The Functions Of Exosomes From Intestinal Epithelial Cells On Maintenance Of Immune Balance Of Intestinal Tract And The Underlying Mechanisms

The intestinal tract is the largest immunotolerant organ in the human body,able to tolerate a host of food antigens and bacteria.However,the underlying mechanisms by which this occurs are still largely unknown.The ingestion of dietary antigens does not result in problematic immune reactions because of the effective creation of an immunotolerant environment in the gut.A complex interplay of factors are involved in the maintenance of this environment,including the participation of regulatory T cells(Tregs),DCs,CD8+ T cells,?? T cells,regulatory B cells,IgA,commensal bacteria and massive cytokines such as TGF-?1 and IL-10.5-9 The breakdown of intestinal immunotolerance can result in autoimmune diseases of the gut such as IBD.IBD,including Crohn's disease and ulcerative colitis,are characterized by a chronic and exacerbated inflammation of the intestinal mucosa.10 A large number of patients suffer from IBD because of the recurrent attacks characteristic of this disease.An understanding of the mechanism of intestinal immunotoleranceis thus required for the development of new effective curative strategies for IBD.Exosomes are lipid-bilayer vesicles,50-100 nm in size,secreted by almost all types of live cells.They are generated by the inward budding and subsequent fusion with the plasma membrane of multi-vesicular bodies,which are then released into the extracellular space.Exosomes have been shown to promote pleiotropic effects on the immune system including immune activation and suppression.The objective of this study was to determine whether exosomes from the murine intestine are involved in the maintenance of intestinal tract immune balance,and to elucidate the mechanism responsible for this process.Exosomes obtained from the intestinal tract were identified,Exosomes from small intestine(Si-EXO)and large intestine(Li-EXO)were all positive for MHC-?,CD11c,and A33,suggesting dendritic cell(DC)and intestinal epithelial cell(IEC)derivations.All exosomes expressed TGF-?1,To further characterize the vesicles,we floated them on a sucrose gradient,and found that A3 3 and HSP70 molecules were enriched in the density of 1.09-1.17 g/ml,corresponding to the expected location of exosomes?Then their immunosuppressive potential was examined in vitro,our study showed similar potential in inhibition of CD4+ T cell proliferation in vitro in a dose-dependent manner.Since dendritic cell(DC)and intestinal epithelial cell(IEC)derivations of exosomes obtained from the intestine,to determine which subset of Li-EXO was responsible for the suppressive effect,we isolated CD11c+ and CD11c-Li-EXO using CD11c+ magnetic beads.Results suggest that Li-EXO from IECs are immunosuppressive.To elucidate the role of TGF-?1 in the immunosuppressive potential of A33+ Li-EXO,we pre-treated the CD4+ T cells with SB525334,a potent and selective inhibitor of TGF-? receptor I.After blocking TGF-?1 signaling,the inhibitory effect of A33+ Li-EXO on CD4+ T cell proliferation was completely abrogated.Using a dextran sulfate sodium salt(DSS)-induced murine inflammatory bowel disease(IBD)model,we determined whether isolated intestinal exosomes were involved in the maintenance of immune balance of the intestinal tract in vivo.After intravenous transfer of A33+ Li-EXO,we observed marked amelioration in body weight loss of IBD mice in a dose-dependent manner.A33+ Li-EXO induced the differentiation of CD4+Foxp3+Tregs both in vitro and in vivo.To determine the role of CD4+Foxp3+ Tregs in the A33+Li-EXO mediated decrease in severity of murine IBD,CD4+Foxp3+ Tregs were depleted by the pretreatment of mice with anti-CD25 mAb.The protective effect of A33+ Li-EXO against DSS-induced weight loss somewhat decreased,which suggests that Tregs contributed to the effect of A33+ Li-EXO in alleviation of the severity of DSS-induced murine IBD.Furthermore,Li-EXO from IBD mice(IBD-Li-EXO)contained higher levels of TGF-?1 than those from control mice(Ctrl-Li-EXO),and IBD-Li-EXO were more immunosuppressive in vitro and in vivo,and A33+ Li-EXO of IBD mice were more effective in the prevention of DSS-induced weight loss compared to control mice.In addition,we found that A33+ Li-EXO significantly inhibited LPS-induced IL-12 secretion and antigen-presenting ability in the mixed lymphocyte reaction of DCs,and the weight loss in mice injected with DT was significantly higher in CDllc-DTR mice in which DC function defects than in WT mice after A33+ Li-EXO treatment.In contrast,decreased secretion of exosomes by Spiroepoxide,a nSmase2 inhibitor,in vivo increased the development of murine IBD.Furthermore,exogenous transfer with Li-EXO abolished the effect of spiroepoxide.These results suggest that inhibition of the secretion of exosomes in vivo can increase the susceptibility of mice to DSS-induced IBD.Furthermore,interestingly,we found that A33+ Li-EXO tended to localize in the intestinal tract.Exosomes from other organs were negative for A3 3 as determined by Western blot,and to further confirm this,we intravenously transferred CFSE-labeled Li-EXO and evaluated the distribution of Li-EXO in vivo.Exogenic Li-EXO tended to traffic to the gastrointestinal tract including the stomach,small intestine,large intestine and mLN.These results indicate that Li-EXO tend to be bound in the gastrointestinal tract.In this study,we found that immunosuppressive exosomes are secreted from IECs.These exosomes can decrease the severity of murine IBD,which suggest that these exosomes might be involved in the maintenance of intestinal tract immune balance.These findings increase our understanding of the mechanisms by which the intestinal tract achieves immunotolerance.A strategy to increase exosome production from IECs represents a potential means of restoring the homeostasis of the immune system in the intestinal tract.This represents a promising treatment strategy of diseases caused by disruption of immune homeostasis including IBD.