CD39~+ Regulatory T Cells Attenuate Lipopolysaccharide-Induced Acute Lung Injury Via Autophagy And The ERK/FOS Pathway

Acute lung injury(ALI)/Acute respiratory distress syndrome(ARDS)is characterized by an uncontrollable cytokine storm,which is associated with high mortality due to lack of effective treatment.Regulatory T cells(Tregs)play an indispensable role in maintaining immune homeostasis and ecto-nucleoside triphosphate diphosphohydrolase 1(ENTPD1 or CD39)is considered as a functional cell marker of Tregs.CD39 catalyze the hydrolysis of pro-inflammatory triphoshpo-and diphosphonucleosides(ATP and ADP)to the monophosphonucleoside(AMP).AMP is further converted into anti-inflammatory adenosine(ADO)by ecto-5’-nucleotidase(CD73).Most of the immunosuppressive and anti-inflammatory effects of Tregs is due to the ADO produced by the ATP/ADP-AMP-ADO pathway and CD39 is the rate-limiting enzyme in this pathway.Previous studies have revealed the role of CD39 on Tregs in limiting tissue injury in myocardial infarction and benign prostate hyperplasia.The role of CD39+Tregs in ARDS has not been reported.In this study,we aimed to investigate the frequency of CD39+Tregs in ARDS patients and evaluate the effect of CD39+Tregs on ALI.First,we analyzed the percentage change of CD39+Tregs in PBMCs of ARDS patients and healthy controls and its correlation with disease severity.Second,we established ALI and control models induced by LPS or PBS instillation in wide type mice or CD39 knockout mice to investigate the survival,lung injury and pulmonary inflammation.Third,we examined the Tregs and CD39+Tregs in PBMCs and the spleen using the flow cytometry.We also detected the change of CD39+Tregs in mouse lung tissues by immunofluorescence.Fourth,we sorted Tregs from the spleens of wild type mice and CD39 knockout mice and co-cultured them with RAW264.7 cells in vitro to compare their anti-inflammatory effects.In vivo,we verified the effects of the sorted Tregs by tail vein reinfusion.At last,we further sought to explore the mechanisms that affect CD39 expression on Tregs.We found that the frequency of CD39+Tregs was decreased in the peripheral blood of ARDS patients and was positively correlated with disease severity.After LPS treatment,CD39-/-mice exhibited more severe inflammation.Consistent with the clinical finding,wild type mice exhibited a decreased frequency of CD39+Tregs in the peripheral blood.Furthermore,CD39+Tregs had a protective effect on LPS-induced inflammation in vitro and the adoptive transfer of CD39+Tregs had a therapeutic effect on ALI in vivo.LPS-induced inflammation in the lung impaired the immunosuppressive effect of Tregs via the autophagy-mediated downregulation of CD39.In addition,CD39 induced the expression of itself in Tregs via activating the ERK1/2-FOS pathway.In brief,the expression of CD39 on Tregs is regulated by both autophagy and the ERK/FOS pathway under LPS induced inflammation.Our results suggested that the adoptive transfer of CD39+Tregs may provide a novel method for the clinical prevention and treatment of ARDS.