【作者】 祝筱梅；

【导师】 姚咏明；

【作者基本信息】 中国人民解放军军医进修学院 ， 烧伤外科学， 2010， 博士

【摘要】 目的：内质网(ER)是真核细胞中非常重要的细胞器,非常敏感,各种应激刺激均可导致内质网功能失调,即内质网应激(ERS)。ERS持续时间及应激水平对于应激细胞的功能与生存均具有重要影响,与多种人类疾病关系密切。我们前期实验结果表明,高迁移率族蛋白B1(HMGB1)对树突状细胞(DC)成熟分化具有重要免疫调节作用。本实验拟对ERS是否参与HMGB1对DC免疫调节机制进行探讨,旨在从新的角度认识ERS在HMGB1介导DC免疫功能障碍中的地位与调节机制,为探索严重烧伤后脓毒症的免疫调控途径提供新思路。方法：采用雄性BALB／c小鼠分离培养脾脏DC,以含10%胎牛血清RPMI-1640调整细胞浓度1-2×106／ml,5%CO237℃培养过夜后随机分组进行如下实验：1.给予HMGB1(0.1-10μg／ml)或衣霉素(TM,ERS特异性诱导剂,0.1.10μg／ml)刺激24-72 h后,应用real time-PCR技术及Western blot分析检测ERS相关的关键分子(GRP78、SEPS1及XBP-1)表达及活化水平的改变。2.给予TM(0.1-10μg／ml)刺激24-72 h后,流式细胞术检测DC表面标志物及共刺激分子的表达情况；应用ELISA和real time-PCR技术检测TNF-α、IL-12的分泌水平。另外,采用MTT试剂盒检测其对T淋巴细胞增殖活性的影响。3.通过RNA干扰技术诱导DC中ERS关键调节分子XBP-1基因沉默后,给予HMGB1刺激(1μg／ml,48h),采用Western blot分析、流式细胞术、ELISA、real time-PCR等技术方法检测抑制ERS反应对HMGB1调节DC成熟分化效应的影响。4.给予TM刺激(1μg／ml,48h)正常DC,或HMGB1(1μg／ml,48h)刺激XBP-1基因沉默型DC后,流式细胞术检测DC表面晚期糖基化终末产物受体(RAGE)表达水平。结果：1.ERS在HMGB1诱导DC成熟分化过程中具有重要作用。HMGB1刺激可明显诱导DC中ERS反应的发生,与正常对照组相比,0.1μg／ml、1μg／ml、10μg／ml的HMGB1作用24～72h后,DC中ERS相关分子GRP78基因及蛋白水平均明显升高,XBP-1表达及活化水平亦有显著上调,其中尤以1μg/ml HMGB1刺激24 h作用效果显著(P<0.05)。但与ERS特异性诱导剂TM作用不同的是,除0.1μg/ml HMGB1作用48 h诱导SEPS1的蛋白表达水平升高外(P<0.05),本实验所观察的其他刺激浓度和作用时间未能引起SEPS1表达的明显上调。2.TM诱导药物性ERS反应对DC功能状态有一定的影响。0.1μg/ml、1μg/ml、10μg/ml的TM作用24～72 h可导致TNF-α、IL-12的分泌增加(P<0.05),但DC表面共刺激分子表达改变不尽一致,CD80表达在0.1μg/ml TM刺激48h、MHC-Ⅱ表达在0.1μg/ml和1μg/ml TM刺激24-48 h可见明显增强,而CD86在本实验观察的TM各刺激浓度和时间点无显著升高。与HMGB1预孵育DC增强T淋巴细胞增殖反应不同,TM预孵育的DC对T细胞增殖活性具有明显抑制效应(P<0.05)。3.小鼠脾脏DC经XBP-1基因沉默后给予HMGB1刺激(1μg/ml,48 h),与非基因沉默细胞(未转染或转染无效序列)相比,其表面标志物及共刺激分子表达上调均明显受抑,对T淋巴细胞增殖反应及Thl功能极化的促进作用亦明显降低(P<0.05)。4.与非基因沉默细胞(未转染或转染无效序列)相比,XBP-1基因沉默阻抑HMGB1诱导的DC表面RAGE受体表达的上调(P<0.05)。TM诱导的药物性ERS反应对DC表面受体RAGE表达具有明显的上调作用(P<0.05)。结论：HMGB1刺激可诱导小鼠脾脏DC发生明显的ERS反应,应用RNA干扰技术抑制ERS反应中关键分子XBP-1的活化可阻抑HMGB1介导的DC成熟分化和表面受体RAGE的表达上调。TM诱导的ERS反应与HMGB1所致ERS反应呈现一定的差异,TM刺激可增加DC细胞因子TNF-α及IL-12的分泌,但对其表型的成熟无明显的诱导效应,TM预孵育DC对T淋巴细胞的增殖反应呈抑制作用。ERS在HMGB1诱导的DC成熟分化中具有重要意义。更多还原

【Abstract】 Background:Endoplasmic reticulum (ER), the most important organelle in eukaryotic cells, is exceedingly sensitive to alterations in homeostasis. Physiological states that increase the demand for protein folding, or stimuli that disrupt proteins folding reactions, create an imbalance between the protein-folding load and the capacity of the ER, causing unfolded or misfolded proteins to accumulate in the ER lumen, a condition referred to as ER stress (ERS). Dendritic cells (DCs) are pivotal regulators of immune reactivity and immune tolerance. The status of DC maturation and function undoubtedly has important influence on the development of septic complications. High mobility group box-1 protein (HMGB1), was recently described as an important late inflammatory cytokine. Our previous study had demonstrated the contribution of HMGB1 to the maturation and differentiation of DCs. Because the ERS response has the function to regulate the balance between homeostasis and apoptosis, we ventured to speculate that the ERS response might contribute to DC maturation and activation. The present study was performed to clarify the involvement of ERS during maturation of mouse splenic DCs induced by HMGB1.Methods:DCs isolated from the spleens of male BALB/c mice were suspended in RPMI-1640 with 10% FCS at 1-2×106 cells/ml on cell culture plates. Cells were then cultured at 37℃in 5% CO2 in humidified air overnight for recovery before subjected to treatments in the following listed experiments.1. DCs were stimulated with HMGB1 or tunicamycin (TM, pharmacological ERS inducer) at increasing dosages (0.1,1,10μg/ml) for different durations (24,48 or 72 hours). The expression levels of two key ER chaperones, glucose regulated protein (GRP) 78 and selenoprotein S (SEPS1), as well as the activation of key molecule of ERS response, X-box binding protein 1 (XBP-1) were determined by real-time RT-PCR and Western blot analysis.2. DCs were stimulated with TM in different concentrations (0.1,1, or 10μg/ml) for different duration (24,48, or 72 hours). Phenotype and cytokine secretion of DCs were analyzed by flow cytometry, real-time