Abstract: 【Abstract】 Objective To investigate the role of Gasdermin E （GSDME）-mediated keratinocyte pyroptosis induced by Cutibacterium acnes （C.acnes） in the pathogenesis of acne. Methods The human immortalized keratinocyte HaCaT cells were stimulated with heat-inactived C.acnes for 15 minutes to 24 hours, and Western blot analysis was performed to determine the expression of cleaved GSDME（GSDME-NT）in HaCaT cells at different time points. Skin tissue samples were collected from 5 acne patients and 4 healthy controls, who visited the Hospital for Skin Diseases, Chinese Academy of Medical Sciences from January 2 to December 1, 2024; additionally, 3 samples of acne cyst contents and 3 samples of normal follicle contents were collected. Immunohistochemical study and Western blot analysis were conducted to determine GSDME-NT expression in the epidermis. Enzyme-linked immunosorbent assay （ELISA） was performed to detect levels of interleukin （IL）-1β and tumor necrosis factor （TNF）-α in acne cyst or normal follicle contents. GSDME-knockdown HaCaT cells were constructed by transfection with lentivirus carrying GSDME-shRNA, and HaCaT cells transfected with lentivirus carrying the nonsense sequence control （NC） served as controls; ELISA was performed to detect the levels of IL-1β and TNF-α in GSDME-knockdown HaCaT cells after C. acnes stimulation （C. acnes + GSDME knockdown group）, as well as in a phosphate-buffered saline （PBS） + NC group, a C. acnes + NC group, and a PBS + GSDME knockdown group. Western blot analysis was conducted to determine the GSDME-NT expression in HaCaT cells pretreated with or without retinol after C. acnes stimulation. Results The cleavage of GSDME in HaCaT cells began at 1 hour after in vitro C. acnes stimulation, with the intracellular detection of GSDME-NT. Compared with the control epidermis, the proportion of GSDME-NT-positive HaCaT cells （9.34% ± 2.92% vs. 3.05% ± 1.14%, t = -3.47, P = 0.026） and GSDME-NT protein expression levels （t = -3.51, P = 0.025） significantly increased in the lesional epidermis of acne patients. The levels of IL-1β （1,337.24 ［1,182.32, 2,230.61］ pg/ml） and TNF-α （811.31 ［438.26, 817.73］ pg/ml） were significantly higher in the acne cyst contents than in the normal follicle contents （IL-1β: 0.00 ［0.00, 108.21］ pg/ml, Z = 1.99, P = 0.046; TNF-α: 46.67 ［12.41, 53.21］ pg/ml, Z = 1.96, P = 0.049）. ELISA showed that the levels of IL-1β and TNF-α were significantly higher in the C. acnes + NC group （12.12 ± 3.07 pg/ml, 26.06 ± 1.57 pg/ml, respectively） than in the PBS + NC group （3.73 ± 2.24 pg/ml, 10.14 ± 0.79 pg/ml, P = 0.003, < 0.001, respectively）; compared with the C. acnes + NC group, the levels of IL-1β and TNF-α significantly decreased in the C. acnes + GSDME knockdown group （3.38 ± 0.93 pg/ml, 12.67 ± 2.10 pg/ml, P = 0.003, < 0.001, respectively）. The GSDME-NT expression was significantly lower in the retinol + C. acnes group than in the C. acnes group （P = 0.029）. Conclusion C. acnes may induce GSDME-mediated pyroptosis in keratinocytes, thereby promoting the release of inflammatory factors and aggravating the inflammatory response in acne. Retinol may inhibit this process.

Key words: Acne vulgaris, Propionibacterium acnes, Pyroptosis, HaCaT cells, GSDME, Interleukin-1beta, Tumor necrosis factor-alpha, Retionl