Tacrolimus reverses pemphigus vulgaris serum-induced depletion of desmoglein in HaCaT cells via inhibition of heat shock protein 27 phosphorylation.

BACKGROUND: Glucocorticoids are the first-line treatment for Pemphigus vulgaris (PV), but its serious side effects can be life-threatening for PV patients. Tacrolimus (FK506) has been reported to have an adjuvant treatment effect against PV. However, the mechanism underlying the inhibitory effect of FK506 on PV-IgG-induced acantholysis is unclear. OBJECTIVE: The objective of this study was to explore the effect of FK506 on desmoglein (Dsg) expression and cell adhesion in an immortalized human keratinocyte cell line (HaCaT cells) stimulated with PV sera. METHODS: A cell culture model of PV was established by stimulating HaCaT cells with 5% PV sera with or without FK506 and clobetasol propionate (CP) treatment. The effects of PV sera on intercellular junctions and protein levels of p38 mitogen-activated protein kinase (p38MAPK), heat shock protein 27 (HSP27), and Dsg were assayed using western blot analysis, immunofluorescence staining, and a keratinocyte dissociation assay. RESULTS: PV sera-induced downregulation of Dsg3 was observed in HaCaT cells and was blocked by FK506 and/or CP. Immunofluorescence staining revealed that linear deposits of Dsg3 on the surface of HaCaT cells in the PV sera group disappeared and were replaced by granular and agglomerated fluorescent particles on the cell surface; however, this effect was reversed by FK506 and/or CP treatment. Furthermore, cell dissociation assays showed that FK506 alone or in combination with CP increased cell adhesion in HaCaT cells and ameliorated loss of cell adhesion induced by PV sera. Additionally, FK506 noticeably decreased the PV serum-induced phosphorylation of HSP 27, but had no effect on p38MAPK phosphorylation. CONCLUSION: FK506 reverses PV-IgG induced-Dsg depletion and desmosomal dissociation in HaCaT cells, and this effect may be obtained by inhibiting HSP27 phosphorylation.

The Role of T Follicular Helper Cells in Clinical Remission and Relapse in Patients with Pemphigus Treated with Rituximab.

BACKGROUND: Pemphigus is a rare chronic autoimmune disease. Recent studies have found that T follicular helper (Tfh) cells may play a role in autoimmune diseases. In this study, Tfh cells frequency, BCL6 gene expression, IL-21, and IL-6 cytokines levels were examined, with the aim of understanding the effect of RTX on these cells in the onset of clinical remission or relapse in patients with pemphigus. METHODS: 20 patients with pemphigus vulgaris and 20 healthy controls without any autoimmune diseases that were admitted to the Dermatology and Venereology Clinic of the Akdeniz University Hospital were included. Peripheral blood sample was taken from all individuals and studied to analyze Tfh cell distribution, IL-21 and IL-6 distribution in CD3+CD4+CXCR5+ lymphocytes with flow cytometry, plasma IL-21 levels with ELISA, and mRNA levels that refer to BCL6 expression with PCR. RESULTS: Circulating Tfh cell distribution and IL-21 and IL-6 distribution in CD3+CD4+CXCR5+ lymphocytes and mRNA levels that refer to BCL6 expression showed no difference between patient and control groups. However, in patients who had received rituximab treatment there was a significant reduction in Tfh cells compared with other groups. Plasma IL-21 levels were significantly higher in the patient group. CONCLUSIONS: We found that plasma concentrations of the cytokine IL-21 were greatly increased in the pemphigus compared with the control group. There were no significant differences in Tfh cell percentages between the patient and control groups. Tfh cells were decreased in patients who received rituximab treatment. Our findings show that the response to RTX in pemphigus causes a reduction in circulating T follicular helper cells, but not in the plasma IL-21 level. Further studies are required to clarify the role of Tfh cells in pemphigus vulgaris.

cAMP: A master regulator of cadherin-mediated binding in endothelium, epithelium and myocardium.