PCR and ELISA, respectively. Modified mixed lymphocyte reaction assay was performed to assess the effect of TM-treated DC on the proliferation rate of T cells.3. DCs were transfected with RNAi lentiviral vector to induce gene silence of XBP-1. Using this cell model, we further investigated the involvement of ERS in immune regulation of HMGB1 on DCs maturation and function.4. XBP-1 silenced DCs were treated with 1μg/ml HMGB1 for 48 hours. The influence of ERS on the expression of the receptor for advanced glycation end products (RAGE) on surface of DCs was detected by flow cytometry.Results:1. Treatment with HMGB1 induced significant up-regulation of ERS-related molecules in mouse splenic DCs. Protein and mRNA levels of GRP 78 were increased after HMGB1 stimulation in a time-and dose-dependent manner. The expression and activity of XBP-1 were also significantly elevated. 2. Pharmacological ERS induced by TM resulted in elevated cytokine release, but failed to induce up-regualation of costimulatory molecules including CD80 and CD86 on the surface of DCs. T cell proliferation in response to Con A was inhibited after co-culture with TM-treated DCs.3. Gene silence of XBP-1 in mouse splenic DCs decreased the levels of CD80, CD86 as well as MHC-II expression and cytokine secretion after HMGB1 treatment, when compared with untransfected or NTi-transfected DC (P<0.05). Further more, XBP-1 silenced DC showed immunosuppressive or tolerogenic DC and failed to stimulate notable proliferation response of T cell, even after treatment with HMGB1.4. Gene silence of XBP-1 resulted in down-regulation of RAGE expression on the surface of mouse splenic DCs after HMGB1 stimulation. The conventionally used pharmacological ERS inducer TM could induce up-regulation of RAGE on the surface of DC in a dose-and time-dependent manner.Conclusion:HMGB1 stimulation induced significant ERS response in mouse splenic DCs. Gene silence of XBP-1 resulted in suppression in activation of DCs and also the up-regulation of RAGE induced by HMGB1. Pharmacological ERS induced by TM demonstrated difference with that induced by HMGB1 in extent of reaction and features and did not effectively arouse DC maturation and activation. Our findings support the notion that ERS response plays an important role in regulation of DC maturation as well as activation induced by HMGB1. INTRODUCTIONSepsis is denoted as a complex clinical syndrome that results from a serious infection followed by an amplified and dysregulated inflammatory response. Despite continuing progress in the development of antibiotics and other supportive care therapies, sepsis remains a leading cause of the high morbidity and mortality in the intensive care unit. Severe complications like multiple organ dysfunctions syndrome (MODS) with high mortality and the lack of knowledge concerning the underlying pathogenetic mechanisms continue to hamper the development of effective therapeutic strategy for septic complications.High mobility group box-1 protein (HMGB1), an evolutionarily ancient non-histone DNA-binding protein within the nucleus, was recently described as a late inflammatory cytokine.Unlike cytokines, such as tumor necrosis factor (TNF)-αand interleukin (IL)-1β, which are released early in the development of a systemic inflammatory response, HMGB1 was released with a delay of 9-16 hours, sustaining for several days after endotoxin exposure by activated macrophages, NK cells, and dendritic cells (DCs) . It was found that patients and animals with sepsis or endotoxemia presented high levels of systemic HMGB1. Numerous evidences indicate that HMGB1 is a necessary late mediator of severe sepsis, and therefore, the delayed kinetic action of HMGB1 provides a wider time window for clinical intervention of sepsis .Extracellular HMGB1 has been shown to be able to provoke inflammation, to regulate the migration of monocytes , and also to contribute to the activation of DCs , which is the pivotal regulator of immune reactivity and immune tolerance. Since its