TLRs介导疟疾免疫记忆的调控作用及机制研究

One of the main difficulties to control Plasmodium infection is the failure to establish long-lasting immune memory. It is required to fully understand the immune mechanism of malaria. However, it is still unclear why the long-lasting immune memory is absent and immune cells are difficult to take effects for long time in the field of malaria. The cooperation of TLRs and multiple receptors is crucial to initiate and regulate antimalarial immune responses. Unfortunately, it is still a mystery that how TLRs participate in and control immune memory in malaria infection. Therefore, it is necessary to confirm the molecular mechanism of TLRs in the formation and maintenance of immune memory cells. It is a scientific problem to be solved how differential identification of Plasmodium by TLRs participate in the regulation of the differentiation, activation, exhaustion and memory of the immune cells. In this project, the Plasmodium vivax infected patients were recruited in the malaria endemic areas along the China-Myanmar border, for the systemic assessment of their cellular and molecular immune functions, TLRs expression profiles, and their associations with immune memory. Furthermore, a set of parallel experiments of murine malaria model will be performed to deeply analyze the possible mechanisms of TLRs mediated immune memory formation and depletion, aiming to provide a new theoretical basis for the development of effective malaria vaccines and antimalarial drugs.

疟疾感染的本质特征是难以建立长效的免疫记忆。充分理解疟疾免疫的相关机制是疟疾防控的前提基础。目前疟疾长效免疫记忆缺失和免疫记忆细胞难以建立长期效应的机制尚不明确。TLRs与多感受器联合作用是启动和调控宿主抗疟免疫应答的关键。疟疾感染中TLRs如何参与并调控免疫记忆仍存在诸多空白。确定TLRs与免疫记忆细胞形成与维持的相关性；解析TLRs对疟原虫的差异识别如何参与调控免疫细胞分化、活化、耗竭和记忆亚群建立是需要解决的科学问题。本项研究以云南中缅边境疟疾流行区患者为主要研究对象，通过对间日疟原虫患者细胞和分子免疫功能的系统评估、TLRs表达谱特点与免疫记忆功能之间相关性的动态分析，明确TLRs多样性识别对疟疾免疫记忆建立与维持的调控作用。进一步开展鼠疟模型的平行试验，深入解析TLRs介导的免疫记忆形成和缺失的相关机制。旨在为有效的疟疾疫苗和抗疟新药的研制开发提供新的理论依据。

TLRs与多感受器联合作用是启动和调控宿主抗疟免疫应答的关键。疟疾感染中TLRs如何参与并调控免疫记忆仍存在诸多空白。本项目利用中缅边境地区的未感染、有症状感染及无症状感染者三种人群，以沈阳本地健康人作为对照，进行血清学、细胞功能学的分析，着重探究中缅边境疟疾流行地区免疫记忆的应答差异，初步确定了流行区人群可能的免疫保护机制及应答模式，并探索了影响免疫记忆建立与维持的相关因素。进一步开展了鼠疟模型的平行试验，深入解析TLRs介导的免疫记忆形成和缺失的相关机制，确定TLRs与免疫记忆细胞形成与维持的相关性。本项目研究发现，沈阳本地健康人群、流行区未感染人群和流行区无症状感染人群和有症状感染人群在原虫血症、体温和症状上具有显著差异。未感染人群CD8+T细胞百分比显著高于有症状感染人群，无症状患者Treg细胞出现显著降低。同时，PvAMA1特异性抗体在有症状感染人群中显著高于流行区未感染人群。T细胞功能亚群中，CD4+记忆T细胞亚群显著高于未感染者，有症状感染人群的CD8+TCM也显著高于未感染人群。有症状感染人群CD4+TCM表面PD-1百分比显著高于未感染及无症状感染者，且在高负荷原虫感染下可导致中枢记忆CD4+T细胞表面的PD-1水平显著高于低虫负荷的人群。进一步，无症状感者初始B细胞和记忆B细胞均显著高于高原虫血症有症状感染者。而高原虫血症有症状感染者活化记忆B细胞比例显著高于低原虫血症有症状感染者。同时低原虫血症有症状感染者非典型记忆B细胞比例显著低于高原虫血症和无症状感染者。利用鼠疟模型研究发现，TLR4、TLR7和TLR9激动剂可改善疟疾的转归。3种TLR激动剂均可促进DC活化，Th1细胞比例增加、促炎细胞因子IFN-γ和TNF-α水平升高，血清IgG1和IgG2a水平升高。同时，3种TLR激动剂可降低Treg细胞的活化，并下调抗炎性细胞因子TGF-β和IL-10水平。TLR激动剂可激活DC介导的固有免疫反应和适应性免疫应答。进一步研究发现，TLR7和TLR9激动剂可增强宿主对疟原虫再感染的抵抗，促进B细胞分化为分泌IgG1和IgG2a的浆细胞，促进中枢记忆T细胞Tcm和记忆效应T细胞Teff的增殖，对Tfh细胞分化增殖亦有促进作用。由此可见，TLR7和TLR9激动剂促进B细胞介导的体液免疫应答的形成对抗疟免疫保护作用的发挥具有重要意义。