Regulation of cadherin-mediated cell adhesion is crucial not only for maintaining tissue integrity and barrier function in the endothelium and epithelium but also for electromechanical coupling within the myocardium. Therefore, loss of cadherin-mediated adhesion causes various disorders, including vascular inflammation and desmosome-related diseases such as the autoimmune blistering skin dermatosis pemphigus and arrhythmogenic cardiomyopathy. Mechanisms regulating cadherin-mediated binding contribute to the pathogenesis of diseases and may also be used as therapeutic targets. Over the last 30 years, cyclic adenosine 3',5'-monophosphate (cAMP) has emerged as one of the master regulators of cell adhesion in endothelium and, more recently, also in epithelial cells as well as in cardiomyocytes. A broad spectrum of experimental models from vascular physiology and cell biology applied by different generations of researchers provided evidence that not only cadherins of endothelial adherens junctions (AJ) but also desmosomal contacts in keratinocytes and the cardiomyocyte intercalated discs are central targets in this scenario. The molecular mechanisms involve protein kinase A- and exchange protein directly activated by cAMP-mediated regulation of Rho family GTPases and S665 phosphorylation of the AJ and desmosome adaptor protein plakoglobin. In line with this, phosphodiesterase 4 inhibitors such as apremilast have been proposed as a therapeutic strategy to stabilize cadherin-mediated adhesion in pemphigus and may also be effective to treat other disorders where cadherin-mediated binding is compromised.

Deregulated phenotype of autoreactive Th17 and Treg clone cells in pemphigus vulgaris after in-vitro treatment with desmoglein antigen (Dsg-3).

The loss of balance between regulatory T (Treg) and T helper 17 (Th17) causes loss of tolerance against desmoglein (Dsg)-3 leading to pemphigus vulgaris (PV), an autoimmune bullous skin disorder associated with autoantibodies against Dsg-3. We aimed to elucidate the complex relationship of Th17 and Treg cells, their molecules, and the underlying mechanism in the development of PV disease. Using cytokine secretion assays, Th17 and Treg cells were sorted by FACS Aria-III within Dsg-3-responsive PBMC population and homogeneous T cell clones were generated in-vitro. Different cell surface molecules like CD25, GITR, CD122, CD152, CD45RO, IL-23R, STAT3, STAT5, CD127, HLA-DR, CCR4, CCR5, CCR6 and CCR7 were studied. The functional response of Th17 and Treg cells were elucidated by measuring the levels of various cytokines released by IL-10 and IL-17 T cells. The mRNA expression of transcription factors (FoxP3 and RORγt) was also analyzed. IL-17 secreting (Th17) cells with phenotype CD4+IL-17+ were greatly increased and IL-10 secreting (Treg) cells with phenotype CD4+IL-10+ were reduced in PV cases than healthy controls. The qPCR analysis showing high expression of retinoic acid receptor-related orphan receptor gamma (RORγt) mRNA in comparison to forkhead box P3 (FoxP3) mRNA confirmed the development of pro-inflammatory Th17 response in PV. Further, the cytokine profile of pro-inflammatory and anti-inflammatory cytokines suggested defective suppressive functions in Treg cells with high inflammatory response. Our findings indicate that autoantigen Dsg-3 specifically allows the proliferation of IL-17 secreting T cells though has a negative effect on IL-10 secreting T cells leading to dysregulation of immunity in PV patients. This antagonistic relationship between Dsg-3-specific Th17 and Treg cells may be critical for the onset and persistence of inflammation in PV cases.

Cytoskeletal anchorage of different Dsg3 pools revealed by combination of hybrid STED/SMFS-AFM.

