Single-cell sequencing reveals distinct immune cell features in cutaneous lesions of pemphigus vulgaris and bullous pemphigoid.

Bullous pemphigoid (BP) and pemphigus vulgaris (PV) are two common subtypes of autoimmune bullous disease (AIBD). The key role of circulating autoreactive immune cells contributing to skin damage of AIBD has been widely recognized. Nevertheless, the immune characteristics in cutaneous lesions remain unclear. Here, we performed single-cell RNA sequencing (scRNA-seq) and single-cell VDJ sequencing (scRNA-seq) to generate transcriptional profiles for cells and T/B cell clonetype in skin lesions of BP and PV. We found that the proportions of NK&T, macrophages/ dendritic cells, B cells, and mast cells increased in BP and PV lesions. Then, BP and PV cells constituted over 75% of all myeloid cell subtypes, CD4+ T cell subtypes and CD8+ T cell subtypes. Strikingly, CD8+ Trm was identified to be expanded in PV, and located in the intermediate state of the pseudotime trajectory from CD8+ Tm to CD8+ Tem. Interestingly, CD8+ Tem and CD4+ Treg highly expressed exhaustion-related genes, especially in BP lesions. Moreover, the enhanced cell communication between stromal cells and immune cells like B cells and macrophages/ dendritic cells was also identified in BP and PV lesions. Finally, clone expansion was observed in T cells of BP and PV compared with HC, while CD8+ Trm represented the highest ratio of hyperexpanded TCR clones among all T cell subtypes. Our study generally depicts a large and comprehensive single-cell landscape of cutaneous lesions and highlights immune cell features in BP and PV. This offers potential research targets for further investigation.

Upregulation of Anti-Desmocollin 3 Antibodies in Pemphigus Diseases: A Case-control Study.

BACKGROUND: Pemphigus diseases are a subgroup of autoimmune bullous diseases characterized by autoantibodies against desmogleins and occasionally desmocollins. Desmocollin 3 is the main desmocollin isoform that contributes to cell adhesion in the epidermis. OBJECTIVE: To evaluate the presence and level of anti-desmocollin 3 antibodies in pemphigus diseases, and to investigate whether their presence is associated with a specific type, presentation, or clinical pattern. METHODS: Forty patients with pemphigus diseases and forty healthy controls were enrolled. Medical history, clinical examination, and pemphigus disease area index (PDAI) scoring were recorded for all patients. Serum samples were collected from both groups for assessment of anti-desmocollin 3 antibody reactivity by ELISA. RESULTS: The presence of anti-desmocollin 3 antibodies was significant among patients with pemphigus compared with controls (P=0.003). The level of anti-desmocollin 3 antibodies was also significantly higher in patients with pemphigus compared with controls (P=0.01). There was no significant relationship between the presence of anti-desmocollin 3 antibodies and any of the clinical presentations of pemphigus (type, severity, duration, activity, presence of annular pattern, or site of affection - mucosal, cutaneous, on the scalp, palmoplantar, or flexural). CONCLUSION: Anti-desmocollin 3 antibodies are upregulated in pemphigus diseases and can contribute to the pathogenesis of pemphigus. No specific clinical type, presentation, or pattern was found to be associated with the presence of anti-desmocollin 3 antibodies.

Paraneoplastic Pemphigus Autoantibodies Against C-terminus of Desmoplakin Induced Acantholysis In Vitro and In Vivo.

