Anti-BP230 IgE autoantibodies in bullous pemphigoid intraindividually correlate with disease activity.

BACKGROUND: Bullous pemphigoid (BP), the most common subepidermal autoimmune blistering disease, is classically defined by the presence of IgG autoantibodies directed against the hemidesmosomal proteins BP180 (type XVII collagen) and BP230 and the predominance of skin lesions. Several studies have addressed the role of anti-BP180 IgE in patients and experimental models, while data on anti-BP230 IgE are scarce. OBJECTIVE: To assess anti-BP230 IgE level by ELISA in BP sera and to correlate it with disease severity and clinical characteristics. METHODS: BP sera underwent anti-BP230 IgE ELISA and Western blotting against human BP230 fragments. RESULTS: We demonstrate that 36/154 (23%) of BP sera were positive for anti-BP230 IgE. Anti-BP230 IgE levels had no correlation with clinical phenotype or disease activity per se. Interestingly, anti-BP230 IgE was significantly associated with disease activity within individuals during the course of the disease. Additionally, anti-BP230 IgE and total IgE levels showed a significant correlation. Notably, anti-BP230 IgG correlated interindividually with disease activity. By Western blotting, the C-terminal domain of BP230 fragments (C2; amino acids 2024-2349 and C3; amino acids 2326-2649), provided the best serological assay for anti-BP230 IgE detection. CONCLUSION: As a complementary tool, IgE immunoblotting is recommended to obtain an optimal serological diagnosis, particularly in patients with severe disease without IgG reactivity by BP180- or BP230-specific ELISA. Although the detection of serum anti-BP230 IgE is not of major diagnostic significance, it may be relevant for therapeutic decisions, e.g., for anti-IgE-directed treatment, which has been successfully used in case series of BP.

Autoreactive Peripheral Blood T Helper Cell Responses in Bullous Pemphigoid and Elderly Patients With Pruritic Disorders.

Bullous pemphigoid (BP) is a prototypic autoimmune disorder of the elderly, characterized by serum IgG autoantibodies, namely anti-BP180 and anti-BP230, directed against components of the basal membrane zone that lead to sub-epidermal loss of adhesion. Pruritus may be indicative of a pre-clinical stage of BP, since a subset of these patients shows serum IgG autoantibodies against BP230 and/or BP180 while chronic pruritus is increasingly common in the elderly population and is associated with a variety of dermatoses. Clinical and experimental evidence further suggests that pruritus of the elderly may be linked to autoimmunity with loss of self-tolerance against cutaneous autoantigens. Thus, the objective of this study was to determine autoreactive T cell responses against BP180 in elderly patients in comparison to patients with BP. A total of 22 elderly patients with pruritic disorders, 34 patients with bullous or non-bullous BP and 34 age-matched healthy controls were included in this study. The level of anti-BP180 and anti-BP230 IgG serum autoantibodies, Bullous Pemphigoid Disease Area Index (BPDAI), and pruritus severity were assessed for all patients and controls. For characterization of the autoreactive T cell response, peripheral blood mononuclear cells were stimulated ex vivo with recombinant BP180 proteins (NH2- and COOH-terminal domains) and the frequencies of BP180-specific T cells producing interferon-γ, interleukin (IL)-5 or IL-17 were subsequently determined by ELISpot assay. Patients with BP showed a mixed Th1/Th2 response against BP180 while autoreactive Th1 cells were identified in a minor subset of elderly patients with pruritic disorders. Furthermore, our T cell characterization revealed that therapeutic application of topical clobetasol propionate ointment in BP patients significantly reduced peripheral blood BP180-specific T cells, along with clinically improved symptoms, strongly suggesting a systemic immunosuppressive effect of this treatment.

Autoantibodies to BPAG1e Trigger Experimental Bullous Pemphigoid in Mice.

Bullous pemphigoid (BP) is an autoimmune blistering disease that targets the hemidesmosomal proteins BP180 and BP230/BPAG1e. Whereas the role of anti-BP180 antibodies has been extensively characterized, the pathogenicity of anti-BPAG1e antibodies remains unclear. The purpose of this study is to elucidate the role of antibodies to BPAG1e in the experimental bullous pemphigoid models. We generated Bpag1 conditional knockout mice, where the knockout of Bpag1 is restricted to keratin 5-expressing epithelial cells. Bpag1 conditional knockout mice were immunized with the C-terminal portion of BPAG1e, and the splenocytes were injected into Rag2-/- mice intravenously. The recipient mice presented with erosion on the feet and tails. Microscopic examination showed subepidermal blisters and a linear deposition of IgG at the dermal-epidermal junction. To assess the potential role of trauma on BP development, we inflicted surface wounds on the dorsum of the Rag2-/- recipient mice after adoptive transfer. The wounded Rag2-/- mice had increased morbidity and severity of BP-like symptoms. Moreover, the depletion of B cells from splenocytes abolished a subepidermal blistering phenotype in vivo. These findings demonstrate that antibodies to BPAG1e might play a pathogenic role in causing subepidermal blistering, and external factors, including trauma, might be a trigger for BP development.

