Role Of HVEM In The Induction Of CD4+CD25+Foxp3+Regulatory T-cell Differentiation From Mouse Bone Marrow-derived Dendritic Cells Treated By1,25-dihydroxyvitamin D3

Backgoud and objectiveAllergic asthma is recognized as a chronic inflammatory disease of the airways inwhich many types of cells play a role, in particular mast cells, eosinophils, B-andT-lymphocytes, neutrophils and epithelial cells. And the pathogenesis of the disease is notclear completely. The pathogenesis of asthma has been considered as the imbalancebetween Th1and Th2cells.And now more and more researches have revealed that thedevelopment of this disease is correlated with over-activated Th2cells and the quantitativeand/or functional insufficiency of regulatory T cells to allergens in allergic asthma.Therefore, the key of intervention strategy is induction and expansion of regulatory T cells,which inhibit over-activated Th2immune response in body.The activated form of vitamin D,1,25-dihydroxyvitamin D3[1,25(OH)2D3],is asecosteroid hormone that regulates bone and calcium/phosphate metabolism. However, anumber of studies have proved that this steroid hormone also has an immunoregulativeeffect. The biologic effects of1,25(OH)2D3are mediated by the vitamin D receptor (VDR).Antigens presenting cells, especially dendritic (DC) cells, express VDR and are key targetsof VDR agonists. Numerous studies have demonstrated that1,25(OH)2D3inhibit thedifferentiation and maturation of DC. These studies have shown that DC treated in vitrowith1,25(OH)2D3experienced down-regulated expression of the co-stimulatory moleculesCD40, CD80, CD86, and MHC class II, decreased IL-12, and enhanced IL-10production,which can cause tolerogenic dendritic cells (tDC), favoring the induction of regulatory T(Treg) cells. These effects are not limited to in vitro activity:1,25(OH)2D3can also induceDC with tolerogenic properties in vivo, as demonstrated in models of Th1related autoimmunity diseases. But, the role in Th2-mediated diseases,such as allergic asthma, isstill controversial. Most of researcher believed that it had a protection role in allergicasthma,but its mechanism is still unknown.Besides co-stimulatory molecules, inhibitory molecules have been found to play acritical role in DC-mediated immune tolerance. Herpesvirus entry mediator (HVEM),known as TNFRSF14, is a member of tumor necrosis factor receptor (TNFR)-TNFsuperfamily. HVEM express on T cells and DCs, can regulate the differentiation of T cellsand DCs. HVEM displays dual functional activity by binding to coinhibitory receptors(such as BTLA or CD160) and attenuating TCR-mediated signaling or acting as a receptorof LIGHT and costimulating T cells.However,the role of HVEM effect on DC mediatedimmunological tolerance is unclear. Rearch showed that HVEM-/-mice were moresusceptible to autoimmunity diseases. APCs from HVEM–/–mice were more active instimulating T cells than those from WT mice. over-expression HVEM on DC produced aregulatory cytokine, IL-10, which had further effects on induction of IL-10producingCD4+T cells. This subset of T cells was then responsible for the protection against EAM.These researches all supported that HVEM as an inhibitor molecular involved in DCmediated immunological tolerance. Therefore,we we hapothesized that1,25(OH)2D3treated mouse tolerogenic bone marrow-derived immature dendritic cells (BMDC) promotethe production of CD4+CD25+Foxp3+T cells through up-regulation of HVEM.MethodsPart one: Phenotypes and function of1,25(OH)2D3-treated BMDC stimulated byovalbumin and lipopolysaccharidePhenotypes of1,25(OH)2D3-treated BMDC stimulated by ovalbumin andlipopolysaccharideMurine bone marrow-derived DC were prepared for different subsets of DC, untreatedimmature DC (imDC) were harvested on day5or day6of culture and used as controlcultures. D3/imDC were generated by