Paraneoplastic pemphigus (PNP) is an autoimmune bullous disease associated with underlying neoplasms and characterized by antibodies against desmoglein 3 (Dsg 3) and plakins. Autoantibodies against desmoglein 3 in sera of patients with PNP have been proven to cause acantholysis in vivo in neonatal mice. As a member of the plakin family, autoantibodies against desmoplakin were detected frequently by immunoprecipitation in the sera of PNP. The recombinant C-terminus of desmoplakin was expressed and purified to adsorb the specific autoantibodies against the C-terminus of desmoplakin. In vitro dispase-dependent keratinocyte dissociation assay and in vivo IgG passive transfer into neonatal mice assay were performed, followed by the electronic microscopy examination and TUNEL assay. We found that anti-C terminus of desmoplakin autoantibodies caused blisters and acantholysis in mice skin at a dose-dependent manner. Moreover, dissociated fragments were observed after incubation with the purified IgG against desmoplakin, compared with normal human IgG (P-value =0.0207). The electronic microscopy examination showed the disconnection of keratin intermediate filaments from desmosomes. Lastly, apoptosis of keratinocytes in the TUNEL assay was all detected in the skins of neonatal mice after injection of the anti-C terminus of desmoplakin autoantibodies. Taken together, the study suggests that autoantibodies against the C-terminus of desmoplakin might be pathogenic in PNP.

Role of ADAM10 and ADAM17 in the Regulation of Keratinocyte Adhesion in Pemphigus Vulgaris.

The severe autoimmune blistering disease Pemphigus vulgaris (PV) is mainly caused by autoantibodies (IgG) against desmoglein (Dsg) 3 and Dsg1. The mechanisms leading to the development of blisters are not fully understood, but intracellular signaling seems to play an important role. Sheddases ADAM10 and ADAM17 are involved in the turnover of the desmosomal cadherin Dsg2 and ADAM10 has been shown to contribute to acantholysis in a murine pemphigus model. In the present study, we further examined the role of ADAM10 and ADAM17 both in keratinocyte adhesion and in the pathogenesis of PV. First, we found that inhibition of ADAM10 enhanced adhesion of primary human keratinocytes but not of immortalized keratinocytes. In dissociation assays, inhibition of ADAM10 shifted keratinocyte adhesion towards a hyperadhesive state. However, ADAM inhibition did neither modulate protein levels of Dsg1 and Dsg3 nor activation of EGFR at Y1068 and Y845. In primary human keratinocytes, inhibition of ADAM10, but not ADAM17, reduced loss of cell adhesion and fragmentation of Dsg1 and Dsg3 immunostaining in response to a PV1-IgG from a mucocutaneous PV patient. Similarly, inhibition of ADAM10 in dissociation assay decreased fragmentation of primary keratinocytes induced by a monoclonal antibody against Dsg3 and by PV-IgG from two other patients both suffering from mucosal PV. However, such protective effect was not observed in both cultured cells and ex vivo disease models, when another mucocutaneous PV4-IgG containing more Dsg1 autoantibodies was used. Taken together, ADAM10 modulates both hyperadhesion and PV-IgG-induced loss of cell adhesion dependent on the autoantibody profile.

Chronic exposure to the anti-M3 muscarinic acetylcholine receptor autoantibody in pemphigus vulgaris contributes to disease pathophysiology.

Pemphigus vulgaris (PV) is a potentially lethal autoimmune mucocutaneous blistering disease characterized by binding of IgG autoantibodies (AuAbs) to keratinocytes (KCs). In addition to AuAbs against adhesion molecules desmogleins 1 and 3, PV patients also produce an AuAb against the M3 muscarinic acetylcholine (ACh) receptor (M3AR) that plays an important role in regulation of vital functions of KCs upon binding endogenous ACh. This anti-M3AR AuAb is pathogenic because its adsorption eliminates the acantholytic activity of PV IgG; however, the molecular mechanism of its action is unclear. In the present study, we sought to elucidate the mode of immunopharmacologic action of the anti-M3AR AuAb in PV. Short-term exposures of cultured KCs to PV IgG or the muscarinic agonist muscarine both induced changes in the expression of keratins 5 and 10, consistent with the inhibition of proliferation and upregulated differentiation and in keeping with the biological function of M3AR. In contrast, long-term incubations induced a keratin expression pattern consistent with upregulated proliferation and decreased differentiation, in keeping with the hyperproliferative state of KCs in PV. This change could result from desensitization of the M3AR, representing the net antagonist-like effect of the AuAb. Therefore, chronic exposure of KCs to the anti-M3AR AuAb interrupts the physiological regulation of KCs by endogenous ACh, contributing to the onset of acantholysis. Since cholinergic agents have already demonstrated antiacantholytic activity in a mouse model of PV and in PV patients, our results have translational significance and can guide future development of therapies for PV patients employing cholinergic drugs.

