Abstract: 【Abstract】 Objective To investigate molecular mechanisms underlying the inflammatory response induced by Cutibacterium acnes （C. acnes） biofilms in human primary keratinocytes. Methods A C. acnes biofilm model was established in vitro, and confocal fluorescence microscopy was performed to examine its three-dimensional structure. The cultured human primary keratinocytes were divided into 3 groups: a dimethyl sulfoxide （DMSO） control group （treated with 0.01% DMSO alone）, a C. acnes suspension group （co-incubated with C. acnes suspensions）, and a C. acnes biofilm group （co-incubated with C. acnes biofilms）. Real-time fluorescence-based quantitative PCR （RT-qPCR） was performed to determine the relative mRNA expression of interleukin （IL）-6, IL-8, and tumor necrosis factor （TNF）-α in the groups after 6-hour culture, enzyme-linked immunosorbent assay to detect the free protein levels of IL-6, IL-8, and TNF-α in the groups after 24-hour culture, and Western blot analysis to determine the protein expression of Toll-like receptor 2 （TLR2） in keratinocytes. In addition, some human primary keratinocytes were pretreated with key molecular blockers targeting the TLR2/mitogen-activated protein kinase （MAPK）/nuclear factor （NF）-κB signaling pathway （C29, ST2825, BAY11-7082, SB203580, U0126-EtOH）, and then co-incubated with C. acnes biofilms; the DMSO control group and the C. acnes biofilm group receiving no pretreatment were simultaneously set as negative and positive controls, respectively. The mRNA and free protein expression levels of IL-6, IL-8, and TNF-α were then detected in the above groups. One-way analysis of variance was used for comparisons among multiple groups, and the Bonferroni method was used for multiple comparisons. Results Confocal fluorescence microscopy demonstrated a three-dimensional C. acnes biofilm structure resembling a lawn, and the biofilm grew well. RT-qPCR and ELISA showed significant differences in the mRNA and free protein expression levels of IL-6, IL-8, and TNF-α among the C. acnes biofilm group, C. acnes suspension group and DMSO control group （mRNA: F = 89.70, 312.17, 46.09, respectively, all P < 0.001; free protein: F = 886.12, 634.25, 307.01, respectively, all P < 0.001）; in detail, the mRNA and free protein expression levels of IL-6, IL-8, and TNF-α were significantly higher in the C. acnes biofilm group than in the C. acnes suspension group and DMSO control group （all P < 0.001）; the C. acnes suspension group showed significantly increased expression levels of IL-6 mRNA and TNF-α free protein compared with the DMSO control group （P < 0.001, = 0.003, respectively）, while there were no significant differences in the expression of IL-6 free protein, TNF-α mRNA, or IL-8 mRNA and free protein between the 2 groups （all P > 0.05）. Western blot analysis showed that the TLR2 protein expression was significantly higher in the C. acnes suspension group and C. acnes biofilm group than in the DMSO control group. After the pretreatment with molecular blockers targeting the MAPK/NF-κB signaling pathway and co-incubation with C. acnes biofilms, the mRNA and free protein expression levels of IL-6, IL-8 and TNF-α were all significantly lower in the C29 group, ST2825 group, BAY11-7082 group, SB203580 group, U0126-EtOH group, as well as in the DMSO control group compared with the C. acnes biofilm group （all P < 0.05）. Conclusion The C. acnes biofilms exhibited a strong ability to induce inflammatory responses in human keratinocytes, possibly through the activation of the TLR2/MAPK/NF-κB signaling pathway.

Key words: Propionibacterium acnes, Biofilms, Keratinocytes, Toll-like receptor 2, Inflammatory reaction, MAPK/NF-κB signaling pathway