Inhibitory Effect And Mechanism Of CD4+CD25-LAG3+T Cells On Pulmonary Fibrosis With Connective Tissue Disorders

ObjectivesPulmonary fibrosis(PF)is a common outcome of diffuse pulmonary inflammatory disease caused by a variety of causes,and interstitial pulmonary disease with connective tissue disorders(CTD-ILD)is an important cause of poor prognosis of pulmonary fibrosis and connective tissue disease.Although the etiology of pulmonary fibrosis is different,its pathological changes are characterized by diffuse alveolitis,myofibroblast proliferation and collagen deposition,which eventually leads to the destruction of lung tissue structure and lung failure.PF is a chronic and progressive irreversible disease with great harm.So far,the pathogenesis of PF is not completely clear,and there is a lack of specific and effective treatment.Immune disorder is an important factor in the progression of tissue inflammation to fibrosis.This study aims to reveal the role of immune regulatory T cells--CD4+CD25-LAG3+Tregs and their secreting factor TGF-β3 in the regulation of pulmonary fibrosis,explore the regulatory mechanism,and determine whether LAG3+T cell subsets have the value of biological markers,so as to provide new targets and new ideas for the mechanism research and treatment of CTD-ILD.MethodsFirst,lung tissue and peripheral blood samples of patients were collected to observe and compare the pathological changes of lung tissue,detect the proportion of CD4+LAG3+Tregs and the expression level of cytokine TGF-β3 in peripheral blood,and collect relevant clinical examination data of these patients for analysis.Secondly,after co-culture,the effects of CD4+LAG3+Tregs on the proliferation,migration and invasion of human lung fibroblasts(HLFs)were observed.And the expression levels of p-Smad3,α-SMA and Fibronectin,m RNA expression levels of Ⅰ type and Ⅲ type collagen.The effect of CD4+LAG3+Tregs on the polarization of macrophages was detected.Finally,the pulmonary fibrosis model of C57BL/6 mice was established by bleomycin(BLM).The extracted CD4+LAG3+Tregs single cell suspension was injected into the mice through the tail vein.Blood samples were collected from the eyeballs on the 28 th day for cytokine analysis,and the mice were killed immediately after that.The effect of CD4+LAG3+Tregs on pulmonary fibrosis was observed by histological detection and biochemical analysis of lung tissues.ResultsThe mean value of C-reactive protein and erythrocytes sedimentation rate in CTD-ILD patients increased,FEV1(%),FVC(%),DLCO(%)and 6-minute walking distance decreased,the proportion of CD4+LAG3+Tregs in peripheral blood and the level of TGF-β3 decreased.CD4+LAG3+Tregs can inhibit the proliferation,migration,invasion and other cellular functions of HLFs,and inhibit the protein expressions of p-Smad3,α-SMA and Fibronectin of HLFs,as well as the m RNA expressions of Ⅰ type and Ⅲ type collagen.CD4+LAG3+Tregs and TGF-β3 can inhibit the polarization of M0 type macrophages to M1 type macrophages,but can promote the polarization of M2 type macrophages.CD4+LAG3+Tregs and TGF-β3 can alleviate these pathological changes in the mice with pulmonary fibrosis model,including the destruction of alveolar structure,the thickening of pulmonary interstitium,the increase of collagen deposition,the increase of hydroxyproline content,and the increase of α-SMA and Fibronectin expression.Neutralizing antibodies against TGF-β3 aggravate these pathological changes.Compared with normal mice,both the proportion of CD4+LAG3+Tregs and the level of TGF-β3 in spleen and peripheral blood of pulmonary fibrosis mice were decreased.ConclusionsCD4+LAG3+Tregs and TGF-β3 levels in peripheral blood were significantly decreased in patients with CTD-ILD,and the mean value of C-reactive protein and erysedimentation rate were increased.Both in vivo and in vitro experiments have demonstrated that CD4+LAG3+Tregs and their secretion of TGF-β3 inhibit the progression of pulmonary fibrosis by inhibiting the activation of lung fibroblasts,and neutralizing antibodies against TGF-β3 attenuated this inhibition.Inhibition of M1-type polarization of macrophages and promotion of M2-type polarization of macrophages may be one of the anti-inflammatory and anti-fibrosis mechanisms of CD4+LAG3+Tregs and their secretion of TGF-β3.CD4+LAG3+Tregs may have the value of biological markers,and these findings will provide new clinical targets and new ideas for the treatment of pulmonary fibrosis.