IDO Overexpression In Airway Epithelialum By Gene Transfection Induce Allograft Tolerance Against Chronic Rejection In Murine HTT Model

Background: Lung transplantation currently is the preferred treatment option for avariety of end-stage pulmonary diseases. Although remarkable progress has been made inimproving outcome, the incidence of acute rejection remains more than50%in the1st year,and the5-year graft survival is still less than50%primarily because of the development ofchronic rejection and graft dysfunction. Chronic rejection is characterized by thedevelopment of obliterative bronchiolitis(OB) in lung allografts and manifests asbronchiolitis obliterans syndrome(BOS) in humans with no effective treatment. Inductionof tolerance may hold the key to improve transplant longevity and quality of life inrecipient and increase the cost-effectiveness of transplant therapy at the same time.Indoleamine2,3-dioxygenase (IDO) is a rate-limiting enzyme for tryptophan metabolism.An increasing body of experimental data in the last decade has shown that IDO is animportant immunosuppressive property for tumor immune resistance, pathogen-inducedimmune surveillance, induction of immune suppression to antigens and prevention ofautoimmune responses. More and more studies have supported the theory thatIDO-overexpression in transplanted cells or organs in vivo was found to significantlyprolong graft survival in animal models. In light of these data on the role of IDO inimmune regulation and tolerance, we set out to overexpress IDO in donor mouse airwayepithelialum using an adenovirus expression vector containing IDO cDNA to investigatethe IDO immune regulatory mechanism on obliterative bronchiolitis of chronic lungallograft rejection in murine HTT model. Part I Construction of Recombinant Adenovirus IDO GeneExpression Vector by Gateway TechnologyObjective: To construct recombinant adenovirus IDO gene expression vector byGateway Technology, then to transfect HEK293A cells for virus packaging, purification,drops testing after identification by PCR screening and sequencing.Methods: According to Gateway Technology scheme, first to synthesis plasmid-attB1-kozak-IDO1/IRES/EGFP-attB2by overlap PCR amplification, then synthesisplasmid-pDown-IDO1/IRES/EGFP by BP respose, finally synthesis recombinantadenovirus IDO gene expression vector-pAV.Ex1d-CMV>IDO1/IRES/EGFP by LRrespose. The vector was transfected into HEK293A cells for virus packaging, purification,drops testing after identification by PCR screening and sequencing.Results: The sequencing reports showed that the sequence of constructedvectors-pDown-IDO1/IRES/EGFP or pAV.Ex1d-CMV>IDO1/IRES/EGFP were matchingwith IDO gene designed and reported in GeneBank. We can detect EGFP fluorescence inadenovirus-transfected HEK293A cells. The virus drops was tested about3.16×1010PFU/ml with TCID50method.Conclusion: The recombinant adenovirus IDO and EGFP gene expression vector wasconstructed successfully, and the virus can be produced with high drops by transfectedHEK293A packing cells.Part II Feasibility on Airway Epithelialum Transfected IDO Geneby Adenovirus Vector in MiceObjective: To observe the expression, efficiency and enzyme activity of IDO gene inthe cultivate mice airway epithelial cells transfected by adenovirus vector. And to explorethe feasibility of IDO gene overexpression in the airway epithelialum by virus airwaytransfecting model in mice. Methods: Adenovirus vector carrying IDO gene was transfected into the cultivatemice airway epithelial cells as IDO transfection group, and setting up the empty vector(GFP) transfection group and negative group as control. The morphology of cells in eachgroup was observed before and after transfection, and the viral transfected efficiency wasevaluated to view EGFP (green) by fluorescent microscope. In each group, qRT-PCR wascarried out to test IDO mRNA levels and Western blot was carried out to detect IDOprotein expression levels in the airway epithelialum. The IDO enzyme activity of theepithelial cells was evaluated.with the kynunine to tryptophan ratio by HPLC in each groupculture supernatant. Then the efficiency and biological safety of IDO gene overexpressionin the airway epithelialum were explored by virus airway transfecting model in mice.Results: At24h after transfection, the viral transfected efficiency in the epithelialumwas near to100%, although low EGFP fluorescence.can be detected at a multiplicity ofinfection (MOI) of50by fluorescent microscope. The morphology of cells was unchangedalthough the fluorescence intensity of epithelialum was increased with MOI. The result ofqPT-PCR showed that IDO mRNA levels in the epithelialum of IDO transfection groupwas significantly higher as compared with other groups (P<0.01), and Western blotanalysis showed that IDO protein levels was expressed higher in IDO transfection group aswell (P<0.01). After transfecting recombinant adenovirus via airway, it was showed thatIDO were mainly overexpressed in airway epithelialum and could be lasting for more