Experimental Research On Effects Of CTLA4IG Modified Dendritic Cells On Asthmatic Airway Inflammation And Th Cell Subset Disfunction

PART ONE A MODIFIED CULTURE METHOD FOR PROLIFERATION LARGE NUMBER DENDRITIC CELLS FROM MOUSE BONE MARROWObjective: To proliferate large number DCs from mouse bone marrow cells by modified culture method.Methods: Mononuclear cells were collected by density gradient centrifugation from mouse bone marrow. Low-density bone marrow mononuclear cells were cultured in 24-well plate and then differentiated and proliferated into DCs by supplementing with low dose rmGM-CSF and rmIL-4 for 6~8 days. Mature DCs were obtained by adding LPS in culture for 24h. DCs were identified by morphology by light microscope and scanning electron microscopy, the surface maker by flow cytometry(FCM), the potential to stimulate allogeneic lymphocytes by mixed lymphocyte reaction (MLR) and the potential to secrete IL-12 by Enzyme-linked immunoadsorbent assay (ELISA).Results: About 2×10~7 DCs with typical dendritic processes were obtained from bone marrow cells of a single mouse after 6~8 days of culture. The bone marrow derived DCs expressed high level of surface markers CD11c (90.01 %), MHCⅡmolecules, CD80, CD86, CD40 and CCR7, and showed strong stimulated proliferation of allogeneic T cells and potential to secrete IL-12.Conclusion: DCs with high purity and function could be simply generated from mouse bone marrow cells using this modified culture method.PART TWO THE EFFECT OF CTLA4IG MODIFIED DENDRITIC CELLS ON TH SUBSET DIFFERENTIATION IN VITROObjective: To investigate the impact on Th subset differentiation from cytotoxic T lymphocyte-associated antigen immunoglobulin (CTLA4Ig) modified dendritic cells (DC-CTLA4Ig) in vitro.Methods: The modified DCs (DC-CTIA4Ig) were prepared by transferring the mouse bone marrow-derived DCs with the constructed adenovirus CTIA4Ig vector.The CTLA4Ig expression and certain cell surface molecules on the DC-CTIA4Ig were detected by FCM, the potential to stimulate allogeneic T cell proliferation of the modified DCs by MLR, the secretion of IFN-γ, IL-4 and IL-10 in the coculture system of antigen presentation by ELISA and the level of Th1, Th2 and Treg in the coculture by FCM.Results: CTLA4Ig gene was successfully transfected into DCs with about 80 % expression of CTLA4Ig. Compared with the controls, DC-CTLA4Ig showed lower surface molecule CD86, inhibited allogeneic lymphocyte proliferation in MLR, decreased secretion of IFN-γand IL-4, increased secretion of IL-10 and ratio of IFN-γ/IL-4 in the coculture system of antigen presentation. Although DC-CTLA4Ig showed both decreased percentage of Th1 and Th2 cells in the coculture, the ratio of Th1/Th2 was increased. Besides, DC-CTAL4Ig increased the percentage of Treg cells in the coculture and decreased the ratio of Th2/Treg.Conclusion: DC-CTLA4Ig were successfully constructed and effectively expressed CTLA4Ig, which could reduce the expression of CD86 on DCs surface. DC-CTLA4Ig inhibited allogeneic T cell proliferation and affected the ratio of Th1/Th2 and Th2/Treg in vitro.PART THREE EXPERIMENTANT RESEARCH ON EFFECTS OF CTAL4IG GENG MODIFIED DENERITIC CELLS ADMINISTRATION DURING THE CHALLENGE PHASE ON ASTHMATIC AIRWAY INFLAMMATION AND TH CELLS SUBSET DISFUNCTIONObjective: To evaluated the therapeutic effects and potential mechanisms of the adoptive transfer of DC-CTLA4Ig into mice during the challenge phase in an experimental model of asthma.Method: SPF level BALB/c mice were randomly divided into 5 groups: healthy na?ve mice sensitized/challenged with Phosphate buffer solution (PBS), OVA sensitized/challenged mice, OVA sensitized/challenged mice intravenously injected with DCs, DC-GFP (green fluorescent protein) or DC-CTLA4Ig 30 