Autoimmune Disease Pemphigus Vulgaris Research Articles

Case Report on Pemphigus Vulgaris

This case report is mainly about the rare auto immune disease pemphigus vulgaris. Its onset appears on the basis of interaction between genetic predisposition and various triggering factors. Mostly it is clinical presented as oral lesions, mucosal lesions, skin blisters that are itchy and painful. It affects people around the age of 50 to 60 mostly Jewish heritages from southeast Europe, India and the Middle East. Basing on clinical features, histopathological and immunopathological findings it has to diagnose and must be treated immediately because, delayed treatment cannot be helpful and may cause fatality to the patient.

Epidermal structure and differentiation.

In this Primer, Moreci and Lechler follow the lifetime of an epidermal cell from its birth to its ultimate death, and detail how this journey is necessary for epidermal function.

The desmosomal cadherin desmoglein-3 acts as a keratinocyte anti-stress protein via suppression of p53

Desmoglein-3 (Dsg3), the Pemphigus Vulgaris (PV) antigen (PVA), plays an essential role in keratinocyte cell–cell adhesion and regulates various signaling pathways involved in the progression and metastasis of cancer where it is upregulated. We show here that expression of Dsg3 impacts on the expression and function of p53, a key transcription factor governing the responses to cellular stress. Dsg3 depletion increased p53 expression and activity, an effect enhanced by treating cells with UVB, mechanical stress and genotoxic drugs, whilst increased Dsg3 expression resulted in the opposite effects. Such a pathway in the negative regulation of p53 by Dsg3 was Dsg3 specific since neither E-cadherin nor desmoplakin knockdown caused similar effects. Analysis of Dsg3−/− mouse skin also indicated an increase of p53/p21WAF1/CIP1 and cleaved caspase-3 relative to Dsg3+/− controls. Finally, we evaluated whether this pathway was operational in the autoimmune disease PV in which Dsg3 serves as a major antigen involved in blistering pathogenesis. We uncovered increased p53 with diffuse cytoplasmic and/or nuclear staining in the oral mucosa of patients, including cells surrounding blisters and the pre-lesional regions. This finding was verified by in vitro studies where treatment of keratinocytes with PV sera, as well as a characterized pathogenic antibody specifically targeting Dsg3, evoked pronounced p53 expression and activity accompanied by disruption of cell–cell adhesion. Collectively, our findings suggest a novel role for Dsg3 as an anti-stress protein, via suppression of p53 function, and this pathway is disrupted in PV.

Role of Src and Cortactin in Pemphigus Skin Blistering.

Autoantibodies against desmoglein (Dsg) 1 and Dsg3 primarily cause blister formation in the autoimmune disease pemphigus vulgaris (PV). Src was proposed to contribute to loss of keratinocyte cohesion. However, the role and underlying mechanisms are unclear and were studied here. In keratinocytes, cell cohesion in response to autoantibodies was reduced in Src-dependent manner by two patient-derived PV-IgG fractions as well as by AK23 but not by a third PV-IgG fraction, although Src was activated by all autoantibodies. Loss of cell cohesion was progredient in a timeframe of 24 h and AK23, similar to PV-IgG, interfered with reconstitution of cell cohesion after Ca2+-switch, indicating that the autoantibodies also interfered with desmosome assembly. Dsg3 co-localized along cell contacts and interacted with the Src substrate cortactin. In keratinocytes isolated from cortactin-deficient mice, cell adhesion was impaired and Src-mediated inhibition of AK23-induced loss of cell cohesion for 24 h was significantly reduced compared to wild-type (wt) cells. Similarly, AK23 impaired reconstitution of cell adhesion was Src-dependent only in the presence of cortactin. Likewise, Src inhibition significantly reduced AK23-induced skin blistering in wt but not cortactin-deficient mice. These data suggest that the Src-mediated long-term effects of AK23 on loss of cell cohesion and skin blistering are dependent on cortactin-mediated desmosome assembly. However, in human epidermis PV-IgG-induced skin blistering and ultrastructural alterations of desmosomes were not affected by Src inhibition, indicating that Src may not be critical for skin blistering in intact human skin, at least when high levels of autoantibodies targeting Dsg1 are present.

