T-bet重组腺病毒调节哮喘小鼠辅助性T细胞1型/2型免疫平衡

Recombinant T-bet adenovirus regulates immune balance of T helper 1/T helper 2 in mice asthma model

目的研究静脉注射T-bet重组腺病毒(AdT-bet)对小鼠哮喘模型过敏性气道炎症及辅助性T细胞1型/2型(Th1/Th2)免疫失衡的影响。方法随机将36只C57BL/6小鼠分为AdT-bet治疗(A)组、模型对照(B)组、正常对照(C)组。以卵蛋白、氢氧化铝免疫建立哮喘模型,A组激发前尾静脉注射1×10~8 pfu/100μL的AdT-bet,各组激发后进行肺泡灌洗分析细胞组分,分离肺T淋巴细胞测定细胞因子分泌水平,以流式细胞仪检测CD3^+、CD4^+T细胞比例及表达γ-干扰素(IFN-γ)和白细胞介素(IL)-4、IL-5的比例,比较各组肺组织学改变。结果A组与B组相比:①明显抑制抗原激发后气道内嗜酸性粒细胞的浸润(0.004±0.003比0.221±0.067,P<0.01);②明显抑制肺T淋巴细胞产生IL-4[(22±12)pg/mL比(170±28)pg/mL]、IL-5[(16±13)pg/mL比(330±30)pg/mL],增加了IFN-γ[(2100±360)pg/mL比(60±50)pg/mL]的产生,差异均有统计学意义(P值均<0.01);③肺T淋巴细胞CD4^+IFN-γ%及IFN-γ^+/IL-4^+明显升高(P值均<0.01),而CD4^+IL-4^+%明显下降(P<0.01);④明显抑制哮喘鼠气道内及肺泡内的过敏性炎症反应。结论激发前静脉应用AdT-bet对哮喘小鼠过敏性气道炎症有明显的防治作用,其机制可能与表达的T-bet上调Th1/Th2比值,从而调整了免疫平衡有关。

Objective To investigate the effect of intravenous injection of recombinant T-bet adenovirus （AdT-bet） on antigen-induced allergic airway reaction and T helper 1/T helper 2 imbalance in mice asthma models. Methods Thirty-six C57BL/6 mice were randomly divided into AdT bet treated group, PBS control group and normal control group. The asthma model was established in C57BL/6 mice by immunization with OVA and Al（OH）2. Recombinant T-bet adenovirus was administrated intravenously into the tail veins（1 × 10^8 pfu/100μL） in the Adt-bet treated group before bronchoprovocation. The cellular composition of bronchoalveolar lavage fluid was analyzed after antigen challenge in each group;; the lung lymphocytes were isolated for cytokines secretion detection and FCM analysis of IFN-γ, IL-4, IL-5 positive CD3^＋ and CD4^＋ T cells. The pulmonary histological changes were compared between the 3 groups. Results Compared with control group, intravenous injection of recombinant T-bet adenovirus markedly inhibited the infiltration of eosinophilia（0. 004 ± 0. 003 vs 0. 221 ± 0. 067, P 〈 0.01） in airways of mice; it also inhibited the IL-4 （[22 ± 12] pg/mL vs [170 ± 28] pg/mL, P 〈 0.01） and IL-5 production （[16 ± 13] pg/mL vs [330 ± 30] pg/mL, P 〈 0. 01） and enhanced the IFN-γ production （[2 100±360] pg/mL vs [60 ± 50] pg/mL, P 〈 0.01） in isolated pulmonary T cells. FCM showed that the proportion of CD4^＋ IFN-γ^＋ lymphocytes and the ratio of IFN-γ^＋to IL-4^＋ lung T cells were significantly increased and the proportion of CD4^＋ IL-4^＋ T cells was significantly decreased in AdT-bet treated group. Histological examination showed that recombinant T-bet adenovirus inhibited the allergic inflammation in the airway and the lung tissues in the mice. Conclusion It is suggested that intravenous injection of recombinant Tbet adenovirus can obviously inhibit the antigen-induced allergic inflammation in airways, which might be associated with the up-regulation of T helper 1/T helper 2 ratio and the regulation of immune balance by T-bet gene delivered by recombinant adenovirus vector.