More Severe Erosive Phenotype Despite Lower Circulating Autoantibody Levels in Dipeptidyl Peptidase-4 Inhibitor (DPP4i)-Associated Bullous Pemphigoid: A Retrospective Cohort Study.

BACKGROUND: The clinical and immunological profile of patients with dipeptidyl peptidase-4 inhibitor (DPP4i)-associated bullous pemphigoid (BP) is inconsistent in the current literature. OBJECTIVES: The aims were to investigate the clinical and immunological features of patients with DPP4i-associated BP and to examine whether there are intraclass differences between different DPP4i agents. METHODS: A retrospective cohort study was conducted, including all consecutive patients diagnosed with BP throughout the years 2009-2019 in a tertiary referral center. RESULTS: The study encompassed 273 patients with BP (mean age at diagnosis 79.1 ± 9.9 years), of whom 24 (8.8%) were associated with DPP4i. Sitagliptin was the prescribed agent for 17 patients (70.8%), and vildagliptin was prescribed in seven patients (29.2%). Relative to other patients with BP, patients with DPP4i-associated BP had more prominent truncal involvement (95.8% vs. 73.9%; P = 0.017), greater erosion/blister Bullous Pemphigoid Disease Area Index (BPDAI) subscore (29.8 ± 17.4 vs. 20.6 ± 14.4; P = 0.018), and lower levels of anti-BP180 NC16A (279.2 ± 346.1 vs. 572.2 ± 1352.0 U/ml; P = 0.009) and anti-BP230 (25.5 ± 47.8 vs. 128.6 ± 302.9 U/ml; P = 0.009) antibodies. Relative to patients with sitagliptin-associated BP, those with vildagliptin-associated BP had a lower seropositivity rate (57.1% vs. 94.1%, P = 0.031) and lower levels (96.7 ± 139.0 vs. 354.5 ± 376.5; P = 0.023) of anti-BP180 NC16A antibodies, and tended to present with higher erosion/blister BPDAI subscore (36.3 ± 9.6 vs. 25.8 ± 19.7; P = 0.095). CONCLUSIONS: DPP4i-associated BP is characterized by a more severe blistering and erosive presentation despite lower levels of typically pathogenic antibodies.

A case of anti-BP230 antibody-positive bullous pemphigoid receiving DPP-4 inhibitor.

Bullous pemphigoid (BP) is a cutaneous autoimmune blistering disorder. Recently, it has been reported that dipeptidyl peptidase-4 inhibitors (DPP4i) is associated with the development of BP (DPP4i-BP). Patients with DPP4i-BP have autoantibodies (autoAbs) preferentially targeting full-length BP180, but not the BP180NC16a domain. In this report, we described a case of anti-BP230 antibody (Ab)-positive BP receiving DPP4i. A 78-year-old male with a medical history of type 2 diabetes receiving vildagliptin at 100 mg daily for 38 months was referred to our hospital with itching blisters on his body and extremities. Skin biopsy showed subepidermal bulla with eosinophil infiltration. Direct immunofluorescence staining revealed a linear staining pattern with complement C3 and IgG at the subepidermal basement membrane zone. By laboratory testing, autoAbs against BP180NC16a and full-length BP180 were negative by enzyme-linked immunosorbent assay (ELISA); however, anti-BP230 Abs were positive by ELISA (index: 123.91). His HLA genotype was DQB1*04:01 and 05:01. Based on these results, we diagnosed the patient with anti-BP230 Abs-positive BP associated with DPP4i. To the best of our knowledge, this is the first case of DPP4i-BP with only anti-BP230 Abs. Further accumulation of DPP4i-BP cases is needed to clarify the relationship between overall features of DPP4i-BP and anti-BP230 Abs.

BIOCHIP mosaic for the diagnosis of autoimmune bullous diseases in Chinese patients.

