The Effects Of Indoxyl Sulfate On The Immune Function Of Hunman Monocyte-derived Dendritic Cells

Background:Cardiovascular disease(CVD)is the major cause of death in patients with chronic kidney disease(CKD)[1].In particular,accelerated atherosclerosis(AS)leading to severe coronary artery disease has been observed in patients with CKD[2,3].In fact,CVD accounts for the premature death of more than 50%of patients undergoing regular dialysis[4].Indoxyl sulfate(IS)is a class of albumin-bound uremic toxins that can not be cleared by current conventional hemodialysis[5].The proteinbound uremic toxins have emerged as important factors associated with cardiovascular disease and the outcome of CKD.It has been reported that IS induces AS mainly by activating vascular endothelial cells and promoting vascular smooth muscle cell calcification[6,7].In recent years,AS is considered as a chronic inflammatory disease in which dendritic cells(DCs)play an impotant role[8,9].Thus,we investigated the impact of IS on phenotypic and functional characteristics of DCs in vitro,which will enable us to further understanding the potential role of IS in atherogenesis.Part ? Isolation,culture and phenotypical analysis of hunman monocyte-derived dendritic cells in vitroObjective:To estabilsh a quick,easy and inexpensive method for the isolation and culture of monocyte-derived dendritic cells(mo-DCs),which can be used to establish the experimental basis for further study the immune function of DCs.Methods:Human peripheral blood mononuclear cells(PBMCs)were isolated from 30ml of heparinized human blood using a double density gradient centrifugation employing Ficoll isopaque and 46%iso-osmotic Percoll,then the adherent cells were treated with rhGM-CSF(1000 IU/mL)and rhIL-4(500 IU/mL)for five days.On the fifth day of culture,the mo-DCs were observed by phase contrast microscope,scanning electron microscopy(SEM)and judged by the expression of the hallmark surface protein CD 11c.Results:1.Yields of mo-DCs ranged from 3 to 6 million(from 30 ml of blood)with a viability of>90.0%.2.The typical form of mo-DCs was observed by phase contrast microscope and SEM.3.FACS analysis of mo-DCs on day 5 shows a homologous population expressing high levels of CD 11c.Conclusion:In conclusion,this protocol is a very fast,easy,and cost-effective way to generate a viable and pure population of mo-DCs.The cells can be easily harvested and used for subsequent studies and functional assays.PRAT ? Indoxyl sulfate exerts an immunomodulatory effect on human monocyte-derived dendritic cellsObjective:To explore the effect of indoxyl sulfate(IS)on differentiation,maturation and function of mo-DCs,which provides the basis to clarify deep mechanisms further.Methods:In vitro,the immature DCs(imDCs)were grown from PBMC with rhGM-CSF(1000 IU/mL)and rhIL-4(500 IU/mL).Then the cells were treated with various concentrations of IS(0,30 ?mol/L,300 ?nol/L,600 ?mol/L)for 24 h.The phenotypic maturation of mo-DCs induced by IS was evaluated by flow cytometry(FCM);The functional maturation of mo-DCs was analyzed by measuring FITC-dextran uptake and ELISA;The structural maturation of mo-DCs was observed under SEM.Results:Our results showed that IS significantly upregulated the expression of CD80,CD83,CD86 and MHC ? key membrane molecules on mature DCs(mDCs),while downregulating phagocytosis and increasing the secretion of IL-12p70 by mDCs(P<0.05).And the mo-DCs cultured for 24 h in the prensence of LPS and IS at 300 pmol/L showed typical morphology with rough surface,long protrusions and fusiform.Among which IS at 300?mol/L is the most appropriate stimulus concentration.Conclusions:Stuctural,phenotypic and functional maturation of mo-DCs can be induced by IS at defined concentrations.This might be one of the mechanisms underlying the progression of atherosclerosis in patients with CKD.