discovery more than 30 years ago, DCs have emerged as the most important potent antigen-presenting cells (APCs), which induce and coordinate host immune response. Interacted with agents including foreign pathogens, such as lipopolysaccharide (LPS), or endogenous signals of cellular injury or damage, DCs are stimulated to mature and play roles in engendering the differentiation of different clones of T helper (Th) cells . The maturation of DC has been implicated as the bridge plank between the innate and adaptive immune systems , and it plays a significant role in the pathogenesis of infection, thus providing a protective mechanism in prevention of infection . Our previous studies had demonstrated HMGB1 to be a potential immunostimulatory signal that induced maturation and differentiation of DCs via the receptor for advanced glycation end products (RAGE), and regulated T cell-mediated immunity . However, the endogenous changes and mechanisms involved in the control of maturation and differentiation of DCs after HMGB1 stimuli are poorly known.The status of DC maturation and function undoubtedly has important influence on the prognosis of sepsis. Recent study revealed that endoplasmic reticulum (ER) stress response led to hepatocyte apoptosis and might mediate the long-term adaptive and deleterious hepatic dysfunction after severe thermal injury in mice, thus providing intensive insights into endogenous sources of cellular stress as the focus of investigation in immune regulatory mechanisms.ER, a membranous network of branching tubules and flattened sacs that extends throughout the cytoplasm of the cell, being contiguous with the nuclear membrane, is one of the most important organelles in eukaryotic cells. The ER is mainly recognized as a protein-folding factory, responsible for the biosynthesis, folding, assembly and modification of numerous soluble proteins and membrane proteins. The ER also functions as a dynamic calcium store, which responds to growth factors, hormones, and stimuli that perturb cellular energy levels, nutrient availability or redox status. Given the importance of this organelle, it is extremely sensitive to alterations in homeostasis. Physiological states that increase the demand for protein folding, or stimuli that disrupt the reactions by which proteins fold, create an imbalance between the protein-folding load and the capacity of the ER, causing unfolded or misfolded proteins to accumulate in the ER lumen, a condition referred to as ER stress (ERS). In response to ERS, mammalian cells trigger unfolded protein response (UPR) signaling pathways to cope with stressful conditions and to monitor the protein folding capacity of the ER and transmit that information to mechanisms that can modulate the ER environment, regulate various aspects of cellular metabolism, and even influence cell fate.Because the ERS response functions to regulate the balance between homeostasis and apoptosis, it was our attempt to determine whether the ERS response and UPR pathway, as induced by HMGB1 might contribute to DC maturation and activation. In the present study, we assessed the expression of two ER resident chaperones, namely glucose-regulated protein (GRP) 78 (BIP) and SEPS1 (selenoprotein S, VIMP, Tanis or SelS), as well as the activity of X-box binding protein 1 (XBP-1), which is the key regulatory molecule of UPR. We demonstrated that treatment of mouse splenic DCs with HMGB1 induced ERS response. The expression of GRP 78 and SEPS1, the two markers of ERS, and the activity of XBP-1 were significantly up-regulated. Surprisingly, pharmacological ERS induced by the use of tunicamycin (TM), an inhibitor of N-linked glycosylation and pharmacological agent that disrupted protein folding and assembly in the ER, failed to activate DC completely, and resulted in tolerance of DC. Furthermore, we found that the reduction of XBP-1 translation by RNA interference (RNAi) in mouse splenic DCs with small hairpin RNA (shRNA) led to a decrease in HMGB1-mediated regulatory effects on DCs.更多还原