The Influence Of Immune Phenotype And Function And The Study Of Pathogenesis Of Vitamin D To Adaptive Immune Response In Mouse Tuberculosis

Backgroμd：As the most common extrapulmonary form of tuberculosis, spinal tuberculosis （TB） isa growing hazard worldwide and has an aggressive behaviour of profound vertebraldestruction and severe complications.1.45million people die of tuberculosis every year,according to WHO reports（2010）. drug-resistant tuberculosis of the spine is more and more,some are multi-drug resistant tuberculosis （MDR-TB）, the treatment of spinal tuberculosisis becoming a serious challenge to the global control of the disease.The chemotherapy ofspinal tuberculosis stems from tuberculosis, the pathogenesis of tuberculosis should beintensively studied,so as to the treatment of spinal tuberculosis.Calciferol （Vitamin D） was used to treat tuberculosis in the late1940s and treatmentwas rationalised based on its role in the calcification of tuberculosis lesions. With theadvent of effective anti-tuberculosis drugs in the mid-1950s, the enthusiasm for treatingtuberculosis with vitamin D subsided. However, increasingly more patients withtuberculosis are now infected with drug-resistant strains, the infection is harder to treat asnormal treatment regimes are ineffective. Thus, why the use of vitamin D may be useful inthe context of drug resistance. Currently, the effects of vitamin D on TB patients remaincontroversial. Morcos et al reported that the addition of vitamin D to the standardantitubercular therapy results in higher rates of clinical and radiological improvement.Nursyam et al also reported that there were more TB patients with radiologicalimprovement in the vitamin D group. Recently, Martineau et al found that administration offour doses of2.5mg vitamin D3significantly hasten sputum culture conversion inparticipants with the tt genotype of the TaqI vitamin D receptor polymorphism. On thecontrary, Wejse et al stated that vitamin D does not improve clinical outcome amongpatients with TB and the trial showed no overall effect on mortality in patients with TB. Furthermore, Sato et al thought that low serum vitamin D level is a good predictor ofprolonged clinical course in patients with active pulmonary TB. Besides, Koo et alconsidered that vitamin D levels do not appear to be associated with the development of TBin the Korean population. The median25（OH）D concentration decreased after treatment forTB. Therefore, more detailed studies are necessary to further elucidate the role of vitamin Din TB infection.Vitamin D has been shown to play an important role in the innate and adaptiveimmune responses to tuberculosis. Recently, many mechanisms by which vitamin Dinfluences the innate immune response to TB have been reported. However, the mechanismby which vitamin D regulates the adaptive immune response remains less wellcharacterized. Dendritic cells （DCs） are the primary cell type that initiates the adaptiveimmune response against Mycobacterium tuberculosis （M. tb） infection. After bacterialuptake, DCs change their behavior, as observed both in vitro and in vivo, by reducing theirphagocytic and/or endocytic capability and initiating expression of immune stimulatorymolecules. This process is termed DC maturation, and it is initiated in nonlymphoid tissueswhere DCs take up antigen （Ag） and continues within secondary lymphoid tissues,particularly the draining lymph nodes （DLNs）. Differentiation of immature DCs in theDLNs leads to phenotypic changes into the mature phenotype resulting in expression ofhigh levels of long-lived MHC I, MHC II molecules and other costimulatory molecules（CD80,CD86）. Furthermore, recent data have shown a critical role for DCs in the primingof the CD4+T cell response to early secretory antigenic target6-kDa （ESAT-6）, therebysuggesting that DCs are essential for the initiation of the adaptive T cell response to humanM. tb infection. The effect of vitamin D on DC maturation has been extensively studied, butvery little is known how vitamin D affects DC during mycobacterial infection.Therefore, the main topic of this study: The Influence of immune phenotype andfunction and the study of pathogenesis of vitamin D to adaptive immunce respone in mousetuberculosis.Methods:1. Construction of mouse models of vitamin D deficiency, normal, sufficient, the levelof25（OH）D and1,25（OH）₂D₃were measured by ELISA method. 