牙龈卟啉单胞菌脂多糖通过髓样细胞触发受体-1调控巨噬细胞极化状态的研究

目的探究牙龈卟啉单胞菌(Porphyromonas gingivalis,Pg)脂多糖(lipopolysaccharide,LPS)刺激巨噬细胞后,髓样细胞触发受体-1(triggering receptors expressed on myeloid cells-1,TREM-1)表达与M1、M2型极化的关系,进一步探讨巨噬细胞TREM-1在牙周炎发生发展中的作用机制。方法使用豆蔻酰佛波醇乙酯诱导人单核细胞系THP-1分化为巨噬细胞,予以0(空白对照组)和1μg/ml的Pg-LPS(LPS组)刺激,同期加入终质量浓度为0.1μg/ml的TREM-1抑制剂LP17(LPS+LP17组)或对照肽(LPS+对照肽组),培养24 h后用实时荧光定量PCR(real-time quantitative-PCR,RT-qPCR)检测TREM-1和巨噬细胞M1、M2型极化标志物(分别为CD86、CD206)及其极化相关细胞因子分别为肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素(interleukin,IL)-1β、IL-10]mRNA表达变化,蛋白质印迹法检测TREM-1、CD86、CD206蛋白含量,酶联免疫吸附测定法检测巨噬细胞培养上清液中TNF-α、IL-1β及IL-10的表达。结果细胞培养24 h后,LPS组巨噬细胞中TREM-1相对mRNA表达量(1.40±0.14)及蛋白表达量(3.85±0.24)均较空白对照组(分别为1.01±0.18、1.00±0.05)显著升高(P<0.05),同时M1型极化标志物CD86 mRNA及蛋白表达(分别为1.42±0.01、1.55±0.07)均较空白对照组(分别为1.00±0.09、1.00±0.10)显著上调(P<0.01),且M1型极化相关细胞因子TNF-α、IL-1βmRNA及蛋白表达量均显著增加(P<0.05);加入TREM-1阻断剂LP17后,与LPS组相比,TREM-1 mRNA及蛋白表达水平差异均无统计学意义(P>0.05),CD86 mRNA(0.96±0.00)和蛋白(1.36±0.02)表达水平均显著下降(P<0.05),TNF-α、IL-1βmRNA及蛋白表达水平均显著降低(P<0.05)。对于M2型极化标志物CD206及极化相关细胞因子IL-10,细胞培养24 h后,CD206 mRNA(0.56±0.05)及蛋白表达(0.25±0.04)均较空白对照组(分别为1.02±0.25、1.00±0.10)显著下调(P<0.01),IL-10 mRNA较空白对照组表达显著上调(P<0.05),其蛋白表达水平与空白对照组相比差异无统计学意义(P>0.05);LPS+LP17组CD206、IL-10 mRNA表达均较LPS组显著下调(P<0.05),CD206、IL-10蛋白表达在两组间差异无统计学意义(P>0.05)。结论TREM-1及其下游信号通路可能参与了Pg-LPS介导的巨噬细胞M1型极化,从而在牙周炎发生发展中发挥促炎作用;尚无证据表明TREM-1是否参与调控Pg-LPS介导的巨噬细胞M2型极化。

Porphyromonas gingivalis lipopolysaccharide regulates macrophage polarization via triggering receptors expressed on myeloid cells-1

Objective To investigate the relationship between pattern recognition receptor triggering receptors expressed on myeloid cells-1(TREM-1)and M1/M2 polarization in macrophages stimulated by Porphyromonas gingivalis lipopolysaccharide(Pg-LPS),so as to explore the mechanism of TREM-1 in periodontitis.Methods Human monocytic cell line THP-1 were induced to differentiate into macrophages by phorbol-12-myristate-13-acetate and stimulated by 0(blank control group)and 1μg/ml Pg-LPS(LPS group),respectively.LP17,the TREM-1 inhibitor(LPS+LP17 group)and its control peptide(LPS+control peptide group)with final concentration of 0.1μg/ml were added at the same time.After 24 hours stimulation,the expression of TREM-1,M1 markers and related cytokinesCD86,tumor necrosis factor-α(TNF-α),interleukin(IL)-1β],M2 markers and related cytokines(CD206,IL-10)mRNA were detected by real-time quantitative-PCR(RT-qPCR),the level of TREM-1,CD86 and CD206 proteins were detected by Western blotting,and TNF-α,IL-1βand IL-10 in the macrophage culture supernatant were detected by enzyme linked immunosorbent assay.Results After 24 h of cell culture,the relative expressions of TREM-1 mRNA(1.40±0.14)and protein(3.85±0.24)in macrophages in the LPS group increased compared with the blank control group(1.01±0.18 and 1.00±0.05,respectively)(P<0.05).Meanwhile,the expression levels of M1 markers CD86 mRNA and proteinLPS group vs blank control group were(1.42±0.01 vs 1.00±0.09)and(1.55±0.07 vs 1.00±0.10),respectively]were up-regulated(P<0.01),and the expressions of mRNA and protein of M1 related cytokines TNF-αand IL-1 increased(P<0.05).After the addition of TREM-1 blocker LP17,the levels of mRNA and protein of TREM-1 showed no significant changes(P>0.05),while the levels of CD86 mRNA(0.96±0.00)and protein(1.36±0.02)decreased(P<0.05),and the levels of TNF-αand IL-1 further decreased(P<0.05).For M2 marker CD206 and related cytokine IL-10,CD206 mRNA(0.56±0.05)and protein(0.25±0.04)were significantly down-regulated(P<0.01)compared with the blank control group(1.02±0.25 and 1.00±0.10,respectively),and IL-10 mRNA was up-regulated compared with the blank control group(P<0.05),with no significant change in protein(P>0.05).After the addition of LP17,the expressions of CD206 and IL-10 mRNA in the LPS+LP17 group were further down-regulated compared with the LPS group(P<0.05),and there was no statistically significant difference between the two groups in protein level(P>0.05).Conclusions TREM-1 and its downstream signaling pathway might be involved in M1 polarization of Pg-LPS-mediated macrophages,thus playing a pro-inflammatory role in the development of periodontitis.There is no obvious evidence that TREM-1 is involved in regulating M2 polarization of Pg-LPS-mediated macrophages.