内毒素打击后小鼠脾脏T淋巴细胞功能恢复动态研究

doi:10.3877/cma.j.issn.1674-1358.2023.01.006

目的

探讨脂多糖（LPS）打击后，小鼠脾脏T淋巴细胞亚群及功能的动态恢复过程。

方法

采用高剂量LPS（10 mg/kg）腹腔单次注射建立小鼠脓毒症模型，在LPS打击后第0 d、7 d，14 d和28 d，使用流式细胞术动态检测脾脏CD4⁺ T和CD8⁺ T细胞中初始T细胞、效应记忆T细胞和中央记忆T细胞的比例以及细胞表面活化分子（CD38和CD69）和共抑制分子（PD-1和TIGIT）的表达水平，分泌细胞因子IL-2和IFN-γ及释放颗粒酶B的能力。正态分布数据采用one-way ANOVA检验进行方差分析，Holm-Sidak’s进行多重比较；偏态分布数据则采用Kruskal-Wallis检验进行分析，Bonferroni法进行多重比较。

结果

LPS打击7 d后，与基线相比，小鼠脾脏初始CD4⁺ T和CD8⁺ T细胞比例均显著降低（F = 52.22、P < 0.0001，F = 10.87、P = 0.0019）；效应记忆CD4⁺ T和CD8⁺ T细胞比例均显著增高（F = 20.54、P < 0.0001，F = 26.03、P < 0.0001）；CD4⁺CD38⁺（F = 35.40、P < 0.0001）、CD4⁺CD69⁺（F = 45.65、P < 0.0001）、CD4⁺PD-1⁺（F = 20.55、P < 0.0001）、CD4⁺TIGIT⁺（F = 19.20、P < 0.0001）、CD8⁺CD38⁺（F = 56.76、P < 0.0001）、CD8⁺CD69⁺（F = 59.47、P < 0.0001）、CD8⁺PD-1⁺（F = 11.15、P = 0.0013）和CD8⁺TIGIT⁺（F = 21.21、P < 0.0001）细胞比例均显著增高，差异均有统计学意义。与基线相比，LPS打击14 d后细胞活化水平、共抑制分子表达水平逐渐恢复，但至28 d时，初始CD4⁺ T细胞（F = 52.22、P < 0.0001）、效应记忆CD4⁺ T细胞（F = 20.54、P = 0.0093）、CD4⁺CD69⁺（F = 45.65、P = 0.0037）、CD4⁺PD-1⁺（F = 20.55、P = 0.0255）和CD4⁺TIGIT⁺（F = 19.20、P = 0.0087）未恢复至基线水平；CD8⁺IFN-γ⁺ T细胞比例（F = 14.33、P = 0.0343）仍高于基线水平，差异均有统计学意义。

结论

LPS打击后28 d小鼠脾脏CD4⁺ T和CD8⁺ T细胞恢复过程存在差异，CD4⁺ T细胞功能总体恢复较CD8⁺ T细胞更为缓慢。

关键词: 脓毒症, 脾脏, 淋巴细胞, 活化, 共抑制分子

Objective

To investigate the dynamic recovery process of splenic T lymphocyte function and their subsets in mice after lipopolysaccharide (LPS) administration.

Methods

High dose LPS (10 mg/kg, once) was administered intraperitoneally to establish septic mouse model. On day 0, day 7, day 14, and day 28 after LPS treatment, flow cytometry was used to check the proportions of naive T cells, effector memory T cells, and central memory T cells in splenic CD4⁺ and CD8⁺ T cells. The expression of cell surface activation molecules (CD38, CD69) and co-inhibitory molecules (PD-1, TIGIT), as well as the ability of splenic CD4⁺ T cells to secrete cytokines IL-2, IFN-γ and CD8⁺ T cells to release granzyme B. Normal distribution data used one-way ANOVA test for variance analysis and used holm-Sidak’s test for multiple comparison. Skewness distribution data used Kruskal-wallis test for variance analysis and used Bonferroni method for multiple comparison.

Results

On day 7 after LPS challenge, compared with the baseline, the proportions of splenic naive CD4⁺ and CD8⁺ T cells in mice were significantly decreased (F = 52.22, P < 0.0001; F = 10.87, P = 0.0019); the proportions of effector memory CD4⁺ and CD8⁺ T cells were significantly increased (F = 20.54, P < 0.0001; F = 26.03, P < 0.0001); the proportions of CD4⁺CD38⁺ (F = 35.40, P < 0.0001), CD4⁺CD69⁺ (F = 45.65, P < 0.0001), CD4⁺PD-1⁺ (F = 20.55, P < 0.0001), CD4⁺TIGIT⁺ (F = 19.20, P < 0.0001), CD8⁺CD38⁺ (F = 56.76, P < 0.0001), CD8⁺CD69⁺ (F = 59.47, P < 0.0001), CD8⁺PD-1⁺ (F = 11.15, P = 0.0013) and CD8⁺TIGIT⁺ (F = 21.21, P < 0.0001) cells were significantly increased. These indicators gradually recovered compared with the baseline within 14 days. On day 28 after LPS challenge, the proportions of naive CD4⁺ T cells (F = 52.22, P < 0.0001), effector memory CD4⁺ T cells (F = 20.54, P = 0.0093), CD4⁺CD69⁺ (F = 45.65, P = 0.0037), CD4⁺PD-1⁺ (F = 20.55, P = 0.0255) and CD4⁺TIGIT⁺ (F = 19.20, P = 0.0087) cells were not returned to the baseline level; the proportion of CD8⁺IFN-γ⁺ T cells (F = 14.33, P < 0.0001) was still higher than the baseline.