IgM to IgG Class Switching Is a Necessary Step for Pemphigus Phenotype Induction in Desmoglein 3-Specific B Cell Receptor Knock-in Mouse.

Pemphigus vulgaris is an autoimmune blistering disease caused by IgG targeting desmoglein 3 (Dsg3), an adhesion molecule of keratinocytes. Anti-Dsg3 IgG production is prevented in healthy individuals, but it is unclear how Dsg3-specific B cells are regulated. To clarify the immunological condition regulating Dsg3-specific B cells, a pathogenic anti-Dsg3 Ig (AK23) knock-in mouse was generated. AK23 knock-in B cells developed normally without undergoing deletion or acquiring an anergic phenotype in vivo. The knock-in B cells showed Ca2+ influx upon IgM cross-linking and differentiated into AK23-IgG+ B cells after LPS and IL-4 stimulation in vitro that induced a pemphigus phenotype after adoptive transfer into Rag2 -/- mice. However, the knock-in mouse itself produced AK23-IgM but little IgG without blisters in vivo. Dsg3 immunization and skin inflammation caused AK23-IgG production and a pemphigus phenotype in vivo. Furthermore, Fcgr2b deficiency or haploinsufficiency spontaneously induced AK23-IgG production and a pemphigus phenotype with poor survival rates in AK23 knock-in mice. To assess Fcgr2b involvement in Ig class-switch efficiency, postswitch transcripts of B cells were quantified and significantly higher in Fcgr2b -/- and Fcgr2b +/- mice than wild-type mice in a gene dose-dependent manner. Finally, RNA sequencing revealed reduced expression of FCGR2B and FcγRIIB-related genes in patient B cells. These results indicated that Dsg3-specific B cells do not spontaneously perform pathogenic class switching in vivo, and pemphigus phenotype induction was prevented under normal conditions. Attenuated FcγRIIB signaling is also one of the drivers for pathogenic class switching and is consistent with immunological features identified from clinical samples. This study unveiled a characteristic immune state silencing autoreactive B cells in mice.

Effects of metformin on autoimmune immunoglobins and interferon-γ in patients with early diagnosed pemphigus vulgaris: a prospective clinical trial.

The management of pemphigus vulgaris (PV) is challenging. This study aimed to evaluate the immunomodulating effects of metformin on PV. The study was conducted in two phases: in the first phase, patients received routine first-line treatment (prednisolone plus azathioprine) for 2 months, then in the second phase, metformin was added to this regimen for another 2 months. After addition of metformin to the first-line medications, significant reductions were seen in serum IgG1 (reduced from 534.92 ± 134.83 mg/dL to 481.58 ± 130.46 mg/dL, P < 0.001), IgG4 (51.83 ± 27.26 mg/dL to 44.50 ± 26.05 mg/dL, P < 0.001) and interferon-γ (277.99 ± 108.71 pg/mL to 45.05 ± 17.080 pg/mL, P = 0.03) concentrations. The suppressant effect of metformin was greatest on IgG4 (coefficient of variation 1.28), the dominant subclass of IgG involved in PV. Metformin could have immunomodulating effects on PV with controlling effects on steroid complications.

Translational implications of Th17-skewed inflammation due to genetic deficiency of a cadherin stress sensor.

