblotting, and ELISAs using BP180 and BP230 detect BP autoantibodies in most BP cases with high specificity; however, BP autoantibodies are sometimes detected in BP patients before the onset of this disease. BP autoantibodies that are detected in patients without typical tense blisters are defined as "preclinical BP autoantibodies". These preclinical BP autoantibodies are detected even in a low percentage of normal healthy individuals. Although the importance of preclinical BP autoantibodies remains elusive, these autoantibodies might be a potential risk factor for subsequent BP development. Therefore, previous comparative epidemiological studies have focused on the prevalence of preclinical BP autoantibodies in populations susceptible to BP (e.g., the elderly) or in diseases with a higher risk of comorbid BP. This mini-review summarizes the literature on the prevalence of preclinical BP autoantibodies in patients with various conditions and diseases, and we discuss the significance of preclinical BP autoantibody detection.

Detection of revertant mosaicism in epidermolysis bullosa through Cas9-targeted long-read sequencing.

Revertant mosaicism (RM) is a phenomenon in which inherited mutations are spontaneously corrected in somatic cells. RM occurs in some congenital skin diseases, but genetic validation of RM in clinically revertant skin has been challenging, especially when homologous recombination (HR) is responsible for RM. Here, we introduce nanopore Cas9-targeted sequencing (nCATS) for identifying HR in clinically revertant skin. We took advantage of compound heterozygous COL7A1 mutations in a patient with recessive dystrophic epidermolysis bullosa who showed revertant skin spots. Cas9-mediated enrichment of genomic DNA (gDNA) covering the two mutation sites (>8 kb) in COL7A1 and subsequent MinION sequencing successfully detected intragenic crossover in the epidermis of the clinically revertant skin. This method enables the discernment of haplotypes of up to a few tens of kilobases of gDNA. Moreover, it is devoid of polymerase chain reaction amplification, which can technically induce recombination. We, therefore, propose that nCATS is a powerful tool for understanding complicated gene modifications, including RM.

Super-resolution imaging detects BP180 autoantigen in immunoglobulin M pemphigoid.

Bullous pemphigoid is generally caused by immunoglobulin (Ig)G autoantibodies against hemidesmosomal BP180 and/or BP230. Recently, the concept of IgM pemphigoid has been proposed. A 23-year-old Japanese woman presented with a 4-month history of severely itchy papules showing subepidermal separations with mild neutrophil infiltration. Direct immunofluorescence (DIF) revealed IgM deposits at the dermoepidermal junction, but neither IgG nor IgA deposits. Indirect immunofluorescence on 1 M NaCl-split skin demonstrated deposits on the epidermal side. The optical density (OD) value of a modified IgM enzyme-linked immunosorbent assay for full-length BP180, but not for BP180-NC16A, was increased. The patient was diagnosed with IgM pemphigoid and was treated with diphenyl sulfone at 50 mg/day without recurrence. To confirm the precise autoantigen, we tried to obtain super-resolution imaging. The deposition pattern of IgM autoantibodies seemed to be oriented parallel to that of BP180. The detailed images detect DIF deposits apart from BP180-NC16A staining, but are close to type VII collagen-NC1 staining. This result suggests that the IgM autoantibodies in the patient might target the C-terminus of BP180. IgM pemphigoid is still not a widely accepted concept, and the clinical course remains unknown. We will carefully follow-up the patient. Super-resolution images may help to detect precise autoantigens in autoimmune blistering diseases.

Hair follicle stem cell progeny heal blisters while pausing skin development.

Injury in adult tissue generally reactivates developmental programs to foster regeneration, but it is not known whether this paradigm applies to growing tissue. Here, by employing blisters, we show that epidermal wounds heal at the expense of skin development. The regenerated epidermis suppresses the expression of tissue morphogenesis genes accompanied by delayed hair follicle (HF) growth. Lineage tracing experiments, cell proliferation dynamics, and mathematical modeling reveal that the progeny of HF junctional zone stem cells, which undergo a morphological transformation, repair the blisters while not promoting HF development. In contrast, the contribution of interfollicular stem cell progeny to blister healing is small. These findings demonstrate that HF development can be sacrificed for the sake of epidermal wound regeneration. Our study elucidates the key cellular mechanism of wound healing in skin blistering diseases.

Preferential Reactivity of Dipeptidyl Peptidase-IV Inhibitor-Associated Bullous Pemphigoid Autoantibodies to the Processed Extracellular Domains of BP180.

Bullous pemphigoid (BP) is a common autoimmune blistering disease in which autoantibodies target the hemidesmosomal components BP180 and/or BP230 in basal keratinocytes. In BP, 80 to 90% of autoantibodies target the juxtamembranous extracellular non-collagenous 16th A (NC16A) domain of BP180. Recently, the administration of dipeptidyl peptidase-IV inhibitors (DPP4i), which are widely used as antihyperglycemic drugs, has been recognized to be a causative factor for BP. DPP4i-associated BP (DPP4i-BP) autoantibodies tend to target epitopes on non-NC16A regions of BP180, and the pathomechanism for the development of the unique autoantibodies remains unknown. To address the characteristics of DPP4i-BP autoantibodies in detail, we performed epitope analysis of 18 DPP4i-BP autoantibodies targeting the non-NC16A domains of BP180 using various domain-specific as well as plasmin-digested polypeptides derived from recombinant BP180. Firstly, Western blotting showed that only one DPP4i-BP serum reacted with the epitopes on the intracellular domain of BP180, and no sera reacted with the C-terminal domain of the molecule. In addition, only 2 DPP4i-BP sera reacted with BP230 as determined by enzyme-linked immunosorbent assay. Thus, DPP4i-BP autoantibodies were found to mainly target the non-NC16A mid-portion of the extracellular domain of BP. Interestingly, Western blotting using plasmin-digested BP180 as a substrate revealed that all of the DPP4i-BP sera reacted more intensively with the 97-kDa processed extracellular domain of BP180, which is known as the LABD97 autoantigen, than full-length BP180 did. All of the DPP4i-BP autoantibodies targeting the LABD97 autoantigen were IgG1, and IgG4 was observed to react with the molecule in only 7 cases (38.9%). In summary, the present study suggests that IgG1-class autoantibodies targeting epitopes on the processed extracellular domain of BP180, i.e., LABD97, are the major autoantibodies in DPP4i-BP.