Role Of Notch Ligand Delta-like 4 In The Induction Of Th17 Cell Differentiation From Ovalbumin-sensitized Mouse Dendritic Cells

Background and research purposesDepending on airway inflammation factor in patients with asthma, the asthma is dividedinto eosinophilicasthma, neutrophilicasthma, mixed cell asthma and few cell asthma.Excessive activation of Th2 cells in eosinophilic asthma, in the past, "Th1 / Th2 imbalance theory" is used to explain its pathogenesis.With the discovery of new T helper cells, we pay more and more attention to the quantity and functional defects ofregulatory T cells. "Th2 / Treg imbalance theory" has been further recognized in eosinophilic asthma.There is excessive Th17 cells differentiation in neutrophils asthma, and is there “Th1 / Th2 imbalance theory” in neutrophils asthma?Dendritic cells are considered the most potent antigen presenting cells in vivo.Different immune microenvironment allows DCs in different states, and different states of DCs can promote T cells into different T-helper cell. Notch ligand on DCs, such as Jagged 1, Jagged 2, Delta-like 1, Delta-like 3 and Delta-like 4 may promote T helper cell differentiation. during in vitro Th17 differentiationconditions, Delta-like 4 was reported to regulate Th17 cell activation: overexpression of Delta-like 4 on DCs increased IL-17 secretion while neutralization decreased it. Furthermore, an impact of Delta-like 4 on Th17 activation was reported in vivo. Neutralization of Delta-like 4 with an anti-Dll4 m Ab during the development of Mycobacterium-induced pulmonary granulomas resulted in the formation oflarger granulomas and secretion of decreased levels of Th17 cytokines.In addition to the involvement of Delta-like 4 in the activation of Th17 cells, theinduction of Jagged 1 m RNA by curdlan, a polysaccharide derived from Candiaalbicans, in human monocyte-derived DCs was shown to increase secretion of Th17 cell cytokines in vitro, and neutralization of Jagged 1 decreased curdlan-induced IL-17 secretion by T cells.1,25-dihydroxyvitamin D3 is the active form of vitamin D in vivo. The active form of vitamin D transcriptionally represses the Smad7 signaling and activates ERK to inhibit the differentiation of a inflammatory T helper cell subset and suppress experimental autoimmune encephalomyelitis. Clinical studies have shown that phenotype modifications of T-cells and their shift toward a Th2 response in patients with systemic lupus erythematosus supplemented with different monthly regimens of vitamin D.The inhibitory effect of 1,25-dihydroxyvitamin D3 on the Th17 response was mediated via both T cells and DCs. DCs pathway is involved in the direct inhibition of 1,25-dihydroxyvitamin D3 on Th17 cell differentiation in young asthmatics.In our experiment, we assumed that 1,25-dihydroxyvitamin D3 can regulate DCs express the Notch ligand Delta-like 4 and inhibit Th17 differentiation, thereby further affecting the Th17 / Treg balance. Weisolate the spleen DCs from sensitized mice, testing Notch ligand expression in DCs in asthma model,stimulate DCs with LPS and test differentiation of T helper cells with 1,25-dihydroxyvitamin D3 and dexamethasone treated DCs,and further study 1,25-dihydroxyvitamin D3 on ratio of Th17 / Treg.Research methodChapter Ⅰ Influence of 1,25 dihydroxyvitamin D3 on LPS treated DCs from OVA-sensitized mouse dendritic cells1.Established OVA sensitized mice modelOn the first day and sixth day, the mice were given of 0.02% OVA-Al(OH) 3 solution 200 ul to sensitize through intraperitoneal injection.On 13 th,14th,15 thdays, 1% OVA solutionis given from airway toestablish asthma model mice.The proportion of eosinophils in bronchoalveolar lavage fluid is used to determine whether the asthma model is seccessful.2.The isolation and culture of dendritic cells from OVA sensitized miceWhen the mice are sacrificed sterilly,we separate the spleen, and make the spleen cells intointo single-cell suspensions with lysed red blood cells.Purify the dendritic cells with CD11 c MACS.The isolated DCs are divided into following