粪菌移植对脓毒症小鼠肠道T细胞免疫稳态和肠道炎症的作用

Effect of fecal bacteria transplantation on immune state of intestinal T cell and intestinal inflammation in septic mice

背景脓毒症是重症患者的主要死亡原因,目前缺乏有效的治疗手段,近年来粪菌移植(fecal microbiota transplantation,FMT)治疗脓毒症受到越来越多关注。目的探讨粪菌移植治疗的免疫学机制,明确粪菌移植治疗对脓毒症小鼠肠道T细胞免疫状态和功能以及肠道炎症状态的影响,为临床治疗提供理论依据。方法8~10周龄C57BL/6小鼠随机分为对照组、脓毒症组和粪菌移植组,后两组通过腹腔注射脂多糖构建脓毒症小鼠模型,粪菌移植组小鼠在脂多糖注射24 h后以灌胃的方式接受来自健康小鼠的粪菌移植,检测各组小鼠肠道固有层T细胞及其亚群以及表面功能性分子的表达,测量结肠长度,检测小肠和结肠组织的病理改变。结果相比对照组小鼠,脓毒症组小鼠小肠T淋巴细胞显著减少(22.03%±1.50%vs 30.78%±1.62%,P<0.01),其中CD4^(+)T细胞(15.08%±1.01%vs 21.58%±1.95%,P<0.01)和CD8^(+)T细胞(5.05%±0.39%vs 6.28%±0.21%,P<0.01)均显著减少,小肠CD4^(+)T细胞(227.25±11.47 vs 266.00±6.27,P<0.01)和CD8^(+)T细胞(73.40±6.82 vs 102.80±4.80,P<0.01)表面CD28的表达显著下调,CD8^(+)T细胞表面PD-1的表达(19.90±1.47 vs 15.00±2.25,P<0.05)显著上调,差异均有统计学意义,说明脓毒症小鼠肠道T细胞的数量和功能受到抑制,结肠长度显著缩短[(6.90±0.08)cm vs(8.18±0.22)cm,P<0.01],组织病理结果显示小肠和结肠均出现明显的炎症和损伤;而与脓毒症组小鼠相比,粪菌移植组小鼠肠道T细胞(26.43%±1.86%vs 22.03%±1.50%,P<0.05),包括CD4^(+)T细胞(17.48±0.85%vs 15.08%±1.01%,P<0.05)和CD8^(+)T细胞(6.74%±0.39%vs 5.05%±0.39%,P<0.01)数量均出现回升,CD4^(+)T细胞(252.25±7.14 vs 227.25±11.47,P<0.05)和CD8^(+)T细胞(95.30±3.63 vs 73.40±6.82,P<0.01)表面CD28的表达出现回升,CD8^(+)T细胞表面PD-1的表达(17.10±0.52 vs 19.90±1.47,P<0.05)出现回降,差异均有统计学意义,提示经粪菌移植治疗的脓毒症小鼠肠道T细胞的数量和功能得到一定恢复,结肠长度明显恢复[(8.13±0.26)cm vs(6.90±0.08)cm,P<0.01],组织病理结果显示小肠和结肠的炎症和损伤得到了明显的缓解。结论经粪菌移植治疗的脓毒症小鼠肠道T细胞数量和功能得到部分恢复,肠道T细胞的免疫稳态得到一定程度的恢复,并且显著缓解了脓毒症小鼠肠道的炎症和损伤。

Background Sepsis is the major cause of death in critically ill patients,but effective treatment strategies for sepsis are still lacking.Fecal microbiota transplantation(FMT)has recently gained concern as a potential treatment for sepsis.Objective To investigate the effects of FMT on the immune status of intestinal T cells and intestinal inflammation in septic mice.Methods C57BL/6 mice aged 8-10 weeks were randomly divided into control group,sepsis group and FMT group.The murine model of sepsis was established by intraperitoneal injection of lipopolysaccharide(LPS).After 24 h of LPS injection,the mice in FMT group received FMT from healthy mice.All mice were sacrificed at 48 h after injection of LPS or PBS.The numerical and phenotypic alterations of T cells in lamina propria of intestinal,colon length and the pathological changes in small intestine and colon were detected.Results Compared with the control group,the number of T lymphocytes(22.03%±1.50%vs 30.78%±1.62%,P<0.01),CD4^(+)T cells(15.08%±1.01%vs 21.58%±1.95%,P<0.01)and CD8^(+)T cells(5.05%±0.39%vs 6.28%±0.21%,P<0.01)in lamina propria of intestinal in sepsis group decreased significantly.The MFIs of CD28 on CD4^(+)T cells(227.25±11.47 vs 266.00±6.27,P<0.01)and CD8^(+)T cells(73.40±6.82 vs 102.80±4.80,P<0.01)were down-regulated significantly,while the MFI of PD-1 on CD8^(+)T cells(19.90±1.47 vs 15.00±2.25,P<0.05)was up-regulated significantly.Colon lengths were significantly shortened([6.90±0.08]cm vs[8.18±0.22]cm,P<0.01),and the small intestine and colon showed obvious inflammation and injury.Compared with the sepsis group,the number of intestinal T cells(26.43%±1.86%vs 22.03%±1.50%,P<0.05),including CD4^(+)T cells(17.48±0.85%vs 15.08%±1.01%,P<0.05)and CD8^(+)T cells(6.74%±0.39%vs 5.05%±0.39%,P<0.01)in the fecal bacteria transplantation group increased significantly.The MFIs of CD28 on CD4^(+)T cells(252.25±7.14 vs 227.25±11.47,P<0.05)and CD8^(+)T cells(95.30±3.63 vs 73.40±6.82,P<0.01)also increased,while the MFI of PD-1 on CD8^(+)T cells(17.10±0.52 vs 19.90±1.47,P<0.05)decreased,and the length of colon increased significantly([8.13±0.26]cm vs[6.90±0.08]cm,P<0.01).The inflammation and injury of small intestine and colon were significantly relieved.Conclusion The immune status and homeostasis of intestinal T cells are partly restored,and intestinal inflammation and injury are significantly alleviated by FMT treatment in septic mice.