Src and cortactin are involved in pemphigus skin blistering

Autoantibodies against desmoglein (Dsg) 1 and Dsg3 primarily cause blister formation in the autoimmune disease pemphigus vulgaris (PV). Src was proposed to contribute to loss of keratinocyte cohesion. However, the role and underlying mechanisms are unclear. In keratinocytes, cell cohesion in response to autoantibodies was reduced in a Src‐dependent manner by two patient‐derived PV‐IgG fractions as well as by AK23, but not by a third PV‐IgG fraction, although Src was activated by all autoantibodies. Loss of cell cohesion was progredient and AK23 similar to PV‐IgG interfered with reconstitution of cell cohesion after Ca2+‐switch, indicating that the autoantibodies also interfered with desmosome assembly. Dsg3 co‐localized along cell contacts and interacted with the Src substrate cortactin. Concomitantly, cell adhesion was impaired in keratinocytes isolated from cortactin‐deficient mice in comparison to keratinocytes isolated from wildtype (wt). AK23‐induced loss of cell cohesion was Src‐dependent only when cortactin was expressed. Similarly, AK23 impaired reconstitution of desmosomal adhesion in Src‐dependent fashion only in the presence of cortactin and AK23‐induced skin blistering was abolished by Src inhibition in wt but not cortactin‐deficient mice. However, in human epidermis, PV‐IgG‐induced skin blistering and ultrastructural alterations of desmosomes were not affected by Src inhibition. Our data suggest that Src and cortactin are involved in pemphigus skin blistering but the contribution of Src is variable.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Autoreactive IgG and IgA B Cells Evolve through Distinct Subclass Switch Pathways in the Autoimmune Disease Pemphigus Vulgaris

SUMMARYLineage analysis of autoreactive B cells can reveal the origins of autoimmunity. In the autoimmune disease pemphigus vulgaris (PV), desmoglein 3 (DSG3) and DSG1 autoantibodies are predominantly of the IgG4 subclass and less frequently of IgG1 and IgA subclasses, prompting us to investigate whether anti-DSG IgG4 B cells share lineages with IgG1, IgA1, and IgA2. Combining subclass-specific B cell deep sequencing with high-throughput antibody screening, we identified 80 DSG-reactive lineages from 4 PV patients. Most anti-DSG IgG4 B cells lacked clonal relationships to other subclasses and preferentially targeted DSG adhesion domains, whereas anti-DSG IgA frequently evolved from or to other subclasses and recognized a broader range of epitopes. Our findings suggest that anti-DSG IgG4 B cells predominantly evolve independently or diverge early from other subclasses and that IgA is most often not the origin of IgG autoreactivity in PV. These data provide insight into how autoreactivity diversifies across B cell subclasses.

Determinants of VH1-46 Cross-Reactivity to Pemphigus Vulgaris Autoantigen Desmoglein 3 and Rotavirus Antigen VP6.

Shared VH1-46 gene usage has been described in B cells reacting to desmoglein 3 (Dsg3) in the autoimmune disease pemphigus vulgaris (PV), as well as B cells responding to rotavirus capsid protein VP6. In both diseases, VH1-46 B cells bearing few to no somatic mutations can recognize the disease Ag. This intriguing connection between an autoimmune response to self-antigen and an immune response to foreign Ag prompted us to investigate whether VH1-46 B cells may be predisposed to Dsg3-VP6 cross-reactivity. Focused testing of VH1-46 mAbs previously isolated from PV and rotavirus-exposed individuals indicates that cross-reactivity is rare, found in only one of seven VH1-46 IgG clonotypes. High-throughput screening of IgG B cell repertoires from two PV patients identified no additional cross-reactive clonotypes. Screening of IgM B cell repertoires from one non-PV and three PV patients identified specific cross-reactive Abs in one PV patient, but notably all six cross-reactive clonotypes used VH1-46. Site-directed mutagenesis studies indicate that amino acid residues predisposing VH1-46 Abs to Dsg3 reactivity reside in CDR2. However, somatic mutations only rarely promote Dsg3-VP6 cross-reactivity; most mutations abolish VP6 and/or Dsg3 reactivity. Nevertheless, functional testing identified two cross-reactive VH1-46 Abs that both disrupt keratinocyte adhesion and inhibit rotavirus replication, indicating the potential for VH1-46 Abs to have both pathologic autoimmune and protective immune functions. Taken together, these studies suggest that certain VH1-46 B cell populations may be predisposed to Dsg3-VP6 cross-reactivity, but multiple mechanisms prevent the onset of autoimmunity after rotavirus exposure.