BACKGROUND: Autoimmune blistering diseases (AIBDs) are a group of fatal diseases with specific autoantibodies. BIOCHIP mosaic is a novel and all-in-one measure used for the rapid diagnosis of AIBDs. OBJECTIVES: To evaluate the diagnostic accuracy based on BIOCHIP mosaic (FA1501-1005-60) in Chinese patients with AIBDs. MATERIALS AND METHODS: Seventy-seven patients with AIBDs and 20 controls were enrolled. The BIOCHIP mosaic was performed using both serum and plasma samples. RESULTS: Based on BIOCHIP mosaic, the data from paired plasma and serum samples demonstrated a high degree of concordance (Cohen's kappa = 0.896-1.000) for autoantibodies against Dsg1, Dsg3, BP180-NC16A-4X, BP230gC, prickle-cell desmosomes, and pemphigoid antigens. Moreover, BIOCHIP mosaic also demonstrated a high degree of consistency for the detection rate of anti-Dsg1, Dsg3, plakins, BP180-NC16A-4X and non-collagenous domain of type VII collagen autoantibodies for the diagnosis of pemphigus foliaceus (77.3%), pemphigus vulgaris (88.6%), paraneoplastic pemphigus (100.0%), bullous pemphigoid (92.8%) and epidermolysis bullosa acquisita (99.0%), respectively. CONCLUSION: Using BIOCHIP mosaic, serum and plasma samples may be used interchangeably at 1/10 dilution. Overall, the BIOCHIP mosaic was shown to be a useful and accurate tool for the diagnosis of AIBDs.

Regulatory T cell subsets in bullous pemphigoid and dipeptidyl peptidase-4 inhibitor-associated bullous pemphigoid.

BACKGROUND: Regulatory T (Treg) cells play an essential role in peripheral immune tolerance. Bullous pemphigoid (BP) is the most common blistering disease and is caused by autoantibodies to two BP antigens: type XVII collagen and BP230. Recently, we reported that Treg cell dysfunction may cause the production of autoantibodies to BP antigens. Several studies have suggested an association between Treg cells and BP pathogenesis. However, Treg cells are heterogeneous in humans, leading to inconsistent results in previous studies. OBJECTIVE: To assess functional Treg subsets in BP. METHODS: We examined three distinct Treg subsets in conventional BP (cBP) patients before versus after systemic corticosteroid treatment, dipeptidyl peptidase-4 inhibitor-associated BP (DPP-4i-BP) patients, younger controls and older controls. RESULTS: We found that total Treg cells and all Treg cell subsets were increased in cBP patients before treatment and decreased by systemic corticosteroid treatment. In contrast, neither total Treg cells nor all Treg subsets were increased in DPP-4i-BP. Notably, CD45RA- Foxp3hi effector Treg cells positively correlated with disease severity in cBP, whereas CD45RA+Foxp3lo naïve Treg cells positively correlated with the disease severity in DPP-4i-BP. CONCLUSION: These findings suggest that Treg cells are differently involved in the pathogeneses of cBP and DPP-4i-BP.

miR-1291 Functions as a Potential Serum Biomarker for Bullous Pemphigoid.

BACKGROUND: Bullous pemphigoid (BP) is a common T helper 2- (Th2-) dominated autoimmune blistering skin disease with significant mortality. MicroRNAs (miRNAs), which are endogenous noncoding RNA molecules, have been reported to be potential biomarkers for some autoimmune diseases; however, to date, there exist no reports on serum expression profiles of miRNAs in BP patients. METHODS: A RNA quantitative PCR- (qPCR-) based array was conducted on sera from 20 active BP patients and 20 healthy controls for screening of miRNAs. Significantly dysregulated miRNAs were validated with use of qPCR as performed on sera samples of 45 active BP patients and 60 healthy controls. Serum CCL17, anti-BP180, and anti-BP230 levels were measured with use of ELISA. RESULTS: Relative baseline expression levels of serum miR-1291 were significantly upregulated in the 45 BP patients as compared with the 60 healthy controls (P < 0.001) and significantly decreased in the disease control stage (n = 13, P = 0.006). In addition, these baseline miR-1291 levels showed a significant positive correlation with the baseline levels of serum CCL17 (P < 0.001) and anti-BP180 (n = 38, P = 0.024). Like that observed for miR-1291, baseline levels of serum CCL17 were also significantly elevated in the 45 BP patients compared with the 60 healthy controls (P < 0.001) and significantly decreased in the disease control stage (n = 13, P = 0.002). However, for anti-BP180, baseline serum levels were significantly elevated in only 38 of the 45 BP patients and significantly decreased in the disease control stage (n = 10, P = 0.004). CONCLUSIONS: Relative expression levels of serum miR-1291 can reflect disease activity of BP. miR-1291 may function as an important new serum biomarker for BP.