four groups, sensitized control group, LPS group, LPS + Dex group and LPS + Vit D3 group. The control group is added 1ul / ml PBS into the culture medium, LPS group is added 1ug / ml lipopolysaccharide into culture medium, LPS + Dex group is added 1ug / ml LPS and 10-8 mol / L dexamethasone into culture medium; LPS + Vit D3 group is added 1ug / ml of LPS and 10-8mol / L 1,25(OH) 2D3 into culture medium. All groups cells are cultured in 5% CO2, 37℃environment for 24 hours.3. The expression ofcostimulatory molecules CD80, CD86, CD40 expression and MHCⅡof DCs with LPS, LPS + Dex and LPS + Vit D3Collectting the four groups cells which culture with LPS, LPS + Dex or LPS + Vit D3 for 24 h, each group cells are detected the expression of cell surface costimulationmolecules MHCⅡ,CD80, CD86 and CD40 by flow cytometry.4. Western Blot detectthe expression of Notch ligand on DCs each with LPS, LPS + Dex and LPS + Vit D3 processingCollectting the four groups cells which culture with LPS, LPS + Dex or LPS + Vit D3 for 24 h, total cellular protein in each group are extracted with protein lysates, which are detected by Western Blot.The Notch ligand is including Jagged 1, Jagged 2, Delta-like 1, Delta-like 3 and Delta-like 4.5. RT-q PCR detectthe m RNA of Notch ligandon DCs with LPS, LPS + Dex and LPS + Vit D3 processingCollectting the four groups cells which culture with LPS, LPS + Dex or LPS + Vit D3 for 24 h, DCs total RNA of each groupare extracted, and the m RNA are reverse transcribed into c DNA.And we detect the m RNA of Notch ligand,including Jagged1, Jagged2, Delta-like 1, Delta-like 3 and Delta-like 4 on the DCs.6. ELISA detect supernatants expressing cytokines secreted by DCsCollecting the culture supernatant which the DCs are culture with for 24 h, the expression of the IL-10, IL-23 and TGF-β in supernatant was detected by ELISA.Chapter Ⅱ The impact of 1,25-dihydroxyvitamin D3 on LPS treated DCs from OVA-sensitized mouse dendritic cells on T cell differentiation1.Isolation of spleen sensitized micesterilly, the spleen is treated as single cell suspension with lysed erythrocytes, and purify CD4 + T lymphocytes with immunomagnetic beads.Collect the isolated dendritic cells which are cultured for 24 h, and washed DCs with the 1640. The cells are resuspended.The treated dendritic cells are co-cultured with T lymphocytes according to the ratio of 1:10.The two cells were co-cultured in a 5% CO2, 37℃for 24 h.2.Collectting the four groups cells which culture with LPS, LPS + Dex or LPS + Vit D3 for 24 h, adjust the concentration of 1 × 106 / ml and stimulates the cells with PMA / lonomycin and BFA / Monensin for 6 hours.Incubation FACS antibodies CD4 + IFN-γ, CD4 + IL-4 and CD4 + IL-17 A, and then detect expression by flow instrument.Collect cellswhich are co-cultured for 24 h, and adjust the concentration of 1 × 106 /ml.Incubation with CD4 + CD25 + Foxp3 directly, detect expression by flow instrument.3.Collecting the culture supernatant which the DCs are culture with for 24 h, the expression of the IL-4, IL-17 A and IFN-γ in supernatant was detected by ELISA.Chapter ⅢThe function of Detal-like 4 ligands in 1,25-dihydroxyvitamin D3 on LPS treated DCs from OVA-sensitized mouse dendritic cells on Th17 cells1.In the co-culture stage, 5ug /ml Detal-like 4 blocking antibody was added into the culture medium, and co-cultured in 5% CO2, 37℃for 24 h.2.Collectting the four groups cells which culture with LPS, LPS+Dex or LPS+Vit D3 for 24 h, adjust the concentration of 1×106 / ml and stimulates the cells with PMA /lonomycin and BFA /Monensin for 6 hours. Incubation FACS antibodies CD4+IFN-γ+, CD4+IL-4+ and CD4+IL-17A+, and then detect expression by flow instrument. Collect cells which are co-cultured for 24 h, and adjust the concentration of 1 × 106 / ml. Incubation with CD4+CD25+Foxp3 directly, detect expression by flow instrument.3. Collecting the culture supernatant which the DCs are culture with for 24 h, the expression of the IL-17 A in supernatant was detected by ELISA.Research ResultsChapter Ⅰ Influence of 1,25 