Different roles of M3 AChR and α9 AChR in the pathogenesis of pemphigus vulgaris

Objective: Pemphigus vulgaris (PV) is a severe autoimmune skin disease, and the role of acetylcholine receptor (AchR) in PV is still unclear. This study aimed to investigate the effects of M3 acetylcholine receptor (AChR) and α9 AChR interference on acantholysis of keratinocytes, which may provide a deeper understanding of PV pathogenesis. Methods: Sera from patient with PV and controls were collected from Hospital for Skin Diseases (Institute of Dermatology), Chinese Academy of Medical Sciences & Peking Union Medical College from January 1, 2016 to December 30, 2016, and were used to extract PV-IgG. RNA interference was used to silence the M3 AChR gene or α9 AChR gene in HaCaT cells, after which the cells were divided into two groups: one treated with PV-IgG and the other without PV-IgG treatment. Changes in the adhesion of HaCaT cells were observed using a cell dissociation assay. Immunofluorescence assay was used to detect the staining patterns of desmosome proteins. Western blot analysis was performed to detect cytoskeleton- or non-cytoskeleton-associated desmosome protein levels. The interactions of desmoglein-3 and plakoglobin (PG) were qualitatively detected using co-immunoprecipitation, and the p38 MAPK and epidermal growth factor receptor (EGFR) phosphorylation levels were examined. Two-tailed Student’s t -test was used to compare the data of the two groups. Results: With M3 AChR silencing, the HaCaT cells treated with PV-IgG induced severe acantholysis ( P <0.001); levels of cytoskeleton-associated desmoglein and PG were significantly reduced ( P<0.001 and P<0.001 ), whereas the phosphorylation of EGFR increased ( P=0.001 ). In contrast, α9 AChR silencing appeared to exert protective effects during acantholysis caused by PV-IgG ( P < 0.001). The coculture of α9 AChR-silenced cells with PV-IgG suppressed the internalization of desmoglein, and decreased levels of cytoskeleton-associated desmoglein and PG ( P <0.001 and P = 0.002 ), and a close interaction between desmoglein-3 and PG was observed ( P = 0.002). The phosphorylation levels of p38 MAPK and EGFR were suppressed by α9 AChR silencing (both P < 0.001). Conclusion: The findings of this study provided evidence that M3 AChR and α9 AChR may play roles in the pathogenesis of PV, demonstrating that M3 AChR exerts a pro-pathogenic effect, whereas α9 AChR plays a protective role in PV IgG-induced acantholysis.