Desmoglein 1 (Dsg1) is a cadherin restricted to stratified tissues of terrestrial vertebrates, which serve as essential physical and immune barriers. Dsg1 loss-of-function mutations in humans result in skin lesions and multiple allergies, and isolated patient keratinocytes exhibit increased proallergic cytokine expression. However, the mechanism by which genetic deficiency of Dsg1 causes chronic inflammation is unknown. To determine the systemic response to Dsg1 loss, we deleted the 3 tandem Dsg1 genes in mice. Whole transcriptome analysis of embryonic Dsg1-/- skin showed a delay in expression of adhesion/differentiation/keratinization genes at E17.5, a subset of which recovered or increased by E18.5. Comparing epidermal transcriptomes from Dsg1-deficient mice and humans revealed a shared IL-17-skewed inflammatory signature. Although the impaired intercellular adhesion observed in Dsg1-/- mice resembles that resulting from anti-Dsg1 pemphigus foliaceus antibodies, pemphigus skin lesions exhibit a weaker IL-17 signature. Consistent with the clinical importance of these findings, treatment of 2 Dsg1-deficient patients with an IL-12/IL-23 antagonist originally developed for psoriasis resulted in improvement of skin lesions. Thus, beyond impairing the physical barrier, loss of Dsg1 function through gene mutation results in a psoriatic-like inflammatory signature before birth, and treatment with a targeted therapy significantly improved skin lesions in patients.

A gene-centric approach to biomarker discovery identifies transglutaminase 1 as an epidermal autoantigen.

Autoantigen discovery is a critical challenge for the understanding and diagnosis of autoimmune diseases. While autoantibody markers in current clinical use have been identified through studies focused on individual disorders, we postulated that a reverse approach starting with a putative autoantigen to explore multiple disorders might hold promise. We here targeted the epidermal protein transglutaminase 1 (TGM1) as a member of a protein family prone to autoimmune attack. By screening sera from patients with various acquired skin disorders, we identified seropositive subjects with the blistering mucocutaneous disease paraneoplastic pemphigus. Validation in further subjects confirmed TGM1 autoantibodies as a 55% sensitive and 100% specific marker for paraneoplastic pemphigus. This gene-centric approach leverages the wealth of data available for human genes and may prove generally applicable for biomarker discovery in autoimmune diseases.

Peripheral tolerance by Treg via constraining OX40 signal in autoreactive T cells against desmoglein 3, a target antigen in pemphigus.

Antigen-specific peripheral tolerance is crucial to prevent the development of organ-specific autoimmunity. However, its function decoupled from thymic tolerance remains unclear. We used desmoglein 3 (Dsg3), a pemphigus antigen expressed in keratinocytes, to analyze peripheral tolerance under physiological antigen-expression conditions. Dsg3-deficient thymi were transplanted into athymic mice to create a unique condition in which Dsg3 was expressed only in peripheral tissue but not in the thymus. When bone marrow transfer was conducted from high-avidity Dsg3-specific T cell receptor-transgenic mice to thymus-transplanted mice, Dsg3-specific CD4+ T cells developed in the transplanted thymus but subsequently disappeared in the periphery. Additionally, when Dsg3-specific T cells developed in Dsg3-/- mice were adoptively transferred into Dsg3-sufficient recipients, the T cells disappeared in an antigen-specific manner without inducing autoimmune dermatitis. However, Dsg3-specific T cells overcame this disappearance and thus induced autoimmune dermatitis in Treg-ablated recipients but not in Foxp3-mutant recipients with dysfunctional Tregs. The molecules involved in disappearance were sought by screening the transcriptomes of wild-type and Foxp3-mutant Tregs. OX40 of Tregs was suggested to be responsible. Consistently, when OX40 expression of Tregs was constrained, Dsg3-specific T cells did not disappear. Furthermore, Tregs obtained OX40L from dendritic cells in an OX40-dependent manner in vitro and then suppressed OX40L expression in dendritic cells and Birc5 expression in Dsg3-specific T cells in vivo. Lastly, CRISPR/Cas9-mediated knockout of OX40 signaling in Dsg3-specific T cells restored their disappearance in Treg-ablated recipients. Thus, Treg-mediated peripheral deletion of autoreactive T cells operates as an OX40-dependent regulatory mechanism to avoid undesired autoimmunity besides thymic tolerance.

Immunocytometric Analysis of Oral Pemphigus vulgaris Patients after Treatment with Rituximab as Adjuvant.