Desmoglein 3 (Dsg3) is a desmosomal cadherin mediating cell adhesion within desmosomes and is the antigen of the autoimmune blistering skin disease pemphigus vulgaris. Therefore, understanding of the complex desmosome turnover process is of high biomedical relevance. Recently, super resolution microscopy was used to characterize desmosome composition and turnover. However, studies were limited because adhesion measurements on living cells were not possible in parallel. Before desmosomal cadherins are incorporated into nascent desmosomes, they are not bound to intermediate filaments but were suggested to be associated with the actin cytoskeleton. However, direct proof that adhesion of a pool of desmosomal cadherins is dependent on actin is missing. Here, we applied single-molecule force spectroscopy measurements with the novel single molecule hybrid-technique STED/SMFS-AFM to investigate the cytoskeletal anchorage of Dsg3 on living keratinocytes for the first time. By application of pharmacological agents we discriminated two different Dsg3 pools, only one of which is anchored to actin filaments. We applied the actin polymerization inhibitor Latrunculin B to modify the actin cytoskeleton and the PKCα activator PMA to modulate intermediate filament anchorage. On the cellular surface Dsg3 adhesion was actin-dependent. In contrast, at cell-cell contacts, Dsg3 adhesion was independent from actin but rather is regulated by PKC which is well established to control desmosome turn-over via intermediate filament anchorage. Taken together, using the novel STED/SMFS-AFM technique, we demonstrated the existence of two Dsg3 pools with different cytoskeletal anchorage mechanisms.

Transcriptomic profiling of pemphigus lesion infiltrating mononuclear cells reveals a distinct local immune microenvironment and novel lncRNA regulators.

Pemphigus is an autoimmune skin disease. Ectopic lymphoid-like structures (ELSs) were found to be commonly present in the pemphigus lesions, presumably supporting in situ desmoglein (Dsg)-specific antibody production. Yet functional phenotypes and the regulators of Lymphoid aggregates in pemphigus lesions remain largely unknown. Herein, we used microarray technology to profile the gene expression in skin lesion infiltrating mononuclear cells (SIMC) from pemphigus patients. On top of that, we compared SIMC dataset to peripheral blood mononuclear cells (PBMC) dataset to characterize the unique role of SIMC. Functional enrichment results showed that mononuclear cells in skin lesions and peripheral blood both had over-represented IL-17 signaling pathways while neither was characterized by an activation of type I Interferon signaling pathways. Cell-type identification with relative subsets of known RNA transcripts (CIBERSORT) results showed that naïve natural killer cells (NK cells) were significantly more abundant in pemphigus lesions, and their relative abundance positively correlated with B cells abundance. Meanwhile, plasma cells population highly correlated with type 1 macrophages (M1) abundance. In addition, we also identified a lncRNA LINC01588 which might epigenetically regulate T helper 17 cells (Th17)/regulatory T cells (Treg) balance via the peroxisome proliferator-activated receptor (PPAR) signaling pathway. Here, we provide the first transcriptomic characterization of lesion infiltrating immune cells which illustrates a distinct interplay network between adaptive and innate immune cells. It helps discover new regulators of local immune response, which potentially will provide a novel path forward to further uncover pemphigus pathological mechanisms and develop targeted therapy.

Downregulation of aquaporin 3 in patients with pemphigus vulgaris.

Pemphigus vulgaris (PV) is an autoimmune bullous skin disease. Aquaporin 3 (AQP3) is a glycerol/water channel involved in several physiological functions. Evaluation of the tissue expression and localization of AQP3 in the skin of PV patients. Twenty-seven PV patients and 30 controls were included. The patients were subjected to history taking, clinical evaluation, Autoimmune Bullous Skin Disorder Intensity Score and 4-mm punch biopsy. The biopsies were stained using anti-human AQP3 antibody and the immunofluorescence pattern and intensity were evaluated using a scoring system and ImageJ software analysis. AQP3 was expressed in the basal epidermis in 27 (100%) and in the suprabasal epidermis in 19 PV patients (70.4%). It was expressed in all controls in basal and suprabasal layers. Intensity of AQP3 immunofluorescence was strong in 2 (7.4%), moderate in 19 (70.4%) and weak in 6 patients (22.2%) while it was strong in 18 (60%) and moderate in 12 controls (40%). AQP3 expression was significantly lower in patients than controls in the suprabasal epidermis (p = 0.001). Patients with extensive disease had significantly weaker AQP3 intensity than those with marked disease (p = 0.005) Downregulation of AQP3 in patients with PV, especially in the suprabasal layers and in extensive clinical disease, suggests a potential role of AQP3 in the pathogenesis of PV.