Reengineering chimeric antigen receptor T cells for targeted therapy of autoimmune disease.

Ideally, therapy for autoimmune diseases should eliminate pathogenic autoimmune cells while sparing protective immunity, but feasible strategies for such an approach have been elusive. Here, we show that in the antibody-mediated autoimmune disease pemphigus vulgaris (PV), autoantigen-based chimeric immunoreceptors can direct T cells to kill autoreactive B lymphocytes through the specificity of the B cell receptor (BCR). We engineered human T cells to express a chimeric autoantibody receptor (CAAR), consisting of the PV autoantigen, desmoglein (Dsg) 3, fused to CD137-CD3ζ signaling domains. Dsg3 CAAR-T cells exhibit specific cytotoxicity against cells expressing anti-Dsg3 BCRs in vitro and expand, persist, and specifically eliminate Dsg3-specific B cells in vivo. CAAR-T cells may provide an effective and universal strategy for specific targeting of autoreactive B cells in antibody-mediated autoimmune disease.

Plakophilins, Desmogleins, and Pemphigus: The Tail Wagging the Dog

SummaryThe importance of desmosomal cell adhesion to human health is evidenced by the autoimmune disease pemphigus vulgaris (PV), in which autoantibodies against the extracellular domain of the desmosomal cadherin desmoglein 3 cause potentially fatal blistering of the skin and mucous membranes. Tucker et al. describe how enhanced expression of a desmosomal cytoplasmic plaque protein, plakophilin-1, protects keratinocytes from PV IgG-induced loss of cell adhesion by inducing calcium-independent hyperadhesive desmosomes. This study beautifully demonstrates that desmosomal adhesion can be modulated by the molecular interactions of the desmoglein tail and suggests that these novel regulatory pathways may possibly be exploited in treating human disease.

Tandem peptide blocks pemphigus vulgaris skin blistering in vivo and identifies a desmoglein receptor function leading to p38MAPK modulation

In the life‐threatening autoimmune disease pemphigus vulgaris, epidermal blisters are caused by autoantibodies primarily targeting the cadherin‐type cell adhesion molecules desmoglein (Dsg) 3 and Dsg1 leading to loss of keratinocyte cohesion. Due to limited deeper insights in disease pathogenesis current therapy is restricted to relatively unspecific long term immunosuppression. Both, direct inhibition of Dsg trans‐interaction as well as altered intracellular signaling likely contribute to loss of cell adhesion. Here, we applied a tandem‐peptide (TP) in vivo consisting of two connected peptide sequences each targeting the Dsg adhesive interface. TP was capable to block autoantibody‐mediated direct interference of Dsg3 transinteraction as revealed by atomic force microscopy and optical trapping. Importantly, it was effective to completely abrogate autoantibody‐mediated skin blistering in mice. Relevant for a possible therapeutic use in patients, TP was effective also when applied topically. In addition, TP blunted autoantibody‐induced activation of p38MAPK, a central signaling pathway in pemphigus pathogenesis. These data indicate that p38MAPK links autoantibody‐induced direct inhibition of Dsg binding to skin blistering. By limiting both loss of Dsg trans‐interaction and subsequent p38MAPK activation, TP may serve as promising treatment option in pemphigus vulgaris. DFG SFB487

An Adult Passive Transfer Mouse Model to Study Desmoglein 3 Signaling in Pemphigus Vulgaris

