Induction Of Asthma Immune Tolerance By Indoleamine2, 3-dioxygenase Gene-modified Immature Dendritic Cells

Asthma is a chronic inflammatory disorder of the airway caused by many inflammatory cells. Th1/Th2 paradigm is known as the main pathogenesis of asthma. Excess activation of Th2 cells evokes Th2 immune response, and it causes elevated Th2 cytokines. Th2 cytokines such as IL-4, IL-5 and IL-13 play a pivotal role on the onset and maintenance of airway allergic inflammation in asthma. Immune tolerance to harmless allergen, harmless allergens do not lead to excess activation of T cells in healthy person. But in asthmatic patients, harmless allergens induce excess activation and proliferation of T cells, and make T cells differentiate into Th2 cells. Therefore, defect of immune tolerance may be an important mechanism for asthma.Indoleamine 2, 3-dioxygenase (IDO) is a rate-limiting enzyme for the tryptophan catabolism to form low- tryptophan environment. The proliferation of actived CD4~+T lymphocyte was inhibited by IDO and IDO induces the immunotolerance. Dendritic cell(DC) is the most powerful of antigen presenting cell(APC) in the present.DC controls immune response and determines tolerance or immunity to encountered allergen. Therefore, DC plays dual task of initiating immune response and maintaining immune tolerance.In the present study, we observe the effect of the ovalbumin allergic mice CD4~+T cells by transfecting a eukaryotic expresn vector containing mouse IDO gene fused with enhanced green fluorescent protein (pEGFP-N1)and immigrate immature DC transfected by pEGFP-N1-IDO into airway of ovalbumin allergic mouse model to observe the effect of airway inflammation for asthma new clinical prevention and cure.Methods:1. The full length cDNA of mouse IDO was cloned by RT-PCR and constructed into eukaryotic expression vector pEGFP-N1-IDO.2. The mouse bone marrow cells were isolated and cultured in complete medium with recombinant murine GM-CSF, then the purity and phenotype of DC was analyzed by flow cytometry(FCM), examining the expression of CD11c, MHC II, CD80and CD86.3. The recombinant pEGFP-N1-IDO was transfected into immature DCs by DOTAP liposome. After screening culture by G418, immature DCs was transfected. The green fluorescence protein expression was confirmed by inverted fluorescence microscope and the transcription and expression of IDO were identified by RT-PCR , Western blotting.4. The expression of CD11c, MHC II, CD80 and CD86 of immature DC transfected pEGFP-N1-IDO analyzed by FCM..5.The mice sensitized by peritoneal injection and challenged by ovalbumin were established. Total cell counts, differentiation cell counts and levels of IL-4, IL-5 and IFN-γin bronchoalveolar lavage fluid(BALF) were detected, and lung histological sections were analyzed.6. To observe the effect of mouse IDO gene on CD4~+T cells by transfecting a eukaryotic expression vector containing mouse IDO gene fused with enhanced green fluorescent protein (pEGFP-N1) . The splenic CD4~+T cells of ovalbumin sensitized mice were isolated and cocultured with immature DCs transfected by pEGFP-N1-IDO, then proliferation response and apoptosis of CD4~+T cells was detected by mixed lymphocyte reaction(MLR) and TUNEL, and the levels of IL-4, IL-5 and IFN-γin cell culture supernatant were detected by ELISA.7. To observe the effect of immature DCs transfected by pEGFP-N1-IDO by immigrating the airway of ovalbumin allergic mouse modelthe.Levels of IL-4, IL-5 and IFNγin bronchoalveolar lavage fluid(BALF) were detected, and lung histological sections were analyzed.Results:1. The IDO gene full length cDNA was amplified by RT-PCR and cloned into pEGFP-N1, and sequencing result showed the sequence of IDO cDNA was in coincidence with the sequence in Genbank.2. The immature DC derived from mouse bone marrow cultured in vitro and identificated.3. The recombinant, pEGFP-N1-IDO, was transfected into immature DCs by DOTAP liposome. The green fluorescence protein expression of pEGFP-N1-IDO and pEGFP-N1 transfected into immature DCs groups were confirmed by inverted fluorescence microscope . the transcription and expression of IDO of pEGFP-N1-IDO transfected into immature DCs group were identified by RT-PCR , Western blotting.4. Transfection with IDO did not affect the expression levels of cell surface molecules.5. The ovalbumin allergic and challenged mouse model was established successfully,with marked lung allergic inflammation, elevated total cells count and eosinophil percentage in BALF, and raised IL-4, IL-5 levels and declined IFN-γlevel in BALF.6. The proliferation of spleen-derived CD4~+T lymphocyte of the allergic and challenged mouse challenged by ovalbumin was inhibited by the imDCs transfected by the IDO gene, the number of CD4~+T lymphocytes treated with the imDCs transfected pEGFP- N1-IDO eukaryotic expression vector decreased significantly compared with the control group (P<0.01). The apoptosis of the CD4~+T lymphocyte was induced by the immature dendritic cells transfected by the IDO gene, the apoptosis rates were 15.3%±2.6%, compared with the imDCs transfected pEGFP- N1 eukaryotic expression vector and control the difference was highly significant respectively (P<0.01). The supernatant of pEGFP-N1-IDO transfected into immature DCs group after co-cultured with CD4~+T cells of IL-4 and IL-5 to be inhibited and the level of IFN-γto be raised,but the control group and pEGFP-N1 transfected into immature DCs group result were opposite.7. After administration of pEGFP-N1-IDO transfected into immature DCs, lung of the ovalbumin allergic and challenged mouse allergic inflammation was ameliorated,and total cell and eosinophil counts and the levels of IL-4, IL-5 in BALF decreased(P< 0.01), and the level of IFN-γin BALF increased(P< 0.01).Conclusions:1. We constructed a modle with high expression vector containing mouse IDO gene transfected into immature DCs.2. Administrating pEGFP-N1-IDO transfected into immature DCs in vitro can induce hyporesponsiveness of spleen CD4~+ T cells from ovalbumin allergic and challenged mice and inhibit Th2 cells activation. The apoptosis of the CD4~+T lymphocyte was induced by the immature dendritic cells transfected by the IDO gene .3. Administrating pEGFP-N1-IDO transfected into immature DCs into airway of ovalbumin allergic and challenged mice airway can inhibit Th2 cells activation and ameliorate airway allergic inflammation, indicating the up-regulation of the express of the IDO gene may induce the immunotolerance in asthma and providing a novel immunointervention strategy for treatment of asthma.