than3weeks without respiratory damage. The result of HPLC showed that the IDO enzymeactivity was higher in IDO transfection group than in the other groups, no matter in culturesupernatant or in airway tissues (P<0.01)Conclusion: The mice airway epithelialum transfected by adenovirus vector canoverexpress IDO gene, protein and enzyme activity in vivo or vitro. The efficiency andsafety were satisfied as virus transfecting airway epithelialum through airway. And thelevels of IDO overexpression in the airway epithelialum can be maintained for more thanthree weeks in vivo. Part III IDO Overexpression in Airway Epithelialum InduceTolerance of CD4~＋T Cells and BMDCsSection A IDO Overexpression in Airway Epithelialum SuppressProliferation of CD4~＋T CellsObjective: Setting up co-culture system of mice airway epithelialum and CD4~＋Tcells to explore the influence of IDO overexpression in airway epithelialum onproliferation, early apoptosis of CD4~+T cells and the role in tolerance induction byup-regulating FoxP3expression in CD4~+CD25~+Treg cells.Methods: Using immune magnetic beads system to abtain the purified CD4~＋T cells.Then Setting up co-culture system of above CD4~＋T cells and airway epithelial cells forfour different groups such as negative group, IDO transfection group, GFP transfectiongroup and1-MT inhibition group. Under stimulation with antibody(CD3/CD28) for72h inco-culture system, the proliferation stimulation index(SI) was examined by CCK-8kit, andthe early apoptosis rate of CD4~+T cells was detected by AnnexinV-FITC and PI kit. Theimmune function of CD4~+T cells was evaluated by MLR and the expression of FoxP3inCD4~+CD25~+Treg cells was determined by FCAS. The enzyme activity of IDO in theepithelialum was detected by HPLC in each group.Results: Under the CD3/CD28stimulation for72h in co-culture system, theproliferation stimulation index(SI) of CD4~+T cells in IDO transfection group was lower ascompared with negative group or GFP transfection group(P<0.01), which was increasedwith treatment of1-MT (P<0.01). The result of AnnexinV-FITC and PI kit test showed thatearly apoptosis rate of CD4~+T cells in IDO transfection group was significantly higher thanin negative group or GFP transfection group(P<0.01), which was decreased with treatmentof1-MT (P<0.01). The result of FCAS showed that the expression of FoxP3inCD4~+CD25~+Treg cells was significantly higher in IDO transfection group than in negative group or GFP transfection group(P<0.01), and the expression was decreased with treatmentof1-MT (P<0.01). The MLR results showed that the inhibition rate of spleen lymphocyteproliferation in IDO transfection group was higher as compared with negative group orGFP transfection groups(P<0.01), which was decreased with treatment of1-MT (P<0.01).The HPLC results showed that the IDO activity was higher than in negative control andGFP transfection groups (P<0.01), which was decreased with treatment of1-MT (P<0.01).Conclusion: In co-culture system, the IDO overexpression in airway epithelialum caninduce CD4~+T cells tolerance by suppressing proliferation, promoting early apoptosis, andup-regulating the expression of FoxP3in CD4~+CD25~+Treg cells. These effects is based onup-regulation of IDO activity in microenvironment, and can be blocked by1-MT.Section B IDO Overexpression in Airway Epithelialum PreventMaturation and Differentiation of BMDCsObjective: To observe the maturation, phenotypic differentiation characteristics ofBMDCs by culturing and expanding them in vitro. Then setting up co-culture system ofmice airway epithelialum and BMDCs under LPS stimulation to explore the influence ofIDO overexpression in airway epithelialum on the maturation and phenotypicdifferentiation of BMDCs and the role in immune tolerance induction.Methods: To culture and expand DCs from mice bone marrow in vitro underinduction of rGM-CSF (10ng/ml), rIL-4(4ng/ml), and observe their morphology andphenotypic differentiation characteristics during culture. On7thday, setting up co-culturesystem of above BMDCs and airway epithelial cells for four different groups such asnegative group, IDO transfection group, GFP transfection group and1-MT inhibitiongroup. Under LPS (1ug/ml) stimulation for72h in co-culture system, phenotypicdifferentiation characteristics such as MHCII, CD86/80molecules on BMDCs surfacewere determined by FCAS. And the immune function of BMDCs was detected by MLR.The IDO activity in the epithelialum were detected by HPLC in each group.Results: Before7thday, the BMDCs cultured and expanded in vitro remain immature, but can be promoted to mature under LPS stimulation for72h. Flow cytometry analysisshowed that the expressions of MHCII,CD80/CD86molecules on BMDCs were lower inIDO transfection group than in negative group or GFP transfection group(P<0.01), and theexpressions were increased with treatment of1-MT (P<0.01). The MLR results showedthat the proliferation rate of spleen lymphocyte in IDO transfection group was significantlylower as compared with negative group or GFP transfection