Up-regulation of ST18 in pemphigus vulgaris drives a self-amplifying p53-dependent pathomechanism resulting in decreased desmoglein 3 expression.

Pemphigus vulgaris (PV) is a life-threatening autoimmune mucocutaneous blistering disease which is to a large extent genetically determined, and results, at least in part, from the deleterious activity of autoantibodies directed against desmoglein (DSG)3, a prominent intra-epidermal adhesion molecule. Those autoantibodies lead to decreased membranal DSG3 expression in keratinocytes (KCs), thereby destabilizing cell-cell adhesion within the epidermis and leading to blister formation. We previously showed that rs17315309, a strong risk variant for PV within the promoter of the ST18 transcription factor gene, promotes epidermal ST18 up-regulation in a p53/p63-dependent manner. Accordingly, ST18 was found to be overexpressed in the skin of PV patients. Increased ST18 expression was then shown to markedly augment PV autoantibodies-mediated loss of KCs cohesion. Here, we demonstrate that ST18 overexpression significantly increases autoantibody-mediated DSG3 down-regulation in keratinocytes. In addition, DSG3 decreased expression boosts p53 function through p38 mitogen-activated protein kinase (p38MAPK) activation and dramatically augments p53-dependent ST18 promoter activity. Finally, the PV risk variant rs17315309 is associated with increased p53 expression in PV skin. Taken collectively, these observations reveal a novel self-amplifying pathomechanism involving ST18, DSG3, p38 and p53, capable of perpetuating disease activity, and therefore indicative of novel actionable molecular targets in PV.

Stabilization of Keratinocyte Monolayer Integrity in the Presence of Anti-Desmoglein-3 Antibodies through FcRn Blockade with Efgartigimod: Novel Treatment Paradigm for Pemphigus?

Pemphigus vulgaris is an autoimmune blistering disease of the epidermis, caused by autoantibodies against desmosomal proteins, mainly desmogleins 1 and 3, which induce an impairment of desmosomal adhesion and blister formation. Recent findings have shown that inhibition of immunoglobulin G binding on the neonatal Fc receptor, FcRn, results in reduced autoantibody recycling and shortens their half-life, providing a valid treatment option for PV. We have here analyzed the role of FcRn in human keratinocytes treated with antibodies isolated from pemphigus vulgaris patient or with recombinant anti-desmoglein-3 antibodies that induce pathogenic changes in desmosomes, such as loss of monolayer integrity, aberrant desmoglein-3 localization and degradation of desmoglein-3. We show that blocking IgG binding on FcRn by efgartigimod, a recombinant Fc fragment undergoing clinical studies for pemphigus, stabilizes the keratinocyte monolayer, whereas the loss of desmoglein-3 is not prevented by efgartigimod. Our data show that FcRn may play a direct role in the pathogenesis of pemphigus at the level of the autoantibody target cells, the epidermal keratinocytes. Our data suggest that in keratinocytes, FcRn may have functions different from its known function in IgG recycling. Therefore, stabilization of keratinocyte adhesion by FcRn blocking entities may provide a novel treatment paradigm for pemphigus.

Immunohistochemical expression of regulatory T cells (CD4 + CD25 + bright FOXP3 + ) in pemphigus patients.

BACKGROUND: Pemphigus is a series of autoimmune skin disorders caused by IgG. Regulatory T cells (Tregs) are a subset of CD4+ T cells that mostly block pathogenic immune responses mediated by self-reactive cells; therefore, a lack of Tregs or a malfunction in their activity could lead to a loss of tolerance and the development of autoimmunity. AIMS: To evaluate the expression of lesional and perilesional Treg markers (CD4 + CD25 + bright FOXP3 + ) in pemphigus patients. PATIENTS AND METHODS: Twenty-three pemphigus patients and 20 healthy controls were included in this study. The expression of CD4 , CD25, and Foxp3 was evaluated by immunohistochemistry. RESULTS: There was statistically significant increase in CD4+ T lymphocytes in lesional skin of pemphigus compared to perilesional skin and control group (p-value: 0.001). There was statistically significant decrease in CD25+ and Foxp3+ cells in lesional skin compared to perilesional and control group (p-value: <0.001, 0.025, respectively). CONCLUSION: The reduction of lesional skin Tregs may play an important role in the pemphigus pathogenesis.