minutes just prior to the first challeng. Airway hyperresponsiveness (AHR) was measured using whole-body plethysmography (WBP) and then bronchoalveolar lavage fluid (BALF) were obtained. Airway inflammation was assessed by histological examinations of lung and white blood differential count in BALF. Cytokines in the BALF and serum were measured by ELISA. CD4+ T cells subsets in lungs and spleens were analysed by FCM.Results: The administration of DC-CTLA4Ig during the challenge phase reduced airway hyperresponsiveness, relieved asthmatic airway inflammation, and decreased the numbers of esosinophils in the BALF in OVA-sensitised/challenged mice. In addition, DC-CTLA4Ig administration altered the balance of Th1/Th2 cytokine production in the lungs with increased IFN-γlevels and decreased IL-4 levels in local airway, increased the level of IL-10 in BALF, decreased the percentage of Th2 and increased both the percentage of Th1 and Treg cells in the lungs of OVA-sensitised/challenged mice. Besides, OVA-sensitised/challenged mice received DC-CTAL4Ig showed no significant difference in the levels of cytokines in serum and Th subset in spleens compared to OVA-sensitised/challenged mice.Conclusion: This research demonstrates that administration of DC-CTLA4Ig during the challenge phase effectively reduces airway hyperresponsiveness and prevents airway inflammation in OVA-sensitised/challenged mice, which most likely due to attenuated secretion of Th2 cytokines and increased secretion of Th1 cytokines in local airway, and the correction of the pulmonary imbalance between Th1/Th2 cells and Th2/Treg cells.PART FOUR EXPERIMENTANT RESEARCH ON EFFECTS OF CTAL4IG GENG MODIFIED DENERITIC CELLS ADMINISTRATION DURING THE SENSITIZATION PHASE ON ASTHMATIC AIRWAY INFLAMMATION AND TH CELLS SUBSET DISFUNCTIONObjective: To evaluated the preventive effects and potential mechanisms of the adoptive transfer of DC-CTLA4Ig into mice during the sensitization phase in an experimental model of asthma.Method: SPF level BALB/c mice were randomly divided into 5 groups: healthy na?ve mice sensitized/challenged with PBS, OVA sensitized/challenged mice, OVA sensitized/challenged mice intravenously injected with DCs, DC-GFP or DC-CTLA4Ig 30 minutes prior to sensitization on day 0 and day 14. AHR was measured using WBP and BALF were obtained. Airway inflammation was assessed by histological examinations of lung and white blood cells differential count in BALF. Cytokines in the BALF and serum were measured by ELISA. CD4+ T cells subsets in lungs and spleens were analysed by FCM.Results: The administration of DC-CTLA4Ig during the challenge phase reduced airway hyperresponsiveness, relieved asthmatic airway inflammation, and decreased the numbers of esosinophils in the BALF in OVA-sensitised/challenged mice. In addition, DC-CTLA4Ig administration altered the balance of Th1/Th2 cytokine production in the lungs with increased IFN-γlevels and decreased IL-4 levels in local airway, increased the level of IL-10 in BALF, decreased the percentage of Th2 and increased both the percentage of Th1 and Treg cells in the lungs of OVA-sensitised/challenged mice. Besides, OVA-sensitised/challenged mice received DC-CTAL4Ig showed no significant difference in the levels of cytokines in serum and Th subset in spleens compared to OVA-sensitised/challenged mice.Conclusion: This research demonstrates that administration of DC-CTLA4Ig during the sensitization phase effectively reduces airway hyperresponsiveness and prevents airway inflammation in OVA-sensitised/challenged mice, which most likely due to attenuated secretion of Th2 cytokines and increased secretion of Th1 cytokines in local airway, and the correction of the pulmonary imbalance between Th1/Th2 cells and Th2/Treg cells.