耻垢分枝杆菌黏膜接种对小鼠过敏性哮喘的免疫调节作用

Immunoregulatory effect of intranasal inoculating M. smegmatis in a murine model of allergic asthma

目的观察耻垢分枝杆菌(Mycobacterium smegmatis,Ms)对过敏性哮喘小鼠防御素β-2(Defb2)、巨噬细胞炎症蛋白(MIP)、IFN-γ、IL-4及过敏原特异性IgE、IgG1、IgG2a表达的影响,探讨Ms黏膜接种干预哮喘的免疫调节机制。方法 SPF级BALB/c小鼠随机分为哮喘组、干预组、对照组3组。以卵清蛋白(OVA)致敏,气道激发建立哮喘模型。OVA致敏前7 d鼻黏膜接种Ms进行干预。以Real-time荧光定量PCR法检测肺组织Defb2、MIP1、MIP2 mRNA的表达,ELISA法检测支气管肺泡灌洗液(BALF)、纵膈淋巴结细胞培养上清中MIP1、MIP2、IFN-γ、IL-4的浓度,ELISA法检测血清总IgE以及OVA特异性IgE、IgG1、IgG2a水平。结果 Ms干预可以显著增高哮喘小鼠Defb-2 mRNA的表达(31.21±1.56对0.78±0.11,P<0.01);Ms干预可使哮喘小鼠表达增高的MIP1 mRNA降低(9.80±1.56对2.44±0.96,P<0.05),但对MIP2 mRNA的影响不明显(P>0.05);Ms干预可使BALF和淋巴结培养上清中MIP1、MIP2降低,使BALF和淋巴结培养上清中IL-4降低(97.43±7.83对153.80±5.20,150.75±54.58对625.81±55.98,P均<0.01),使BALF中IFN-γ升高(78.92±11.41对44.88±3.26,P<0.01);Ms干预可以显著降低哮喘小鼠血清总IgE、OVA-IgE、OVA-IgG1,升高OVA-IgG2a,降低哮喘小鼠OVA特异性IgG1/IgG2a比值(1.09±0.03对1.42±0.04,P<0.01)。结论 Ms黏膜免疫可以通过促进Defb-2表达增强哮喘小鼠抗感染能力,通过降低巨噬细胞炎症蛋白表达、调节Th1/Th2平衡、抑制过敏原特异性IgE表达等途径干预哮喘,耻垢分枝杆菌有望继卡介苗之后成为哮喘预防的另一候选疫苗。

Objective To evaluate the effect of the intranasal inoculation with Mycobacterium smegmatis（Ms）on the expressions of β-Defensin-2（Defb-2）,macrophage inflammatory protein（MIP）and allergen-specific immunoglobulin（Ig）in the murine models of asthma.Methods Eighteen BALB/c mice were randomly divided into the asthma group,the Ms interventional group and normal control group（n=6 per group）.The mouse was sensitized and challenged with ovalbumin（OVA）for the establishment of the asthma model.Seven days before sensitizing,the mouse in Ms interventional group was inoculated with Ms intranasally.The expressions of Defb-2,MIP1 and MIP2 mRNA in lung tissues were detected by real-time quantitative PCR.ELISA was used to detect the concentrations of MIP1,MIP2,interferon-γ（IFN-γ）and interleukin-4（IL-4）in Bronchoalveolar Lavage Fluid（BALF）and supernatants of mediastinal lymph node cells,and the levels of total IgE（TIgE）,OVA-IgE,OVA-IgG1 and OVA-IgG2a in serum.Results With the Ms intranasal inoculation,the up-regulated level of Defb-2 mRNA（31.21±1.56 vs.0.78±0.11,P0.01）and the down-regulated level of MIP1 mRNA（2.44±0.96 vs.9.80±1.56,P0.05）were observed in the lungs of asthmatic mice,while the down-regulated expression of MIP2 mRNA was not significant（P0.05）.The lower levels of MIP,MIP2 and IL-4 in BALF and lymph node cell culture supernatants（P0.01）and the higher level of IFN-γ in BALF（P0.01）were found in the Ms treated asthmatic mice compared with those in the non-Ms treated mice.There significantly were the lower levels of serumal TIgE,OVA-IgE,OVA-IgG1,the higher level of OVA-IgG2a,and the decreased ratio of OVA-specific IgG1/IgG2a in the asthmatic mice inoculated with Ms intranasally compared with those in the asthmatic mice without Ms inoculation.Conclusions The intranasal inoculation of Ms can enhance the host defense against microbial infection through increasing the expressions of Defb-2 and IFN-γ,and suppress allergen induced inflammation by regulating the expressions of MIP1 and MIP2,and the balance of Th1/Th2.