CXCR2胞内信号分子调控脓毒症幼稚和成熟粒细胞趋化能力的机制研究

Sepsis, a type of systemic inflammatory response to infection，is still one of the leading causes of death in Intensive Care Unit. As the most abundant immune cells in circulation, neutrophils are mobilized from bone marrow and migrated into injured tissues by specific chemokines to eliminate the invaded microorganisms. There was a marked impairment in neutrophil recruitment during sepsis, especially severe sepsis and septic shock patients, characterized by failure of migration into the site of infection and failure of obliterating the microorganisms. Several chemokines combined with co-receptor CXCR1/2, activated intracellular signal molecules by phosphorylation, and induced globular actin (G-actin) to aggregate into fibrous actin (F-actin); finally, neutrophils migrated into tissues. In the normal condition, neutrophils are produced and matured in the bone marrow, and more than 99% neutrophils are mature in the peripheral blood. In the recent years, distinct neutrophil subsets with functional and phenotypic heterogeneity have been explored. Mature neutrophils showed enhanced anti-microbial function and ROS generation, while immature neutrophils may account for increased susceptibility to infections and exhibit suppressive effects on T-cell proliferation. In our preliminary research, we found that immature neutrophils were more prone to migrate into injured tissues during bacterial infection (published in European Journal of Immunology, 2014). Thus we have detected the expression of CXCR1/2 on immature and mature neutrophils by flow cytometry and found that the expression of CXCR1/2 on immature neutrophils was not higher than that on mature neutrophils as expected. We propose the hypothesis that “the difference of cell signal phosphorylation and F-actin polymerization induced by CXCR1/2 activation between immature and mature neutrophils might affect their migration in the site of infection, and further influence the distribution and function of immature and mature neutrophils in the tissue during sepsis”. First, we prepared sepsis mouse model, analyzed the distribution of immature and mature neutrophils in the liver and lung, and identified the function (oxidative burst, phagocytosis, suppressive ability). Next, in order to explore the difference of migration between immature and mature neutrophils, we will activate IL-8-CXCR2 signal pathway by activating neutrophils from normal or septic mice bone marrow in vitro, detect the expression of phosphor-signal pathway molecules by flow cytometry and Western blot, and analyze the F-actin polymerization by co-focal. In addition, we will verify the migration mechanism of immature and mature nerutrophils by induced differentiation by GM-CSF in vitro and cell transfer in vivo. Our results might replenish the migration mechanism and functions in sepsis of different neutrophils subsets and provide the references for prevention and intervention sepsis in the clinic.

脓毒症时粒细胞趋化至局部组织，清除入侵的病原体。粒细胞的趋化功能与脓毒症病情进展及预后密切相关。CXCR2是多种粒细胞趋化因子的共同受体，通过磷酸化级联活化胞内信号分子，引起细胞骨架改变而完成趋化过程。既往将粒细胞作为整体研究其趋化机制，但幼稚和成熟粒细胞具有不同的免疫功能。申请人发现趋化因子对幼稚粒细胞具有更强的趋化作用，但是幼稚细胞的CXCR2受体的表达低于成熟细胞。根据上述发现和前期转录组测序结果，申请人提出：“CXCR2胞内信号分子磷酸化水平及下游细胞骨架调控成熟和幼稚粒细胞趋化能力，进而影响上述细胞的组织分布和功能”的科学问题。本研究拟通过体外活化正常或脓毒症小鼠幼稚和成熟粒细胞，在细胞内信号分子磷酸化水平以及细胞骨架水平验证上述假说，采用体外培养诱导分化和细胞回输从体内、体外两方面进一步证实上述机制及与脓毒症预后的关系，为临床干预和治疗脓毒症提供新的思路和细胞靶点。

脓毒症是由感染引起的全身炎性反应综合征，严重脓毒症和脓毒症休克患者的死亡率高达40-70%。在感染情况下，不同功能和分化状态（幼稚和成熟）的粒细胞在趋化因子的作用下，通过何种途径进入感染部位，是影响组织损伤的发生、发展和转归的关键因素。粒细胞与趋化因子受体CXCR2结合，活化下游信号通路磷酸化和肌动蛋白的聚合，完成趋化过程。前期工作提示：幼稚粒细胞具有更强的趋化进入感染组织的能力，但其表面CXCR2表达水平较低。因此，本研究中我们通过对正常和脓毒症小鼠（CLP）骨髓和肺组织幼稚和成熟粒细胞比例变化，功能检测，IL8 -CXCR2信号传导通路中Akt、ERK、Stat3和NF-kB等分子磷酸化水平的检测，及细胞骨架F-actin的聚合进行分析，明确幼稚和成熟粒细胞的趋化机制差异。.在CLP小鼠外周血和肺组织均可见Gr-1low幼稚和Gr-1high成熟粒细胞的明显聚集。功能鉴定显示，与正常对照相比，CLP骨髓幼稚和成熟粒细胞活性氧功能显著增强，幼稚粒细胞活性氧功能与成熟粒细胞接近，脓毒症小鼠幼稚粒细胞吞噬能力明显增高。对照小鼠骨髓和肺组织成熟粒细胞CXCR2表达明显高于幼稚粒细胞，脓毒症明显上调肺组织幼稚粒细胞CXCR2受体的表达水平。与骨髓幼稚粒细胞相比，IL8刺激后成熟粒细胞胞内MAPK和STAT3通路明显磷酸化，幼稚粒细胞通路相关分子未见明显磷酸化。正常对照幼稚和成熟粒细胞几乎不表达F-actin，IL8刺激后，成熟粒细胞表现为明显的变形和F-actin的聚集增强表达现象。脓毒症情况下，成熟粒细胞出现明显的F-actin 聚集现象，IL8刺激下幼稚粒细胞可出现为F-actin表达上调。微丝相关蛋白检测未见明显变化。这些结果表明，成熟粒细胞通过经典通路，即与CXCR2结合，活化下游MAPK和STAT3通路，进一步引起F-actin的聚合，进而完成趋化，而幼稚粒细胞在脓毒症组织聚集，也表现为F-actin的聚集，但可能并不通过经典通路发挥作用，其趋化机制尚需进一步研究。本研究采用经典趋化通路对幼稚和成熟粒细胞趋化通路在进行了阐述，为临床干预和调控成熟粒细胞在脓毒症提供理论依据和实验室数据。