【作者】 钟河江；

【导师】 蒋建新；

【作者基本信息】 第三军医大学 ， 外科学， 2009， 博士

【摘要】 感染是严重创伤常见并发症,目前对其救治仍缺乏有效的措施。因此,在创伤早期预防和治疗损伤诱发的器官功能不全具有十分重要的意义。创伤可导致免疫反应异常,而这与创伤后严重感染易感性增加有关。神经内分泌反应是创伤后最早出现的一种机体反应。大量研究表明神经内分泌系统对免疫功能具有重要的调控作用。严重创伤后,下丘脑-垂体-肾上腺(hypothalamic-pituitary-adrenal,HPA)轴激活是创伤早期主要的神经内分泌反应。糖皮质激素(glucocorticoids, GCs)是HPA轴的最终效应激素,对免疫系统具有重要调控作用。因GCs具有广泛而重要的免疫抑制特性,被临床上作为抗炎药物。大量研究表明,除了免疫抑制作用外,内源性或天然GCs也可产生免疫刺激作用。本课题组前期研究表明一定剂量的内源性GCs (皮质酮)可增强巨噬细胞免疫功能。然而,对于GCs免疫刺激作用的机制尚不清楚。内质网是参与钙调节和蛋白折叠的一种重要细胞器,对维持细胞内稳态十分重要。一旦内质网内稳态受各种应激干扰而发生紊乱,新合成的未折叠蛋将在内质网腔内蓄积,诱发内质网应激(endoplasmic reticulum stress,ERS)。为应对蓄积的未折叠蛋白,哺乳动物细胞可触发一种特异的适应性反应,即未折叠蛋白反应(unfolded protein response,UPR),UPR对ERS时细胞恢复,以及活跃的分泌细胞(如免疫细胞)的功能与存活相当重要。巨噬细胞广泛分布于哺乳动物组织,在天然免疫和获得性免疫中起重要作用。近年来研究发现免疫细胞发生ERS在细胞功能调控中起重要作用,如抗原提呈、浆细胞分化、抗体生成以及T细胞对抗原反应等。另外,也有大量研究表明ERS与免疫炎症反应关系密切。ERS相关的转录因子X盒结合蛋白1(X-box binding protein 1, XBP1)和激活转录因子6 (activating transcription factor 6, ATF6)在ERS诱导细胞功能变化中具有重要作用。然而,GCs对巨噬细胞ERS的影响及其与GCs免疫刺激作用的关系目前尚未明确。基于本课题组前期研究发现及目前研究现状,我们推测ERS可能在介导GCs免疫刺激中具有重要作用。由此,本课题主要围绕GCs免疫刺激作用与免疫细胞ERS的关系及其可能的分子机制,分别从四个方面进行了系统研究:(1)应用不同浓度内源性GCs (皮质酮)处理小鼠腹腔巨噬细胞,观察ERS相关分子GRP78、XBP1和ATF6表达变化,首先明确GCs对巨噬细胞ERS的诱导作用。为了进一步阐明ERS与免疫功能变化之间的关系,我们观察了ERS诱导剂毒胡萝卜素对小鼠腹腔巨噬细胞免疫功能的影响。此外,对GCs免疫抑制作用与ERS的关系进行了研究。(2)应用CRH基因敲除小鼠,观察急性束缚应激后HPA轴活性对免疫细胞ERS的影响及其与免疫功能变化的关系。(3)应用慢病毒介导的RNA干扰技术,通过选择性沉默小鼠XBP1基因,观察XBP1在低浓度皮质酮免疫刺激中的作用。(4)应用GR拮抗剂RU486,观察低浓度皮质酮诱导ERS与GR的关系。通过上述系统性研究,明确ERS介导GCs免疫刺激的作用及其分子机制,为深入阐明神经内分泌系统对免疫功能的调控提供实验依据。主要结果如下:1.前期研究提示低浓度GCs对巨噬细胞有免疫刺激作用。为了检测GCs诱导小鼠腹腔巨噬细胞内质网应激的作用,以及明确ERS在低浓度GCs诱导免疫刺激中的作用。我们应用不同浓度皮质酮(啮齿动物的主要内源性GCs)处理小鼠腹腔巨噬细胞,观察GCs诱发ERS的作用,同时,检测UPR信号通路中两个重要的转录因子XBP1和ATF6的表达。结果发现,低浓度皮质酮(10 ng/ml and 50 ng/ml)诱导小鼠腹腔巨噬细胞ERS同时,可激活UPR,并且观察到XBP1 mRNA发生剪接和ATF6蛋白分解。为了进一步明确ERS与巨噬细胞免疫变化之间的关系,我们观察了ERS诱导剂毒胡萝卜素对巨噬细胞免疫功能的影响。结果显示,与低浓度皮质酮的免疫刺激作用一样,毒胡萝卜素可增强巨噬细胞趋化、吞噬功能及TNF-α生成。并且,高浓度皮质酮(1000 ng/ml)对LPS刺激的小鼠巨噬细胞(RAW264.7细胞)产生免疫抑制效应的同时能抑制LPS诱导的RAW264.7细胞ERS,提示GCs免疫抑制作用也与ERS有关。2.促肾上腺皮质素释放激素(Corticotropin-releasing hormone,CRH)是HPA轴最近端的应激激素,是应激时神经内分泌反应的中枢性协调分子。为了进一步检测HPA轴活性对免疫细胞ERS的影响及其与免疫功能变化的关系。我们应用CRH基因敲除小鼠,观察在心理应激(束缚应激) 1 h后免疫细胞GRP78、XBP1和ATF6的表达及免疫功能的变化。结果显示,与CRH-/-小鼠相比,CRH+/+和CRH+/-小鼠在急性束缚应激后,免疫细胞GRP78的mRNA和蛋白表达水平均显著增加,同时,也观察到IRE1/XBP1和ATF6信号转导通路激活,并且,发现与CRH-/-小鼠相比,CRH+/+和CRH+/-小鼠腹腔巨噬细胞的免疫功能均显著增强。3.观察了XBP1在GCs调控巨噬细胞功能中的作用,通过构建并筛选小鼠XBP1基因的RNA干扰慢病毒载体,应用XBP1-siRNA慢病毒感染小鼠腹腔巨噬细胞,选择性失活XBP1基因,发现低浓度皮质酮(50 ng/ml)不能增强小鼠腹腔巨噬细胞的吞噬功能及分泌TNF-α的能力。4. GCs的活性主要是由糖皮质激素受体(glucocorticoid receptor,GR)所介导。因此,我们观察了GR在低浓度皮质酮诱导巨噬细胞ERS中的作用。结果显示,应用GR拮抗剂RU486预处理巨噬细胞能显著地抑制低浓度皮质酮(50 ng/ml)诱导的GRP78、XBP1 mRNA及蛋白表达水平的增加。此外,应用RU486阻断GR能部分抑制低浓度皮质酮的免疫刺激作用。主要结论:1.低浓度皮质酮可诱导巨噬细胞ERS,激活UPR。与低浓度皮质酮产生的免疫刺激作用相似,毒胡萝卜素诱导巨噬细胞内质网应激也具有免疫刺激作用。GCs免疫抑制作用也与ERS有关。总之,这些结果表明低浓度皮质酮对巨噬细胞免疫刺激作用可能与诱导细胞内ERS相关。2.适度HPA轴活化可诱导免疫细胞ERS,增强免疫细胞功能。因而从整体水平证实内源性GCs免疫刺激作用可能与诱导免疫细胞ERS有关。3. XBP1在调控低浓度皮质酮免疫刺激作用中具有重要作用。4.低浓度皮质酮诱导巨噬细胞免疫刺激作用至少部分由GR所介导,而且,在巨噬细胞低浓度皮质酮是通过GR诱导ERS。更多还原