group(P<0.01), which wasincreased with treatment of1-MT (P<0.01). The HPLC results showed that the IDOactivity in IDO transfection group was higher than in negative group or GFP transfectiongroups (P<0.01), which was decreased with treatment of1-MT (P<0.01).Conclusion: In co-culture system, IDO overexpression in the airway epithelialum caninduce BMDCs tolerance by inhibiting the expression of MHCII, CD80/CD86molecules,preventing further maturation. These effects is based on up-regulation of IDO activity inmicroenvironment, and can be blocked by1-MT.Part IV Reproduction of Chronic Lung Allograft Rejection inMurine Heterotopic Tracheal Transplant ModelObjective: Setting up murine heterotopic tracheal transplant(HTT) model toreproduce chronic rejection characteristic as obliterative bronchiolitis(OB) after lungtransplantation, as to provide the platform for the study of chronic lung allograft rejection.Methods: Thirty receptors mice were randomly average divided into two groups asthe experimental group and the control group for heterotopic tracheal transplantationmodel. The receptors in the experimental group were from C57BL/6(H-2b) mice, whilethose in the control group were from BALB/c(H-2d) mice, and all donor transplanttracheals were from BALB/c mice. Every five receptor mice in each group were randomlysacrificed on postoperation7th,14th,28thday to harvest tracheal allografts for pathologyevaluation, CD3~+T cells immunohistochemical staining, and determining the degree ofluminal occlusion.Results: In the experimental group, the immune pathological damage of transplant tracheals were more severe with time, and the tracheal lumen fated to obliteration totallyby granulation tissue on postoperation28thday as similar to pathological process ofobliterative bronchiolitis. The score of pathology, the degree of CD3~+T cells infiltrationand luminal occlusion in tracheal allografts were significantly higher in the experimentalgroup than in the control group(P<0.01).Conclusion: Murine heterotopic tracheal transplant model can be used to reproduceobliterative bronchiolitis of chronic rejection after lung transplantation. The model istechnically simple and reproducibility for research on chronic lung allograft rejection.Part V IDO Overexpression in Airway Epithelialum AttenuateObliterative Bronchiolitis (Chronic Rejection) in miceObjective: To explore the role and mechanism of IDO overexpression in transplantairway epithelialum attenuating chronic lung allgraft rejection-obliterative bronchiolitis inmurine HTT model.Methods: Sixty receptors from C57BL/6(H-2b) mice were randomly average dividedinto four groups as negative group, IDO transfection group, GFP transfection group and1-MT inhibition group for heterotopic tracheal transplantation model. All donor transplanttracheals were from BALB/c(H-2d) mice, and the donor tracheals in IDO transfectiongroup were transfected with adenovirus IDO vector7days before transplantation, and toGFP transfection group were transfected with adenovirus GFP vector as control. Onpostoperation7th,14th,28thday, five receptor mice in each group were randomly sacrificedto harvest tracheal allografts for pathology evaluation, CD3~+T cells immunohistochemicalstaining, and determining the degree of luminal occlusion. The expression of IDO gene intracheal allografts was detected by fluorescence microscope.And on postoperation7thday,the T cells from tracheal allografts and drainage lymph node were repaired for detectinglymphocytes proliferation activity by MLR and the rate of Th17cells and Treg cells wereanalyzed by FCAS. In the end, the IDO activity in tracheal allografts and peripheral serumwere detected by HPLC in each group. Results: On each checkpoint, the immune pathological damage of tracheal allograftsin IDO transfection group was slighter as compared with the other groups. Thepathological score, and the degree of CD3~+T cells infiltration and luminal occlusion intracheals allografts were all significantly lighter in IDO transfection group than in the othergroups (P<0.05). The IDO gene expression in tracheal allografts can be detected for4weeks by fluorescence microscope in IDO transfection group. On postoperation7thday, theresults of Flow cytometry analysis showed that the proliferation activity of T cells intracheal allografts and drainage lymph node were suppressed, accompanying with thelower rate of Th17cells and the higher rate of Treg cells in IDO transfectiongroup(P<0.01). The HPLC results showed that IDO activity in tracheal allografts weresignificantly higher in IDO transfection group than in the other groups (P<0.01), however,which was no diffrence in all peripheral serum samples(P>0.05).Conclusion: IDO overexpression in the allografts airway epithelialum can preventobliterative bronchiolitis of chronic rejection. The mechnism may be associated withreducing T cells infiltration of allograft, inhibiting T cells proliferation, and steering initialdifferentiating T cells more to Treg cells than to Th17cells by increasing local IDOactivity.