Bruton's tyrosine kinase inhibition-An emerging therapeutic strategy in immune-mediated dermatological conditions.

Bruton's tyrosine kinase (BTK), a member of the Tec kinase family, is critically involved in a range of immunological pathways. The clinical application of BTK inhibitors for B-cell malignancies has proven successful, and there is strong rationale for the potential benefits of BTK inhibitors in some autoimmune and allergic conditions, including immune-mediated dermatological diseases. However, the established risk-to-benefit profile of "first-generation" BTK inhibitors cannot be extrapolated to these emerging, non-oncological, indications. "Next-generation" BTK inhibitors such as remibrutinib and fenebrutinib entered clinical development for chronic spontaneous urticaria (CSU); rilzabrutinib and tirabrutinib are being studied as potential treatments for pemphigus. Promising data from early-phase clinical trials in CSU suggest potential for these agents to achieve strong pathway inhibition, which may translate into measurable clinical benefits, as well as other effects such as the disruption of autoantibody production. BTK inhibitors may help to overcome some of the shortcomings of monoclonal antibody treatments for immune-mediated dermatological conditions such as CSU, pemphigus, and systemic lupus erythematosus. In addition, the use of BTK inhibitors may improve understanding of the pathophysiological roles of mast cells, basophils, and B cells in such conditions.

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.

Electron microscopy reveals that phospholipase C and Ca2+ signaling regulate keratin filament uncoupling from desmosomes in Pemphigus.

Pemphigus vulgaris (PV) is a severe autoimmune blistering skin disease caused primarily by autoantibodies (PV-IgG) against the desmosomal cadherins desmoglein (Dsg) 1 and Dsg 3. Pemphigus is a model disease to study desmosome regulation because patient lesions are characterized by ultrastructural hallmarks including loss, shrinkage and splitting of desmosomes as well as by retraction of keratin filaments. The mechanisms underlying the disease are not completely understood but involve several intracellular signaling pathways triggered by autoantibody binding. Recently, we demonstrated that Phosphoinositid-Phospholipase C (PLC) and Ca2+ signaling are required for acantholysis in human epidermis. Here, we used transmission electron microscopy to characterize the role of PLC and Ca2+ signaling with regard to the pathogenic effects of PV-IgG on desmosome ultrastructure in human ex vivo skin model. First, we observed that the PV-IgG used in this study significantly reduced desmosome length and caused uncoupling of desmosomes from keratin filaments. Moreover, PV-IgG enhanced the number of split desmosomes but did not cause a significant loss of desmosomes. We found that inhibition of PLC and Ca2+ signaling significantly blocked keratin filament uncoupling but not shrinkage of desmosomes. Blocking Ca2+ flux prevented desmosome splitting. The ultrastructural analysis revealed that for preventing skin blistering it is sufficient to enhance keratin filament insertion, which is regulated by PLC/ Ca2+. Here, we underscore the unique role of electron microscopy to investigate the underlying mechanisms by which a signaling pathway regulates desmosome ultrastructure in pemphigus.

Mechanisms of Trx2/ASK1-Mediated Mitochondrial Injury in Pemphigus Vulgaris.

