RESUMEN
Objective To examine the expression profile of programmed death-ligand 1 (PD-L1) on lung endothelial or epithelial cells,and to determine the specific role of PD-L1 in mouse model of indirect acute lung injury (i-ALI).Methods Eighty male C57BL/6 mice were randomly divided into two parts (both n =40).The effects of different administration routes on the expression of PD-L1 were observed.The mice in each part were randomly divided into sham,i-ALI,i-ALI+small interfering RNA (siRNA) random sequence control,and i-ALI+PD-L1 siRNA which could specifically inhibit PD-L1 expression groups,with l0 mice in each group.i-ALI was reproduced in a mouse model of hemorrhagic shock in combination with a subsequent cecal ligation and puncture (CLP).In sham group,only bilateral femoral arteries were ligated without catheterization or bleeding,and only cecum was separated but perforation was not ligated.Intravenous or intratracheal delivery of PD-L1 siRNA was performed 2 hours following the resuscitation to suppress the expression of PD-L1 on lung endothelial or epithelial cells.The mice in i-ALI+siRNA random sequence control group were given siRNA random sequence without inhibition effect on PD-L1 expression,and those in sham group and i-ALI group were given 100 μL phosphate buffered saline (PBS).The mice were sacrificed at 24 hours after CLP,and samples of blood,lung tissue and bronchoalveolar lavage fluid (BALF) were harvested.Expressions of PD-L1 were determined with flow cytometry.Cytokines and chemokines in plasma,lung tissue and BALF were determined by enzyme linked immunosorbent assay (ELISA).The protein concentration in plasma and BALF and the activity of myeloperoxidase (MPO) in lung tissue were quantitatively measured.The pathological changes in lung tissue were observed under light microscope.Results ① Compared with sham group,PD-L1 expression on lung endothelial or epithelial cells were significantly elevated in i-ALI group [endothelial cells:(27.88 ± 1.53)％ vs.(19.64 ± 1.03)％,epithelial cells:(58.70 ± 8.21)％ vs.(29.23 ± 3.94)％,both P ＜ 0.05].② Mice received intravenous delivery of liposomal-encapsulated siRNA had significantly lower expression of PD-L1 on lung endothelial cells as compared with that of i-ALI group [(21.37 ± 0.76)％ vs.(27.88 ± 1.53)％,P ＜ 0.05].Intratracheal delivery of naked PD-L1 siRNA mainly inhibited the PD-L1 expression on epithelial cell as compared with that of i-ALI group [(31.23±4.71) ％ vs.(58.70±8.21) ％,P ＜ 0.05].The expression of PD-L1 in pulmonary microvascular endothelial cells or pulmonary epithelial cells of i-ALI mice was not affected by siRNA random sequence.③ PD-L1 silencing on pulmonary endothelial cells induced by intravenous delivery of PD-L1 siRNA led to a lower protein ratio of BALF/plasma [(4.48 ± 0.35) × 10-3 vs.(6.11 ± 0.56) × 10-3,P ＜ 0.05] and a decreased MPO activity in lung tissue (U · μg-1 · min-1:2.48 ± 0.47 vs.4.56 ± 0.52,P ＜ 0.05) as compared with that of i-ALI group.Moreover,inflammatory mediator levels such as interleukin-6 (IL-6),monocyte chemoattractant protein-1 (MCP-1),macrophage inflammatory protein-2 (MIP-2) and tumor necrosis factor-α (TNF-α) in lung tissue or plasma were significantly reduced following PD-L1 suppression on endothelial cells as compared with those of i-ALI group [IL-6 (ng/g):177.4±23.2 vs.287.9±57.3,MCP-1 (ng/g):839.6±91.7 vs.1 395.7±211.9,MIP-2 (ng/g):923.7± 107.3 vs.1 700.9±240.2 in lung tissue;IL-6 (ng/L):950.2±192.7 vs.1 828.2±243.6,TNF-α (ng/L):258.7±29.1 vs.443.0 ± 58.1,MCP-1 (ng/L):2 583.8±302.3 vs.4 328.1 ±416.4,MIP-2 (ng/L):1 512.9± 165.6 vs.2 005.9 ± 85.7 in plasma,all P ＜ 0.05],however,there was no significant change in the levels of inflammatory factors in BALF.It was shown in lung tissue histology that PD-L1 silencing on pulmonary endothelial cells induced by intravenous delivery of PD-L1 siRNA led to lessened pulmonary edema and reduced immune cells emigration.Intratracheal delivery of PD-L1 siRNA for PD-L1 suppression on epithelial cells had minimal effects on protein ratio of BALF/plasma,MPO activity,inflammatory mediator expressions in lung tissue,plasma,and BALF as well as lung tissue histology.Conclusion PD-L1 silencing on endothelial cells but not epithelial cells protected mice against hemorrhagic shock-sepsis induced i-ALI.