| Background and ObjectiveAcute lung injury(ALI)/Acute respiratory distress syndrome(ARDS)is a rapidly progressive lung disease characterized by increased vascular permeability,pulmonary edema,extensive inflammation and severe hypoxemia.The causes of ARDS can be divided into two categories: 1)direct lung injury mainly caused by pneumonia;2)indirect lung injury(i ALI)caused by sepsis,hemorrhagic shock,trauma and pancreatitis,etc.Since significant progress has been achieved in organ support therapy,the mortality rate of ARDS remains as high as 40%.Currently,it is generally accepted that the pathogenesis of i ALI involves the activation of neutrophils,pulmonary endothelial cells(EC)and / or epithelial cells(Epi C)and their interaction in the immune system.However,the precise mechanism underlying the loss of endothelial/epithelial barrier integrity caused by neutrophil infiltration or inflammatory mediators remains unclear.PD-L1 is the main ligand of programmed cell death receptor-1(PD-1 or CD279),which is widely expressed on immune cells and non-immune cells.Studies have shown that PD-1/PD-L1 pathway might play a special role in the regulation of i ALI pathogenesis,but little is known about how the expression of PD-L1 would directly affects the function of the non-immune cells,especially the endothelial cells and epithelial cells.How the expression of PD-L1 on lung EC/Epi C changes during i ALI and whether this change affects the integrity or permeability of EC / Epi C monolayer cells required further investigation.In this study,we firstly examined the expression profile of PD-L1 on pulmonary endothelial cells and epithelial cells during i ALI.Then,PD-L1 siRNA was administered through caudal vein or trachea,PD-L1 expression on different cells was measured and its effect on the occurrence and development of i ALI was determined.Finally,the role and mechanisms of PD-L1 siRNA in regulating endothelial cell barrier function was investigated in vitro.Materials and methods1.The expression of PD-L1 m RNA and protein on endothelial cell line(CRL-2167)and epithelial cell line(LA4)with or without IFN-γ stimulation was determined by rt-PCR or flow cytometry.Ten male wild-type C57 BL / 6 mice aged 8-12 weeks were randomly divided into sham-operated group(sham group,4 mice)or i ALI model group(Hem/ CLP Group,6 mice).Mice were sacrificed at24 hours after CLP,and lung tissue samples were collected and examined.2.Endothelial cell line(CRL-2167)and epithelial cell line(LA4)were cultured and treated with PD-L1 siRNA in the presence of IFN-γ stimulation.RT-PCR and flow cytometry were used to detect the expression of PD-L1 m RNA and protein respectively.Gene silencing efficiency of PD-L1 siRNA on the expression of PD-L1 was determined in cultured cells.44 male wild-type C57BL/6 mice aged 8-12 weeks were randomly divided into sham operated group(sham group,4 mice),i ALI model + PBS group(Hem/CLP Group + PBS,6 mice),i ALI model + control siRNA group(Hem/CLP Group + control siRNA,6 mice)and i ALI model + PD-L1 siRNA group(Hem/CLP Group + PD-L1 siRNA group,6 mice).PD-L1 siRNA was administered through caudal vein or trachea 2 hours after Hem resuscitation.Lung samples were collected 24 hours after CLP procedures.The expression of PD-L1 m RNA and protein in mouse pulmonary vascular endothelial cells and epithelial cells were detected by rt-PCR or flow cytometry respectively.3.72 male wild-type C57BL/6 mice aged 8-12 weeks were randomly divided into sham-operated group(sham group,6 mice),i ALI model + PBS group(Hem/CLP Group + PBS,10 mice),i ALI model + control siRNA group(Hem/CLP Group + control siRNA,10 mice)and i ALI model + PD-L1 siRNA group(Hem/ CLP Group + PD-L1 siRNA group,10 mice).PD-L1 siRNA was administered through caudal vein or trachea 2 hours after HEM resuscitation.Mice were sacrificed 24 hours after CLP,and the samples of peripheral blood,lung tissue and bronchoalveolar lavage fluid(BALF)were collected.The pathological changes of lung tissue were measured under light microscope.The expression of cytokines and chemokines in peripheral blood,lung homogenate and BALF were detected by enzyme-linked immunosorbent assay(ELISA).The concentration