细胞自噬在上生殖道沙眼衣原体感染中的作用机制研究

Upper genital tract (UGT) inflammation resulted from chlamydial sexually transmitted infection is the number one cause of infertility in women. Ectopic pregnancy, miscarriage and premature birth are among other complications of chlamydial STI. Currently, an incomplete understanding of the interaction between the pathogen chlamydia trachomatis and the human host hinders the development of an effective preventive means for the infection and its complications. The goal of this application is to elucidate the role of autophagy in the regulation of chlamydial infection and in the pathogenesis of UGT inflammation using a well-established mouse model for chlamydial STI. Autophagy is a cellular process leading to the degradation of cytoplasm contents in response to diverse signals. A number of drugs that are used or being tested for cancer and other diseases are known to modify autophagy. Autophagy can either serve as an innate defense mechanism against pathogens or facilitate the intracellular growth of other invading microbes. Autophagy has also been recognized as a critical regulator of inflammation. Interestingly, we have observed a nearly 10 fold increase in bilateral hydrosalpinx, a severe inflammatory condition leading to infertility, in mice with ATG5-knockout and thus autophagy-deficient myeloid cells, as compared to control mice with autophagy-proficient myeloid cells, following chlamydial infection. We hypothesize that myeloid autophagy inhibits the development of UGT pathology by suppressing chlamydia-induced inflammatory response. We will test this hypothesis by three Specific Aims: 1) determining how chlamydial growth/survival is affected by autophagy in Mø and PMN, 2) investigating how chlamydiae suppress autophagy to cause inflammation in the UGT, and 3) assessing if and how chlamydial infection-induced UGT inflammation is affected by autophagy in the urogenital epithelium. Our work may provide rationales for using autophagy-modulators for the prevention and treatment of not only chlamydial STI and consequent UGT inflammation but also complications of chlamydial respiratory infection including atherosclerosis and late-onset Alzheimer disease.

沙眼衣原体（Ct）感染引起的上生殖道（UGT）炎症可导致严重并发症，但作用机制不完全清楚。自噬是宿主抗微生物感染的一种天然防御机制，细胞自噬在胞内病原体感染过程中发挥着双刃剑的作用，一方面自噬可抑制杀灭胞内病原体，保护机体免受侵害；另一方面，一些胞内病原体可抑制阻断自噬的发生或成熟，甚至利用自噬逃避机体的免疫反应以利于其在体内存活和增殖。我们研究发现，Ct感染可引起宿主细胞发生自噬，在髓系细胞ATG5-KO 的小鼠中，Ct感染引起的UGT 炎症反应较野生型小鼠严重，提示当宿主的自噬反应系统缺陷时，Ct诱发的炎症反应更严重。自噬是否影响Ct的生存和/或生长及其在UGT炎症中的作用机制尚不明确。本项目以组织特异性基因敲除小鼠及自噬相关基因沉默的人传代细胞为模型，采用免疫荧光共聚焦、Bioplex等方法，研究细胞自噬与Ct生长、炎症反应、UGT病损进展之间的相互关系和作用，为Ct防治提供新思路。

本课题探究自噬相关基因Atg5、P62在沙眼衣原体感染的HeLa229细胞炎症反应中所起的作用，为探索沙眼衣原体致病机制及临床上防治沙眼衣原体感染提供理论指导与数据支撑。. 我们以髓系细胞自噬相关基因ATG5基因敲除（myeloid ATG5-knockout，mATG5-KO）小鼠和对照小鼠为对象，建立沙眼衣原体（Chlamydia trachomatis，Ct）MoPn型小鼠阴道感染模型，研究发现髓系细胞ATG5敲除小鼠，Ct上行感染导致的上生殖道（upper genital tract，UGT）炎症更加严重。我们还发现Ct感染人宫颈上皮HeLa细胞和THP-1来源的人巨噬细胞后能够诱导宿主细胞自噬。通过基因沉默技术建立了自噬相关基因atg5、p62沉默的Hela、THP1细胞株，运用免疫印迹、免疫荧光等方法证实在Ct感染的细胞中，与细胞自噬或炎症反应有关的蛋白Atg5、LC3、NF-kB等的表达增多，而自噬活动的负相关蛋白P62表达量减少。这些数据表明：一方面，宿主能通过细胞自噬的方式抵抗Ct感染；另一方面，Ct也能利用细胞自噬为自己在宿主细胞内存活提供条件。细胞自噬在Ct感染的过程中起着双刃剑的作用。上述研究为后续自噬相关基因（atg5和p62）沉默的细胞中Ct生长情况与细胞炎症反应的研究奠定理论与实践基础。. 通过Atg5沉默的HeLa229-shAtg5细胞系感染沙眼衣原体，证明Atg5是调控沙眼衣原体感染的HeLa229细胞发生自噬的重要分子，沉默Atg5基因可显著降低HeLa229细胞自噬水平；HeLa229-shAtg5细胞在应对Ct感染时，炎症反应强于HeLa229-shNC细胞，表现为促炎因子IL-1β、IL-6、IFN-γ分泌量显著增多，抗炎因子IL-10分泌量显著降低，表明细胞自噬具有一定的抗炎作用. 而后，本课题组成功构建p62沉默的HeLa229-shp62细胞系以及HeLa229-shNC细胞系，并使其稳定传代。以电子显微镜技术均检测到两种细胞中的自噬小体和沙眼衣原体包涵体，发现HeLa229-shp62细胞中的沙眼衣原体包涵体形态差别不大，但自噬小体数量比HeLa229-shNC细胞明显减少，差异具有统计学意义（p＜0.05）。Ct感染后，与HeLa229-shNC相比，HeLa229-shp62细胞的自噬水平