| Backgoud and objectiveBronchial asthma is an airway chronic inflammatory disease with many of the inflammatory cells involved in. Allergic asthma is mediated by Th2 immune response. The development of this disease is correlated with dysfunction of immune tolerance to the foreign'harmless'antigen. Regulatory T cells (Tregs) , a subset of T cells, can modulate the immune response in an active suppression manner. Recent studies have demonstrated that dysfunction and low frequency of Tregs maybe exist in the allergic asthma. So it has been hypothesized that the imbalance of Th2 and Tregs plays an important role in the development of allergic asthma. Tregs , especially the CD4+ CD25+ Foxp3+Tregs subset play a major role in the control of asthma onset through inhibition of inflammatory cells, such as Th1, Th2,CD8+T cell and dendritic cells(DCs). Promoting in vivo expansion of Tregs may be a promosing intervention strategy of allergic asthma. It has been reported that 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] can induce the generation of CD4+CD25+T cells, but its mechanism is still unknown.1,25(OH)2D3, the biologically activated form of vitamin D, has recently been confirmed that, besides its role in metabolism of bone and calcium and cell proliferation and differentiation,it is involved in the modulation of immune response. Most of the immune cells are the target of 1,25(OH)2D3,especially DCs and T cells. The inhibition of T cells'activation and secretion of IL-2 and IFN-γand induction of IL-10 production is the direct role of 1,25(OH)2D3 on T cells. The inhibitory expression of costimulatory molecular CD80,CD86,MHCⅡon DC as well as the decrease of IL-12 production and the increase of IL-10 production leading to DCs with tolerogenic phenotype and function plays an important role in the immunoregulatory activity of 1,25(OH)2D3. The protection role of 1,25(OH)2D3 in Th1-mediated diseases ,such as autoimmune diseases and allograft regection has been confirmed by basic experiments and clinic research. But, its role in Th2-mediated diseases, such as allergic asthma, is still in dispute. Most of the researchers believe that it has a protection role in allergic asthma, but its mechanism is still unknown.Notch signal pathway plays an important role in development of organs and differentiation of cells. Recently it has been found that Notch signal pathway directs the differentiation of T cells towards Th1,Th2 and Tregs. It has been reported that expression of Notch3 on CD4+CD25+ T cells is higher than that on CD4+CD25- T cells. Transgenic mice with Overexpression of Notch IC promote the expansion of Tregs. Human APC with overexpression of Jagged1 promotes the development of IL-10-Tregs, which inhibit the immune response and mediate the antigen-specific immune tolerance. Hassen Karred et al have found that jagged2-expressing hemotapoietic progenitors promote regulatory T cell expansion in the periphery through Notch signaling. Taken together, Notch signal pathway plays an important role in development of Tregs.In summary, we hypothesize that 1,25(OH)2D3 treated dendritic cells promote the expansion of CD4+CD25+ FoxP3+T cells through Notch signaling , restoring the periphery immune tolerance and attenuating the allergic airway inflammation.MethodsPart One1.Dendritic cells were generated from mouse bone barrow in the presence of GM-CSF(we named this kind of cells BMDC);2.BMDC cultured in the presence of 10-6,10-7,10-8 mol/L 1,25(OH)2D3 for 72 h, respectively. PBS substitute for 1,25(OH)2D3 in blank control group. The expression of surface molecule CD80,CD86 and MHCⅡon BMDC was examined by FACS.3.The mRNA level of Notch ligands Jagged1,Jagged2,Delta1,Delta3 and Delta4 expressed by BMDC was examined by semi-quantitive RT-PCR and the protein of Notch ligands (Jagged1 and Jagged2) expressed by BMDC was examined by Western Blotting.4.Blank control, 10-6,10-7,10-8 mol/L 1,25(OH)2D3-treated BMDC co-cultured with spleen CD4+T cells from normal mice spleen and 10ug/ml OVA for 48 h, respectively. The percentage of CD4+CD25+FoxP3+T cells in CD4+ T cells was detected by FACS.5.Blank control ,10-8 mol/L 1,25(OH)2D3 treated BMDC co-cultured with spleen CD4+T cell from allergic airway inflammation mouse model and 10ug/ml OVA for 48h. The percentage of CD4+CD25+FoxP3+T cells in CD4+ T cells was detected by FACS. 6.The percentage of CD4+CD25+FoxP3+T cells in spleen CD4+ T cells from normal mouse and allergic airway inflammation mouse model was detected by FACS, respectively.7.10-8 mol/L1,25(OH)2D3 treated BMDC co-cultured with CD4+T cells from normal mouse in the presence of Jagged1 or Jagged2 polyclonal antibody at a concentration of 10 ug/ml. PBS substitute for polyclonal antibody in blank control groups. The percentage of CD4+CD25+FoxP3+T cells in CD4+T cells was detected by FACS.Part Two1. Mice were intraperitoneally sensitized with OVA on day 0,7 and 14 and then randomly subdivided into two groups, 1,25(OH)2D3 BMDC group and PBS-BMDC group. 