Effect of fecal bacteria transplantation on immune state of intestinal T cell and intestinal inflammation in septic mice
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摘要:
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。 Abstract: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. -
Key words:
- sepsis /
- fecal microbiota transplantation /
- intestines /
- immunity /
- T-lymphocytes /
- mice
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图 2 经与未经粪菌移植治疗的脓毒症小鼠结肠组织病理学改变(HE染色，标尺=200 μm)
Figure 2. Representative images of colon tissues sections in septic mice with or without FMT treatment (HE staining, scale bar=200 μm)
图 3 经与未经粪菌移植治疗的脓毒症小鼠小肠组织病理学改变(HE染色，比例尺=200 μm)
Figure 3. Representative images of small intestine tissues sections in septic mice with or without FMT treatment (HE staining, scale bar=200 μm)
图 4 各组小鼠小肠固有层T细胞及其亚群数量变化(n=4)
Figure 4. Relative numbers of T cells, CD4 + T cells and CD8 + T cells in lamina propria of small intestine in each group (n=4)
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