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Title: [Study on the protection of gingival epithelial barrier by interleukin-22 through regulating microbiota and E-cadherin expression].
Author: Zhang C, Zhang L, Ren JX, Li JY, Tan LP, Gao L, Zhao CJ.
Journal: Zhonghua Kou Qiang Yi Xue Za Zhi; 2024 Jul 09; 59(7):653-662. PubMed ID: 38949133.
Abstract:
Objective: To investigate the regulatory effect and mechanism of interleukin-22 (IL-22) on the gingival epithelial barrier in the context of periodontal inflammation. Methods: IL-22 knockout (IL-22 KO) mice were constructed, and periodontitis mice models were established through oral gavage with polymicrobial inoculation. DNAs were extracted from the oral plaques of IL-22 KO periodontitis mice group (n=7) and their wild-type littermates periodontitis group (n=7) to establish a periodontitis-related oral microbiota database"PD-RiskMicroDB", determining the relationship between changes in oral microbiota and microbial function in two groups using 16S rRNA sequencing results. Gingival epithelial cells (GEC) were cultured by modified trypsinization method, and were stimulated with 100 μg/L IL-22, Porphyromonas gingivalis (Pg) (multiplicity of infection:100), separately or together for 3 and 12 hours. The experimental groups were as follows: control group (no stimulation), IL-22 group, Pg group and Pg+IL-22 group. The expression of barrier protein E-cadherin in each group at 3 h was detected by immunofluorescence, real-time fluorescence quantitative PCR (RT-qPCR) and Western blotting. Fluorescein isothiocyanate-dextran-mediated epithelial cell permeability experiment was conducted to clarify the changes in permeability of GEC in each group at 3 and 12 h. The mRNA expressions of E-cadherin in the gingival epithelium of wild-type littermates periodontitis group and IL-22 KO periodontitis group were detected by RT-qPCR. Fifteen C57BL/6 wild-type mice were randomly divided into control group (n=5), periodontitis group (n=5) and periodontitis+IL-22 treatment group (n=5). RT-qPCR and immunohistochemistry (IHC) staining were used to detect the expression level of E-cadherin in the gingival epithelium of each group. Results: 16S rRNA sequencing results showed that the composition of oral microbiota changed in IL-22 KO periodontitis group, of which the abundance of bacterial genera related to periodontal tissue invasion was significantly increased (linear discriminant analysis score: 2.22, P=0.009), compared with wild-type littermates periodontitis group. In vitro cell experiments showed that after Pg infection for 3 hours, the cell connections of GEC in Pg group were interrupted, and the fluorescence intensity of E-cadherin was reduced in Pg group compared with the control group. Meanwhile, the mRNA and protein expression levels of E-cadherin (mRNA: 0.69±0.12; protein: 0.60±0.12) were downregulated compared with the control group [mRNA: 1.00±0.00 (P=0.043); protein: 1.04±0.08 (P=0.003)], respectively. The fluorescence intensity of E-cadherin in the Pg+IL-22 group was enhanced compared with Pg group, and expression levels of E-cadherin mRNA (1.16±0.10) and protein (0.98±0.07) in Pg+IL-22 group showed a significant increase compared with Pg group [mRNA: 0.69±0.12 (P=0.005); protein: 