SIRT6介导的3型天然淋巴样细胞调控肠源性脓毒症的分子机制研究

Sepsis is a common and devastating syndrome, and is associated with high risks of morbidity and mortality in ICU. However, there is no effective therapy because the underlying mechanisms remain unclear. Recent studies reveal that group 3 innate lymphoid cells (ILC3) play an immune regulatory role in gut, which may become the novel therapeutic target of sepsis. Data from our prvious studies showed that intestinal flora disorder was associated with the dysfunction of ILC3 in the process of sepsis. In addition, sepsis was attenuated in SIRT6 knockout mice which was associated with upregulating ILC3 function and increasing the secretion of IL-22. Therefore, we hypothesize that SIRT6 interferes with the pathological process of gut-derived sepsis by regulating the function of ILC3. In present study, we will use the gut-derived sepsis mouse model to investigate the effects and mechanisms of ILC3 on maintaining intestinal immune barrier and micro-ecological balance, preventing intestinal bacterial translocation and enhancing intestinal anti-infective ability by Flow Cytometry and Epigenetics methods et al. Using 16sRNA to detect the changes of intestinal flora and to study the regulation of intestinal microecological disturbance on SIRT6. Constructing SIRT6 knockout mice to explore the effects of SIRT6 on the differentiation and function of ILC3 subsets. Whole-transcriptome sequencing will be used to study the signaling pathways in regulating ILC3 by SIRT6. All results from present study will elucidate the molecular mechanisms of SIRT6-mediated ILC3 regulation in gut-derived sepsis and find the key regulatory factor targeting ILC3. Meanwhile, we try to provide a novel therapeutic target for sepsis and subsequent multiple organ dysfunction syndrome based on this study.

脓毒症死亡率高，机制和治疗靶点不明。新近研究显示3型天然淋巴样细胞(ILC3)在肠道发挥免疫调控作用，有望成为治疗靶点。我们研究显示脓毒症时肠道菌群紊乱、ILC3功能受抑，而敲除表观修饰调控因子SIRT6后ILC3功能上调，IL-22分泌增加，抗脓毒症能力增强。我们推测SIRT6通过调控ILC3干预肠源性脓毒症的病理进程。下一步拟运用肠源性脓毒症模型，通过流式细胞术和表观遗传组学等方法，研究ILC3如何维护肠道免疫屏障及微生态平衡、阻止肠道菌群移位、增强肠道抗感染能力；应用16sRNA检测肠道菌群变化，研究肠道微生态破坏对SIRT6调控作用；构建SIRT6敲除小鼠，研究SIRT6对ILC3亚群分化和功能的影响；采用全转录组测序研究SIRT6调控ILC3的信号通路，阐明SIRT6介导ILC3调控肠源性脓毒症的分子机制，旨在建立靶向ILC3关键调节分子，为脓毒症和多器官功能障碍治疗提供新靶点。

目的：脓毒症是重症患者死亡的重要原因，寻找脓毒症的有效治疗靶点和策略迫在眉睫。本项目明确3型天然淋巴样细胞（ILC3）在脓毒症肠损伤中的作用，阐明SIRT6对ILC3增殖及功能的调控作用，探究SIRT6调控ILC3功能的分子机制。.方法：体内实验探究脓毒症对ILC3的影响。野生型（WT）小鼠行盲肠结扎穿孔术（CLP）构建脓毒症模型组，分为CLP术后1、2、4天及假手术1天四组，分别评估术后小鼠临床症状评分、生存率，检测全身炎症因子、肝肾功能，HE检测各脏器病理；免疫组化评估肠上皮紧密连接蛋白（TJP），PCR评估肠道炎症，流式细胞技术分析肠道粘膜固有层ILC3的增殖、表型及功能变化。利用ILC3特异性敲除（KO）SIRT6基因工程小鼠，及WT小鼠，分别行CLP构建脓毒症模型，评估小鼠临床症状评分、存活率、全身炎症、肝肾功能及肠道病理、炎症等。体外探究SIRT6调控ILC3分泌GM-CSF的分子机制。流式分选KO及WT小鼠肠道ILC3，体外小肠类器官与LPS共培养，进一步联合ILC3“三重共培养”，检测类器官TJP，炎症因子及抗菌肽的表达情况；回收“三重共培养”体系中KO及WT来源的ILC3，PCR检测MAPK及NF-κB信号相关分子表达情况。.结果：脓毒症中，肠道ILC3数量明显减少，分泌GM-CSF、IL-17A及IL-22的ILC3比例明显升高，增殖标志物Ki67及NCR、CCR表型无明显改变。CLP术后1天即出现血清炎症指标明显升高及各脏器病理损伤。脓毒症时，与WT组相比，KO组小鼠大肠ILC3分泌GM-CSF比例减少，生存率提高，血清GM-CSF、IL-6分泌减少，肠组织IL-1β、GM-CSF mRNA表达下调，Reg 3g mRNA表达上调。体外实验发现，与WT来源ILC3相比，KO来源ILC3的“三重共培养”体系中，分泌GM-CSF及IL-1β较少，肠上皮TJP表达较高，肠上皮炎症因子表达水平较低，Reg 3g mRNA表达水平较高。探究分子机制，KO组ILC3的ERK1、ERK2、NF-κB p65 mRNA表达量与WT组相比明显下调。.结论：脓毒症中，肠道ILC3通过分泌的炎症相关细胞因子GM-CSF、IL-17A及IL-22参与其病理进程，发挥保护作用；SIRT6可能通过ERK1/2及NF-κB信号通路调控肠道ILC3分泌GM-CSF。