Evidence has accumulated that changes in intracellular signaling downstream of desmoglein 3 (Dsg3) may play a significant role in epithelial blistering in the autoimmune disease pemphigus vulgaris (PV). Currently, most studies on PV involve passive transfer of pathogenic antibodies into neonatal mice which have not finalized epidermal morphogenesis, and do not permit analysis of mature hair follicles (HFs) and stem cell niches. To investigate Dsg3 antibody-induced signaling in the adult epidermis at defined stages of the HF cycle, we here developed a model with passive transfer of the monospecific pathogenic Dsg3 antibody AK23 into adult 8-week-old C57Bl/6J mice. Validated using histopathological and molecular methods, we found that this model faithfully recapitulates major features described in PV patients and PV models. Two hours after AK23 transfer we observed widening of intercellular spaces between desmosomes and EGFR activation, followed by increased Myc expression and epidermal hyperproliferation, desmosomal Dsg3 depletion and predominant blistering in HFs and oral mucosa. These data confirm that the adult passive transfer mouse model is ideally suited for detailed studies of Dsg3 antibody-mediated signaling in adult skin, providing the basis for investigations on novel keratinocyte-specific therapeutic strategies.

Deregulation of PERK in the autoimmune disease pemphigus vulgaris occurs via IgG-independent mechanisms

Serum and IgG isolated from patients with the autoimmune blistering disease pemphigus vulgaris (PV) trigger complex intracellular pathways in keratinocytes, including alterations of the cell cycle and metabolism, which ultimately lead to cell-cell detachment (acantholysis). We have shown previously that one of the earliest pathogenic events in PV is the activation of protein kinases, including the PKR-like endoplasmic reticulum (ER) kinase PERK. In the present study we investigated in more detail the role of PERK in the pathogenesis of PV. PERK levels were assessed by Western blotting and in-cell enzyme-linked immunosorbent assay, and PERK expression was silenced by siRNA technology. The effects of PV sera/IgG on keratinocyte cultures were investigated by flow cytometry, MTT and adhesion assays. We show that PERK is activated in keratinocytes exposed to PV serum, as demonstrated by an increase in phosphorylated PERK levels and phosphorylation of eIF2α. Decreased expression of PERK by siRNA reduced the effects of PV serum on the cell cycle and keratinocyte viability, two key events in PV pathophysiology. As impairment of metabolic activity in PV is partially due to non-IgG serum factors, we then investigated the activation of PERK in keratinocytes incubated with whole PV serum, purified PV IgG and IgG-depleted PV serum. The data demonstrated that PV sera depleted of IgG, but not PV IgG, triggered PERK phosphorylation and this correlated with a marked reduction of metabolic activity in keratinocytes exposed to IgG-free serum. Knockdown of PERK by siRNA abrogated the changes in the cell cycle and apoptosis induced by IgG-depleted PV serum. Finally, the reduction of metabolic activity observed in keratinocytes exposed to IgG-depleted PV serum was almost absent in PERK-deficient cells. Taken together, the results demonstrate that activation of PERK participates in the reduction of metabolic activity and cell viability seen in PV and that this phenomenon depends on non-IgG factors. PERK activation may represent a novel signalling mechanism linking ER stress and acantholysis in PV.

Desmosomal interactome in keratinocytes: a systems biology approach leading to an understanding of the pathogenesis of skin disease.

We provide the first description of the desmosome network in keratinocytes using a systems level approach. The desmo-adhesome consists of 59 proteins connected by 128 direct interactions and forms different functional subnets. Whilst the structure appears to be extremely robust against random perturbations, network fragmentation analysis suggests that the desmo-adhesome is susceptible to targeted attacks. To confirm this prediction, we applied this model to the autoimmune disease Pemphigus Vulgaris (PV), a paradigm of external perturbation of the desmosome. Our analysis showed that the adaptor protein plakophilin (Pkp) 3 was in the highest percentile group for both connectivity rate and gene expression changes in experimental PV. This observation led us to speculate that Pkp3 was crucial in desmosomal remodelling, and therefore we designed the experiments to verify this hypothesis. Our data demonstrate that, whilst Pkp3 is important in conferring adhesive strength to keratinocytes, it also acts as a central molecule mediating cell-cell detachment induced by PV IgG.