of BALF protein and the activity of myeloperoxidase(MPO)and caspase-3 in lung tissue were detected quantitatively.4.Endothelial cell barrier model was established using endothelial cell line(CRL-2167).Cells were divided into control group,IFN-γ treatment group(PBS+ r IFN-γ Group),IFN-γ+ control siRNA treatment group(control siRNA +r IFN-γ Group),IFN-γ + PD-L1 siRNA treatment group(PD-L1 siRNA + r IFN-γGroup).The permeability of the cells to FD4 in different groups was detected at72 hours after treatment.Western-blot and immunofluorescence staining were used to detect the effects of PD-L1 siRNA on the expression and distribution of tight junction proteins ZO-1 or occludin in monolayer cells.Result1.Endothelial cell line(CRL-2167)and epithelial cell line(LA4)constitutively expressed PD-L1.The expression of PD-L1 m RNA and protein increased after IFN-γ stimulation.PD-L1 was constitutively expressed on pulmonary microvascular endothelial cells and epithelial cells in WT mice.The expression of PD-L1 on pulmonary microvascular endothelial cells and pulmonary epithelial cells in i ALI mice was significantly higher than that in sham-operated mice(P < 0.05).2.PD-L1 siRNA significantly reduced the expression of PD-L1 m RNA and protein in CRL-2167 cells and LA4 cells(P < 0.05).PD-L1 siRNA administered via caudal vein could effectively inhibit the expression of PD-L1 m RNA and protein in mouse pulmonary vascular endothelial cells(P < 0.05).Intratracheal administration of PD-L1 siRNA could effectively inhibit the expression of PD-L1 m RNA and protein in lung epithelial cells(P < 0.05).3.Administration with PD-L1 siRNA through caudal vein inhibited the expression of PD-L1 in endothelial cells,following which pathological examination showed that pulmonary interstitial edema and inflammatory cell infiltration were significantly reduced.Compared with the control siRNA group,several inflammatory factors(IL-6,MCP-1,MIP-2)in lung tissue and(IL-6、MCP-1、MIP-2、IL-10 和 TNF-α)in plasma were significantly decreased(P <0.05).However,there was no significant change in the level of inflammatory factors in BALF.The BALF protein/plasma protein ratio was significantly lower than that in the control siRNA group(P < 0.05),and the MPO and caspase-3activities in lung tissue were significantly lower than that in the control siRNA group(P < 0.05).Intratracheal administration of PD-L1 siRNA inhibited the expression of PD-L1 in lung epithelial cells,but no significant changes were seen in lung histopathology,inflammatory factors expression(lung tissue,peripheral blood or BALF),BALF protein / plasma protein ratio,MPO activity or caspase-3activity.4.CRL-2167 cells were cultured to establish a monolayer endothelial cell barrier.Then,PD-L1 siRNA was used to suppress the expression of PD-L1 under stimulation with inflammatory factors.As compared with the control siRNA group,treatment with PD-L1 siRNA resulted in decreased FD4 influx and increased expression of tight junction proteins(ZO-1),and improved morphological alteration of tight junction.Conclusion1.PD-L1 was constitutively expressed on pulmonary microvascular endothelial cells and epithelial cells,and the expression on both cells was significantly increased following i ALI.2.The siRNA administered through different ways may target specific tissues and cells.Intratracheal administration of bare(non liposome encapsulated)siRNA mainly targeted lung epithelial cells,but not endothelial cells;On contrast,intravenous administration of liposome encapsulated siRNA mainly targeted vascular endothelial cells rather than lung epithelial cells.3.Suppressing the expression of PD-L1 on pulmonary microvascular endothelial cells rather than pulmonary epithelial cells protected mice from HEM/ CLP induced i ALI.4.PD-L1 siRNA up-regulated the expression of tight junction protein on endothelial cells and improved the endothelial barrier function under stimulation with pro-inflammatory factors.5.PD-L1 plays an non-redundant regulatory role in the pathophysiological process of acute lung injury and may be a promising immunotherapeutic target for patients with ARDS. |
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