The role of PD-L1 mediating lymphocyte YAP phosphorylation in viral acute lung injury

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.