IL-12诱导的microRNAs对NK细胞功能及脓毒症形成的调控机制

Sepsis is a state of disrupted inflammatory homeostasis that is often initiated by overwhelming infection and results in high mortality of infected animals. Recent studies have demonstrated that excessive activation and dysfunction of natural killer (NK) cells are associated with the development and progression of sepsis. Our previous studies have demonstrated that IL-12 tightly control the changes of NK cell activity through regulation of miRNA expression, IL-12 treatment initially activates NK cells, with prolonged IL-12 treatment resulting in NK cells hyporesponsiveness. However, it is still needed to further study whether there are some miRNAs that are involved in the development and progression of sepsis via regulation of NK cells. Therefore, firstly, this project are proposed to investigate the changes of cell number, cell subsets percentage, cell activity, key signaling pathways activities and miRNA expression profiles of NK cells using flow cytometry and miRNA array after IL-12 administration of normal mouse or IL-12 teatment of NK cells, to determine the regulation patterns and mechanisms of IL-12 on normal mouse NK cells and key miRNAs involved in this regulation. Then, to develop endotoxin-induced sepsis mice model by administration of lipopolysaccharide (LPS), and to detect the changes of cell counts of different tissue, cell subsets percentage, cell activity and responsivity, miRNA profiles and key molecules of IL-12 and TLR4 pathways in NK cells in the different phase of sepsis, to determine the changes of the activity, responsivity, and functional subsets distribution of NK cells in the development and progression of sepsis. To screen miRNAs involved in the development of sepsis. Administration of targeted miRNA mimics and inhibitors into sepsis mice to measure their effects on NK cells activity, and inflammatory reaction and mice mortality, to determine the effects of these miRNAs on promoting the development and progression of sespsis, and their relationship with IL-12. The prospective results will help us to clarify mechanisms of NK cell dysfunction in the development and progression of sepsis, and provide a new insight into understanding sepsis pathogenesis.

脓毒症是由感染诱发机体炎症机制失控并导致动物急性死亡的主要原因。近来的研究认为NK细胞的功能异常与脓毒症的形成密切相关。我们前期研究证实，IL-12通过miRNA调控NK细胞的活性变化，然而是否有miRNA分子通过调控NK细胞进而参与脓毒症的形成还有待于进一步的研究。本项目拟首先通过流式和miRNA芯片检测IL-12作用正常小鼠后NK细胞的数量、亚群比例、活性、关键途径活性和miRNA表达谱的变化，确定IL-12诱导的miRNAs及其对NK细胞的调控作用和机制；然后复制细菌内毒素诱导的脓毒症小鼠，检测脓毒症不同时期NK细胞的数量、亚群比例、活性、关键途径活性和miRNA表达谱的变化，筛选与脓毒症形成相关的miRNAs，检测并明确这些miRNAs在调控NK细胞和促进脓毒症形成中的作用以及与IL-12之间的关系，探讨脓毒症形成过程中NK细胞功能异常的分子机制。为认识脓毒症发病机制提供理论支撑。

脓毒症是由严重感染或创伤诱发机体炎症失控所导致的临床综合征，自然杀伤（NK）细胞的功能异常与脓毒症的形成密切相关。前期研究证实，IL-12通过miRNA调控NK细胞活性，然而miRNA是否参与脓毒症形成中NK细胞功能活性的调控还有待研究。本项目首先研究了连续IL-12作用是否引起小鼠NK细胞功能低下；通过注射脂多糖（LPS）建立了脓毒症免疫抑制小鼠模型，检测了LPS注射后不同时间小鼠的免疫状态、NK细胞的数量、亚群比例、活性、关键途径活性和miRNA表达谱的变化；筛选了与脓毒症形成相关的miRNAs，检测并明确这些miRNAs在促进脓毒症后期NK细胞功能低下中的作用以及这些miRNAs与IL-12之间的关系；检测了Treg细胞和IL-10在脓毒症后期NK细胞功能低下形成中的作用。研究结果表明，连续的IL-12刺激在体外实验中不会诱导NK细胞功能低下，但在体内引起NK细胞数量减少和功能低下，提示在脓毒症小鼠中高水平IL-12的持续存在可导致NK功能低下；通过对比试验确认5 mg/kg LPS注射可建立脓毒症免疫抑制小鼠模型，此模型小鼠抗二次感染能力显著降低；在5 mg/kg LPS注射小鼠24h后，NK细胞对IL-12和PAM3反应性显著降低，而低剂量LPS注射不引起NK细胞反应性低下；在Rag1-/-小鼠和Treg缺失小鼠中，高剂量LPS注射并不会引起NK细胞功能低下，而在il10-/-小鼠中，高剂量LPS注射仍能诱导NK细胞功能低下；高剂量LPS注射可上调NK细胞中miR-34a-5p、miR-19b-5p、miR-7a-5p、miR-222-3p和miR-223-3p的表达，进而抑制NK细胞IFN-γ的产生和杀伤活性，而其中miR-34a-5p和miR-223-3p的上调又与IL-12密切相关。此研究工作为进一步阐明脓毒症形成过程中NK细胞功能异常的分子机制提供了新的理论数据。