addition of10-81,25(OH)2D3on day3controlculture. Cells were harvested on day8. OVA-D3/imDC and LPS-D3/imDC were generatedby addition of LPS (1μg/ml) and OVA(100μg/ml) on day7D3/imDC culture lasted24hours and cells were harvested on day8. LPS activation of immature DC (LPS/imDC) andOVA activation of immature DC (OVA/imDC) were stimulated by exposure to LPS (1 μg/ml) and OVA(100μg/ml) on day7. Cells were cultured for24hours and harvested onday8.Morphology of1,25(OH)2D3-treated BMDC activated by ovalbumin andlipopolysaccharideMorphology of different subsets of DC were observed by inverted microscope.Phagocytic ability analysis of1,25(OH)2D3-treated BMDC activated byovalbumin and lipopolysaccharideFluid-phase uptake was assessed by incubation of1,25(OH)2D3-treated BMDCactivated by ovalbumin and lipopolysaccharide with100ug/ml FITC-OVA at either4°C or37°C for30minutes. The relative number of positive cells was determined using flowcytometry with a FACS Calibur.Flow cytometric analysisDC were triple-stained with APC-anti-CD11c and either PE-CD80,-MHC class II, orFITC-CD86,-CD40for phenotypic analysis. The relative number of positive cells wasdetermined using flow cytometry with a FACS Calibur. Appropriately conjugatedisotype-matched control antibodies were used as negative controls.The levels of IL-12p70, IL-10,IL-6analyzed by Enzyme-linked immunosorbentassayDifferent subsets of DC culture supernatants were stored at-80°C. The levels ofIL-12p70, IL-10and IL-6were measured using ELISA kits according to the manufacturer’sinstructions.Mixed leukocyte reactionDC were purified by using magnetic-activated cell sorting (MACS) according to themanufacturer’s instructions. Na ve CD4+T cells were purified from Balb/c spleen cellsusing magnetic-activated cell sorting (MACS) according to the manufacturer’s instructions.1,25(OH)2D3-treated BMDC stimulated by ovalbumin and lipopolysaccharide co-culturewith T cells (1×10~5) from BALB/c mice for4days at different ratio of DC:T cell, andpulsed with1μCi of [3H] thymidine for the last18h of culture. Cells were harvested ontoglass fiber filters, and the radioactivity incorporated was quantitated using a Beckmanliquid scintillation counter. In vitro differentiation assessment of CD4+CD25+Foxp3+Treg cellsNa ve CD4+CD25-T cells were purified from Balb/c spleen cells using magnetic-activated cell sorting (MACS) according to the manufacturer’s instructions. AllogeneicCD4+CD25-T cells (1×10~6) were cocultured with1,25(OH)2D3-treated BMDCstimulated by ovalbumin and lipopolysaccharide for4days.co-culture cells were stained,and the incidence of CD4+CD25+Foxp3+Treg was determined using flow cytometry.Expression of chemokine receptor CCR5, CCR7and CXCR4on1,25(OH)2D3-treated DCs stimulated by ovalbumin and lipopolysaccharideDC were stained with APC-anti-CD11c and PE-CCR5,-CCR7and–CXCR4forchemokine receptor analysis. The relative number of positive cells was determined usingflow cytometry with a FACS Calibur. Appropriately conjugated isotype-matched controlantibodies were used as negative controls.Part two: Role of HVEM in the induction of CD4+CD25+Foxp3+regulatoryT-cell differentiation from1,25(OH)2D3-treated DCThe level of IL-2secreted by1,25(OH)2D3-treated DC stimulated by ovalbuminand lipopolysaccharide analyzed by Enzyme-linked immunosorbent assayDifferent subsets of DC culture supernatants were stored at-80°C. The levels of IL-2was measured using ELISA kits according to the manufacturer’s instructions.Expression of HVEM, PD-L1and PD-L2on1,25(OH)2D3-treated DC stimulatedanalyzed by flow cytometric analysis and quantitative real-time polymerase chainreactionFlow cytometric analysisDifferent subsets of DC were stained with APC-anti-CD11c, PE-HVEM,-PDL1,-PDL2for FACS analysis. The relative number of positive cells was determined using flowcytometry with a FACS Calibur. Appropriately