The Role of Non-Neuronal Acetylcholine in the Autoimmune Blistering Disease Pemphigus Vulgaris.

The importance of acetylcholine (ACh) in keratinocyte adhesion and acantholysis has been investigated over the last three decades, particularly in the pathophysiology of autoimmune blistering dermatoses. Pemphigus vulgaris (PV) is an autoimmune blistering skin disease where autoantibody-mediated suprabasilar intraepidermal splitting causes flaccid blisters and non-healing erosions of the oral mucosa and sometimes also of the skin. Historically, acantholysis in PV was thought to be driven by anti-desmoglein (Dsg) antibodies. Herein, we describe the role of autoantibodies against keratinocyte muscarinic and nicotinic acetylcholine receptors, as well as the annexin-like molecule pemphaxin that also binds ACh, in the immunopathogenesis of PV. The identification of targets in this disease is important, as they may lead to novel diagnostic and therapeutic options in the future for this potentially deadly disease.

Botulinum Neurotoxin Type A Directly Affects Sebocytes and Modulates Oleic Acid-Induced Lipogenesis.

Excess sebum (seborrhea) results in oily skin and is associated with large pore size and acne. Studies in healthy, seborrheic volunteers have reported that intradermal injection of commercial preparations of botulinum neurotoxin type A (BoNT/A) (onabotulinumtoxinA, abobotulinumtoxinA, and incobotulinumtoxinA) reduced sebum production, and thus, skin oiliness and pore size. The mechanism for these effects has not been fully elucidated; however, several theories involving direct or indirect effects of BoNT/A on neuronal and/or dermal cells (e.g., sebocytes) have been proposed. In the present study, we evaluated the direct effect of native research grade BoNT/A complex, a commercial preparation of BoNT/A (onabotA), and BoNT/A variants on sebocyte lipogenesis using an in vitro sebocyte cell model. We show that picomolar concentrations of BoNT/A (BoNT/A complex: half maximal effective concentration [EC50] = 24 pM; BoNT/A 150 kDa: EC50 = 34 pM) modulate sebocyte lipogenesis and reduce oleic acid-induced sebocyte differentiation, lipogenesis, and holocrine-like secretion. Comparative studies with the binding domain of BoNT/A, which lacks enzymatic activity, show that this effect is independent of the enzymatic activity of BoNT/A and likely occurs via sebocyte cell surface receptors (e.g., fibroblast growth factor receptors). Overall, these results shed light on the potential mechanism of action and rationale for use of BoNT/A for treatment of sebum-related conditions.

Mitochondrial Autoantibodies and the Role of Apoptosis in Pemphigus Vulgaris.

Pemphigus vulgaris (PV) is an IgG autoantibody-mediated, potentially fatal mucocutaneous disease manifested by progressive non-healing erosions and blisters. Beyond acting to inhibit adhesion molecules, PVIgGs elicit a unique process of programmed cell death and detachment of epidermal keratinocytes termed apoptolysis. Mitochondrial damage by antimitochondrial antibodies (AMA) has proven to be a critical link in this process. AMA act synergistically with other autoantibodies in the pathogenesis of PV. Importantly, absorption of AMA inhibits the ability of PVIgGs to induce blisters. Pharmacologic agents that protect mitochondrial function offer a new targeted approach to treating this severe immunoblistering disease.

The M3 Muscarinic Acetylcholine Receptor Promotes Epidermal Differentiation.

The M3 muscarinic acetylcholine receptor is predominantly expressed in the basal epidermal layer where it mediates the effects of the autocrine/paracrine cytotransmitter acetylcholine. Patients with the autoimmune blistering disease pemphigus develop autoantibodies to M3 muscarinic acetylcholine receptor and show alterations in keratinocyte adhesion, proliferation, and differentiation, suggesting that M3 muscarinic acetylcholine receptor controls these cellular functions. Chmr3-/- mice display altered epidermal morphology resembling that seen in patients with pemphigus vulgaris. In this study, we characterized the cellular and molecular mechanisms through which M3 muscarinic acetylcholine receptor controls epidermal structure and function. We used single-cell RNA sequencing to evaluate keratinocyte heterogeneity and identify differentially expressed genes in specific subpopulations of epidermal cells in Chmr3-/- neonatal mice. We found that Chmr3-/- mice feature abnormal epidermal morphology characterized by accumulation of nucleated basal cells, shrinkage of basal keratinocytes, and enlargement of intercellular spaces. These morphologic changes were associated with upregulation of cell proliferation genes and downregulation of genes contributing to epidermal differentiation, extracellular matrix formation, intercellular adhesion, and cell arrangement. These findings provide, to our knowledge, previously unreported insights into how acetylcholine controls epidermal differentiation and lay a groundwork for future translational studies evaluating the therapeutic potential of cholinergic drugs in dermatology.

