| BackgroundAcute lung injury(ALI)is an early form of acute respiratory distress syndrome(ARDS),which is mainly characterized by progressive lung inflammation,enhanced permeability of alveolar capillary barrier and pulmonary edema.Despite significant advances in clinical treatment strategies for ALI/ARDS,there is no pharmacological treatment to date.The inhospital mortality of patients with ARDS was still up to 40%.V-domain immunoglobulin suppressor of T cell activation(VISTA),a novel immune checkpoint molecule,is playing an increasingly important role in tumor and autoimmune diseases.Previous studies have shown that VISTA knockout at the animal or cellular level shows enhanced anti-tumor properties and promotes inflammatory responses.In view of the important role of VISTA in regulating inflammatory response,its role in ALI deserves further exploration;however,the exact role of VISTA in ALI was not reported in the literature currently.The purpose of this study was to investigate the role of VISTA in lipopolysaccharide(LPS)-induced ALI in mice,and to investigate its potential mechanism.Methods1.Two kinds of ALI models were constructed using LPS to explore the effects of VISTA gene knockout on the survival rate,severity of lung tissue injury and inflammatory factor levels(1)ALI model induced by intratracheal injection of LPS in mice(direct model): A single intratracheal injection of LPS was used to establish ALI model in mice.VISTA KO mice were randomly divided into KO Sham group and KO LPS group,and WT mice were randomly divided into WT Sham group and WT LPS group.Male mice with C57 BL/6genetic background were selected for each group,and all mice were 8～12 weeks old and weighed 20 g～25 g.The ALI model was established by intratracheal injection of LPS(20 mg/kg)to analyze the 7 d survival rate.The ALI model was established by intratracheal injection of LPS(10 mg/kg),and pathological features of lung tissues,lung tissue permeability and inflammatory factors level were studied 6 h after modeling.All mice in control groups were intratracheally injected with equal volume of PBS buffer.(2)ALI model induced by endotoxemia constructed by intraperitoneal injection of LPS in mice(indirect model): Single intraperitoneal injection of LPS was used to establish endotoxemia,and the groups were the same as above.A single intraperitoneal injection of LPS(20 mg/kg)was used to construct an endotoxemia model to analyze the 7 d survival rate.The ALI model induced by endotoxemia was established by intraperitoneal injection of LPS(40 mg/kg),and pathological features of lung tissues,lung tissue permeability and inflammatory factors level were studied 6 h after modeling.All mice in control groups were intraperitoneally injected with equal volume of PBS buffer.2.To explore the effects of different types of anti-VISTA antibodies on ALI induced by intratracheal injection of LPS in mice(1)ALI model induced by intratracheal injection of LPS in mice: Male C57 BL/6 mice aged 8～12 weeks(20 g～25 g)were selected for a single intratracheal injection of LPS(10 mg/kg)to establish ALI model.WT mice were randomly divided into PBS control group(Sham group),homotype control antibody injection group(LPS+Ig G group),13F3 antibody injection group(LPS+13F3 group),and MH5 A antibody injection group(LPS+MH5A group)after modeling.Immediately after intratracheal injection of LPS(10 mg/kg),mice in LPS+Ig G group,LPS+13F3 group and LPS+MH5A group were injected with 100 μg Ig G,100 μg 13F3 and 20 μg MH5 A antibodies via tail vein,respectively.Mice in Sham group were intratracheally injected with equal volume of PBS buffer.(2)The effects of VISTA-antagonistic antibody(clone 13F3)and agonistic antibody(clone MH5A)on the severity of lung tissue injury and inflammatory factors level of ALI model mice by intratracheal injection of LPS were investigated by lung histopathological analysis,lung tissue permeability study and inflammatory factors level detection.3.Preliminary study on the mechanism of VISTA gene regulating LPS-induced ALI(1)Histopathological,immunohistochemical and immunofluorescence methods were used to investigate the effects of VISTA gene knockout on the pathological features and infiltrating cell types of skin and lung tissues of mice.(2)The mice ALI model induced by endotoxemia was established by intraperitoneal injection of LPS.The methods and groups were the same as above.Transcriptome analysis of lung tissues in each group was performed to explore the related genes and pathways of VISTA knockout regulating ALI in mice.(3)Related proteins in the lung tissues of LPS-induced ALI mice were detected by WB method to verify the relevant mechanism of VISTA gene knockout in regulating ALI in mice.Results1.In the LPS induced direct ALI and endotoxemia induced indirect ALI models,VISTA knockout significantly reduced the 7 d survival rate of both ALI mice,and promoted the release of IL-1β,IL-6 and TNF-α in alveolar lavage,fluid lung tissue or serum.The W/D value and BALF protein concentration of lung tissue were significantly increased,and the severity of pathological injury of lung tissue was aggravated in in KO LPS group.2.VISTA-antagonistic antibody(clone 13F3)promoted the release of IL-1β,IL-6 and TNF-α in lung tissue,increased W/D value of lung tissue and significantly aggravated the severity of lung injury induced by LPS in mice.In contrast,the VISTA-agonistic antibody(clone MH5A)reduced the release of IL-1β,IL-6 and TNF-α,decreased the W/D value of lung tissue and effectively alleviated the severity of lung injury induced by LPS in mice.3.Macrophage recruitment was observed in lung tissues of VISTA knockout mice.Transcriptomic sequencing of lung tissues of ALI mice induced by endotoxemia constructed by intraperitoneal injection of LPS showed that VISTA knockout promoted the expression of chemokines CCL2 and CXCL3,NF-κB signaling pathway factors NF-κB1 and Rel,and inflammatory factors IL-1β and IL-6.WB results of lung tissues showed that VISTA knockout significantly increased the phosphorylation of P65 and P38 and the expression of CCL2 in the lung tissue of LPS-induced ALI mice.Vista-antagonistic antibody 13F3 can promote P38 phosphorylation and CCL2 expression.In contrast,the VISTA-agonistic antibody MH5 A inhibited P38 phosphorylation and CCL2 expression.ConclusionThe presence of VISTA gene has a protective effect against LPS-induced ALI in mice,and the VISTA knockout can aggravate lung injury and reduce the 7 d survival rate of ALI mice.VISTA-antagonistic antibody can increase the severity of LPS-induced ALI in mice,while VISTA-agonistic antibody will alleviate the severity of LPS-induced ALI in mice.VISTA knockout can activate NF-κB and MAPK signaling pathways to promote the release of chemokine CCL2 and inflammatory factors,thereby aggravating the severity of LPSinduced ALI in mice.This study preliminarily indicated that VISTA gene has inhibitory effects on NF-κB,MAPK and other inflammatory signaling pathways,chemokines such as CCL2 and macrophage recruitment,which may be one of the important mechanisms of VISTA regulating LPS-induced ALI in mice. |
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