Alteration Of CD4~+CD25~+ T Regulatory Cell Pathway Of Neonatal Repeat BCG Vaccinations In A Mouse Model Of Asthma And Study On Its Transcription Factor Foxp3 Function

BackgroundAllergic asthma is a chronic inflammatory disease characterized by reversible airway obstruction,airway hyperreactivity,airway infiltration by inflammatory cells, particularly eosinophils and T lymphocytes,and high levels of allergen-specific IgE. Although current asthma therapy is effective and well tolerated in most patients, these drugs do not change the underlying immunological mechanisms,and there is a clear medical need for the development of new therapies capable of changing or reprogramming the underlying immune processes to produce a long term protective immune response.Epidemiological and clinical studies have provided compelling evidence that suggest a link between the relative lack of infectious diseases and the increase in allergic disorders.It has been demonstrated that a positive tuberculin test result suggestive of past infections with mycobacterium tuberculosis was inversely related to the subsequent development of atopy and asthma.Several animal studies showed that vaccination with mycobacteria prevented or treated allergic and asthmatic manifestations.However,the mechanism by which mycobacteria inhibit the inflammatory response remains unclear.The mechanism by which induction of Th1 cytokines such as IFN-γ,and thus reversion of the imbalance between Th1/Th2 had been generally proposed.Zuany-Amorim demonstrated that Mycobacterium vaccae is effective in blocking allergic inflammation by a mechanism independent of IFN-γ. Similarly,our colleague found that neonatal Bacillus Calmette-Guérin(BCG) vaccinations significantly inhibited allergic inflammation,but did not significantly change the low IFN-γresponses in late-challenged mice.We thought that mechanisms other than simple changes in the balance between Th1 and Th2 may account for observed effects of vaccination with mycobacteria.T regulatory(Treg)cells are thought to be crucial in the immunotolerance that are capable of suppressing adaptive immune responses.CD4~+CD25~+ Treg cell regulatory T cells are the most important population of regulatory T cells that constitutively express CD25,with suppressive properties inhibiting functions of effector CD4~+ and CD8~+ T cells.Foxp3 is a master gene governing the development and function of CD4~+CD25~+ Treg cell,and is expressed on the CD4~+CD25~+ Treg cell specifically.The suppressive mechanisms of CD4~+CD25~+ Tregs are not clear,but there is evidence that cell-cell contact by CTLA-4(cytotoxic lymphocyte associated antigen-4)is required.And there are also conflicting evidences concerning roles for interleukin(IL)-10 and transforming growth factor(TGF)-β.There are increasing evidences that there are functional defect and/or number abnormity of CD4~+CD25~+Treg cell in asthma and CD4~+CD25~+Treg cell plays an essential role in limiting the development of immune response in allergy and asthma.Therapeutic opportunities to exploit the immunosuppressive effects of Tregs for prophylactic or therapeutic treatment may become feasible.The Treg may be involved in the inhibitory effect of BCG upon asthma inflammatory response.Vaccination with mycobacteria is associated with persistent chronic intracellular infection in vivo that maintains a sustained immune response. The immune system has regulatory mechanisms for suppressing the effector response to persistent infection.It has been demonstrated that tuberculosis(TB) infection and BCG vaccination induced increased FOXP3 expression and CD4~+CD25~+Treg cells in human peripheral blood.Given the role of Treg cell in asthma and the effect of mycobacteria infection on induction of Treg cells,we conducted this study to investigated whether the protective effects on allergic pulmonary inflammation of BCG vaccinations are associated with the alteration of CD4~+CD25~+Treg cell in murine asthma model and through which mechanisms the Treg may play,and study the expression and function of Foxp3 in vitro using the Foxp3 eukaryotic expression plasmid. Part 1 Neonatal repeat Bacillus Calmette-Guérin vaccinations inhibits de novo allergic inflammatory response in mice via alteration of CD4~+CD25~+ T regulatory cell pathwayObjective:To investigate whether the inhibitory effect of BCG vaccinations on allergic pulmonary inflammation is associated with the alteration of CD4~+CD25~+ Treg cells in murine asthma model and through which mechanisms the T regulatory cell may play.Method:New born C57BL/6 mice were divided into 3 groups:asthma group,BCG vaccination group and control group.BCG group mice were vaccinated with BCG on days 0,7 and 14 for three times,while asthma and control groups mice