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.

Transient lymphadenopathy during intravenous immunoglobulin (IVIg) in a patient with pemphigus vulgaris.

This is the first case of transient cervical lymphadenopathy as an adverse event during IVIg infusion. IVIg plays a vital role in the treatment of many dermatological conditions and identification of adverse events can facilitate patient counseling.

Atypical pemphigus: autoimmunity against desmocollins and other non-desmoglein autoantigens.

In this review, we recap current knowledge about non-desmoglein autoantigens in atypical forms of autoimmune pemphigus. More than 50 keratinocyte proteins, including adhesion molecules, receptors and enzymes as well as mitochondrial proteins can be targeted, leading to alterations in numerous intracellular signaling pathways. Patients with pemphigus herpetiformis feature various combinations of antibodies to desmogleins 1 and 3 and desmocollins 1-3. Pemphigus vulgaris patients who do not have antibodies to desmogleins develop typical clinical and histological features of pemphigus. Experimental results revealed synergy of different autoantibodies. Alterations of the keratinocyte adhesive function caused by a single antibody alone are reversible due to self-repair. Since composition of the pool of the most common pathogenic antibodies appears to be similar among pemphigus patients with or without anti-desmoglein antibodies, the atypical pemphigus represents a unique model for elucidation of the molecular mechanisms of autoimmunity against non-desmoglein antigens. Further studies of the immunopathology of atypical pemphigus should shed new lights on the pathophysiology of conventional variants of autoimmune pemphigus.

Protective Effects of Pinus halepensis Bark Extract and Nicotine on Cigarette Smoke-induced Oxidative Stress in Keratinocytes.

BACKGROUND/AIM: Cigarette smoke (CS) is a major environmental health threat. The oxidative stress induced by CS on keratinocytes and the possible protective effect of nicotine, its receptor inhibitors, and Pinus halepensis bark extract in relation to known antioxidants were investigated. MATERIALS AND METHODS: Primary mouse keratinocytes were exposed to cigarette smoke in the presence and absence of Pinus halepensis bark extract (1 µg/ml), rutin (50 µM) and ascorbic acid (250 µM), nicotine (1 µM) with or without mecamylamine (5 µM) and α-bungarotoxin (0.1 µM). Keratinocyte viability and oxidative stress were evaluated by MTT and fluorescence assays. RESULTS: Pinus halepensis bark extract decreased the oxidative stress and increased the viability of keratinocytes, and moreover, these effects were more pronounced compared to the mixture of rutin and L-ascorbic acid. Nicotine significantly enhanced the viability potentiation of the beneficial effect induced by Pinus halepensis bark extract. Mecamylamine and α-bungarotoxin showed no specific effect. CONCLUSION: Pinus halepensis bark extract in combination with nicotine may successfully reverse skin damage induced by cigarette smoke.

Rationale for including intravenous immunoglobulin in the multidrug protocol of curative treatment of pemphigus vulgaris and development of an assay predicting disease relapse.

Analysis of reported outcomes of treatment of pemphigus vulgaris (PV) patients demonstrated that the multidrug approach offers a lower relapse rate compared to the FDA-approved prednisone/rituximab regimen. The multidrug protocol protects keratinocytes from autoantibody attack by systemic corticosteroids and mitochondrion-protecting drugs, selectively eliminates pathogenic autoantibodies by intravenous immunoglobulin (IVIg) and inhibits autoantibody production by cytotoxic immunosuppressors. Therefore, IVIg should be always added to the prednisone/rituximab regimen that does not eliminate circulating autoantibodies. To decrease risk for relapse to a minimum, PV should be maintained in full clinical remission until the critical mass of autoreactive plasma cells dies off. The two major factors that determine patient's risk for a relapse are the composition of the pool of pathogenic autoantibodies and the innate abilities of keratinocytes to sustain an autoantibody attack. As it is currently impossible to evaluate the risk for a relapse, development of a biomarker assay that could do so would be helpful in a long-term management of PV patients. We compared the magnitude of cytochrome c (CytC) release in keratinocytes by serum from PV patients in acute disease stage vs. remission and identified very strong positive correlation with disease severity. PV patients whose serum contained autoantibodies requiring higher amounts of normal IgG to neutralize their ability to release CytC were found to be at a higher risk for disease relapse. However, lack of very strong statistical correlation suggested that CytC is not an ideal biomarker to predict disease relapse, which should prompt a search for alternative candidates.

