梅毒螺旋体介导的抗体应答缺陷在梅毒持续感染中的细胞与分子机制

Spirochete Treponema pallidum/Tp, as major infectious pathogens of sexually transmitted disease, causes severe chronic disease syphilis, threatening public health in human. The incidence of Syphilis is generally hard to be naturally cleared by immune system in most cases. Tp infection,causing progression of central nervours system disease-neurosiphilis,leads to clinical complex for their therapy, but the mechanism behind the pathogenesis remain elusive. We hypothesize that Tp infection could deteriorate impairment including damage of lymphoid tissues and dysfunctional T/ B cells. In particular, once the function of follicular T helper (TFH) cells are impaired, the counterpart humoral immune responses and antibody production for opsonization and neutralization are thus reduced due to loss help from TFH cells, resulting in failure of pathogens clearance. Our preliminary data show that CXCR5+TFH cells at least contain two populations: PD-1int and PD-1high CD4+ T cells in lymph nodes. The latter predominantly resies in germinal center of lymph nodes (not found in peripheral blood), which dispay stronger help for B cell maturation and IgG production, compared PD-1int populations. These PD-1highTFH cells are significantly reduced in neurosyphilis, accompanided by increased CXCR5 ligand-CXCL13 in serum and attenuated ability of TFH cells to migrate and reside in germianl center of lymph nodes. These findings suggest that PD-1highTFH cells in lymphoid tissues are critical for maintainance of B cell development and antibody responses, and the functional PD-1high TFH cells might be impaired in patients with syphilis, which contributes to impairment of humoral immune responses.To our knowledge, the role of TFH cells in siphilis has not been currently reported. In this proposal, we will comprehensively examine the changes of the numbers and function of TFH cells in lymph nodes in syphilis patients following-up treatment and/or at different stage, investigate effects of this infection on B-cell differentiation, maturation and antibody production, determine the pathological changes of lymph nodes by routine histopathology,Immunohistochemistry and confocal microscopy image analysis, validate TFH cells function in animal models, and evaluate the correlation functional TFH cells /B cell responses with outcome of clinical therapy and disease progression. Insight into deficiency of antibody responses in syphilis will be better understanding the mechanism of pathogenesis, and provide hopeful guidance for clinical diagnosis, therapy strategy and disease prediction.

机体感染梅毒螺旋体后其自然病程可出现自愈、潜伏和严重组织损伤三种临床结局，其发病机制不清。基于初始感染过程中抗体应答的重要性及我们的初步研究结果，推测螺旋体感染可能损伤了滤泡辅助性T(TFH)细胞的B细胞辅助功能，引起抗体应答缺陷，引发感染持续和疾病恶化。我们的前期研究证实分布于淋巴结滤泡生发中心PD-1high TFH细胞具有更强的促B细胞成熟和IgG分泌能力。预实验显示梅毒螺旋体持续感染引发淋巴结病变、外周血CXCR5趋化因子-CXCL13水平升高以及定向游走至淋巴结组织的TFH细胞减少，提示TFH细胞失能可能是螺旋体感染中抗体应答缺陷的关键。本研究将通过检测各期梅毒外周血及淋巴结中TFH细胞的变化和功能特点以及B细胞分化成熟和抗体应答特性，探索梅毒感染过程中抗体应答缺陷的细胞与分子机制，并进一步通过体外和动物实验加以证实。这一研究可能对梅毒的临床治疗和疫苗研制均有重要的理论意义。

研究背景 .机体感染梅毒螺旋体慢性化发病机制不清。本研究已完成各种梅毒感染阶段中外周血Tfh样细胞数量和功能的改变，并分析了外周血Tfh样细胞与疾病进展的相关性，相关的数据正在收集整理，准备撰写成文以便投稿发表。需要特别指出的是，梅毒螺旋体感染引起显著的淋巴结病变，因此淋巴结生发中心 (GC) 结构以及GC Tfh细胞功能的改变十分重要。遗憾的是，由于获得临床病人的淋巴结十分困难，本初步研究只能局限于梅毒感染过程中的外周血Tfh样细胞功能及其迁移潜能研究。考虑到天然免疫及其调节在梅毒感染过程中的作用，本研究同时也开展了临床感染病人中天然免疫，尤其是全面分析梅毒螺旋体感染对NK细胞功能及细胞杀伤功能相关的分子表达的影响。探讨NK细胞在梅毒螺旋体慢性化感染中的可能机制。.方法 .流式细胞术检测梅毒患者外周血中NK细胞及其亚型；分选外周血CD3-CD56+NK细胞，用K562细胞刺激NK细胞，用流式细胞术检测IFN-γ+NK细胞和Granzyme B+NK细胞的百分比。进一步将NK细胞与K562细胞以不同的比例 (分别为1:1, 10:1, 30:1)混合培养8小时后，用流式细胞术检测不同效靶比下NK细胞对K562细胞的杀伤效率。.结果 .1.与正常对照组相比，二期梅毒患者外周血中CD3+CD56+NKT细胞百分比显著低于正常对照组；并且以CD3-CD56dimNK和CD3-CD56dim CD16- NK细胞百分比下降最为显著。而与之相反，二期梅毒患者外周血CD3-CD56-CD16dim NK细胞和CD3-CD56-CD16brightNK细胞百分比显著高于正常对照组。.2.用K562刺激外周血PBMC后，与正常对照组相比，二期梅毒和神经梅毒患者外周血IFN-γ+ NK细胞显著降低。二期梅毒和神经梅毒患者IFN-γ+的CD3-CD56dimNK细胞、CD3-CD56brightNK细胞比例低于正常对照组。在NK细胞杀伤功能实验中发现，在效靶比1:1、10:1和30:1时，二期梅毒杀伤K562细胞的能力与正常对照组相比均无统计学差异。.结论 .1. 梅毒患者外周血NK细胞总的百分比减少，并且以细胞毒性NK细胞亚型百分比减少为主，但免疫功能不完善的NK细胞亚型百分比增多，提示梅毒螺旋体感染可降低机体内NK细胞的功能。.2. 梅毒患者外周血IFN-γ+NK细胞百分比降低，提