Immune Regulation Of Dendritic Cell Transfected By Gene TLR4 For Bronchial Asthma

BackgroundBronchial asthma(BA)refers to a chronic inflammatory disorder of the airways,resulting from mostly hypersensitivity.Its pathogenesis and development are considered related to diverse inflammatory cells, mediators and cell factors,and the imbalance of T cell subgroup (Th1/Th2),increase in Th2 cell number and its activated function may play a key role.Dendritic cell(DC)is up to now the most potent antigen presenting cell(APC)and is also the unique one to activate initial T cell, especially it is a critical factor to recognize exterior and interior antigen and regulate innate and adaptive immunity.DCs distributed in airways serve as the guard cells of respiratory system and turn mature after taking in inhaled antigen by pinocytosis,pahgocytosis and receptor-mediated endocytosis,manifested with increasing expression of chemotatic factors,costimulatory molecules and MHCII molecules, and decreasing expression of Fcγ～RII,FcγRI and Fcε1,which would move to mediastinal lymph node from airways within 24 hours to produce primary immune response,promoting initial Th cells(Th0)to transform into Th1 or Th2.The antigen recognition signal and costimulatory signal related to DC are necessary to Th0 activation because it is controlled by the cytokines produced by DCs stimulated by antigen.For example,IL-12 and IFN-γare key factors to induce Th0 into Th1.A great many studies indicated that DC's immune regulation was related to the expression of TLR on the surface of cell membrane. It is generally known that PAMP may activate DCs by TLR4,resulting in considerable secretion of cytokines such as IL-12p70 and IFN-γmediated protein(IP-10).In use of this mechanism,that is,TLR4 of DCs may promote the differentiation of T cells,we made a trial to induce gene TLR4 via adenovirus vector into DCs stimulated by antigen such as LPS,so that DCs could produce much more cytokines like IFN-γto up-regulate the expression of IL-12Rβ2 and TLR4 from Th0,which may induce Th0 into Th1.ObjectiveWe infused DCs(transfected by gene TLR4 and stimulated by LPS)into the mice with allergic asthma induced by OVA in order to promote the differentiation of Th1,and reverse the imbalance of T cell subgroup caused by allergic asthma.From this study,we expect to find a new gene therapy for asthma through intervention of DC transfected with TLR4.Method1.The full length cDNA of TLR4 gene was obtained by using RT-PCR method from mRNA of Balb/c mouse spleen cells and then was connected with pcDNA3.1(+)carrier for sequencing and nucleotide analysis.2.With the aid of AdMax package system,293 cells were transfected with both adenovirus shuttle plasmid(with exogenous gene cloned)and packaging plasmid(with most adenovirus genomes),and then they were recombined via Cre/loxP system into recombinant adenovirus.AdV-TLR4 mRNA expression level in passage DCs was detected by RT-PCR assay and AdV-TLR4 protein level was detected by Western Blot method.3.The expression of AdV-TLR4-transfected DCIL-12mRNA,IL-4mRNA and IFN-γmRNA were detected by RT-PCR;the expression of IL-12,IL-4 and IFN-γin culture supernatant of AdV-TLR4-transfected DCs were detected by ELISA assay;specific immunity marker proteins on the surface of AdV-TLR4-transfected DCs including Ia,CD40,CD80 and CD 86 were detected by flow cytometry.4.Asthma mouse model was prepared with hypodermical injection of 0.1 %OVA Al(OH)3 solution and atomization of 1%OVA normal saline solution; tissue fixation with formalin,embedment,slice,HE stain and AB/PAS re-stain for mouse lung tissue morphologic observation.Eos in BALF was observed after Gimsa—Wright stain;the DCs were obtained through bone marrow cells separation,red blood cells treatement with lysate,induction of GM-CSF and IL-4,and RMPI 1640 culture,the expression level of both DCTLR4 mRNA and TLR4 protein of asthma mice were detected;IL-4,IL-5,IFN-γ,serum IgE level of mice spleen cells,human serum IL-4 and IgE,and IL-4 and IgE in BALF were detected via ELISA;CD4+ and CD25+ spleen cells percentage were detected with flow cytometry.Result1.Mouse TLR4 gene cDNA obtained from PCR has a full length of 2508bp and encodes 536 amino acids.The result of blast indicated this cDNA was 100%homologous with the one(sequence NO.NM021297)reported in Genbank.2.PCR and enzyme digestion identification of recombinant plasmid showed:gene TLR4 was set correctly;pBHGF35 backbone plasmid was transfected into 293 cells and was packaged into virus.PCR assay proved the package of recombinant virus was finishend;the recombinant virus was transfected into mouse DCs and the TLR4 mRNA and TLR4 proteins were highly expressed.3.1.L-12 and IFN-γ(inducing Th1 differentiation)were highly expressed 48 hours after DCs were transfected by Ad-TLR4,while,IL-4(inducing Th2 differentiation)showed a decreasing expression;PCR assay of transfected DC showed that the changes in IL-12 mRNA,IL-4 mRNA and IFN-γmRNA were consistent with the changes in corresponding proteins expression;LPS test indicated that LPS could effect the cytokines produced by transfected cells which were changed more greatly in LPS(+)group than that in LPS(-). LPS could also enhance some bionomics of Ad-TLR4-DC.2.Cytometry assay showed that CD86 was expressed more highly in Ad5F35-mTLR4 group than that in both control and Ad5F35-mCMV-EGFP group. Simultaneously,Ia,CD40 and CD80 of DC transfected by Ad5F35-mTLR4 were expressed much more highly in LPS(+)group than that in LPS(-)group; CD80 and CD86 in LPS(+)were more highly expressed than that in LPS(-) group.LPS may promote and strengthen the change of specific immunity marker proteins on the surface of Ad-TLR4-DCs.4.When TLR4 DC suspension was given to the mice with allergic asthma,the result showed a decrease in beaker cell proliferation,mucosal fluid in airways,WBC,Eso ratio and IL-4 level.However the percentage of CD4+CD25+ cells increased,which indicating TLR4DC could resist bronchial asthma.The mechanism is possibly related to the cytokines induced by TLR4DCs and the former can promote T cells differentiation,for instance, IFN-γmay induce T11 differentiation;TLRC4DC may also increase immune-regulating T cells CD4+CD25+ that enhance the toleration of allergen and minimize the inflammation.Conclusion1.Full-length coding region cDNA of mouse TLR4 gene was successfully established.2.Recombinant viral infection titer is 2.3×108 IU/ml and it can be utilized in animal experiment in vivo and in vitro.3.LPS can lead to the change of specific immunity marker proteins on the surface of AdV-TLR4- DC including Ia,CD40,CD80 and CD 86,and it also result in the change of expression of IL-12,IL-4 and IFN-γsecreted by DC.These changes are presumed to be related to signal conduction of DCTLR4.4.TLR4DC may inhibit the bronchial inflammation induced by OVA. Plus,LPS and TLR4 show a coordination which is possibly related to immune regulation of DC.