A historic perspective on the current progress in elucidation of the biologic significance of non-neuronal acetylcholine.

The "5th International Symposium on Non-neuronal Acetylcholine: from bench to bedside" was held on September 27-29, 2019 in Hyatt Regency, Long Beach, CA, USA. Approximately 50 scientists from 11 countries over 6 continents participated in this meeting. The major topics included an overall biologic significance of non-neuronal acetylcholine (ACh) and the roles of the non-neuronal cholinergic systems in mucocutaneous, respiratory, digestive, immunologic, endocrine, cardiovascular, musculoskeletal and kidney diseases, and cancer. This meeting facilitated continued work to advance the fundamental science and translational aspects of the interdisciplinary studies on non-neuronal ACh. The progress made has opened a new chapter in the field of cholinergic pharmacology, and advanced our knowledge beyond regulation of individual cell- and tissue-types, defining a new paradigm of selective pharmacological regulation of vital function of practically all types of non-neuronal cells. It is now clear that the autocrine and paracrine control of non-neuronal cells by non-neuronal ACh is implemented through synergistic, additive, and reciprocal effects triggered by two different cholinergic receptor classes. Each biologic effect of ACh is determined by a unique combination of cholinergic receptors subtype expressed at each stage of cell development and differentiation. The plasticity of the non-neuronal cholinergic system helps adjust homeostasis to new environmental conditions.

Mechanisms of synergy of autoantibodies to M3 muscarinic acetylcholine receptor and secretory pathway Ca2+/Mn2+-ATPase isoform 1 in patients with non-desmoglein pemphigus vulgaris.

Pemphigus vulgaris (PV) is a potentially lethal mucocutaneous blistering disease characterized by IgG autoantibodies (AuAbs) binding to epidermal keratinocytes and inducing a devastating blistering disease affecting oral and/or esophageal surfaces and, sometimes, also the skin. Anti-keratinocyte AuAbs developed by the desmoglein (Dsg) 1/3 AuAb-negative acute PV patients are pathogenic, as they induced acantholysis and epidermal split in the experimental models of PV in vitro and in vivo. These PV patients have various combinations of AuAbs to keratinocyte muscarinic acetylcholine receptor subtype M3 (M3AR), the secretory pathway Ca2+/Mn2+-ATPase isoform 1 (SPCA1), and desmocollin 3 whose relative concentrations correlate with the disease activity. In this study, we identified new molecular mechanisms of the synergistic cooperation of AuAbs to M3AR and SPCA1 in inducing acantholysis in the anti-Dsg 1/3 AuAb-negative PV patients. Anti-M3AR AuAb was found to play an important role in determining the level of intraepidermal split just above the basal cells, caspase to mediate early pro-apoptotic events triggered by anti-SPCA1 AuAb, and the neonatal Fc receptor (FcRn) to contribute to the pathobiological actions of both anti-M3AR and anti-SPCA1 AuAbs. Altogether, these novel results support our original hypothesis that pemphigus acantholysis is a complex disease process (also known as apoptolysis) initiated by AuAbs directed against different keratinocyte proteins that play important roles in supporting cell viability and regulating vital cell functions.

Diagnosis and management of pemphigus: Recommendations of an international panel of experts.

