Hypoxia Enhanced Dendritic Cells Producing Immune Negative Regulatory Molecule Indolamine 2,3-Dioxygenase

Objective:Hypoxia-associated metabolic reprogramming modulates the biological functions of many immune and non-immune cells,and affects types and intensities of immune response.Dendritic cells(DCs),as a bridge connecting innate and adaptive immunity,play a key role in the occurrence,development and even Outcomes of inflammation.DCs not only present antigens to stimulate T cells,but also suppress T cell-mediated immune response.For example,DCs exhibit suppressive effects on T cells by secreting indocyanine 2,3-Indoelamine(IDO).During the process of inflammation,monocytes migrate to the tissue to differentiate into inflammatory DC,which is accompanied by a decrease in oxygen content caused by enhanced oxygen consumption in inflamed tissues.It is not clear that whether and how hypoxia affects the differentiation and function of monocyte-derived DCs as well as their synthesis of IDO.In this study,we investigated the effect of hypoxia on DC development and IDO synthesis and the underlying molecular mechanisms.Methods:The peripheral blood of the volunteers was collected and CD14+ monocytes were purified by immunomagnetic beads sorting.GM-CSF and IL-4 were used to stimulate monocytes to develop in vitro for 7 days,during which 9-5-2-1% gradient(9% for 3days,5% for 2days,2% for 1day and 1% for 1day)or 2-1% gradient(2% for 5days and 1% for1day)O2 concentrations was provided to mimic hypoxic environment in blood circulation system and tissue.Normal oxygen content(21%O2)culture conditions was used as the control.At certain time point,RNA of DCs in each group was extracted,and q PCR was performed to analyze the expression of IDO m RNA,and the phenotype of DCs in each group was detected by flow cytometry.During the incubation of DCs,addition of a specific si RNA interfering with the expression of Hypoxia-inducible factor(HIF)alpha was used to observe whether the HIF-alpha mediated pathway was involved in the regulation of thesynthesis of IDO.During the process of DC development,expression of adenosine receptors was dynamicly detected,and the effects of A2 b R and A3 R inhibitors or A2 a R agonists on IDO expression and DCs phenotye weas examined.Further experiments to investigate the underlying molecular mechanisms at the micro RNA level were performed.RNA of DC cultured in different oxygen contents was extracted,then high throughput chip was used to detect and analyze micro RNA expression profiles for selection of differential expressed micro RNA,and judge whether its expression is regulated by HIF pathway.Differentially expressed miro RNA was then transfected into DCs,and DCs cultured under hypoxic condition were used as controls.High throughput chips were used to detect and analyze m RNA transcriptional expression profiles in micro RNA-transfected DC,hypoxia-induced DC and controls respectively,and GO enrichment analysis as well as KEGG enrichment analysis was further carried out.Finally,key micro RNA and its inhibitor were transfected into DCs to evaluate their effects on expression of IDO and A3 R.Results : During the process of DC differentiation from monocytes,hypoxia can upregulate the expression of IDO m RNA in DCs in a concentration-dependent pattern.Our results showed that the lowest concentration of O2(1%O2)responded to the strongest stimulating effects of IDO on DCs.Hypoxia culture significantly promoted MHC-II,CD86,CD54,CD274 expression and inhibited CD40,CD83 expression when compared with normal oxygen condition.Additionally,upregulation of MHC II,CD86,CD54,CD274 by hyoxia could be reverse if synthesis of IDO on DCs was blocked,whereas CD40,CD83 expression still greatly decreased on DCs.These data indicated that IDO was involved in the regulation of DC phenotype.We also found that addition of si RNA specific to HIF-alpha in the culture significantly inhibited the expression of IDO m RNA in DCs under normal oxygen condition,and the inhibition rate was close to 50%.However,the inhibitory effect on IDO expression by si RNA was not obvious under hypoxic condition,indicating that the upregulation of IDO induced by hypoxia was not dependent on HIF-alpha signaling pathway.In addition,under hypoxic condition,there was an increased expression of adenosine receptors on DCs including A1 R,A2a,A2 b R and A3 R.Blockade of A3 R could significantly inhibit IDO expression,blocking A2 b R could significantly promote IDO expression,and A2 a R agonist has no significant effect on IDO expression on DCs under hypoxia condition.We also found that A3 R inhibitors could reverse the upregulated expression of CD86,MHC-II,CD54 and CD274,and had no effects on theexpression of CD40 and CD83 on DCs.By analyzing the expression of micro RNA,it was found that upregulation of mi R-210 was most obvious and independent of HIF-1 pathway.Results from transcriptional analysis and GO/KEGG enrichment analysis performed on hypoxia-induced DC and their controls,suggesting that mi R-210 was the key micro RNA in the regulation of DC biological functions under hypoxia condition.Although transfection of mi R-210 could increase IDO and A3 R m RNA expression on DCs,IDO1,IDO2 and A3 R were not the target genes directly regulated by mi R-210.Conclusion:In the process of DC differentiation,hypoxia regulates monocyte-derived DC to express IDO in A3R-dependent signaling pathway,and IDO regulates the phenotype of DCs.The cause is that hypoxia could alter the micro RNA expression of DCs,and increased expression of mi R-210 contributes to the regulation of DC biological functions under hypoxia condition.Upregulation of IDO and A3 R by mi R-210 was in an indirect pattern,as IDO2 and A3 R were not the target genes directly regulated by mi R-210.Altogether,the results in this study provide a theoretical basis for the clinical application of adenylate receptor and IDO agonists or antagonists in the future.