1,25(OH)2D3 BMDC group was adoptively transferred with 1,25(OH)2D3- treated BMDC and PBS-BMDC group was adoptively transferred with PBS- treated BMDC. Those treated BMDC suspension, corresponding to 1 x 106 cells, was administered intratracheally through the opening vocal cords using a 23-gauge metal catheter connected to the outlet of a micropipette . Then they were challenged with OVA once daily for 6 consecutive days2.24 hours after last OVA exposure, the mice were accessed lung allergic inflammatory response.3.24 hours after last OVA exposure, the level of IL-4,IL-13 ,IL-5 and IFN-γin BALF was examined by ELISA and count of EOS in BALF were accessed .4.24 hours after last OVA exposure, CD4+CD25+FoxP3+ Tregs in mice spleen were examined by FACS.ResultsPart one1.BMDC was obtained successfully with immature phenotype.2.The expression of surface molecule CD80,CD86 and MHCⅡon BMDC treated by different concentration of 1,25(OH)2D3 was significantly decreased(P<0.01);3.The mRNA expression of Jagged1 and Jagged2 was significantly increased(P<0.01) and that of Delta4 wasn't changed(P>0.05) on different concentration of 1,25(OH)2D3 treated BMDC as well as that of Delta1 and Delta3 was undetectable on all groups of BMDC;4.The protein of Jagged1 and Jagged2 was significantly increased on different concentration dose of 1,25(OH)2D3 treated BMDC(P<0.01); 5.The percentage of CD4+CD25+FoxP3+T cells in normal mouse spleen induced by different dose of 1,25(OH)2D3 treated BMDC was significantly increased(P <0.01),compared with blank control.6.The percentage of CD4+CD25+ FoxP3+T cells was significantly increased in asthma mouse spleen induced by 10-8 mol/L 1,25(OH)2D3-treated BMDC, compared with blank control (P<0.01).7.The percentage of CD4+CD25+FoxP3+T cell induced by 1,25(OH)2D3-treated BMDC with treatment of Jagged1 poly-antibody wasn't changed whereas that induced by1,25(OH)2D3-treated BMDC with treatment of Jagged2 poly-antibody was significantly decreased, compared with blank control group(P<0.01).Part Two1.In PBS-DC group mice, OVA challenge led to a dense inflammatory infiltrate of lymphocytes, mononuclear and eosinophils as well as to epithelial shedding. However, in 1,25(OH)2D3-DC group mice, tissue inflammation after OVA challenge was greatly attenuated with significantly less cellular infiltration.2. In BAL fluids from 1,25(OH)2D3-DC group mice, the numbers of eosinohpils and the level of IL-4, IL-5 ,IL-13 and IFN-γproduction were significantly decreased compared with that in BAL fluids from PBS-DC groups.3.The percentage of CD4+CD25+FoxP3+ T cells in spleen CD4+ T cells from1,25(OH)2D3-DC group was significantly increased, compared with that from PBS-DC group.Conclusion1. 1,25(OH)2D3 inhibits the expression of MHCⅡand co-stimulatory molecule CD80 and CD86 on DCs,rendering them as an immature phenotype called tolerogenic DCs which induce the generation of CD4+CD25+ Foxp3+ T cells in vivo and in vitro. Molecular mechanism by which 1,25(OH)2D3-treated DCs induce the generation of CD4+CD25+ Foxp3+ T cells is correlated with Jagged2-mediated Notch signaling pathway.2. Adoptive transfer of 1,25(OH)2D3-treated DCs to OVA-sensitized mice marked inhibits not only Th2-type immune response but also Th1-type immune response, promoting the expansion of CD4+CD25+ Foxp3+ T cells in vivo. It suggests that CD4+CD25+Foxp3+T cells may be an alternative regulation mechanism of Th2-type immune response, independent of Th1/Th2 balance.3. 1,25(OH)2D3-treated DCs attenuate allergic airway inflammation in the experimental mice, which make it have a potency for clinic application and provide a new strategy for treatment of asthma. |
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