Background: B-cell depletion therapy was demonstrated to be a valid and safe alternative as an adjuvant in oral-pharyngeal pemphigus vulgaris (OPV) patients. We aimed to assess its effects on anti-desmoglein (Dsg) 1 and 3 and leukocytes subsets profile in these patients' population. Methods and Materials: We evaluated the immunologic profile of 10 OPV patients treated with RTX as adjuvant by using the ELISA testing for anti-Dsg-1 and -3 titers and the immunophenotyping for B and T-cell lymphocyte subpopulations and compared them with the PDAI score for clinical remission. Results: A significant difference in medians between baseline, end of RTX therapy, and 6 months after RTX therapy was observed in Dsg-3 titer (p < 0.001), in the CD8 (p = 0.009), and CD20 counts (p < 0.001). Multiple comparisons after Bonferroni adjustment confirmed such significant differences mainly between baseline and the end of RTX therapy and baseline and 6 months after RTX therapy. Only the anti-Dsg-3 titer at the end of RTX therapy demonstrated a slight positive correlation with the PDAI score at baseline (p = 0.046, r = 0.652). Conclusions: B-cell depletion adjuvant therapy in OPV patients demonstrated a significant impact on anti-Dsg-3 titer and B and T-cell lymphocyte subpopulations profile.

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.

A Scoping Review of the Role of Metalloproteinases in the Pathogenesis of Autoimmune Pemphigus and Pemphigoid.

Pemphigus and pemphigoid diseases are potentially life-threatening autoimmune blistering disorders that are characterized by intraepithelial and subepithelial blister formation, respectively. In both disease groups, skin and/or mucosal blistering develop as a result of a disruption of intercellular adhesion (pemphigus) and cell-extracellular matrix (ECM) adhesion (pemphigoid). Given that metalloproteinases can target cell adhesion molecules, the purpose of the present study was to investigate the role of these enzymes in the pathogenesis of these bullous dermatoses. Studies examining MMPs (matrix metalloproteinases) and the ADAM (a disintegrin and metalloproteinase) family of proteases in pemphigus and pemphigoid were selected from articles published in the repository of the National Library of Medicine (MEDLINE/PubMed) and bioRxiv. Multiple phases of screening were conducted, and relevant data were extracted and tabulated, with 29 articles included in the final qualitative analysis. The majority of the literature investigated the role of specific components of the MMP family primarily in bullous pemphigoid (BP) whereas studies that focused on pemphigus were rarer. The most commonly studied metalloproteinase was MMP-9 followed by MMP-2; other MMPs included MMP-1, MMP-3, MMP-8, MMP-12 and MMP-13. Molecules related to MMPs were also included, namely, ADAM5, 8, 10, 15, 17, together with TIMP-1 and TIMP-3. The results demonstrated that ADAM10 and MMP-9 activity is necessary for blister formation in experimental models of pemphigus vulgaris (PV) and BP, respectively. The data linking MMPs to the pathogenesis of experimental BP were relatively strong but the evidence for involvement of metalloproteinases in PV was more tentative. These molecules represent potential candidates for the development of mechanism-based treatments of these blistering diseases.

Anti-Desmocollin Autoantibodies in Autoimmune Blistering Diseases.

The presence of anti-desmocollin (Dsc) antibodies is rarely described in autoimmune blistering diseases patients. Moreover, several clinical phenotypes of pemphigus may be associated with these antibodies. In this review we analyze clinicopathological, immunologic and outcome features of anti-Dsc autoimmune blistering diseases patients, to improve their diagnosis and management. We conducted a systematic search of PubMed and Embase (1990-present) for studies reporting cases of autoimmune blistering diseases with anti-Dsc antibodies. We classified the selected patients as patients with exclusively anti-Dsc autoantibodies, and patients with anti-Dsc and other autoantibodies. Of 93 cases with anti-Dsc autoantibodies included, 38 (41%) had exclusively these antibodies. Only 18% of patients presented with the typical clinicopathological phenotype of pemphigus vulgaris or pemphigus foliaceous. Mucosal involvement was seen in approximately half of the patients. Up to 18% of cases were associated with neoplasms. Acantholysis was described in 54% of cases with histopathological information. Treatments and outcomes vary in the different clinical phenotypes. The presence of anti-Dsc antibodies must be suspected mainly in those patients with either atypical pemphigus, in special with clinical pustules, or in cases showing intraepithelial or dermal neutrophilic/eosinophilic infiltrate on histological examination and dual pattern by direct immunofluorescence examination.