0.60±0.12 (P=0.007)]. The result of epithelial permeability test showed that there was no statistical difference in epithelial permeability among control group, Pg group, IL-22 group and Pg+IL-22 group with treatment for 3 hours (F=0.20, P=0.893). While when the treatment time turned to be 12 hours, the epithelial barrier permeability showed a significant increase in Pg group (1.39±0.15) compared with control group (1.00±0.00, P=0.027), and a decrease in Pg+IL-22 group (1.02±0.18) compared with Pg group (1.39±0.15, P=0.034). In vivo, the mRNA expression of E-cadherin in the gingival epithelium of IL-22 KO periodontitis group decreased significantly (0.32±0.21) compared with wild-type littermates periodontitis group (1.01±0.01) (t=5.70, P=0.005). Moreover, RT-qPCR and IHC staining results showed that the mRNA expression level of E-cadherin (0.40±0.07) and absorbance value of E-cadherin positive expression (0.02±0.00) in gingival epithelial tissue of periodontitis group were both significantly down-regulated compared with control group [mRNA: 1.00±0.00 (P=0.005); absorbance value of E-cadherin positive expression: 0.04±0.01 (P=0.006)]. Meanwhile, the mRNA expression level of E-cadherin (1.06±0.24) and the absorbance value of E-cadherin positive expression (0.03±0.01) were both observed increase in periodontitis+IL-22 treatment group compared with periodontitis group (P=0.003, P=0.039). Conclusions: IL-22 may exert a protective effect on the gingival epithelial barrier in an inflammatory environment by regulating the invasiveness of oral microbiota and the expression of host barrier protein. 目的： 研究白细胞介素-22（IL-22）在牙周炎症环境下对牙龈上皮屏障的调控作用及其可能机制。 方法： 构建IL-22敲除小鼠，采用混合菌液口腔灌洗法构建牙周炎模型。收集同窝生野生型牙周炎组及IL-22敲除牙周炎组小鼠（每组7只）口腔菌斑并提取DNA，建立牙周炎相关风险微生物数据库“PD-RiskMicroDB”并结合16S rRNA测序结果，测定两组小鼠口腔菌群变化和微生物功能的关系。通过改良胰酶消化法培养牙龈上皮细胞（GEC），以100 μg/L IL-22、感染复数为100的牙龈卟啉单胞菌（Pg）分别或联合刺激GEC，实验分组为：对照组（细胞未加刺激）、IL-22组、Pg组及Pg+IL-22组，处理时间为3和12 h；采用细胞免疫荧光、实时荧光定量PCR（RT-qPCR）及蛋白质印迹法检测各组细胞3 h后上皮屏障蛋白E-钙黏蛋白（E-cadherin）的表达；通过异硫氰酸荧光素-葡聚糖（FITC-D）介导的上皮细胞通透性实验明确各组GEC 3和12 h细胞通透性的变化。RT-qPCR检测同窝生野生型牙周炎组及IL-22敲除牙周炎组小鼠牙龈上皮E-cadherin表达水平。将15只C57BL/6野生型小鼠按随机数字表法随机分为对照组、牙周炎组和牙周炎+IL-22处理组，每组5只。RT-qPCR及免疫组织化学（IHC）染色法检测各组小鼠牙龈上皮E-cadherin的表达水平。 结果： 16S rRNA测序结果显示，与同窝生野生型牙周炎组小鼠相比，IL-22敲除牙周炎组小鼠口腔菌群组成发生改变，其中与牙周组织侵袭相关的菌属丰度显著增高（线性判别分析得分为2.22，P=0.009）。体外细胞实验显示，Pg感染GEC 3 h后，Pg组GEC的细胞连接中断，Pg组E-cadherin荧光强度较对照组减弱，E-cadherin的mRNA（0.69±0.12）和蛋白（0.60±0.12）表达水平均显著低于对照组（分别为1.00±0.00、1.04±0.08）（P=0.043，P=0.003）；而Pg+IL-22组的E-cadherin荧光强度较Pg组稍增强，Pg+IL-22组E-cadherin的mRNA（1.16±0.10）和蛋白（0.98±0.07）表达水平均显著高于Pg组（分别为0.69±0.12、0.60±0.12）（P=0.005，P=0.007）；上皮通透性实验结果显示，Pg处理GEC 3 h后，对照组、IL-22组、Pg组及Pg+IL-22组各组间总体比较上皮屏障通透性差异无统计学意义（F=0.20，P=0.893）；处理12 h后，相较于对照组（1.00±0.00），Pg组GEC上皮屏障通透性（1.39±0.15）显著增加（P=0.027），而Pg+IL-22组上皮屏障通透性（1.02±0.18）显著低于Pg组（P=0.034）；体内RT-qPCR结果显示，IL-22敲除牙周炎组小鼠牙龈上皮E-cadherin的mRNA表达水平（0.32±0.21）显著低于同窝生野生型牙周炎组（1.01±0.01）（t=5.70，P=0.005）。RT-qPCR及IHC染色结果显示，牙周炎组小鼠牙龈上皮组织E-cadherin的mRNA表达水平（0.40±0.07）和E-cadherin阳性表达吸光度值（0.02±0.00）均显著低于对照组（分别为1.00±0.00、0.04±0.01）（P=0.005，P=0.006）；牙周炎+ IL-22处理组E-cadherin的mRNA表达水平（1.06±0.24）和E-cadherin阳性表达吸光度值（0.03±0.01）均显著高于牙周炎组（P=0.003，P=0.039）。 结论： IL-22可能通过调节口腔菌群的侵袭性以及宿主屏障蛋白表达，发挥其对炎症环境下牙龈上皮屏障的保护作用。.

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