Role of BP230 autoantibodies in bullous pemphigoid.

Bullous pemphigoid (BP) is an autoimmune disease associated with subepidermal blistering due to autoantibodies directed against BP180 and BP230. BP180 is currently considered as the major pathogenic autoantigen. However, previous clinical findings suggested that anti-BP230 autoantibodies alone can cause skin lesions in animal models and many BP patients. The characteristics of BP230 and the pathogenic roles of anti-BP230 antibodies have been proposed. First, at the molecular level, BP230 mediates the attachment of keratin intermediate filaments to the hemidesmosomal plaque and interacts with other constituents of hemidesmosomes. Second, the presence of BP230 autoantibodies may correlate with specific clinical features of BP. The immunoglobulin (Ig)G autoantibodies from BP patients react mainly against the C-terminus of BP230, while the IgE autoantibodies are still inconclusive. Third, in vivo, autoantibodies against BP230 involved in the disease may not only induce the inflammatory response but also impair the structural stability of hemidesmosomes. This article reviews recently published work about the role of BP230 and its antibodies, including IgG and IgE, aiming to find clues of its clinical association and lay the foundation for the research on the pathogenicity of antibodies against BP230.

Validation of the BIOCHIP test for the diagnosis of bullous pemphigoid, pemphigus vulgaris and pemphigus foliaceus.

BACKGROUND: The BIOCHIP is a novel multiplex indirect immunofluorescence technique used in the serological diagnosis of bullous pemphigoid and pemphigus. The BIOCHIP method combines the screening of autoantibodies and target antigen-specific substrates in a single miniature incubation field. OBJECTIVE: To evaluate the diagnostic accuracy of the new immunofluorescence BIOCHIP multiplex tool in pemphigus and bullous pemphigoid. METHODS: For the validation of the BIOCHIP, sera from patients with BP (n = 38), PF (n = 8) and pemphigus vulgaris (PV) (n = 23) were used. In addition, sera from disease control patients (n = 63) and healthy volunteers (n = 39) were used. The multiplex BIOCHIP and direct immunofluorescence (DIF) were performed for all BP, PF and PV patients. Additional indirect immunofluorescence (IIF) was performed on patients with BP, and ELISA was performed on patients with pemphigus. RESULTS: The BIOCHIP mosaic showed a sensitivity of 86.8% and specificity of 85% for BP180 or BP230 being positive in BP. It demonstrated a sensitivity of 75% and specificity of 97.7% for Dsg1 in PF. The BIOCHIP was found to have a sensitivity of 60.9% and specificity of 73.6% for Dsg3 in PV. CONCLUSION: The BIOCHIP mosaic-based immunofluorescence test is potentially a simple, time and effort saving test that can aid in the diagnosis and screening of BP, PV and PF. However, there is potential for interpretation bias and a learning curve that needs to be taken into consideration.

Polymorphisms in the Mitochondrial Genome Are Associated With Bullous Pemphigoid in Germans.

Bullous pemphigoid (BP) is the most prevalent autoimmune skin blistering disease and is characterized by the generation of autoantibodies against the hemidesmosomal proteins BP180 (type XVII collagen) and BP230. Most intriguingly, BP is distinct from other autoimmune diseases because it predominantly affects elderly individuals above the age of 75 years, raising the question why autoantibodies and the clinical lesions of BP emerges mostly in this later stage of life, even in individuals harboring known putative BP-associated germline gene variants. The mitochondrial genome (mtDNA) is a potential candidate to provide additional insights into the BP etiology; however, the mtDNA has not been extensively explored to date. Therefore, we sequenced the whole mtDNA of German BP patients (n = 180) and age- and sex-matched healthy controls (n = 188) using next generation sequencing (NGS) technology, followed by the replication study using Sanger sequencing of an additional independent BP (n = 89) and control cohort (n = 104). While the BP and control groups showed comparable mitochondrial haplogroup distributions, the haplogroup T exhibited a tendency of higher frequency in BP patients suffering from neurodegenerative diseases (ND) compared to BP patients without ND (50%; 3 in 6 BP with haplogroup T). A total of four single nucleotide polymorphisms (SNPs) in the mtDNA, namely, m.16263T>C, m.16051A>G, and m.16162A>G in the D-loop region of the mtDNA, and m.11914G>A in the mitochondrially encoded NADH:ubiquinone oxidoreductase core subunit 4 gene (MT-ND4), were found to be significantly associated with BP based on the meta-analysis of our NGS data and the Sanger sequencing data (p = 0.0017, p = 0.0129, p = 0.0076, and p = 0.0132, respectively, Peto's test). More specifically, the three SNPs in the D-loop region were negatively, and the SNP in the MT-ND4 gene was positively associated with BP. Our study is the first to interrogate the whole mtDNA in BP patients and controls and to implicate multiple novel mtDNA variants in disease susceptibility. Studies using larger cohorts and more diverse populations are warranted to explore the functional consequences of the mtDNA variants identified in this study on immune and skin cells to understand their contributions to BP pathology.