【Abstract】 Infection is a common complication after severe trauma. there are still no effective therapeutic strategies so far. Therefore, the prevention and treatment of injury-induced organ dysfunction is very important at the early stage of injury. Major trauma results in massive impairment of immunologic reactivity, which has been demonstrated to correlate clinically with increased susceptibility to serious infection. It has been found to elicit neuroendocrine responses immediately after trauma. There is increasing evidence revealing that the neuroendocrine system is important in the regulation of immune function. The key neuroendocrine response to stress is activation of the hypothalamic-pituitary-adrenal (HPA) axis, The glucocorticoids (GCs) are the final effectors of the HPA axis. These GCs play an important role in the modulation of the immune system. GCs are regarded widely as being immunosuppressive and are used clinically as antiinflammatory agents. A growing body of literature suggests that, far from being suppressive, endogenous or natural GCs are also known to exert immunostimulatory effects. Previous study showed that GCs can enhance the immune functions of peritoneal macrophages, specially at low concentrations. However, the precise cellular mechanisms underlying the immunostimulatory effects of GCs have not been fully elucidated.The endoplasmic reticulum (ER) is a critical organelle involved in intracellular calcium regulation and protein folding, which are crucial for cellular homeostasis. Once ER homeostasis is perturbed by various stressors, newly synthesized unfolded proteins accumulate in the ER, resulting in ER stress (ERS). To cope with accumlated unfolded ER proteins, mammalian cells trigger a specific adaptive response called the unfolded protein response (UPR). UPR is crucial both for cell recovery under condition of ERS and for the function and survival of active secretory cells, such as immune cells. Macrophages are ubiquitous in mammalian tissues and play a central role in both innate and acquired immune responses. Recently, emerging evidence indicates that ERS in immune cells has been shown to play an important role in the regulation of the cellular functions, such as antigen presentation, plasma cell differentiation and antibody production, and T cell response to antigen. In addition, there is also increasing evidence indicating a strong link between ERS and immune inflammatory response. ERS-induced transcription factors, such as X-box binding protein 1 (XBP1), activating transcription factor 6 (ATF6) have been demonstrated to play an essential role in ERS-induced changes in cellular functions. However, the effects of GCs on macrophage ERS and its relationship with the immunostimulatory effects have yet to be defined.Based on our previous findings and current researches, the aim of this work was to investigate whether the immunostimulatory effects of GCs mediated by ERS and their potential molecular