Pemphigus vulgaris identifies plakoglobin as key suppressor of c-Myc in the skin

The autoimmune disease pemphigus vulgaris (PV) manifests as loss of keratinocyte cohesion triggered by autoantibody binding to desmoglein (Dsg)3, an intercellular adhesion molecule of mucous membranes, epidermis, and epidermal stem cells. Here we describe a so far unknown signaling cascade activated by PV antibodies. It extends from a transient enhanced turn over of cell surface-exposed, nonkeratin-anchored Dsg3 and associated plakoglobin (PG), through to depletion of nuclear PG, and as one of the consequences, abrogation of PG-mediated c-Myc suppression. In PV patients (6/6), this results in pathogenic c-Myc overexpression in all targeted tissues, including the stem cell compartments. In summary, these results show that PV antibodies act via PG to abolish the c-Myc suppression required for both maintenance of epidermal stem cells in their niche and controlled differentiation along the epidermal lineage. Besides a completely novel insight into PV pathogenesis, these data identify PG as a potent modulator of epithelial homeostasis via its role as a key suppressor of c-Myc.

Dysfunction of keratinocyte adhesion.

Dysfunction of keratinocyte adhesion.

Replicative senescence enhances apoptosis induced by pemphigus autoimmune antibodies in human keratinocytes

We have recently shown that skin lesions of the autoimmune disease pemphigus vulgaris are associated with Fas-mediated apoptosis. Here, we describe the induction of the Fas-dependent apoptosis pathway in cultured keratinocytes by pemphigus vulgaris autoantibodies (PV-IgG), as seen from a variety of cellular, morphological and biochemical parameters. All apoptotic characters appear stronger and faster in aged cultures than in young, showing increased susceptibility of senescent keratinocytes to PV-IgG-mediated apoptotic death and culture lesions. Together with immunosenescence, this phenomenon may explain the late onset of pemphigus disease.

Postnatal Lethality of P-Cadherin/Desmoglein 3 Double Knockout Mice: Demonstration of a Cooperative Effect of these Cell Adhesion Molecules in Tissue Homeostasis of Stratified Squamous Epithelia

To investigate the cooperativity of different cell adhesion molecules in maintaining the structural integrity of the epidermis, we have generated mice deficient for both a classical cadherin, P-cadherin, and a desmosomal cadherin, desmoglein 3. In epithelial cells, P-cadherin is localized to the adherens junction, whereas desmoglein 3 is found in desmosomes. Previous studies have shown that these two junctional complexes are important for keratinocyte cell-cell adhesion. Both P-cadherin and desmoglein 3 expression are restricted to the basal and most immediate suprabasal cells of the epidermis, whereas both proteins are found throughout the oral mucosal epithelium. Although P-cadherin mutant mice have no apparent defect in epithelial cell adhesion, the desmoglein 3 mutant phenotype resembles that of patients with the autoimmune disease pemphigus vulgaris, in that the mice develop spontaneous mucous membrane blisters and trauma-induced skin blisters. The oral lesions in DSG3-/- mice reduce their food intake, resulting in a runted phenotype; however, most animals recover and live past weaning age. In contrast, animals mutant for both P-cadherin and desmoglein 3 die before weaning. The majority of the double mutant animals die around 1 wk after birth, apparently due to malnutrition. These studies suggest that loss of P-cadherin leads to a more severe desmoglein 3 mutant phenotype in the double knockout mice. This is the first in vivo evidence of possible synergism between a classical and desmosomal cadherin.

Adhesion molecules and related proteins in Darier's disease and Hailey-Hailey disease.

We have used antibodies to plakoglobin and E-cadherin: the lectins, peanut agglutinin (PNA) and soybean agglutinin (SBA); and sera from patients with the autoimmune diseases pemphigus vulgaris (PV) or pemphigus foliaceus (PF), in an immunohistological study of Darier's disease and Hailey-Hailey disease. There was normal expression of plakoglobin, E-cadherin, lectins and pemphigus antigens at the periphery of keratinocytes in uninvolved skin. Clumps of plakoglobin were detected within acantholytic cells in Hailey-Hailey disease, whereas expression was diffuse in acantholytic cells in Darier's disease. This difference may reflect differences in the pathogenesis of acantholysis. E-cadherin expression was weak or absent at the periphery of some acantholytic cells; lectin binding was sometimes reduced around acantholytic cells, and pemphigus antibodies did not bind to the acantholytic cells involved skin in either disease. Internalization, conformational changes or proteolysis may alter the expression of extracellular epitopes by acantholytic cells.