conjugated isotype-matched controlantibodies were used as negative controls.Quantitative real-time polymerase chain reactionTotal RNA from different subsets of DC were extracted using TRIzol reagent(Invitrogen) according to the manufacturer’s instructions. The integrity of the total RNAwas examined using1%agarose gel electrophoresis. The quantity was determined based onabsorbance at260nm (A260), and the purity was analyzed based on the absorbance ratio at260and280nm (A260/280). cDNA was synthesized from1μg of total RNA using an All-in-one First-strand cDNA Synthesis Kit according to the manufacturer’srecommendations. We used SYBR Green for all the tested genes. The thermal cyclingconditions were as follows: an initial denaturation at95°C for10min followed by40cyclesat63°C for20s,72°C for10s, followed by melting from72°C to95°C in0.5°C,10sincrements. GAPDH and HVEMwere tested using real-time polymerase chain reaction(RT-PCR). Quantitative RT-PCR was performed using a Bio-Rad IQ5Real-time PCRsystem. Glyceraldehyde phosphate dehydrogenase (GAPDH) served as an internal control.Data analysis was performed using a Sequence Detector System software (Bio-Rad IQ5),and the results are expressed as fold changes in relative mRNA expression level. This levelwas calculated using the△△Ct method with GAPDH as the reference gene and the imDC orLPS/imDC as the baseline.Effects of anti-HVEM on tolerogenic characteristics of1,25(OH)2D3-treated DCsby MLRTo investigate the importance of HVEM in the tolerogenic capacity of OVA-D3/imDCand LPS-D3/imDC, we measured the ability of OVA-D3/imDC and LPS-D3/imDC tostimulate CD4+T cell proliferation by blocking HVEM expression in OVA-D3/imDC andLPS-D3/imDC before co-culture with na ve CD4+T cells.Effects of anti-HVEM on tolerogenic characteristics of1,25(OH)2D3-treated DCsby quantitative real-time polymerase chain reaction and Enzyme-linkedimmunosorbent assayQuantitative real-time polymerase chain reactionTotal RNA from CD4+CD25-T cells co-cultured with OVA-D3/imDC andLPS-D3/imDC for96hours were extracted using TRIzol reagent according to themanufacturer’s instructions. OVA/imDC and LPS/imDC as positive control. In blockexperiment, OVA-D3/imDC and LPS-D3/imDC treated with10ug/ml anti-HVEM mAb orisotype controls IgG for48h, and then unbound antibodies were removed by washing priorto addition of na ve CD4+CD25-T cells. cDNA was synthesized from1μg of total RNAusing an All-in-one First-strand cDNA Synthesis Kit according to the manufacturer’srecommendations. We used SYBR Green for all the tested genes. GAPDH and T-bet,GATA-3, Foxp3were tested using real-time polymerase chain reaction (RT-PCR).Quantitative RT-PCR was performed using a Bio-Rad IQ5Real-time PCR system. Glyceraldehyde phosphate dehydrogenase (GAPDH) served as an internal control. Dataanalysis was performed using a Sequence Detector System software (Bio-Rad IQ5), and theresults are expressed as fold changes in relative mRNA expression level. This level wascalculated using the△△Ct method with GAPDH as the reference gene and the imDC as thebaseline.Enzyme-linked immunosorbent assayDifferent subsets of DC co-culture with na ve CD4+CD25-T cells supernatants werestored at-80°C. The levels of IFN-γ, IL-4and IL-10was measured using ELISA kitsaccording to the manufacturer’s instructions.Result:Part one: Phenotypes and functions of1,25(OH)2D3-treated BMDC stimulated byovalbumin and lipopolysaccharideOVA-D3/imDC and LPS-D3/imDC exhibited characteristic immature DC with shortdendrites compare with mature DC (OVA/imDC and LPS/imDC) with prominent dendrites.D3/imDC displayed more highly efficient at uptake of soluble FITC-OVA than that ofimDC, mature DC (OVA/imDC and LPS/imDC) showed low efficient at uptake of solubleFITC-OVA, OVA-D3/imDC and LPS-D3/imDC shown highly efficient at uptake of solubleFITC-OVA