Analysis of the Specificity of Auto-Reactive Antibodies to Individual Fragments of the Extracellular Domain of Desmoglein 3 in Patients with Pemphigus Vulgaris.

A method for the analysis of the epitope specificity of auto-reactive antibodies to desmoglein 3 (Dsg3) using competitive ELISA has been developed. It is based on a two-stage solid-phase ELISA with initial "depletion" of auto-reactive antibodies against the studied epitope and subsequent quantitative assessment of antibodies against full-length extracellular domain Dsg3. The proposed approach for assessing the specificity of the autoimmune response in patients with pemphigus vulgaris can provide in the future the possibility to personalize the therapy using plasmapheresis by preliminary selection of the antigenic composition of the extracorporeal immunosorbent.

Pathogenic Activation and Therapeutic Blockage of FcαR-Expressing Polymorphonuclear Leukocytes in IgA Pemphigus.

Pathomechanisms in IgA pemphigus are assumed to rely on Fc-dependent cellular activation by antigen-specific IgA autoantibodies; however, models for the disease and more detailed pathophysiologic data are lacking. In this study, we aimed to establish in vitro models of disease for IgA pemphigus, allowing us to study the effects of the interaction of anti-keratinocyte IgA with cell surface FcαRs. Employing multiple in vitro assays, such as a skin cryosection assay and a human skin organ culture model, in this study, we present mechanistic data for the pathogenesis of IgA pemphigus, mediated by anti-desmoglein 3 IgA autoantibodies. Our results reveal that this disease is dependent on FcαR-mediated activation of leukocytes in the epidermis. Importantly, this cell-dependent pathology can be dose-dependently abrogated by peptide-mediated inhibition of FcαR:IgA-Fc interaction, as confirmed in an additional model for IgA-dependent disease, that is, IgA vasculitis. These data suggest that IgA pemphigus can be modeled in vitro and that IgA pemphigus and IgA vasculitis are FcαR-dependent disease entities that can be specifically targeted in these experimental systems.

A systematic review on efficacy, safety, and treatment-durability of low-dose rituximab for the treatment of Pemphigus: special focus on COVID-19 pandemic concerns.

BACKGROUND: Rituximab is a FDA-approved monoclonal antibody for adults with moderate to severe potentially life-threatening pemphigus vulgaris. Recent studies have focused on assessments of efficacy and safety of low-dose rituximab (<2 gram in each cycle). METHOD: Databases were searched from 2010 to 2020 (last update: 1 June 2020). RESULT: Nine studies were entered; including180 cases (92: women, 88: men, age range: 9-83 years). The dosages of each Rituximab cycle varied between ultra-low-dose (≤500 mg for a cycle, either multiple infusions or a single infusion), low-dose (2 × 375 mg/m2 or 2 × 500 mg) and modified-dose (3 × 375 mg/m2 or 3 × 500 mg). The efficacy and safety of Rituximab in the studies are known by the recovery time, relapse time, and side events. According to the studies, 2 × 500 can lead to complete remission in a broad range, from 35 to 82%. These differences might be explained by different end-points and variable cumulative corticosteroid dosage after RTX administration. Although the studies showed that low dose RTX is efficient, there are some controversies regarding the choosing low-dose for severe patients. CONCLUSION: Considering the effectiveness of low-dose, intermediate dose, and ultra-low-dose protocols of Rituximab in inducing remission in pemphigus disease and considering factors such as cost of therapy, and the need to induce a minimum of immunosuppression for a minimum duration in the COVID-19 pandemic, suggested to use low-dose Rituximab protocol (2 infusions of 500 mg Rituximab: interval of 2 weeks) to induce the remission in mild-to-moderate pemphigus patients.