炎性复合体-细胞焦亡通路介导钩端螺旋体免疫逃避的分子机制

Leptospirosis, a zoonosis of worldwide distribution and recognized as an emerging natural focus infectious diseases, is caused by pathogenic bacteria of the genus Leptospira. Leptospires evade the host innate immune response and colonization in kidneys in resistant animals and chronically excrete the leptospiruria into the environment. Humans or animals become infected directly through exposure to urine and/or indirectly through fresh water contaminated with urine. To clarify the obscure mechanism of immune evasion in leptospirosis, we focus on pyroptosis which is contribute to bacterial clearance, and our previous studies showed that the expression of NLRP3 has been proved suppression by the survival of leptospires. Base on these data, we propose a scientific hypothesis that the immune evasion in leptospirosis is correction with the inhibition of inflammasome-pyroptosis pathway. In this study, we will focus on the interaction between leptospires and inflammasome, and explore the role and the control mechanism of inflammasome in immune evasion of leptospires. Specifically, we will figure out the impacts of leptospires on inflammasome-pyroptosis pathway; and validate the role of inflammasome in leptospirosis by using the gene deficient cells and mouse; and further reveal the molecular mechanism of the interaction between PAMPs and inflammasome through stimulating mice and cells with leptospiral PAMPs or knockout strains and detect by Co-IP and shotgun proteomics assay. The results will not only help to elucidate the mechanism of immune evasion in leptospires, also provide basis for the effective control, prevention and treatment of the disease.

钩端螺旋体（钩体）病是由致病性钩体引起的一种全球性自然疫源性人兽共患传染病。针对钩体逃避宿主免疫系统，长期定殖于动物肾脏，并不断从尿液排出钩体污染环境，造成敏感动物和人类钩体病多发难控。为了明确钩体免疫逃避，依据细胞焦亡是清除胞内菌保护机体关键环节的理论，基于本课题组发现肾脏钩体的定殖与抑制NLRP3炎性体表达有关的研究基础，推论钩体免疫逃避与其抑制炎性复合体-细胞焦亡通路激活有关。本研究拟从钩体与炎性复合体的互作入手，探讨其在钩体免疫逃避中的作用及调控机制。首先明确钩体对宿主细胞焦亡和炎性复合体表达的影响；再利用基因缺陷细胞和小鼠，验证炎性复合体在钩体感染中的作用及机能；最后通过钩体PAMPs分子及基因敲除株刺激细胞和小鼠，经免疫共沉和蛋白质组学等方法，揭示PAMPs与炎性复合体通路互作的分子机制。这不仅有助于阐明钩体在肾脏中的免疫逃避机制，也为控制该病源头提供了新的研究思路和理论指导。

钩端螺旋体（钩体）病是由致病性钩体引起的一种全球性自然疫源性人兽共患传染病。钩体能逃避宿主免疫系统，长期定殖于动物肾脏，并不断从尿液排出钩体污染环境，造成敏感动物和人类钩体病多发难控。为了明确钩体免疫逃避，本研究从钩体与炎性复合体的互作入手，探讨其在钩体免疫逃避中的作用及调控机制，并为钩体病的新型疗法提供思路。.首先，利用体内体外实验，明确了钩体主要通过激活NLRP3炎性体来引起细胞焦亡，此过程需要两个信号。第一个信号是钩体激活TLR2和TLR4，进而使IL-1β的前体形式大量表达，第二个信号是钩体激活NLRP3炎性体，对IL-1β的前体进行切割，使之成为成熟的IL-1β，并引起细胞焦亡。接着，应用NLRP3缺陷小鼠，发现NLRP3炎性体缺陷使小鼠对钩体更加耐受。由于NLRP3炎性体的激活可以抑制细胞自噬水平，检测体内自噬水平后，发现NLRP3缺陷后提高了小鼠体内的自噬水平。用雷帕霉素提高自噬水平后，显著抑制了胞内钩体的存活，同时，用3-MA来处理小鼠以降低自噬水平，发现3-MA处理后的小鼠感染钩体后，体内的钩体载量更高，说明自噬有助于清除体内的钩体。以上结果表明NLRP3炎性体缺陷可以通过提高自噬水平来清除钩体。随后，选择可以抑制炎性体激活的药物来治疗钩体感染，如多西环素、大黄素、百里香酚、黄芪甙，减轻了钩体引起的病理损伤。解释了多西环素具有良好抗钩体功效的免疫学机制，为从调节炎性体-细胞焦亡通过角度开发设计抗钩体药物奠定了实验基础。.在探索治疗钩体病的新型疗法中，发现低剂量的喹诺酮类药物（环丙沙星、诺氟沙星）可以影响机体免疫反应，恶化钩体病。钩体感染后，若没能及时治疗，等出现症状后，即使治疗，患病动物的死亡率也较高。鉴于此，我们应用兔源抗钩体多克隆抗体，成功提高了感染晚期金黄地鼠的存活率，为钩体病晚期治疗提供了思路。此外，应用LPS、TLR2激动剂和β-葡聚糖来治疗钩体急性感染都提高了金黄地鼠的存活率，β-葡聚糖有望成为钩体疫苗的良好佐剂。