BACKGROUND: Several European countries recently developed international diagnostic and management guidelines for pemphigus, which have been instrumental in the standardization of pemphigus management. OBJECTIVE: We now present results from a subsequent Delphi consensus to broaden the generalizability of the recommendations. METHODS: A preliminary survey, based on the European Dermatology Forum and the European Academy of Dermatology and Venereology guidelines, was sent to a panel of international experts to determine the level of consensus. The results were discussed at the International Bullous Diseases Consensus Group in March 2016 during the annual American Academy of Dermatology conference. Following the meeting, a second survey was sent to more experts to achieve greater international consensus. RESULTS: The 39 experts participated in the first round of the Delphi survey, and 54 experts from 21 countries completed the second round. The number of statements in the survey was reduced from 175 topics in Delphi I to 24 topics in Delphi II on the basis of Delphi results and meeting discussion. LIMITATIONS: Each recommendation represents the majority opinion and therefore may not reflect all possible treatment options available. CONCLUSIONS: We present here the recommendations resulting from this Delphi process. This international consensus includes intravenous CD20 inhibitors as a first-line therapy option for moderate-to-severe pemphigus.

Real-world experience of dupilumab treatment for atopic dermatitis in adults: a retrospective analysis of patients' records.

BACKGROUND: Clinical trial data for dupilumab, a monoclonal antibody against the interleukin-4 receptor (IL-4Rα), have shown that it is safe and effective for the treatment of moderate to severe atopic dermatitis in patients whose disease is resistant to other therapies. However, little real-world experience with dupilumab use has been reported thus far. The aim of this retrospective study was to assess overall outcomes in adult patients with atopic dermatitis (AD) treated with dupilumab. METHODS: A retrospective review of electronic medical records was conducted for patients treated with dupilumab in the Department of Dermatology at the University of California, Irvine. RESULTS: We analyzed the medical records of 77 AD patients who received dupilumab according to standard dosing and had at least one documented follow-up visit. In 66 patients (86%), dupilumab improved clinical disease severity, with 23 patients (30%) experiencing complete clearance on dupilumab. Dupilumab was generally well-tolerated and caused no serious adverse events. The most common side effects included dry eyes, conjunctivitis, and keratitis. The most common reason for discontinuation of treatment was lack of substantial clinical improvement or progression of disease severity, followed by ophthalmologic side effects. CONCLUSIONS: Overall, dupilumab was well-tolerated and resulted in clinical improvement in our patient population. These results provide additional important information on the safety and utility of dupilumab treatment for moderate to severe atopic dermatitis in the real-world clinical setting.

Synergy among non-desmoglein antibodies contributes to the immunopathology of desmoglein antibody-negative pemphigus vulgaris.

Pemphigus vulgaris (PV) is a potentially lethal mucocutaneous blistering disease characterized by IgG autoantibodies (AuAbs) binding to epidermal keratinocytes and inducing this devastating disease. Here, we observed that non-desmoglein (Dsg) AuAbs in the sera of patients with Dsg1/3 AuAb-negative acute PV are pathogenic, because IgGs from these individuals induced skin blistering in neonatal mice caused by suprabasal acantholysis. Serum levels of AuAbs to desmocollin 3 (Dsc3), M3 muscarinic acetylcholine receptor (M3AR), and secretory pathway Ca2+/Mn2+-ATPase isoform 1 (SPCA1) correlated with the disease stage of PV. Moreover, AuAb absorption on recombinant Dsc3, M3AR, or SPCA1 both prevented skin blistering in the passive transfer of AuAbs model of PV in BALB/c mice and significantly decreased the extent of acantholysis in a neonatal mouse skin explant model. Although acantholytic activities of each of these immunoaffinity-purified AuAbs could not induce a PV-like phenotype, their mixture produced a synergistic effect manifested by a positive Nikolskiy sign in the skin of neonatal mice. The downstream signaling of all pathogenic non-Dsg AuAbs involved p38 mitogen-activated protein kinase (MAPK)-mediated phosphorylation and elevation of cytochrome c release and caspase 9 activity. Anti-Dsc3 and anti-SPCA1 AuAbs also activated SRC proto-oncogene, nonreceptor tyrosine kinase (SRC). Of note, although a constellation of non-Dsg AuAbs apparently disrupted epidermal integrity, elimination of a single pathogenic AuAb could prevent keratinocyte detachment and blistering. Therefore, anti-Dsg1/3 AuAb-free PV can be a model for elucidating the roles of non-Dsg antigen-specific AuAbs in the physiological regulation of keratinocyte cell-cell adhesion and blister development.

