目的

探讨在念珠菌感染导致的致死性脓毒症中，哺乳动物雷帕霉素靶（mTOR）介导T-bet表达对CD4⁺ T细胞增殖分化的调控作用。

方法

4-5周龄，体重16-18g的雄性健康C57BL/6 N野生型（WT）、T细胞特异性mTOR/TSC1基因敲除小鼠（以抑制/增强T细胞的mTOR通路活性），分为感染组（n=30）和对照组两个大的组别（n=10），通过经典的念珠菌孢子尾静脉注射法建立念珠菌血症小鼠模型。造模后12小时根据小鼠脓毒症评分（MSS）进一步将感染组小鼠按照脓毒症严重程度分成MSS 4-7，MSS8-10，MSS＞10三个亚组（n=10），比较组间小鼠生存曲线，取小鼠脾脏应用流式细胞技术分选出CD4⁺ T细胞进行计数并应用Western Blotting检测细胞中mTOR通路下游蛋白p-mTOR和p-p70S6激酶表达水平以及特异性转录因子T-bet的表达水平。分析mTOR介导T-bet表达对T细胞免疫的影响。

结果

和对照组相比，感染组小鼠CD4⁺ T细胞计数减低，并随着脓毒症的加重持续下降(p值＜0.0001)；感染组小鼠mTOR通路被激活，其下游活性指标p-mTOR与p-P70S6K的表达水平随着脓毒症的进展而增强(p值＜0.0001)；感染组小鼠T-bet表达虽高于对照组，但不同MSS组小鼠的T-bet表达水平随脓毒症的恶化而降低(p值＜0.05)。在相同的MSS组中，Lck-mTOR小鼠的T-bet表达和CD4⁺ T细胞计数高于WT小鼠(p值＜0.0001)，而Lck-TSC1小鼠的T-bet表达和CD4⁺ T细胞计数低于WT小鼠(p值＜0.0001)。

结论

mTOR介导T-bet表达影响念珠菌感染导致的致死性脓毒症小鼠的CD4⁺ T细胞存活。这一结论为改善念珠菌感染导致脓毒症引起的宿主免疫功能受损提供新的靶点和潜在的治疗策略。

Objective

To investigate the regulation effect of mTOR on CD4⁺ T cell differentiation in lethal candida sepsis by modulating T-bet expression.

Methods

Healthy male C57BL/ 6N wild-type (WT), T-cell specific knockout mTOR (LCK-mTOR) and T-cell specific knockout TSC1 (LCK-TSC1) mice, aged 4-5 weeks and weighing 16-18g were used to establish a model of Candida sepsis by injecting Candida albicans SC5314 suspension through tail vein. 12 hours after modeling, the Murine Sepsis Score was used to group mice with different disease severity and CD4⁺ T lymphocytes were selected from spleens of mice by flow cytometry. The expression levels of p-mTOR and p-p70S6 kinase in the downstream of mTOR signaling pathway and T-bet, a transcription factor specific to CD4⁺ T lymphocytes, were detected by Western Blotting. The kidneys, lungs, liver and spleen were taken to make Gomori's methenamine silver staining sections to observe whether the organs were invaded by Candida albicans spores and hyphae.

Results

Compared with the control group, CD4⁺ T cell count decreased in infected mice and continued to decrease with the aggravation of sepsis. The mTOR signaling pathway was activated in infected mice and its activity increased with the progression of sepsis. Although the expression of T-bet in infected group was higher than that in control group, the expression of T-bet in different MSS groups decreased with the deterioration of sepsis. In the same MSS group, the T-bet expression and CD4⁺ T cell count of LCK-mTOR mice were higher than those of WT mice, while of which in LCK-TSC1 mice were lower than those of WT mice.

Conclusion

T-bet expression mediated by the mTOR pathway influences the CD4⁺ T cell count in mice with Candida sepsis. This conclusion provides a new target and a potential treatment strategy for improving the compromise of host immunity caused by candida sepsis.