The Intersection of IgE Autoantibodies and Eosinophilia in the Pathogenesis of Bullous Pemphigoid.

Bullous pemphigoid (BP) is an autoimmune blistering disease characterized by autoantibodies targeting cellular adhesion molecules. While IgE autoantibodies are occasionally reported in other autoimmune blistering diseases, BP is unique in that most BP patients develop an IgE autoantibody response. It is not known why BP patients develop self-reactive IgE and the precise role of IgE in BP pathogenesis is not fully understood. However, clinical evidence suggests an association between elevated IgE antibodies and eosinophilia in BP patients. Since eosinophils are multipotent effector cells, capable cytotoxicity and immune modulation, the putative interaction between IgE and eosinophils is a primary focus in current studies aimed at understanding the key components of disease pathogenesis. In this review, we provide an overview of BP pathogenesis, highlighting clinical and experimental evidence supporting central roles for IgE and eosinophils as independent mediators of disease and via their interaction. Additionally, therapeutics targeting IgE, the Th2 axis, or eosinophils are also discussed.

Effects of Omalizumab on FcεRI and IgE Expression in Lesional Skin of Bullous Pemphigoid.

Recent studies suggest an important role of immunoglobulin E (IgE) as an alternative pathogenic pathway in the development of bullous pemphigoid (BP), as the most frequent subepidermal blistering disease of the skin Use of IgE targeted therapies, such as omalizumab, has been shown promising in recent studies. The aim of this study was to assess the effect of omalizumab on FcεRI and IgE expression on circulating basophils and on lesional intradermal cells in BP to generate insight into the immunological effects of omalizumab in BP. We report two cases of BP patients treated with omalizumab. Efficacy of treatment was assessed clinically 4 months after initiation of the therapy. Lesional and non-lesional skin biopsies where taken before and 4 weeks after initiation of omalizumab therapy. In addition, FcεRI expression on circulating cells and IgE levels in serum and in the skin samples, as well as anti-BP180 and anti-BP230 in serum and eosinophils and basophils counts in blood were assessed before and during treatment. Both patients showed a marked improvement after 4 months, with no adverse effects. Down-regulation of FcεRI, IgE in lesional skin and on circulating basophils were observed in parallel with clinical improvement. The current case study supports the role of omalizumab in the treatment of a subset of BP patients. Our observations suggest that omalizumab represents a valuable therapeutic option in the management of BP patients. Its efficacy might be related to reduction in FcεRI+ and IgE+ basophils and intradermal cells.

New Insights Into the Pathogenesis of Bullous Pemphigoid: 2019 Update.

There are several lines of evidence indicating that the physiopathological bases of bullous pemphigoid (BP), the most common subepidermal autoimmune bullous disease, are hallmarked by the production of autoantibodies directed against the hemidesmosomal anchoring proteins BP180 and BP230. In contrast to the robustness of the latter assumption, the multifaceted complexity of upstream and downstream mechanisms implied in the pathogenesis of BP remains an area of intense speculation. So far, an imbalance between T regulatory cells and autoreactive T helper (Th) cells has been regarded as the main pathogenic factor triggering the autoimmune response in BP patients. However, the contributory role of signaling pathways fostering the B cell stimulation, such as Toll-like receptor activation, as well as that of ancillary inflammatory mechanisms responsible for blister formation, such as Th17 axis stimulation and the activation of the coagulation cascade, are still a matter of debate. In the same way, the pathomechanisms implied in the loss of dermal-epidermal adhesion secondary to autoantibodies binding are not fully understood. Herein, we review in detail the current concepts and controversies on the complex pathogenesis of BP, shedding light on the most recent theories emerging from the literature.

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.