Clinical And Experimental Studies Of Transcription Factor T-bet On Immunomodulatory Role In Bronchial Asthma

Bronchial asthma is a common respiratory tract disease in human being,which is associated with chronic airway inflammation caused by many kinds of cells and cell component.The important immunopathological characteristics of asthma are the imbalance differentiation of T helper cell subset(Th1/Th2),dominant Th2 immune response and the loss of immune tolerance induced by airway tract to allergen. Wheezing bronchitis which ranged to asthma by nelson pediatrics is a kind of special bronchitis having asthmatic clinical manifestation during infancy.T-bet(T box expressed in T cells)is the specific transcription factor for Th1 cell, and plays a crucial role in its polarization.By intensively activating the transcription of IFN-γgene,T-bet not only induces expression of itself and IL-12Rβ2 on Th cells,so as to enhance the sensitivity to IL-12 and accelerate the differentiation of naive Th cell to Th1 cell,but also reverses the developing or developed Th2 cell to Th1 cell with intervention of retrovirus vector,following much IFN-γproduction and the secretion of Th2 type cytokine to be inhibited.T-bet also represses GATA-3,the specific transcription factor of Th2 cell,and promotes Th1 polarizing.T-bet knock-out mice show spontaneous bronchial hyperreactivity and airway inflammation of asthma-like.In this study,one objective was to explore the immunopathological features of wheezing bronchitis and relationship to asthma,through the clinical research effect of T-bet on wheezing bronchitis in infancy.Another was to construct adeno-associated virus(AAV)vector of mouse T-bet gene,which was applied to mouse model of asthma by nose,and to investigate the immunomodulatory effects of T-bet delivery on asthma.In clinical research,the mRNA and protein levels of T-bet and GATA-3 of peripheral blood lymphocytes in patients with wheezing bronchitis were detected by reverse transcription-polymerase chain reaction(RT-PCR)and western-blot.The levels of IFN-γ,IL-4 and IL-5 in the supernatant of the lymphocytes incubated were examined by enzyme-linked immunosorbent assay(ELISA).The relationship between that transcription factors and cytokines was analyzed by simple linear correlation.In experimental study,firstly,the cDNA of T-bet was amplified from splenic cells of Balb/c mouse by RT-PCR and inserted into pGEM-T vector,and the sequence of the DNA was determined.Secondly,the T-bet gene cutting from pGEM-T vector by two enzyme was inserted into the expressive plasmid pzac2.1 of adeno-associated virus,and come into being recombinant plasmid pzac2.1-T-bet that together with trans plasmid p5E18 and helper plasmid pAddeltaF6 were packed to form recombinant AAV carrying T-bet gene (rAAV-T-bet)in HEK293 cells by the calcium phosphate sedimentation method.Then the rAAV-T-bet suspension was purified by heparin agarose affinity chromatography and concentrated by Milipore Amicon Ultra-15 100k centrifugal filter device.The titer of rAAV-T-bet was detected by real-time PCR.Some kinds of cells including D2SC were transfected by rAAV-T-bet in vitro so that the expression of T-bet was observed.Thirdly,Balb/c mice were sensitized by intraperitoneal injections with OVA and challenged by nebulized OVA.The sensitized mice were given twice intranasal delivery of AAV-T-bet at 3 to 4 days before the inhalation challenge.After 24h of last challenging,the mice were sacrificed and the samples were obtained.Transcription factors T-bet and GATA-3 in lungs,T-bet in splenic cell,airway histology,and other inflammatory parameters,such as the development of eosinophils,cytokines in bronchoalveolar lavage fluid(BALF),total IgE and OVA specific IgE in serum,and B lymphocyte stimulator(Blys)in body fluids were assayed.The results in clinical study showed that in infants with wheezing bronchitis there was a predominant differentiation of Th2 type cells accompanying with lower production of T-bet,IFN-γand over-expression of GATA-3,IL-4.Two positive correlations were found between the levels of IFN-γand T-bet as well as IL-4 and GATA-3.Two negative correlations were also proved in the levels of IFN-γwith IL-4 and T-bet with GATA-3.The experimental study results were that the full-length cDNA of T-bet has been amplified.T-bet gene recombinant expressive plasmid pzac2.1-T-bet was constructed, and recombinant AAV vector,rAAV-T-bet was packed successfully in HEK293 cells. The pure higher titer rAAV-T-bet was made through purifying and concentrating.T-bet was expressed in some kinds of cells such as D2SC etc in vitro when rAAV-T-bet was transfected. On the basis of building mouse model of asthma,intranasal administration of rAAV-T-bet could deliver T-bet gene to airway successfully and efficiently play a immunomodulatory role to immune confusion of asthma.Firstly,the overproduction of Th2 lymphocyte signature cytokine IL-4,IL-5 and specific transcription factor GATA-3 were suppressed while the Th1 signature cytokine IFN-γand specific transcription factor T-bet showed opposite increase after rAAV-T-bet treatment.Secondly,remarkable airway inflammatory cells infiltration and mucus gland hypersecretion were inhibited and the number of eosinophil in BALF decreased.Thirdly,the levels of specific transcription factor Foxp3 of CD4+CD25+ T regulatory cell were distinctly lower in asthma model,which increased significantly after intranasal administration of rAAV-T-bet.In addition,the rAAV-T-bet delivery decreased IgE levels,but enhanced the expression of B lymphocyte stimulator(Blys)and T-bet outside of lung.The conclusions were as follows:(1)There were asthma-like immunological changes and predominant Th2 type cell immune response in infants with wheezing bronchitis.The imbalance of Th1/Th2 type cytokines IFN-γ/IL-4 profiles was regulated by transcription factors T-bet/GATA-3 respectively.The action of T-bet and GATA-3 was mutual inhibition.(2)Pzac2.1-T-bet was constructed,and recombinant AAV vector, rAAV-T-bet was packed successfully in HEK293 cells.T-bet was expressed in D2SC cell line when transfected by rAAV-T-bet,which laid the groundwork for studies on immunomodulatory role of T-bet gene transfecting antigen presenting cell and intervening asthma model.(3)The intranasal transfer of T-bet gene by rAAV-T-bet infection could promote Th1 immune response and suppress Th2 immune response,and restore Th1/Th2 immune responses balance.And that airway inflammation was inhibited in mouse model of allergic asthma.(4)The expression of Foxp3 was lower in lung of mouse model of asthma.It was suggested that there may be decreasing number and dysdifferentiation of T regulatory cell,and the ability descended for inducement immune tolerance in the model.After treatment of rAAV-T-bet,the activity of T regulatory cell expressed Foxp3 was boosted up,and imbalance of Th1/Th2 gained farther regulation.Therefore,the intensively Th1 response was avoided.(5)In addition, the rAAV-T-bet treatment also played a indirect role to B lymphocyte.With the suppression of Th2 type immune response,IL-4 secretion decreased,and the ability of IgE production from B cell declined.Similarly,with the enhancement of Th1 type immune response,the production of IFN-γincreased and that resulted in of Blys profiles going up.