肺炎衣原体借助TLR2/HS“共受体”入侵血管平滑肌细胞进而促进动脉粥样硬化形成

Although accumulating evidence indicates that Chlamydia pneumoniae (C.pn) infection is closely associated with atherosclerosis (AS), the exact mechanisms for C.pn infection-induced AS remain unclear. Toll-like receptor 2 (TLR2) and heparan sulfate (HS) have been shown to play important roles in the development of AS. And HS requires the membrane receptor as a "co-receptor" for successful invasion of some microorganisms into their host cells. Our preliminary results showed that TLR2 is closely related to C.pn infection of vascular smooth muscle cells (VSMCs) and the subsequent changes in VSMC migration ability. Therefore, we hypothesize that TLR2/HS "co-receptor" is required for C.pn's invading into VSMCs and promoting cell migration, thereby leading to the development of AS. Accordingly, our research will be carried out in the following two aspects: at the cellular level, co-immunoprecipitation assay will be used to determine the interaction of TLR2 with HS in the infected VSMCs; Then the entry of C.pn into cells and the ability of cell migration will be detected after HS and TLR2 are respectively upregulated and downregulated, or after HS and TLR2, either alone or in combination, are overexpressed. At the whole-animal level, the TLR2 knockout mice and the HS-overexpressing mice will be both inoculated with C.pn, and then the number of C.pn inclusions in the mouse aortic VSMCs will be determined; After the downregulation of the HS expression in TLR2 knockout mice or suppression of TLR2 activation in HS-overexpressing mice, C.pn inclusions in the mouse aortic VSMCs will be detected, and then histological staining with the Movat's pentachrome stain will be used to measure the areas of the AS lesions in the aorta. Thus, TLR2/HS "co-receptor" will be demonstrated to mediate the entry of C.pn into VSMCs, and then promote the development of AS. Our research findings will not only present a new molecular mechanism by which C.pn invades into VSMCs, and then results in the initiation of AS, but also provide a new target for preventing AS.

肺炎衣原体（C.pn）感染与AS关系密切，但机制不清。TLR2和硫酸乙酰肝素（HS）均对AS发病起重要作用，且后者可与膜受体形成共受体来介导微生物入侵细胞。前期研究发现C.pn感染VSMC及引起其迁移功能改变与TLR2密切相关，故推测C.pn可能借助TLR2/HS共受体入侵VSMC并促进其迁移进而参与AS发病。我们拟从细胞水平：免疫共沉淀确定TLR2与HS之间的相互作用；分别改变TLR2和HS表达或共表达TLR2和HS，检测C.pn感染率和细胞迁移能力。动物水平：C.pn分别感染TLR2缺陷和HS高表达的小鼠，或降低TLR2缺陷小鼠的HS表达或抑制HS高表达小鼠的TLR2功能后行感染，观察主动脉内VSMC感染情况；五色套染法评价AS病变程度。由此，确证TLR2/HS共受体介导C.pn入侵VSMC并促进AS发病。研究结果将阐明C.pn入侵VSMC及其导致AS的分子机制，提供防治AS的新靶点。

肺炎衣原体（C. pn）感染与动脉粥样硬化（AS）关系密切，但机制不清。首先，我们发现，C. pn感染可促进ApoE-/-小鼠AS病变形成并使病变中血管平滑肌细胞（VSMC）的数量明显增多。那么，C. pn感染如何促使VSMC迁移至内膜？C. pn入侵VSMC是其发挥促进细胞迁移作用的先决条件。为此，我们进一步探讨了TLR2/CXCR4共受体在C. pn入侵VSMC中的作用：Western blot和免疫荧光实验结果显示，C. pn入侵过程中TLR2表达增多，且TLR2由胞浆向细胞膜转移，而后又向胞浆转移；应用RNA干扰技术分别沉默TLR2与CXCR4的表达后，免疫荧光实验结果显示，C. pn感染率均明显降低；免疫共沉淀和邻位连接技术结果显示，C. pn入侵过程中，TLR2与CXCR4之间的相互作用增强，且TLR2和CXCR4的蛋白分布呈现重合状态。β-arrestin2可介导受体内吞，很可能参与C. pn入侵VSMC。基于此，我们利用RNA干扰和免疫荧光技术观察了β-arrestin2在C. pn入侵VSMC中的作用，结果显示，沉默β-arrestin2的表达使C. pn感染率明显降低；进一步的免疫共沉淀和邻位连接技术结果显示，C. pn感染可促进CXCR4对β-arrestin 2的募集，且此过程受TLR2调控。TLR2/CXCR4共受体是否还与C. pn感染诱导VSMC迁移有关？随后，我们利用RNA干扰技术分别沉默TLR2与CXCR4的表达后，发现感染诱导的VSMC迁移均被明显抑制，同时感染增强FAK-c-Jun通路活性的效应也明显减弱。那么，TLR2和CXCR4是否在C. pn感染促进AS形成及促使AS病变内VSMC数量增多中发挥作用？于是，我们将C. pn分别感染TLR2缺陷和CXCR4抑制剂处理的ApoE-/-小鼠，结果显示，TLR2基因敲除和CXCR4抑制可减轻感染诱导的AS病变，同时，感染诱导的主动脉流出道病变中的VSMC数量也明显减少。综上，本项目证实了C. pn经由TLR2/ CXCR4共受体通过激活β-arrestin2通路入侵VSMC，同时还通过激活FAK-c-Jun通路诱导VSMC迁移，进而促使AS病变内VSMC数量增多，促进AS病变形成，揭示了C. pn入侵VSMC并导致AS发生发展的新机制，为研发防治AS的药物提供了新靶点。