Role And Mechanism Of IL-17 In LPS-induced Acute Lung Injury

Background and Objective:Acute respiratory distress syndrome（ARDS）induced by sepsis is a common clinical critical disease,alveolar epithelial cell injury is one of the main pathological changes.As a proinflammatory factor secreted by Th17 cell,interleukin 17（IL-17）activates target cells to produce a variety of inflammatory cytokines through combining with the corresponding receptor（IL-17R）,then mediates inflammatory cells to cause local tissue damage.At present the study about the role of IL-17 in acute lung injury（ALI）is relatively less.This study selected human alveolar type II epithelial cell（A549）and mouse as the research object,lipopolysaccharide（LPS）as stimulating factor,to explore the impact about inflammatory reaction and mechanism of IL-17 on A549 and mouse ALI model,moreover analyzed the expression and significance of IL-17 in ARDS patients,provided more theoretical basis for clinical treatment and evaluation of sepsis-ARDS.Method:PartⅠ:the effect of LPS on the expression of IL-17RC in human alveolar type II epithelial cell1.Firstly examined the expression of IL-17RC on A549 by agarose gel electrophoresis and Western blot.2.Different concentrations of LPS（0.01、0.1、1、10μg/ml）stimulated A549 for 3hours;0.1μg/ml LPS stimulated A549 for different time（1、3、6、12 hours）,detected IL-17RC mRNA level with RT-PCR.3.0.1μg/ml LPS stimulated A549 for different time（1、3、6、12、24 hours）,Flow cytometry and Western blot were used to detect IL-17RC protein level.PartⅡ:the effect and mechanism of IL-17 on the function of human alveolar type II epithelial cell1.Different concentrations of IL-17（0.1、0.5、1μg/ml）stimulated A549 for 3 hours;0.5μg/ml IL-17 stimulated A549 for different time（1、3、6、12、24 hours）.Extracted A549RNA after stimulattion in 1、3、6 h.Cell supernatant and A549 protein were collected in 6、12、24 h.2.Control、LPS（0.1μg/ml）、IL-17（0.5μg/ml）、LPS（0.1μg/ml）+IL-17（0.5μg/ml）.Cell supernatant was collected in 6、24 h and A549 protein was collected in 24 h.3.Control、IL-17（0.1μg/ml）、IL-17（0.2μg/ml）、IL-17（0.5μg/ml）、IL-17（0.5μg/ml）+anti-IL-17RC Ab（0.1μg/ml）.Cell supernatant was collected in 6、24 h and A549 protein was collected in 24 h.4.Control、AG490（50μmol/L）、IL-17（0.5μg/ml）、IL-17（0.5μg/ml）+AG490（50μmol/L）.Cell supernatant was collected in 6、24 h and A549 protein was collected in 24 h.5.SPA、ENaC-α、TNF-αand IL-8 mRNA levels were detected by RT-PCR.SPA、TNF-αand IL-8 protein levels were detected by ELISA.ENaC-α、p-STAT3 and p-Akt levels were detected by Western blot.PartⅢ:the role and mechanism of IL-17 in LPS induced mouse ALI model1.Mouse ALI model was established by intratracheally instilling with LPS（3 mg/kg）.Control group、ALI model（6、12、24 h after LPS stimulation）.IL-17 level of BALF and plasma was detected by ELISA.2.Control group（PBS）、control antibody group（1μg）、LPS（3 mg/kg）、LPS+IL-17Ab group（3 mg/kg LPS+1μg IL-17Ab injected intravenously after 6 h）.3.HE staining was used to assess the degree of lung pathological injury,measured the lung wet/dry ratio,total protein concentration in BALF was determined by BCA,counted total BALF cells,TNF-αand IL-10 levels of BALF and plasma were detected by ELISA.The expression of RORγt in lung tissue was detected by immunohistochemistry,and the expression of RORγt and p-Akt in lung tissue was detected by Western blot.PartⅣ:the value of IL-17 in evaluating the condition of patients with ARDS1.IL-17 level of the plasma was detected by ELISA in 18 healthy control group and35 patients with sepsis-ARDS.2.The relationship between IL-17 level and oxygenation index（PaO₂/FiO₂）and prognosis was analyzed.Result:PartⅠ:the effect of LPS on the expression of IL-17RC in human alveolar type II epithelial cell1.IL-17RC mRNA and protein were detected on A549.2.The expression of IL-17RC mRNA was