mechanisms, which includes the following four aspects: (1) To observe the expression changes of ERS-associated molecules GRP78, XBP1 and ATF6 in mice peritoneal macrophages treated with various concentrations of endogenous GCs (corticosterone). In order to further elucidate the relationship between ERS and altered immune functions, we investigated the effects of thapsigargin, an ERS inducer, on immune functions of mice peritoneal macrophages. Furthermore, to investigate the relationship the immunosuppressive effects of GCs between ERS in vitro. (2) We used CRH knockout (CRH-/-) mice, to observe in vivo the effects of the HPA axis activation on ERS and immune functions of immune cells of mice exposed to acute restraint stress. (3) Using lentiviral-mediated RNAi (RNA interference) technology, selectively silenceing of the XBP1 gene in mice, to investigate the role of XBP1 in the immunostimulatory effects of low concentration of corticosterone in vitro. (4) To use the GR antagonists RU486 (mifepristone) to explore the relationship between low concentration of corticosterone-induced ERS and GR. The purpose of this study is to further elucidate the molecular mechanisms of the immunostimulatory effects of GCs, and then provide more evidence for deepening the understanding of the regulation of the neuroendocrine system on the immune function.The main results are shown as follows:1. Previous works indicate that low concentrations of GCs exert immunostimulatory effects on macrophages function. In order to test the hypothesis that GCs induces ERS in mice peritoneal macrophages, and to elucidate the role of ERS in low concentrations of GCs-induced immunostimulation. We treated mice peritoneal macrophages with various concentrations of corticosterone, a major type of endogenous GCs in rodents, to observe the GCs-induced ERS. Meanwhile, we further examined the expression of XBP1 and ATF6, two key transcription factors of UPR signaling pathway. In parallel to induction of ERS, low concentrations of corticosterone (10 ng/ml and 50 ng/ml) could also activate the UPR. Splicing of XBP1 mRNA and ATF6 cleavage were observed in mice peritoneal macrophages. In order to further elucidate the relationship between ERS and altered immune functions of macrophages, we investigated the effects of thapsigargin, an ERS inducer, on macrophage function. Similar to the immunostimulatory effects of low concentrations of corticosterone, thapsigarin could enhance macrophages chemotaxis, phagocytosis and TNF-αproduction. However, high concentrations of corticosterone (1000 ng/ml) exert immunosuppressive effects on mouse macrophages (RAW264.7 cells) following stimulation with lipopolysaccharide (LPS), and inhibit LPS-induced ERS of RAW264.7 cells.2. Corticotropin-releasing hormone (CRH) is the most proximal element of the HPA axis, and it acts as central coordinator for neuroendocrine response to