A new ex vivo human oral mucosa model reveals that p38 MAPK inhibition is not effective in preventing autoantibody‐induced mucosal blistering in pemphigus

Abstract

Summary Background Pemphigus vulgaris (PV) is an autoimmune disease characterized by blister formation in the epidermis and oral mucosa due to loss of keratinocyte cohesion. Autoantibodies present in patients with PV (PV‐IgG) are known to primarily target desmoglein (Dsg)1 and Dsg3 in desmosomes. The mucosal‐dominant subtype of PV (mdPV) is caused by PV‐IgG autoantibodies against the cadherin‐type adhesion molecule Dsg3. p38 mitogen‐activated protein kinase (p38MAPK) signalling has been characterized as an important pathway downstream of PV‐IgG binding and its inhibition is protective in ex vivo human skin. However, the role of p38MAPK signalling in mdPV is unknown as no experimental model has been available. Objectives To establish a human ex vivo oral mucosa culture, and evaluate the p38MAPK dependency of blister formation and of ultrastructural alterations of desmosomes induced by mdPV‐IgG. Methods Human labial mucosa was injected with mdPV‐IgG as well as AK23, a pathogenic mouse monoclonal Dsg3 antibody, in the presence or absence of p38MAPK inhibitors. Viability was evaluated by 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay and apoptosis by terminal deoxynucleotidyl transferase dUTP nick‐end labelling assay. Blister score was determined following haematoxylin and eosin staining and Dsg3 distribution by immunostaining. Samples were processed for transmission electron microscopy to analyse desmosome ultrastructure. Results Both AK23 and mdPV‐IgG induced blisters and caused reduction in desmosome size and number in labial mucosa. Inhibition of p38MAPK was not effective in preventing these alterations. Conclusions In contrast with human epidermis, PV‐IgG and AK23 induce blisters and desmosome ultrastructural changes in labial mucosa via a mechanism not dependent on p38MAPK. What's already known about this topic? Pemphigus vulgaris IgG (PV‐IgG) induces blistering as well as a reduction in desmosome number and size mediated by p38 mitogen‐activated protein kinase (p38MAPK) signalling in ex vivo human skin. What does this study add? This study establishes a new human ex vivo mucosa model to test pathomechanisms mediated by PV‐IgG. The study demonstrates that both AK23 and mucosal‐dominant PV induce blisters and associated ultrastructural changes in labial mucosa via a mechanism not dependent on p38MAPK signalling. What is the translational message? This study highlights the respective tissue‐specific responses of oral mucosa and skin related to PV pathogenesis, similar to the patient situation.

Role of PKC and ERK Signaling in Epidermal Blistering and Desmosome Regulation in Pemphigus

“…Using a similar model, blister formation in human skin has been sufficiently blocked by inhibition of p38MAPK signaling (18) whereas in mucosa explant culture inhibition of p38MAPK was not protective against PV-IgG-induced blistering (19). Previous experiments have demonstrated that inhibition of PKC and ERK pathways sufficiently abrogated cell dissociation in keratinocyte cell cultures (17, 20, 25) and deactivation of PKC signaling in neonatal pemphigus mouse model effectively blunted blister formation (30, 31).…”

“…It is activated secondary to PV-IgG binding and its phosphorylation is detected in lesioned skin of PV patients (14) and in keratinocyte cell cultures treated with PV-IgG (15, 16). Interestingly, pharmacologic inhibition of p38MAPK effectively prevented all PV-IgG-induced features of the disease in keratinocyte cell cultures (15, 17), animal models (8), and in ex vivo human skin (18) but not in an ex vivo model of human mucosa (19). As a result, p38MAPK is believed to be central in PV pathogenesis, at least with respect to skin blister formation.…”

Milestones in Personalized Medicine in Pemphigus and Pemphigoid

“…Upon targeting of Dsg1, Ca 2+ influx is induced and the ERK pathway is activated. In contrast, after binding of Dsg3-specific IgG, signaling via p38MAPK occurs in the epidermis but not in mucosal tissues, and SRC family of protein tyrosine kinases and EGFR pathways are activated ( 51 , 52 ). Current data also strongly suggest that, in addition to Dsg1/3 autoantibodies, non-Dsg antibodies, as well as soluble Fas ligand contribute to the pemphigus phenotype ( 53 – 56 ).…”

Tissue‐specific personalized medicine: the next level of individualized treatment

“…While in oncology personalized medicine approaches are becoming relatively well established, personalized treatment decisions have just begun to emerge in chronic inflammatory diseases. One such example is the article by Egu and colleagues in this issue of the BJD . Herein the authors first established a new model of mucosal pemphigus vulgaris (PV) and then contrasted the molecular pathways leading to blistering in this disease in skin and mucosa.…”

“…However, despite the fact that both the skin and mucosa are affected by pemphigus, most research in pemphigus is carried out using keratinocytes – hence, these insights are limited to the skin lesions of pemphigus. In this issue, Egu and colleagues, following their work on p38 MAPK in the skin, investigated the role of p38 MAPK in mucosal pemphigus . For this purpose, they established a human ex vivo mucosal cell culture system to evaluate the p38 MAPK dependency of mucosal blister formation.…”