Conclusions

After LPS challenge, differences exist during the recovery process of splenic CD4⁺ T and CD8⁺ T cells in mice within 28 days. The overall recovery process of splenic CD4⁺ T cells function in septic mice was slower than that of CD8⁺ T cells after LPS treatment.

Key words: Sepsis, Spleen, Lymphocytes, Activation, Co-inhibitory molecules

表1 脂多糖（10 mg/kg）打击后小鼠脾脏细胞数量（ ± s，× 10⁷/脾）

组别	只数	脾脏细胞数	CD3⁺ T细胞数	CD4⁺ T细胞数	CD8⁺ T细胞数
LPS 0 d组	8	9.81 ± 1.89	2.59 ± 0.17	1.76 ± 0.23	0.78 ± 0.27
LPS 7 d组	8	10.3 ± 1.74	2.55 ± 0.46	1.63 ± 0.24	0.79 ± 0.16
LPS 14 d组	8	11.5 ± 1.74	2.62 ± 0.54	1.69 ± 0.29	0.72 ± 0.16
LPS 28 d组	8	11.0 ± 1.78	2.42 ± 0.18	1.56 ± 0.18	0.78 ± 0.15
F值		0.28	0.19	0.62	0.27
P值		0.64	0.82	0.27	0.47

表1 脂多糖（10 mg/kg）打击后小鼠脾脏细胞数量（ ± s，× 10⁷/脾）

组别	只数	脾脏细胞数	CD3⁺ T细胞数	CD4⁺ T细胞数	CD8⁺ T细胞数
LPS 0 d组	8	9.81 ± 1.89	2.59 ± 0.17	1.76 ± 0.23	0.78 ± 0.27
LPS 7 d组	8	10.3 ± 1.74	2.55 ± 0.46	1.63 ± 0.24	0.79 ± 0.16
LPS 14 d组	8	11.5 ± 1.74	2.62 ± 0.54	1.69 ± 0.29	0.72 ± 0.16
LPS 28 d组	8	11.0 ± 1.78	2.42 ± 0.18	1.56 ± 0.18	0.78 ± 0.15
F值		0.28	0.19	0.62	0.27
P值		0.64	0.82	0.27	0.47

图1 LPS（10 mg/kg）腹腔注射后小鼠脾脏T细胞亚群比例注：小鼠脾脏T细胞亚群：CD62L⁺CD44^－初始T细胞、CD62L^－CD44⁺效应记忆T细胞和CD62L⁺CD44⁺中央记忆T细胞。A：LPS打击7 d、14 d和28 d后CD4⁺ T细胞亚群比例的变化；B：LPS打击7 d、14 d和28 d后CD8⁺ T细胞亚群比例的变化

图2 LPS（10 mg/kg）腹腔注射后小鼠脾脏T细胞表面CD38和CD69阳性细胞比例注：A：LPS打击7 d、14 d和28 d后CD4⁺T细胞CD38和CD69阳性细胞比例变化；B：LPS打击7 d、14 d和28 d后CD8⁺ T细胞CD38和CD69阳性细胞比例变化

图3 LPS（10 mg/kg）腹腔注射后小鼠脾脏T细胞表面PD-1和TIGIT阳性细胞比例注：A：LPS打击7 d、14 d和28 d后CD4⁺ T细胞PD-1和TIGIT阳性细胞比例变化；B：LPS打击7 d、14 d和28 d后CD8⁺ T细胞PD-1和TIGIT阳性细胞比例变化

图4 LPS（10 mg/kg）腹腔注射后小鼠脾脏T细胞细胞因子分泌能力变化注：A：LPS打击7 d、14 d和28 d后用PMA和离子霉素体外刺激脾脏CD4⁺ T细胞，检测分泌IL-2和IFN-γ能力的变化；B：LPS打击7 d、14 d和28 d后用PMA和离子霉素体外刺激脾脏CD8⁺ T细胞，检测分泌IL-2子IFN-γ能力的变化；C：LPS打击7 d、14 d和28 d后用PMA和离子霉素体外刺激脾脏CD8⁺ T细胞，检测释放Granzyme B能力的变化

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Dynamic recovery of splenic T lymphocytes function in mice with endotoxin-induced sepsis

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Dynamic recovery of splenic T lymphocytes function in mice with endotoxin-induced sepsis