similarly to imDC. FACS analysis of D3/imDCs showed immature phenotypescharacterized by down-regulated expression of the co-stimulatory molecules (CD80, CD86,and CD40) and MHC class II,especially expression of CD86. Upon activation by LPS andOVA, imDCs displayed mature phenotypes, characterized by up-regulated expression of theco-stimulatory molecules (CD80, CD86, and CD40) and MHC class II. However,OVA-D3/imDC and LPS-D3/imDC showed semi-mature phenotypes, which expressedmore CD86and MHC class II than imDC (all P<0.05), but less than OVA/imDC andLPS/imDC (mature DC)(P<0.01and0.001respectively).Part two: Role of HVEM in the induction of CD4+CD25+Foxp3+regulatoryT-cell differentiation from1,25(OH)2D3-treated DCThe level of IL-10was significant higher in the culture supernatant fromOVA-D3/imDC and LPS-D3/imDCs relative to imDC and OVA/imDC and LPS/imDC (allP<0.001). The levels of IL-12p70and IL-6were significantly lower than in OVA/imDC andLPS/imDC (all P<0.001). OVA-D3/imDC and LPS-D3/imDC displayed poorer T cell stimulatory capacity than OVA/imDC and LPS/imDC at different DC:T cell ratios. MoreCD4+CD25+Foxp3+Treg cells were produced by OVA-D3/imDC and LPS-D3/imDCco-cultured with na ve CD4+T cells than by OVA/imDC and LPS/imDC.OVA-D3/imDC and LPS-D3/imDC secreted low levels IL-2. The results of FACSshowed that the levels of HVEM were very low in imDC and LPS/imDC, but21.0foldenhancement in HVEM expression was observed in D3/imDC, not counting a4.1and5.8fold up-regulation after LPS and OVA activation.relative to imDC,OVA/imDC andLPS/imDC (P<0.001). OVA-D3/imDC and LPS-D3/imDC showed up to a1.0fold and0.9fold up-regulation of PDL1relative to OVA/imDC and LPS/imDC. However, expression ofPDL2was down-regulated in OVA-D3/imDC and LPS-D3/imDC. The results ofquantitative real-time polymerase chain reaction showed the levels of relative mRNAexpression of HVEM was significant up-regulated on OVA-D3/imDC and LPS-D3/imDC(P<0.05and0.01). The capacity of HVEM-blocked OVA-D3/imDC and LPS-D3/imDC tostimulate CD4+T cell activation was significantly lower than that of control cells (P<0.01).Next, we investigated the role of HVEM on OVA-D3/imDC and LPS-D3/imDC withrespect to differentiation toward CD4+CD25+Foxp3+Treg cells and Th2/Th1cells asindicated by both mRNA and cytokine levels. we found LPS-D3/imDC andOVA-D3/imDC could promote na ve CD4+cells toward Treg cells differentiation byenhance Foxp3transcript and and production of IL-10in culture supernatants.OVA-D3/imDC could inhibit na ve CD4+cells toward Th2cells differentiation by impairGATA-3transcript and and production of IL-4in culture supernatants Similarly,LPS-D3/imDC could inhibit na ve CD4+cells toward Th1cells differentiation by impairT-bet transcript and and production of IFN-γ in culture supernatants.,too. Blockage ofHVEM on LPS-D3/imDCs and OVA-D3/imDC prior to co-cultured with na ve CD4+Tcells led to DC-driven differentiation towards Treg cells was impaired by decreasing therate of Foxp3transcription and the production of IL-10in culture supernatants.Conclusion1.1,25(OH)2D3-treated BMDC renders dendritic cells (DCs) tolerogenic, favoring theproduction of CD4+CD25+FoxP3+Treg cells and inhibit CD4+T cells activation in vitro.2.1,25(OH)2D3-treated BMDC expressed significant high levels of HVEM. Blockageof HVEM on LPS-D3/imDCs and OVA-D3/imDC prior to co-cultured with na ve CD4+T cells led to DC-driven differentiation towards Treg cells was impaired, furthermore, theability of1,25(OH)2D3-treated BMDC suppress T-cell proliferation was attenuated. Allthese indicated that the critical role of HVEM in the induction of CD4+CD25+Foxp3+regulatory T-cell differentiationIn this study, HVEM play key role in the induction of CD4+CD25+Foxp3+regulatoryT-cell differentiation from mouse bone marrow-derived dendritic cells treated by1,25-dihydroxyvitamin D3.