significantly increased after stimulation with 0.1μg/ml LPS.IL-17RC mRNA was highest in 3 h after stimulation.3.The level of IL-17RC protein began to increase in 6 h after stimulation,and to the high level in 24 h.PartⅡ:the effect and mechanism of IL-17 on the function of human alveolar type II epithelial cell1.SPA、ENaC-αmRNA expressed highly on A549 and decreased significantly after IL-17 intervention,especially with 0.5μg/ml concentration stimulation in 3 h.TNF-αmRNA was not detected,IL-8 mRNA expression was low on A549,the expression of TNF-αand IL-8 mRNA were significantly increased after IL-17 intervention especially with 0.5μg/ml concentration stimulation in 3 h.2.After A549 was stimulated by IL-17,the level of SPA was significantly decreased in 6 h,ENaC-αlevel was reduced to the lowest in 24 h,TNF-αand IL-8 expression increased and reached the high level in 24 h.3.After A549 was stimulated by LPS and IL-17 in combination,SPA level decreased more significantly in 6 h compared to IL-17 and LPS alone.ENaC-αexpression decreased,TNF-a and IL-8 levels increased in 24 h,the changes of LPS+IL-17 group were more significant than LPS and IL-17 alone.4.After A549 was treated with IL-17,SPA level reduced in 6 h,ENaC-αlevel decreased in 24 h,TNF-αand IL-8 levels increased in 24 h,the degree of changes was correlated with the concentration of IL-17.Anti-IL-17RC Ab could partially reverse the effect induced by IL-17 in A549.5.After A549 was stimulated with IL-17,p-STAT3 and p-AKT levels were elevated with a dosage dependent manner,which could be partially blocked by anti-IL-17RC Ab.AG490,an inhibitor of STAT3,could alleviate the damage of IL-17 on A549.PartⅢ:the role and mechanism of IL-17 in LPS induced mouse ALI model1.After mouse ALI model was established,IL-17 levels of BALF and serum were significantly higher than those in control group,and to the highest peak in 12 h.2.IL-17Ab intervention could significantly alleviate lung injury score and wet/dry ratio,reduce the total number of cells and protein level in BALF.IL-17Ab intervention could also decrease TNF-αlevel and increase IL-10 level of BALF and plasma.3.IL-17Ab intervention could reduce the expression of Th17 related transcription factor RORγt in the lung tissue by immunohistochemical.Western blot detection showed that IL-17Ab intervention could reduce the expression of RORγt and p-Akt in the lung tissue of mouse ALI model.PartⅣ:the value of IL-17 in evaluating the condition of patients with ARDS1.IL-17 level in plasma of the 35 patients with sepsis-ARDS was significantly higher than those of the healthy control group.2.There was a negative relationship between IL-17 and PaO₂/FiO₂.Compared with survival group,IL-17 level in death group was significantly higher.Conclusion:1.IL-17RC was distributed in A549,and there was a a time and dosage dependent manner after LPS stimulation.The expression of IL-17RC mRNA was highest after the stimulation of 0.1μg/ml LPS in 3 h,and IL-17RC protein level was high in 24 h.2.IL-17 could lead to A549 damage through JAK/STAT3 and PI3K/Akt signaling pathway,manifested as decreased SPA and ENaC-αexpression,increased TNF-αand IL-8secretion,which had synergistic effects with LPS,could be partially alleviated by anti-IL-17RC Ab and AG490.3.IL-17 levels of BALF and plasma were significantly increased in LPS induced ALI.IL-17Ab administation could reduce inflammatory cell infiltration and lung permeability,precipitate Th17/Treg inflammatory recation deviation toward Treg cells by affecting the expression of RORγt and PI3K/Akt,so as to alleviate the symptoms of ALI.4.IL-17 level in plasma of patients with sepsis-ARDS was significantly increased,and there was a negative correlation with PaO₂/FiO₂ and survival rate.IL-17 may be an indicator of evaluating severity and predicting prognosis of ARDS.