Retrospective analysis of a single-center clinical experience toward development of curative treatment of 123 pemphigus patients with a long-term follow-up: efficacy and safety of the multidrug protocol combining intravenous immunoglobulin with the cytotoxic immunosuppressor and mitochondrion-protecting drugs.

BACKGROUND: Pemphigus vulgaris (PV) is a life-long IgG autoantibody-mediated blistering disease affecting the mucosal surfaces lined by the stratified epithelium (oral, nasal, genital) and sometimes also the skin. While corticosteroid treatment is life saving, the high dose and prolonged courses required for disease control are associated with significant adverse effects, including death. Although introduction of rituximab (RTX) provided for a favorable outcome, the high relapse rate, that is, up to 80%, precludes successful use of RTX as a monotherapy. Intravenous immunoglobulin (IVIg) is being increasingly utilized as off-label therapy for a variety of autoimmune and inflammatory diseases, including PV and pemphigus foliaceus (PF). AIMS: The goal of pemphigus research is to develop an effective treatment modality that would allow patients to achieve and maintain a stable clinical remission without the need for additional treatments, or cure. MATERIALS AND METHODS: This article summarizes clinical outcome of 123 pemphigus patients treated with a combination of IVIg, an immunosuppressive cytotoxic drug (ICD) and mitochondrion-protecting drugs in the Blistering Disease Clinic at the University of California, Irvine from 2007 to 2017. RESULTS: The mean time to disease control was 0.2 months and time to complete remission - 1.7 months. Duration of complete remission on drugs until relapse or end of treatment was 19.3 months. The mean duration of complete remission off drugs until relapse was 15.8 months. That until end of follow up was 48.4 months, with a minimum of 14 and a maximum of 91 months. The overall complete remission rate off all drugs was 100%, with 12% overall relapse rate. Most relapses, 8.1 vs. 3.3%, occurred during the time of treatment, compared to posttreatment. No patients had more than a single relapse. The duration of the posttreatment follow-up ranged from 9 to 97 months with a mean of 64.8 months, or 5.4 years. The total number of IVIg cycles ranged from 26 in patients without a relapse to 37 in patients with a relapse. The clinical outcome in patients that received IVIg with RTX or another ICD were found to be very similar. DISCUSSION: Thus, the multidrug IVIg regimen allowed to achieve three principal treatment objectives: (i) rapid control of pemphigus symptoms; (ii) stable disease remission; and (iii) overall safety of treatment. CONCLUSIONS: While the individualized therapeutic approaches to eradicate the autoreactive B cell clones causing disease in each particular PV or PF patient are being developed, all pemphigus patients can benefit from the treatment protocol described in this study.

Mechanisms of pathogenic effects of eosinophil cationic protein and eosinophil-derived neurotoxin on human keratinocytes.

Cutaneous deposition of eosinophil degranulation proteins is a major feature of eosinophil-rich cutaneous diseases including bullous pemphigoid (BP). We sought to better understand the effect of two of these proteins - eosinophil cationic protein (ECP) and eosinophil-derived neurotoxin (EDN), on human keratinocytes using the Het-1A cell line. To evaluate expression of key cytokines and chemokines observed in BP as well as metal metalloprotease 9 (MMP9), we performed qPCR and in-cell Western assays on cells treated with either ECP or EDN. We further evaluated the effect of ECP and EDN on keratinocyte survival, generation of reactive oxygen species (ROS) and apoptosis. Lastly, we assessed ECP and EDN's ability to induce keratinocyte detachment from provisional matrix. Treatment of keratinocytes with ECP and EDN resulted in significant increases in IL-5, eotaxin-1 and CCL5 (RANTES) expression at both mRNA and protein levels, but not IL-17 or IL-31. ECP and EDN also upregulate MMP9 production. Inhibiting MMP9, we confirmed that keratinocyte expression of IL-5, eotaxin-1 and RANTES was independent from MMP9. Both ECP and EDN were cytotoxic to keratinocytes, inducing ROS formation and apoptosis through a mitochondrion-dependent pathway as evidenced by results of terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) and cytochrome c release assays, respectively. ECP but not EDN led to significant keratinocyte detachment from provisional matrix. These findings demonstrate that the pathogenic effects of ECP and EDN in BP may result from their direct action on keratinocytes, and as such may became a target for future therapies in eosinophil-rich cutaneous diseases.