沙眼衣原体持续感染中TLRs/NLRs信号通路异常活化的机制及作用研究

The goal of this research proposal is to investigate the role of innate immune molecules, toll-like receptors (TLRs) and nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs), in persistent Chlamydia trachomatis (CT) infection. CT is the most prevalent sexually transmitted bacterial pathogen. Since most infected women are asymptomatic, they do not seek medical treatment, As a result, they have a high tendency to develop persistent infection, and to suffer increased risk of tubal factor infertility, pelvic inflammatory disease and other devastating complications. Certain pattern recognition receptors including TLRs and NLRs are known to initiate antichlamydial immune response. However, previous studies in the literature mainly focused on the acute infection model. Given the importance of persistent infection, we propose to study how the PRRs regulate chlamydial persistence. Our preliminary studies have shown contrasting TLRs/NLRs activation profiles in acute and persistent chlamydial infection models. We have also discovered that chlamydial infection affects the expression of certain NK cell receptor ligand (NKRL), including MICA molecules, which play a crucial role in natural killer lymphocyte (NK cell) activation. We hypothesize that the alterations in TLRs/NLRs signaling in epithelial cells persistently infected with chlamydiae as well as in uninfected epithelial cells, affect NK cell activation. To test this hypothesis, we propose to co-culture infected and uninfected epithelial cells by employing the Transwell system to determine TLRs/NLRs signaling in Chlamydia-infected cells, co-cultured uninfected cells that resemble uninfected cells in infected host as well as naive uninfected cells not co-cultured with infected cells during acute and persistent infections. The cells will be subjected to analyses of TLRs/NLRs mRNA, TLRs/NLRs protein, downstream effector molecules of the receptor including (but not limited to) cell surface MICA and MHC-I protein and cytokine production using PCR Array, Pathway Array, flow cytometry, and ELISA, respectively. Susceptibility of infected and uninfected cells to the cytotoxicity of NK cells will also be determined. Furthermore, we will seek to selectively activate or suppress critical molecules in the TLRs/NLRs pathways and determine the effects of these manipulations on intracellular chlamydial loading, cytokine production, MICA expression and NK cell activation. Elucidation of the role of TLRs/NLRs pathways in persistent CT infection may be instrumental for the design of novel therapeutic strategies.

沙眼衣原体（Chlamydia trachomatis，Ct）持续感染状态下,炎症的持续存在可导致机体泌尿生殖道多种疾病。我们观察到Ct感染后,人类生殖道上皮细胞的某些TLRs/NLRs信号通路分子在不同感染状态下（未感染/急性感染/持续性感染）出现转录水平的明显改变，受染细胞在细胞因子分泌谱、表面NKRL表达量、对NK细胞杀伤敏感性等方面都表现出与TLRs/NLRs信号通路密切相关的变化。已有研究发现，TLRs/NLRs信号通路与下游细胞因子之间存在反馈调控网络；某些胞内感染病原体可调控宿主细胞的TLRs/NLRs信号转导，影响固有免疫细胞的活化状态，逃避免疫杀伤。据以上结果，拟建立Ct不同感染模型，采用qPCR、RNAi、western blot等方法，在不同的靶细胞中研究TLRs/NLRs信号通路在Ct持续感染中异常活化的机制，为研究机体防御反应提供新的理论和实验依据。

沙眼衣原体（Ct）持续感染状态下，炎症的持续存在可导致机体泌尿生殖道多种疾病。我们前期研究发现，Ct感染后人类生殖道上皮细胞的某些TLRs/NLRs信号通路分子在不同感染状态下出现转录水平的明显改变，受染细胞在细胞因子分泌谱、对NK细胞杀伤敏感性等方面都表现出与TLRs/NLRs信号通路密切相关的变化。为进一步阐明在Ct持续感染中TLRs/NLRs信号通路异常活化的机制。我们在前期实验结果的基础上，通过建立Ct不同感染模型，采用qPCR、RNAi、western blot等方法，证实在IFN-γ诱导的沙眼衣原体持续感染状态下，炎性细胞因子IL-6、IL-1α的分泌以及STAT3、TLR2的表达明显增加；STAT3在炎性细胞因子IL-6和IL-1α的异常分泌中起关键作用；STAT3在NK细胞对沙眼衣原体的杀伤中起重要作用；在沙眼衣原体持续感染状态下，存在着STAT3-TLR2信号轴的活化。本结果为进一步研究机体防御Ct感染提供新的理论和实验依据。