The Intervention Study Of Enhanced Expression Of Foxp3in The Pathogenesis Of Asthma

Bronchial asthma is a serious global health problem with300million individuals suffered in this disease and250,000deaths caused by it every year all over the world. Asthma is a chronic inflammatory disorder of the airway characterized by infiltration of lymphocytes, eosinophils, mast cells and macrophages, airway hyperresponsiveness (AHR) and airway remodeling. The social and family issues caused by asthma have put a heavy burden on our human society.There are several distinct types of T-helper lymphocytes:Th1, Th2, Th17and so on. Among the numerous mechanisms of asthma, the generation of Th2cytokine response to environmental allergens is predominantly considered. As for patients with allergic diseases the Th1/Th2balance is in favour of Th2which produces a characteristic repertoire of inflammatory factors, including IL-4, IL-5, IL-13, etc. In asthmatic subjects Th2cells are activated and they release a Th2cytokine profile that regulates IgE production, inflammatory cell recruitment, mucus hypersecretion and structural changes in the airways. On the other hand, the deficiency of immunological tolerance induced by allergen also accounts for the development of asthma. Healthy individuals can avoid Th2response by generating immunological toleranc when exposed to allergens while asthmatic individuals can not.Regulatory T cells (Tregs), especially CD4+CD25+Tregs accounting for approximately5-10%of the peripheral CD4+T cells in normal unimmunized individuals play an important role in the tolerance induction in the murine model of asthma. CD4+CD25+Tregs can produce inhibitory cytokines IL-10and TGF-P to suppress the differentiation, proliferation or cytokine production of Th2cells. The transcription factor forkhead box protein3(Foxp3), is specifically expressed in CD4+CD25+Tregs and is crucial for the differentiation of naive T cells towards the Tregs’phenotype.To date, a lot of factors have been found out to be able to recruit CD4+CD25+Tregs or induce Foxp3expression in CD4+CD25-T cells and reduce the Th2immune response in asthmatic individuals. But all of these studied used systematically methods and may cause some potential problems due to side effects on other organs in vivo. So we intended to enhance the expression of Foxp3in the lung tissue locally and hypothesized this method may relieve the symptoms of asthma. We successfully constructed Foxp3/PMX retroviral vectors containing mouse Foxp3gene and converted these viruses into the murine model of asthma by intratracheal instillation, and the lung inflammation, AHR, the percentage of Th2and Tregs and the level of cytokines in the BALF were characterized. Possible roles of IL-10and TGF-β in mediating this process were investigated as well.Objective:To investigate the effect of enhanced expression of Foxp3locally in the airway by Foxp3/PMX retroviruses intratracheal instillation on the airway inflammation, AHR, and the percentage of Th2and Tregs in allergic asthma.Methods:BALB/c mice were used in this study and divided into4groups, including Saline Group, OVA Group, Foxp3Group and Control Group. Foxp3/PMX retroviruses which contained the mouse Foxp3gene were constructed and administrated into asthmatic mice through intratracheal instillation before ovalbumin challenging, 50μl/day,3days, while the Control Group were treated with Empty/PMX retroviruses as well.24hours later the AHR, airway inflammatory responses, the total cell numbers in the BALF and the Eos numbers in the BALF were characterized. The Foxp3mRNA in the lung tissue was measured by Q-PCR while the Foxp3protein in the lung tissue was measured by Western blot. The percentages of CD4+Foxp3+T cells and CD4+IL-4+Tcells in the totlal CD4+T cells were measured by FACS. Cytokines IL-4, IL-13, IL-17, IL-10and TGF-β in the BALF were measured by ELISA. IL-2, IFN-γ, TNF-α, IL-4, IL-5, IL-13, IL-17A, IL-10and TGF-β mRNA in the lung tissue were measured by Q-PCR.Results:①The level of Foxp3mRNA and protein in the lung tissue of OVA Group was increased than Saline Group, as well as the percentages of CD4+Foxp3+Tregs and Th2in the lung tissue. Enhanced expression of Foxp3locally by Foxp3/PMX retroviruses intratracheal instillation further increased the level of Foxp3mRNA and protein in the lung tissue, accompanied by increased CD4+Foxp3+Tregs and decreased CD4+IL-4+T cells.②The AHR, inflammatory cell infiltration and mucous secretion were increased in OVA Group than Saline Group. While decreased AHR, inflammatory cell infiltration and mucous secretion were detected in Foxp3Group than Control Group.③Decreased IL-4、IL-5、IL-13、IL-17A mRNA in the lung tissue were detected when the asthmatic mice were administrated with Foxp3/PMX retroviruses as well as the level of cytokines IL-4、IL-13、L-17in the BALF.④Increased IL-10and TGF-β mRNA in the lung tissue were detected when the asthmatic mice were administrated with Foxp3/PMX retroviruses as well as the level of cytokines IL-10and TGF-β1in the BALF.Conclusions:This study demonstrates that enhanced expression of Foxp3in the airway can attenuate the Th2and Th17immune response and reduce the allergic airway inflammation, AHR and mucus overproduction probably through increasing the CD4+Foxp3+Tregs and the suppressing the Th2cells in the lung tissue, accompanied by the upregulation of the immunologic suppressors IL-10and TGF-β.