OBJECTIVE: Apoptotic events mediated by mitochondrial injury play an important role on the onset of Pemphigus vulgaris (PV). The thioredoxin-2 (Trx2)/apoptosis signal-regulating kinase 1 (ASK1) signaling pathway is considered a key cascade involved on the regulation of mitochondrial injury. Hence, we have investigated the regulatory mechanism of the Trx2/ASK1 signaling in PV-induced mitochondrial injury. METHODS: Serum and tissue samples were collected from clinical PV patients to detect the oxidative stress factors, cell apoptosis, and expression of members from Trx2/ASK1 signaling. HaCaT cells were cultured with the serum of PV patients and transfected with Trx2 overexpression or silencing vector. Changes in the levels of reactive oxygen species (ROS), mitochondrial membrane potential (△ψm), and apoptosis were further evaluated. A PV mouse model was established and administered with Trx2-overexpressing plasmid. The effect of ectopic Trx2 expression towards acantholysis in PV mice was observed. RESULTS: A series of cellular and molecular effects, including (i) increased levels of oxidative stress products, (ii) destruction of epithelial cells in the skin tissues, (iii) induction of apoptosis in keratinocytes, (iv) reduction of Trx2 protein levels, and (v) enhanced phosphorylation of ASK1, were detected in PV patients. In vitro experiments confirmed that Trx2 can inhibit ASK1 phosphorylation, alleviate ROS release, decrease △ψm, and lower the apoptotic rate. Injection of Trx2-overexpressing vectors in vivo could also relieve acantholysis and blister formation in PV mice. CONCLUSION: The Trx2/ASK1 signaling pathway regulates the incidence of PV mediated by mitochondrial injury.

Arginase 1+ IL-10+ polymorphonuclear myeloid-derived suppressor cells are elevated in patients with active pemphigus and correlate with an increased Th2/Th1 response.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells, which are characterized by their capability to suppress T-cell responses. While MDSCs have been traditionally associated with cancer diseases, their role as regulators of autoimmune diseases is emerging. Pemphigus is a chronic autoimmune blistering skin disease characterized by dysregulated T-cell responses and autoantibody production. The role of MDSCs in pemphigus disease has not been defined yet. The aim of this study was to characterize MDSCs in pemphigus patients and to dissect their relationship with CD4+ T-cell subsets and clinical disease assessments. For this purpose, we performed a cross-sectional analysis of 20 patients with pemphigus. Our results indicate that a population of CD66b+ CD11b+ polymorphonuclear-like MDSCs (PMN-MDSCs) is expanded in the peripheral blood mononuclear cell fraction of pemphigus patients compared to age-matched healthy donors. These PMN-MDSCs have the capability of suppressing allogeneic T-cell proliferation in vitro and show increased expression of characteristic effector molecules such as arginase I and interleukin-10. We further demonstrate that PMN-MDSCs are especially expanded in patients with active pemphigus, but not in patients in remission. Moreover, MDSC frequencies correlate with an increased Th2/Th1 cell ratio. In conclusion, the identification of a functional PMN-MDSC population suggests a possible role of these cells as regulators of Th cell responses in pemphigus.

Immunophenotyping in pemphigus reveals a TH17/TFH17 cell-dominated immune response promoting desmoglein1/3-specific autoantibody production.

BACKGROUND: TH2 cells were thought to be a pivotal factor for initiation of the autoimmune blistering disease pemphigus. However, the role of other T-cell subsets in pemphigus pathogenesis remained unclear. OBJECTIVE: We aimed to characterize the exact phenotype of T cells responsible for the development of pemphigus. METHODS: Whole transcriptome shotgun sequencing was performed to determine differential gene expression in pemphigus lesions and skin of healthy individuals. The cutaneous cytokine signature was further evaluated by real-time quantitative PCR. In peripheral blood, the distribution of TH cell and folliclular helper (TFH) cell subsets was analyzed by flow cytometry. Finally, the capacity of TH and TFH cell subsets to induce desmoglein (Dsg)-specific autoantibodies by memory B cells was evaluated in coculture experiments. RESULTS: Transcriptome analysis of skin samples identified an IL-17A-dominated immune signature in patients with pemphigus, and Kyoto Encyclopedia of Genes and Genomes pathway analysis confirmed the dominance of the IL-17A signaling pathway. Increased expression of IL17A and associated cytokines was also detected by real-time quantitative PCR comparing lesional with perilesional or healthy skin. Interestingly, utilization of flow cytometry showed that patients with active pemphigus had elevated levels of circulating IL-17+, TH17, TFH17, and TFH17.1 cells. Notably, levels of TH17 and TFH17 cells correlated with levels of Dsg-specific CD19+CD27+ memory B cells, and patients with acute pemphigus showed higher levels of Dsg3-autoreactive TFH17 cells. Coculture experiments revealed TFH17 cells as primarily responsible for inducing Dsg-specific autoantibody production by B cells. CONCLUSION: Our findings show that TFH17 cells are critically involved in the pathogenesis of pemphigus and offer novel targets for therapeutic intervention.