stress. To further examine the effects of HPA axis activation on ERS of immune cells and its relationship with altered immune functions in vivo, we investigated the expression of GRP78, XBP1 and ATF6 and altered immune functions of immune cells of CRH knockout mice exposed to the psychological stress (restraint stress) for 1 h. Our results show that following acute restraint stress, compared to CRH-/- mice, the mRNA and protein expression levels of GRP78 were significantly increaed in immune cells of CRH+/+ or CRH+/- mice. Meanwhile, IRE1/XBP1 and ATF6 signaling pathway were activated. Furthermore, we show here that the immune functions of primary peritoneal macrophages of CRH+/+ or CRH+/- mice was greatly enhanced, compared to those of CRH-/- mice.3. To explore the role of XBP1 in modulation of macrophage functions by GCs, after construction and screening of lentiviral vector of RNA interference of mouse XBP1 gene, we selectively inactivated the XBP1 gene in mice peritoneal macrophages infected with XBP1-siRNA lentivirus. We found that low concentration of corticosterone (50 ng/ml) did not increase the cellular phagocytosis and TNF-αproduction.4. The activities of GCs are mostly mediated by the glucocorticoid receptor (GR). Thus, we investigated the possible role of GR in the induction of ER stress in macrophages by low concentration of corticosterone. Our results showed that pretreatment of macrophages with the classical GR antagonist RU486 (mifepristone) could significantly inhibit the increased expression of GRP78 and XBP1 at both mRNA and protein levels induced by low concentration of corticosterone. Furthermore, blocking of the GR by RU486 partly abolished the immunostimulatory effects of low concentration of corticosterone.Taken together, we concluded that:1. In parallel to induction of ERS, low concentrations of corticosterone could also activate the UPR. Similar to the immunostimularoty effects of low concentrations of corticosterone, thapsigarin could enhance macrophages chemotaxis, phagocytosis and TNF-αproduction. high concentrations of corticosterone exert immunosuppressive effects related to ERS. Together, these results suggest that the immunostimulatory effects of low concentrations of corticosterone on macrophages might be related to its induction of intracellular ERS.2. HPA axis activation could induce ERS and enhance immune functions of immune cells, and also highlight a mechanistic association between the moderate activation of the HPA axis elicited immunostimulatory effects and induced ERS of immune cells.3. XBP1 might play an important role in modulation of the immunostimulatory effects of low concentration of corticosterone.4. GR was at least partly responsible for the immunostimulatory effects of macrophages induced by low concentration of corticosterone. Furthermore, low concentration of corticosterone induces ERS via GR in macrophages.更多还原