The Protective Effect Of Regulatory T Cells And Related Mechanism In LPS Induced Acute Respiratory Distress Syndrome

Objective:to explore the effect and related mechanism of Regulatory T cells(Tregs)in LPS induced Acute respiratory distress syndrome(ARDS).Methods:male,6-8 weeks,BALB/c mice were randomly divided into 5 groups:Sham group,LPS 1 day group,LPS 2 day group,LPS 4 day group,LPS 7 day group.In sham group,0.9%saline were given intratracheally(1.5ml/kg).In LPS group,mice were received intratracheal administration with LPS(3mg/kg).Then mice were sacrificed on day 1,2,4,7 day.The right lung and spleen were removed and Tregs in single cell suspension were detected by flowcytometry.Based on the results above,we determine the optimal time of Tregs variation in LPS induced ARDS,and regroup BALB/c mice.Mice were divided into 2 groups:2 day group and 4 day group.Every group were divided into 4 subgroups:Saline+IgG,Saline+anti-CD25,LPS+IgG,LPS+anti-CD25.Left Bronchoalveolar lavage fluids(BALFs)were collected by lavaging the lung and total cells counts in BAL fluids(BALFs)were enumerated.Flowcytometry was used to detect the proportion of neutrophils,macrophages and lymphocytes in lung tissue.The middle lobe of the right lung was fixed in 4%paraformaldehyde.Pulmonary sections in HE staining were used to observe the lung inflammation under microscope,and the pulmonary pathological score was obtained.IFN-?+Thl cells,IL-17a+Th17 cells,IL-4+Th2 cells in right lung and spleen single cell suspension were determined by flowcytometry.Release of Secreted soluble protein(IFN-y,IL-17a,IL-6,IL-4,TNF-a,IL-10)in BALF was analysed by bead-based immunoassays using a Th panel kit.The concentration of TGF-?1 in BALF was also determined using ELISA kit.Pulmonary gene expression of IFN-y,IL-17 a,IL-6,IL-4,TNF-?,IL-10 was detected by qPCR.Results:Tregs aggregation was observed in lung and spleen on ARDS mice.Intraperitoneal injection of anti-CD25 antibody depleted Treg cells in mice,and then established the mouse model of ARDS.Compared with the ARDS group,although the ratio of lymphocytes was not affected,the total cell count of BALF,the proportion of neutrophils and were decreased,the proportion of macrophages were increased in Treg depleted ARDS mice.In addition,depletion of Treg cells down-regulated the number of CD326+ epithelial cells,destroyed the proliferation of CD31+endothelial cells,and impaired Thl and Th17 immune responses.Conclusion:Treg cells contribute to the resolution of lung inflammation in ARDS,promote the repair of lung injury,and play a protective role in ARDS by regulating the Th immune responses.