The role of PD-L1 mediating lymphocyte YAP phosphorylation in viral acute lung injury
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摘要 目的:探讨程序性死亡受体配体1(PD-L1)介导淋巴细胞yes相关蛋白(YAP)磷酸化在病毒性急性肺损伤中的作用。方法:将SPF级C57BL/6J小鼠随机分为对照组、根据小鼠气管内滴注聚肌苷—聚胞苷酸(即Poly I:C)的时间分为4 h、8 h、1 d、3 d、7 d组,在这五组中选取炎症损伤最严重的组作为Poly I:C组;利用PD-1/PD-L1抑制剂BMS-1 10mg/kg腹腔注射预处理后气管内滴注Poly I:C,作为Poly I:C+BMS-1组。野生型小鼠Poly I:C组和PD-L1敲基因小鼠Poly I:C组处理同Poly I:C组;野生型小鼠对照组和PD-L1敲基因小鼠对照组腹腔注射麻醉后气管内给予等量生理盐水。相应时间点将小鼠安乐死收集标本。采用苏木精—伊红(HE)染色法评估小鼠肺组织损伤程度;通过检测支气管肺泡灌洗液(BALF)中总蛋白浓度、总细胞数以及酶联免疫吸附法(ELISA)检测BALF中肿瘤坏死因子(TNF)-α水平评估炎症情况;蛋白免疫印迹(western blotting, WB)检测程序性死亡受体1(PD-1)、PD-L1、YAP、p-YAP蛋白表达。结果:与对照组比较,8 h、1 d、3 d、7 d组肺组织病理学损伤评分及BALF总蛋白浓度显著升高,8 h、1 d、3 d组BALF中总细胞数明显增多,4 h、8 h、1 d、3 d组BALF中TNF-α水平显著上调,差异均有统计学意义(P<0.05);其中1 d组小鼠肺组织病理学损伤评分、BALF中总蛋白浓度、BALF总细胞数以及BALF中TNF-α水平均高于4 h、8 h、3 d、7 d组。与对照组比较,1 d、3 d、7 d组小鼠肺组织中PD-1和PD-L1蛋白水平均明显升高(P<0.05)。Poly I:C组小鼠肺组织病理损伤评分和BALF中TNF-α水平明显高于对照组,与Poly I:C组相比,Poly I:C+BMS-1组小鼠肺组织病理损伤评分和BALF中TNF-α水平显著降低(P<0.05)。与对照组相比,Poly I:C组和Poly I:C+BMS-1组小鼠淋巴细胞中YAP蛋白表达明显下调,p-YAP蛋白表达显著上调(P<0.05);与Poly I:C组相比,Poly I:C+BMS-1组小鼠淋巴细胞中YAP蛋白表达上升,p-YAP蛋白表达下降(P<0.05)。与对照组相比,Poly I:C组小鼠淋巴细胞中YAP蛋白表达明显下调而p-YAP蛋白表达显著上调,PD-L1基因敲除后Poly I:C组小鼠淋巴细胞中YAP蛋白表达有所上升而p-YAP蛋白表达明显下降,差异均有统计学意义(P<0.05)。结论:病毒性急性肺损伤小鼠肺组织中高表达PD-L1蛋白,PD-L1可能通过激活淋巴细胞的YAP磷酸化加重病毒性急性肺损伤。Abstract Objective: To investigate the role of programmed death receptor ligand 1(PD-L1) in mediating the phosphorylation of lymphocyte yes-associated protein(YAP) in viral acute lung injury. Methods: SPF-grade C57BL/6J mice were randomly divided into control groups, and divided into groups of 4 h, 8 h, 1 d, 3 d, and 7 d according to the time of intratracheal drip of polyinosinic-polycytidylic acid(Poly I: C). The group with the most severe inflammatory injury among these five groups was selected as the Poly I: C group; PD-1/PD-L1 inhibitor BMS-1 10mg/kg intraperitoneal injection pretreated with intratracheal drip of Poly I: C, as Poly I: C + BMS-1group. The Poly I: C group of wild-type mice and the Poly I: C group of PD-L1 knockout mice were treated as the Poly I: C group; the control group of wild-type mice and the control group of PD-L1 knockout mice were anesthetized by intraperitoneal injection and given an equal amount of saline intratracheally. Mice were euthanized at the corresponding time points and specimens were collected. Hematoxylin-eosin(HE) staining was used to assess the degree of lung tissue injury in mice; inflammation was assessed by measuring the total protein concentration, total cell number in bronchoalveolar lavage fluid(BALF), and tumor necrosis factor(TNF)-α levels in BALF using enzyme-linked immunosorbent assay(ELISA). Western blotting was used to detect the expression of PD-1, PD-L1, YAP, and p-YAP proteins. Results: Compared with the control group, the lung histopathological injury score and total protein concentration in BALF were significantly increased in the 8 h, 1 d, 3 d, and 7 d groups, the total cell number in BALF was significantly increased in the 8 h, 1 d, and 3 d groups, the TNF-α level in BALF was significantly up-regulated in the 4 h, 8 h, 1 d, and 3 d groups, and the differences were statistically significant(P<0.05); among them, the lung histopathological injury score, total protein concentration in BALF,total cell number in BALF and TNF-α level in BALF in the 1d group were higher than those in the 4 h, 8 h, 3 d and 7 d groups. Compared with the control group, the levels of PD-1 and PD-L1 proteins in the lung tissues of mice in the 1 d, 3 d, and 7 d groups were significantly increased(P<0.05). The scores of pathological injury in the lung tissues of mice in the Poly I: C group and the levels of TNF-α in BALF were significantly higher than those in the control group. Compared with the Poly I: C group, the lung histopathological injury score and the levels of TNF-α in BALF were significantly lower than those in the Poly I: C+BMS-1 group(P<0.05). Compared with the control group, YAP protein expression was significantly down-regulated and p-YAP protein expression was significantly up-regulated in the lymphocytes of mice in the Poly I: C and Poly I: C+BMS-1 groups(P<0.05). Compared with the Poly I: C group, YAP protein expression in the lymphocytes of mice in the Poly I: C+BMS-1 group was increased and p-YAP protein expression was decreased(P<0.05). Compared with the control group, YAP protein expression was significantly down-regulated, and p-YAP protein expression was significantly up-regulated in the lymphocytes of mice in the Poly I: C group; YAP protein expression in the lymphocytes of mice in the Poly I: C group was increased, while p-YAP protein expression was significantly decreased after the knockdown of the PD-L1 gene, and the differences were all statistically significant(P<0.05). Conclusion: PDL1 protein is highly expressed in the lung tissues of mice with viral acute lung injury, and PD-L1 may aggravate viral acute lung injury by activating YAP phosphorylation in lymphocytes.
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Keywords
- programmed death ligand /
- yes-associated protein /
- acute lung injury /
- lymphocyte
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