肺泡上皮细胞与巨噬细胞互作对牛结核分枝杆菌感染的免疫调节机制研究

Pulmonary tuberculosis of bovine is a chronic fatal zoonotic infectious disease caused by an infection of Mycobacterium bovine (M. bovine), which has significantly hampered a normal and stable development of animal production industry, as well as negatively impacted on the safety of products of milk and meats, and public health. Alveoli of lung are the solo sites for gas-exchanges and the terminals of the airway in the lung，which are consisted of alveolar epithelial cells (AECs) and alveolar macrophages (AMs), the targets and immune cells of mycobacterial infections. Both of AECs and AMs have been demonstrated to play key roles in maintenances of the immunological homeostasis and normal lung function during an infection. Using a 3D model of in vitro cell co-culture, we recently found that an interaction of AECs and AMs could either positively or negatively regulate the inflammatory responses of cells in response mycobacterial infections. The regulatory role of interaction was largely depended on the model of infection. However, the underlying mechanism of signaling remains elusive. Therefore, the goal of present proposal is aimed to dissect the underlying signaling pathways involved in regulations of inflammatory responses mediated by the interaction of AECs and AMs of bovine in response to M. bovine infection，using a 3D bovine AEC/AM co-culture model. These studies will provide novel insights into the underlying mechanisms of pathogenesis of pulmonary tuberculosis of bovine.

牛结核病是由牛结核分枝杆菌引起的慢性致死性人兽共患传染病，不仅严重影响畜牧业的健康稳定发展，而且严重威胁乳肉产品及公共卫生安全。肺泡作为呼吸道的终端是实现气体交换的唯一场所，组成肺泡的上皮细胞和巨噬细胞都是结核分枝杆菌的靶细胞和免疫反应细胞，在抗结核分枝杆菌感染过程中对维系机体免疫反应的动态平衡和肺脏正常功能发挥着关键作用。研究发现，利用体外3D细胞共培养模型，在不同的感染条件下，肺泡上皮细胞和巨噬细胞之间能够相互促发或抑制结核分枝杆菌诱导的炎症反应，从而发挥相互免疫调节作用，但是这种细胞之间相互作用的调控信号机制目前还一无所知。因此，本项目旨在在建立牛肺泡上皮细胞体外气液相培养模型的基础上，利用巨噬细胞-上皮细胞3D共培养模型和RNA-seq转录组分析技术，剖析牛结核分枝杆菌感染过程中牛肺泡上皮细胞与巨噬细胞相互作用的信号调控机制，对进一步开展牛结核病免疫机理研究具有重要意义。

牛结核病是由牛结核分枝杆菌引起的慢性致死性人兽共患传染病，不仅严重影响畜牧业的健康稳定发展，而且严重威胁乳肉产品及公共卫生安全。肺泡作为呼吸道的终端是实现气体交换的唯一场所，组成肺泡的上皮细胞和巨噬细胞都是结核分枝杆菌的靶细胞和免疫反应细胞，在抗结核分枝杆菌感染过程中对维系机体免疫反应的动态平衡和肺脏正常功能发挥着关键作用。因此，本项目在建立牛肺泡上皮细胞体外气液相培养模型的基础上，利用巨噬细胞-上皮细胞共培养模型和RNA-seq转录组分析技术，剖析牛结核分枝杆菌感染过程中牛肺泡上皮细胞与巨噬细胞相互作用的信号调控机制，对进一步开展牛结核病免疫机理研究具有重要意义。. 通过本项目实施，在分离和培养牛原代肺脏上皮细胞（AEC）和肺泡巨噬细胞（BAM）的基础上，成功建立了AEC /AM气液相共培养模型；RNA-Seq及生物信息学分析结果表明，BCG感染BAM后，激发宿主细胞产生强烈的免疫反应，引起LncRNA和mRNA表达谱的改变；RT-PCR、Western-blot检测表明，BCG感染BAM后，激活了TLR信号介导的炎性反应；流式检测结果表明，BCG诱导BAM发生凋亡；以BAM及RAW264.7细胞为材料，揭示了酯酰辅酶 A胆固醇酰基转移酶1（ACAT1）与双特异性磷酸酶5（DUSP5）对BCG诱导的巨噬细胞自噬的调控作用，即：ACAT1介导细胞内胆固醇水平，并通过与PI3K/Akt/mTOR信号通路的相互作用参与BCG诱导的巨噬细胞自噬；DUSP5可通过抑制p-ERK1/2磷酸化进而抑制BCG诱导的RAW264.7细胞自噬；ERK1/2途径对BCG感染巨噬细胞自噬和脂肪酸代谢具有调控作用，为脂肪酸代谢在巨噬细胞抗结核分枝杆菌中的免疫应答机制提供了理论依据。