2.To observe the difference of the CD4+T, CD8+T、the ratio of CD4+T/CD8+T inthree group mouse after stimulation with Bacillus Calmette-Guerin （BCG）. IFN-γ and IL-10in the spleen lymphocyte suspension were measured by ELISA method after they werecultured using Tuberculin-Purified Protein Derivative （TB-PPD）.3. The culture and identification of bone marrow-derived dendritic cells（BMDCs）. Theexpression of CD11C, MHC-II,CD80,CD86were measured by flow cytometry analysis,themorphous of dendritic cells were observed by light microscope, scanning electronmicroscope, transmission electron microscope.4. Measure the differential expression of CD11C, MHC-II, CD86, CD80of dedriticcells under different vitamin D levels.5. Measure the ability of the Dendritic cells to stimulate proliferation of allogeneicmixed lymphocytes under different vitamin D levels.6. Measure the proliferation of BMDCs under different vitamin D levels.7. Measure the cytokine of BMDCs under different vitamin D levels.8. Measure the differental expression of VDR and CYP27B1of BMDCs underdifferent vitamin D levels.Results:1.The levels of25（OH）D and1,25（OH）₂D₃in plasma were11.6±3.56nmol/l and20±3.46pmol/l respectively in the–D group,76.5±9.13nmol/l and120±10.28pmol/lrespectively in the N group, and78.1±9.16nmol/l and130±12.15pmol/l respectively inthe+D group.2. The percentage of CD4+Tcells was27.1±0.6in the–D group,23.62±0.42in the Ngroup, and19.46±0.32in+D group. Additionally, there were significant differencesamong the three groups. The percentage of CD8+T cells was12.15±0.61in the–D group,8.7±0.64in the N group, and7.12±0.48in the+D group with significant differences amongthe three groups. The CD4+T/CD8+T lymphocyte ratios were2.23±0.15,2.71±0.21, and2.73±0.31, respectively, and statistical differences were noted among the three groups.TheIFN-γ level was416.42±16.42pg/ml in the–D group,325.41±11.16pg/ml in the N group,and276.26±25.32pg/ml in the+D group. There were significant differences among thethree groups. The IL-10level was16.45±1.58pg/ml in the–D group,24.31±2.16pg/ml inthe N group, and26.28±0.42pg/ml in the+D group, and significant differences were noted among the three groups.3. The method of bone marrow-derived dendritic cells was successful.The percentagesof cells in the cultures that were positive for the expression of various costimulatorymolecules were as follows: CD11C:84.64%±4.00; MHCII:88.75%±4.14; CD80:84.51%±8.63, and CD86:57.08%±9.55. Non-adherent DCs with large veils are visible inthe culture.4.The percentages of CD11C-positive cells in the control,25（OH）D3（100nM）,1,25（OH）₂D₃（10pM）,1,25（OH）₂D₃（100nM） cultures were84.64%±4.00,82.28%±7.27,81.17%±6.15and32.25%±3.02respectively. The percentages of cells in the control,25（OH）D3（100nM）,1,25（OH）₂D₃（10pM）,1,25（OH）₂D₃（100nM） cultures that were positivefor the expression of various costimulatory molecules were as follows: MHC-II:88.75%±4.14,85.92%±4.05,83.37%±3.96and46.65%±1.24respectivelyï¼›CD80:84.51%±8.63,84.75%±9.37,83.15%±8.48（p=0.61） and65.67±7.12respectively; and CD86:57.08%±9.55,51.32%±9.21,50.15%±8.76and15.69%±3.12respectively.5.25（OH）D3and1,25（OH）₂D₃（10pM）-treated BMDCs had a reduced capacity tostimulate T cell proliferation.1,25（OH）₂D₃（100nM）-treated BMDCs had a significantlyreduced capacity to stimulate T cell proliferation. This was evident for all the DC to T cellratios tested.6. The Optical density （OD） value of the-/+25（OH）D3,1,25（OH）₂D₃（10pM） and1,25（OH）₂D₃（100nM）-treated cultures were0.61±0.25,0.54±0.25,0.49±0.24and0.21±0.15respectively in the WST-1proliferation assay, indicating a reduction in the rate ofproliferation with all vitamin D metabolites.7. The levels of IFN-γ, IL-2, IL-6and IL-10were significantly higher and IL-4lowerin the media from25（OH）D3and1,25（OH）₂D₃（10pM）-treated cells when compared tocontrol cells. IL-12levels were not different between the25（OH）D3and1,25（OH）₂D₃（10pM）-treated cells and control cells. However, the levels of IFN-γ, IL-2,IL-6, IL-10and IL-12were significantly lower in the1,25（OH）₂D₃（100nM）-treated cellsthan control cells, the level of IL-4was significantly higher in the1,25（OH）₂D₃（100nM）-treated cells than control cells.8. The BMDCs can express the genes of CYP27B1and VDR. The expression ofCYP27B1in25（OH）D3-treated cells was higher than control-treated cells; But not significantly different between1,25（OH）₂D₃（100nM）-treated cells and control-treatedcells.The expression of VDR in25（OH）D3-treated cells was lower than control-treatedcells; But not significantly different between1,25（OH）₂D₃（100nM）-treated cells andcontrol-treated cells.Conclusion:1. It is feasible to establish mouse model of vitamin D deficiency by controlling thesources of vitamin. Vitamin D deficiency affects the immunity against Mycobacteriumtuberculosis infection in mice. Vitamin D deficiency results in the susceptibilty oftuberculosis.2.25（OH）D3influnces the the maturation and function of DCs during mycobacterialinfection by controlling the expresstion of VDR and CPY27B1, so as to avoid excessiveimmune response.