Dsg2 Upregulation as a Rescue Mechanism in Pemphigus.

In pemphigus vulgaris (PV), autoantibodies directed against the desmosomal cadherin desmoglein (Dsg) 3 cause loss of intercellular adhesion. It is known that Dsg3 interactions are directly inhibited by autoantibody binding and that Dsg2 is upregulated in epidermis of PV patients. Here, we investigated whether heterophilic Dsg2-Dsg3 interactions occur and would modulate PV pathogenesis. Dsg2 was upregulated in PV patients' biopsies and in a human ex vivo pemphigus skin model. Immunoprecipitation and cell-free atomic force microscopy (AFM) experiments demonstrated heterophilic Dsg2-Dsg3 interactions. Similarly, in Dsg3-deficient keratinocytes with severely disturbed intercellular adhesion Dsg2 was upregulated in the desmosome containing fraction. AFM revealed that Dsg2-Dsg3 heterophilic interactions showed binding frequency, strength, Ca2+-dependency and catch-bond behavior comparable to homophilic Dsg3-Dsg3 or homophilic Dsg2-Dsg2 interactions. However, heterophilic Dsg2-Dsg3 interactions had a longer lifetime compared to homophilic Dsg2-Dsg2 interactions and PV autoantibody-induced direct inhibition was significantly less pronounced for heterophilic Dsg2-Dsg3 interactions compared to homophilic Dsg3 interactions. In contrast, a monoclonal anti-Dsg2 inhibitory antibody reduced heterophilic Dsg2-Dsg3 and homophilic Dsg2-Dsg2 binding to the same degree and further impaired intercellular adhesion in Dsg3-deficient keratinocytes. Taken together, the data demonstrate that Dsg2 undergoes heterophilic interactions with Dsg3, which may attenuate autoantibody-induced loss of keratinocyte adhesion in pemphigus.

Distorted frequency of dendritic cells and their associated stimulatory and inhibitory markers augment the pathogenesis of pemphigus vulgaris.

The objective of this study was to investigate the frequency and functionality of DCs and its associated stimulatory and inhibitory markers in the pathogenesis of PV Active PV patients (n = 30) having both skin and oral lesions, and 30 healthy controls were recruited in the study. The frequency of DCs was determined by flow cytometry followed by the primary culture by using recombinant IL-4 (250 IU/ml) and GM-CSF (600 IU/ml). The culture supernatant was used for ELISA. RNA was isolated from sorted DCs and used for the mRNA expression of DC-associated stimulatory (CD40 and CD80) and inhibitory (PSGL1 and ILT3) markers. Tissue localization of Langerhans cells was done by immunohistochemistry. In this study, altered frequency of myeloid DC (mDC) and plasmacytoid DC (pDC) was seen in the circulation of PV patients. The primary culture of patient-derived DCs showed anomalous cytokine profiling. In the culture supernatant of DCs, elevated levels of TNF-ɑ and IL-12 were detected in PV patients. Meanwhile, reverse trend was found in the case of IFN-ɑ and IL-10 cytokine levels. Similarly, a discrepancy in the expression of DC-associated stimulatory (CD40 and CD80) and inhibitory (PSGL1 and ILT3) markers suggested their possible involvement in the immunopathogenesis of PV. An elevated number of tissue localizing Langerhans cells was also observed in the perilesional skin. This study indicates the distorted frequency and functionality of DCs in the immunopathogenesis of PV. Targeting these functional markers in the future may generate novel therapeutic options for better management of PV.