with the same volume of saline solution.From the sixth week,asthma and BCG groups mice were sensitized and challenged with OVA to schedule,while control group mice with saline solution.Eosinophil accumulations in the airway and peribronchial/perivascular tissue in the lungs of the mice were investigated. Proportion of spleen CD4~+CD25~+ Treg cells/CD4~+ cells were measured by flowcytometry.Foxp3 expression in spleen was analyzed with RT-PCR and Western Blot.CTLA-4 and GITR expression in spleen were also determined with RT-PCR. Cytokines IL-4,IFN-γ,IL-10 and TGF-βlevels in the cultured splenocytes supernatant were examined with ELISA.Results:Administration of BCG at birth prevented the eosinophil accumulation in BALF and peribronchial and perivascular area in the lungs of asthma mice.OVA group mice have a lower spleen CD4~+CD25~+Treg cells/CD4~+ cells proportion compared with control group.Treatment with BCG increased the proportion markedly.Foxp3 expression in OVA mice spleens was significantly higher than that in control group,and there was a more higher level due to the treatment with BCG. Also BCG vaccination increased the expression of CTLA-4 and GITR in the mice spleen.OVA group mice had higher IL-4 and lower IFN-γlevels in the cultured splenocytes supematant than control group,and BCG vaccination corrected the polarization respectively.There were significantly higher levels of IL-10 and TGF-βwith the BCG vaccination.Conclusion:Neonatal repeat BCG vaccinations ameliorate de novo local eosinophilic inflammation induced by allergen.Insufficiency of CD4~+CD25~+ regulatory T cell number and function and the three-polar imbalance among Treg, Th1 and Th2 may play a key role in the pathogenesis of asthma.BCG vaccinations correct the imbalance of Th1,Th2,and promote the activation of the CD4~+CD25~+ Treg cells and up-regulate the expression of the master transcription factor Foxp3 to conduct immuno-tolerance.The cell-cell contact inhibition mediated by CTLA-4, GITR and regulatory cytokines IL-10,TGF-βmay be involved in the regulatory mechanisms of CD4~+CD25~+ Treg cells induced by BCG.Part 2Effects of transcription factor Foxp3 transfection on the asthma mice splenocytes functionsObjective:To transfect the Foxp3 eukaryotic expression plasmid into the asthma mice splenocytes,then to study its expression and the effects on the asthma mice splenocytes functions.Method:The mice models were sensitized and challenged to make the asthma model and then the splenocytes were harvested and cultured.The Foxp3 expression vector pcDNA3.1(-)-Foxp3 was transfected into the splenocytes with electroporation. The splenocytes transfected with empty vector[pcDNA3.1(-)]and control splenocytes(non-transfected)were also studied.Foxp3 mRNA and protein expressions were detected by RT-PCR and Western Blot respectively.Proportion of CD4~+CD25~+ Treg cells/CD4~+ cells were measured by flowcytometry.Proliferation of the splenocytes was analyzed with MTT.ELISA was used to determined the levels of IL-4,IFN-γin the supernatant of the splenocytes..Results:Foxp3 mRNA and protein expression in transfected group were significantly higher than those in empty vector and control groups.Transfected group has a higher CD4~+CD25~+Treg cells/CD4~+ cells proportion compared with empty vector and control groups.Proliferation of transfected cell was marked inhibited compared with empty vector and control groups.IL-4 and IFN-γlevels were significantly lower in transfected group than in empty vector and control groups.Conclusions:The transfected Foxp3 gene over-expresses in the asthma mice splenocytes.Foxp3 can increase the number of CD4~+CD25~+ T cells and inhibit the splenocytes proliferation and production of Th1 and Th2 cytokines.Summary1.Neonatal repeat BCG vaccinations ameliorate de novo local eosinophilic inflammation induced by allergen.2.Insufficiency of CD4~+CD25~+ regulatory T cell number and function and the three-polar imbalance among Treg,Th1 and Th2 may play a key role in the pathogenesis of asthma.3.BCG vaccinations correct the imbalance of Th1,Th2,and promote the activation of the CD4~+CD25~+ Treg cells and up-regulate the expression of the master transcription factor Foxp3 to conduct immuno-tolerance. Consequently,BCG can inhibit the allergic inflammation of asthma.4.The cell-cell contact inhibition mediated by CTLA-4,GITR and regulatory cytokines IL-10,TGF-βmay be involved in the regulatory mechanisms of CD4~+CD25~+ Treg cells induced by BCG.5.The transfected Foxp3 gene over-expresses in the asthma mice splenocytes. Foxp3 can increase the number of CD4~+CD25~+ T cells and inhibit the splenocytes proliferation and production of Th1 and Th2 cytokines. Induction of Foxp3 expression may develop new approach to the treatment of asthma.