呼吸道菌群失调加重小鼠过敏性呼吸道炎症反应

Airway Dysbacteriosis Exacerbated Murine Airway Allergic Inflammation

【目的】探讨呼吸道菌群失调对小鼠过敏性呼吸道疾病发病的影响。【方法】C57BL/6雌性小鼠给予万古霉素雾化吸入10 d后处死,应用16S rRNA高通量测序技术分析支气管肺泡灌洗液(BALF)菌群组成,探索建立小鼠呼吸道菌群失调模型的方法。建模成功后,应用相同的方法建立小鼠呼吸道菌群失调模型,在此基础上,通过腹腔注射致敏及雾化吸入卵清蛋白(OVA)激发,诱导小鼠过敏性呼吸道炎症反应,计数擦鼻频率,检测BALF中细胞总数及嗜酸性粒细胞百分比,肺组织病理分析炎症反应及杯状细胞增生情况,ELISA法检测血清中IgE水平,BALF中IFN-γ、IL-4、IL-5水平,血清、BALF及肠组织中IL-33水平,从而分析呼吸道菌群失调对过敏性呼吸道炎症反应的影响。【结果】万古霉素雾化吸入使小鼠呼吸道Bradyrhizobium、Sphingopyxis、Cupriavidus、Pelomonas等菌属增加,而Akkermansia及Prevotella_6等菌属显著降低,发生明显的菌群失调。利用此动物模型进一步研究发现,呼吸道菌群失调加重了卵清蛋白诱导的过敏性呼吸道炎症反应,表现为擦鼻频率增加,BALF中细胞渗出增加、嗜酸性粒细胞百分比增高,肺组织炎症及杯状细胞增生加重,血清中IgE水平增加。另外,与卵清蛋白组相比,菌群失调联合卵清蛋白组小鼠BALF中Th1型细胞因子IFN-γ水平降低,Th2型细胞因子IL-4、IL-5水平升高,发生更为严重的Th1/Th2平衡失调;BALF中IL-33水平增加,而血清及肠组织中IL-33水平差异无统计学意义,表明呼吸道菌群失调仅影响局部IL-33的产生。【结论】应用万古霉素雾化吸入成功建立了小鼠呼吸道菌群失调模型。呼吸道菌群失调可能通过增加局部IL-33产生,激活Th2及固有样淋巴细胞(ILC),诱发Th1/Th2平衡失调,从而促进过敏性呼吸道疾病发病。

[ Objective ] To investigate the effects of airway dysbacteriosis on the development of murine allergic airway diseases （AAD） [Methods] Female C57BL/6 mice were neubulized with Yancomycin for 10 days and then were sacrificed. The bacterial population in bronchoalveolar lavage fluid （BALF） were evaluated using 16S rRNA high-throughput sequencing technology, exploring the method of establishing an airway dysbacteriosis mouse model. After the mouse model was established successfully, airway dysbacteriosis mouse models were established by the same method, and based on that, the mice were sensitized and challenged with ovalburain （OVA） to induce airway allergic inflammation. The frequency of nasal rubbing behaviors per mice was counted; the total cell number and eosinophil relative abundance in BALF were evaluated ; the lung tissue inflammation and goblet cell metaplasia were as- sessed according to histopathological features ; and the IgE level in serum, IFN-y, IL-4 and IL-5 levels in BALF, and IL-33 levels in serum, BALF and intestine tissue were measured by ELISA. [Results] Nebulization of Vancomycin increased Bradyrhizobium, Sphingopyxis, Cupriavidus, Pelomonas, and decreased Akkermansia and Prevotella_6 in airway, inducing significant airway dysbacte- riosis. Using the animal model, further study found that airway dysbacteriosis exacerbated OVA-induced airway allergic inflammation, including increased nasal rubbing frequency, higher serum IgE level, more total cell count especially eosinophil infiltration, more serious lung tissue inflammation and goblet cell metaplasia. Additionally, compared to OVA group, mice in Dysbacteriosis and OVA group had significantly increased level of Th2 cytokine IL-4 and IL-5, and significantly decreased Thl cytokine IFN-γin BALF, which revealed that mice in Dysbacteriosis and OVA group had more remarkable Th 1/Th2 imbalance. Furthermore, IL-33 level showed a significant increase in BALF, but didn＇t change in serum or intestine tissue in Dysbacteriosis and OVA group compared to OVA group. Indicating that airway dysbacteriosis may only affect the local production of IL-33. [ Conclusions ] An airway dysbacteriosis mouse model was established by Vancomycin nebulization successfully. Airway dysbacteriosis may activate innate lymphoid cells （ILC） and Th2 cell by inducing local IL-33 secreting, which leads to the imbalance of Th1/Th2, and in turn promotes the development of AAD.