dihydroxyvitamin D3 on LPS treated DCs from OVA-sensitized mouse dendritic cells1. The proportion of eosinophils in BALF of asthma model mice is significantly higher than the control group, cells count also increase significantly compared with the control group. The purity of isolated spleen dendritic cell is greater than 80% detected by flow cytometry.2.Compared with the control group,in the LPS-stimulated DCs group, the expression of CD80, CD86 molecule and MHCⅡhave not increase, and only the expressionof CD40 is significantly increased(P <0.05).The data is CD80(61.5 ± 11.7% vs 58.5 ± 8.4%), CD86(54.4 ± 8% vs 65 ± 2.4%), CD40(16.9 ± 3.4% vs 31.2. ± 7.7%), MHC molecule(27.7 Ⅱ± 8.1% vs 27.3 ± 4.3%), respectively. Dex or Vit D3 treated with LPS-stimulated DCs groups compared with the LPS stimulation DCsgroup, the expression of costimulatory molecules CD80, CD86, CD40, and MHCⅡare not significantly lower.The data is CD80(64.4 ± 10.5%, 65.4 ± 8.0% vs 58.5 ± 8.4%),(56.4 ± 2.3%, 58.9 ± 1.6% vs 65 ± 2.4%) CD86, CD40(21.6 ± 2.3%, 22.4 ± 8.9% vs 31.1 ± 7.7%),MHC molecule(36.4 ± 4.9%, 25.8 ± Ⅱ7.9% vs 27.3 ±4.3%), respectively. These findings suggest that LPS, 1,25(OH) 2D3 and dexamethasone treated DCs do not affect DCs maturation.3. LPS-stimulated DCs group compared with the control group, Jagged1 protein expression increase slightly, and while adding 1,25(OH) 2D3 treatment, Jagged1 protein expression increased further(P <0.05). Jagged2 protein expression is consistent with the Jagged1 protein. After stimulated with LPS, Detal-like 1 expression increase, and adding 1,25(OH) 2D3 treatment, Detal-like 1 expression in DCs increased further. With the dexamethasone treatment, Detal-like1 decrease compared with LPS group. Detal-like 3 stimulated with LPS do not increase, and while adding 1,25(OH) 2D3 treated, the expression of Detal-like 3 decrease slightly. Compared with the control group, LPS-stimulated DCs Detal-like 4 expression is significantly increase(P <0.05), and while adding dexamethasone treatment,theexpression of Detal-like 4 decrease slightly, and while adding 1,25(OH) 2D3 treatment, compared with LPS-stimulated group, the expression of Detal-like 4 decrease significantly(P <0.05).4. LPS-stimulated DCs group,compared with control group, Jagged1 m RNA increase 0.3 times, Jagged 2 m RNA increase by 0.2 times, Detal-like1 m RNA increase by 3.5-fold(P <0.01), Detal-like3 m RNA rose 0.4-fold(P <0.01), Detal-like 4 m RNA increase 6.5-fold(P <0.01); after LPS stimulated and Vit D3 or Dex treatment, Jagged1 m RNA in Vit D3 group is 1.5 times than the LPS group(P <0.05), Jagged 2 m RNA in Vit D3 group is 1.4 times than the LPS group(P <0.01), Delta-like 1 m RNA in Vit D3 group is 2.4 times than the LPS group(P <0.01), Detal-like3 m RNA in Vit D3 group is 0.85 times than the LPS group(P <0.05), Detal-like 4 m RNA in Vit D3 group is 0.54-foldthan LPS group(P <0.05). Dex has no effect on Jagged1, Jagged2, Delta-like 1, Delta-like 3, but only on Detal-like 4 m RNA is 0.8 times than LPS group(P <0.05).5. Spleen DCs secrete low level of IL-10(10.35 ± 1.85) pg / ml and IL-23(4.28 ± 0.95) pg / ml, hypersecretion of TGF-β(592.59 ± 60.16) pg / ml.With LPS treatment,there is high secretion of IL-10(110.72 ± 9.16) pg / ml, TGF-β(840.44 ± 109.3) pg / ml, and LPS has no effect on the secretion of IL-23(3.56 ± 1.05) pg / ml. With LPS-stimulated DCs and adding dexamethasone treatmenr, the secretion of IL-10(72.69 ± 5.05) pg /ml and TGF-β(615.54 ± 9.56) pg / ml is decreaed than LPS group, andthere is no effect on IL-23 secretion.With LPS-stimulated and adding 1,25(OH) 2D3 treated, the secretion of IL-10(92.72 ± 15.06) pg / ml and TGF-β(734.44 ± 57.58) pg / ml is decreased slightly than LPS group, and there is no effect on IL-23(4.19 ± 1.20) pg/ml secretion.Chapter Ⅱ The impact of 1,25-dihydroxyvitamin D3 on LPS treated DCs from OVA-sensitized mouse dendritic cells on T cell differentiation1.Compared with the control group, LPS-treated group can stimulate T cells differentiated to CD4 + IFN-γ(8.93 ± 2.18 vs 15.77 ± 2.23), CD4 + IL-4(9.32 ± 2.36 vs 12.6 ± 1.07), CD4 + IL- 17A(9.27 ± 1.35 vs 16.06 ± 1.83) and CD4 + CD25 + Foxp3(16.4 ± 1.57 vs 10.93 ± 1.43). LPS-stimulated DCs and adding 1,25(OH) 2D3 treatment compared to the control group, may also make T cells into CD4 + IFN-γ(17.33 ± 2.32 vs 8.93 ± 2.18), CD4 + IL-4(13.62 ± 1.38 vs 9.32 ± 2.36) differentiation, and the differentiation of CD4 + IFN-γ, CD4 + IL-4 in LPS-stimulated group increased slightly, but there was no statistically difference.Compared with LPS group, 1,25(OH) 2D3 treatment DCs group can suppresse CD4+IL-17A+differentiation(7.61 ± 1.72 vs 16.06 ± 1.83)which is induced by LPS, partially inhibit the CD4 + CD25 + Foxp3(14.7 ± 1.52 vs 16.4 ± 1.57) differentiation. Compared with LPS group, the Dex+LPS group can suppresse CD4 + IFN-γ(8.93 ± 1.13 vs 15.77 ± 2.23),CD4 + IL-4(7.10 ± 0.81 vs 12.6 ± 1.07), CD4 + IL-17A(8.86 ± 0.96 vs 16.06 ± 1.83) and CD4 + CD25 + Foxp3(9.46 ± 0.85 vs 16.4 ± 1.57) differentiation which is induced by LPS.2. Comparison with the control group,in LPS treatment supernatant, the IL-17A(12.01 ± 2.2 vs 4.46 ± 0.11), IL-4(2.5 ± 0.14 vs 1.66 ± 0.12) increase, and IFN-γ(1.88 ± 0.57 vs 1.34 ± 0.37) secretion also increased, but there was no statistical difference. 1,25(OH) 2D3-treated group compared to LPS group, IL-17A(3.11 ± 0.25 vs 12.01 ± 2.2) decrease, IL-4(3.15 ± 0.3 vs 2.5 ± 0.14) rise, and IFN-γ(2.21 ± 0.5 vs 1.88 ± 0.57) increase, but there was no statistical difference. Dex group compared with LPS group, both IL-17A(3.89 ± 0.75 vs 12.01 ± 2.2), IL-4(1.3 ± 0.24 vs 2.5 ± 0.14) and IFN-γ(0.56 ± 0.2 vs 1.88 ± 0.57) decrease.Chapter Ⅲ The function of Detal-like4 ligands in 1,25-dihydroxyvitamin D3 on LPS treated DCs from OVA-sensitized mouse dendritic cells on Th17 cells1.During the co-culture period,Detal-like 4 blocking antibody was added, compared with the control group, the LPS-treatment DCs can not make T cells into CD4 + IL-17A(9.4 ± 1.41 vs 10.03 ± 0.73), and adding 1,25(OH) 2D3 or dexamethasone treatment LPS-stimulated DCs can not decrease CD4 + IL-17 A further(8.96 ± 0.22,9.36 ± 0.67 vs 9.4 ± 1.41). Compared with the non-blocking LPS group, the blocking LPS group CD4 + IL-17 A differentiation decreased significantly(16.06 ± 1.83 vs 9.4 ± 1.41). After blocking Detal-like 4, the CD4+CD25+Foxp3in the control group, LPS group, LPS + Dex group and LPS + Vit D3 group differentiation expressed the same trend of non-blocking.2.After blocking Detal-like 4,compared with the control group and LPS group,the IL-17 A in LPS + Dex group decreased significantly(3.08 ± 0.5,3.33 ± 0.17 vs 1.50 ± 0.36), and LPS + Vit D3 group compare to the control group and LPS group is not significantly lower(3.47 ± 0.63 vs 3.08 ± 0.5,3.33 ± 0.17).Research conclusions1. Sensitized mice isolated splenic DCs are in a more mature state, and LPS, dexamethasone or 1,25(OH)2D3 can not affect DCs mature state. 1,25(OH)2D3 can be adjusted the expression of protein and m RNA of Notch ligand, such as Jagged1, Jagged2, Delta-like1, Delta-like3 and Delta-like4, and dexamethasone only decrease the Delta-like 4 m RNA.2. LPS treated DCs can stimulate T cells to differentiate into helper T cell, including Th1, Th2, Th17 and Treg, and LPS treated DCs can stimulate the Th1 improving secretion of IFN-γ,stimulate the Th2 improving secretion of IL-4,and stimulate the Th17 improving secretion of IL-17 A.Adding dexamethasone treated DCs, can inhibit Th1, Th2, Th17 and Treg differentiation induced by LPS, also inhibited Th1, Th2 and Th17 cytokine secretion. 1,25(OH)2D3 treatment DCs, can further promote Th1 and Th2 differentiation, related to increased Jagged1, Jagged2 and Detal-like1. 1,25(OH)2D3 suppress Th17 differentiation, and has no effect on Treg differentiation.3.1,25(OH)2D3 can regulate Th17 differentiation and function, but can not regulate Treg cell differentiation; 1,25(OH)2D3 can inhabit decrease the Th17 differentiation by inhibiting the expression of Detal-like 4 ligand. Dexamethasone can regulate Th17 and Treg differentiation, and not only through the Notch ligand.