严重烧伤后高迁移率族蛋白B1诱导树突状细胞内质网应激相关性细胞凋亡及其免疫学效应

Impaired immune function and associated immunosuppression are hallmarks of septic syndrome, a major cause of mortality in intensive care units. Deactivation and apoptosis of dendritic cells (DCs) is potentially associated with immunosuppression and plays an important role in the pathogenesis of sepsis. However, fundamental questions regarding the mechanisms of DC dysfuntion and apoptosis remain unresolved. In our previous study, we have proved that high mobility group box-1 protein (HMGB1), a late inflammatory cytokine described recently, was a potential immunostimulatory signal and could induce significant endoplasmic reticulum stress (ERS) in mice splenic DCs when used at some concertration, and related to DCs maturation as well as activation. We suspect wether ERS-mediated apoptosis, activated by excessive ERS, is related to DCs dysfunction and apoptosis after severe burns. The current study was conducted to clarify the effects of continued stimulation with high level of HMGB1 on DCs apoptosis and also the intrinsic relation with ERS. Firstly, we plan to assess the relationship between ERS and apoptosis of DCs, by detecting expression/activation of ERS markers and key molecules including GRP 78, XBP-1, SEPS1, CHOP and caspase-12 in DCs, as well as DC function and apoptosis after HMGB1 stimulating in vitro, or in HMGB1-treated or severe burn mice model. Secondly, pharmacological ERS will be induced by tunicamycin (TM) to observe the direct influence of ERS response on dysfunction and apoptosis of DCs. Thirdly, we will use salubrinal, a selective inhibitor of eIF2alpha dephosphorylation that can protect cells from ER stress, to moculate ERS respone in HMGB1-treated DC and assess the alteration of DC immune function as well as apoptosis. Fourthly, expression of XBP-1, SEPS1, CHOP, and caspase-12 in DCs will be modulated both in vitro and in vivo, to investigate the intrinsic influence of ERS-mediated apoptosis in molecule mechanisms of DC immune disorder. Our findings will help to clarify the underlying mechanisms of ERS response in immune regulation of DCs induced by HMGB1, thus shed a light to the development of novel therapeutic strategies for improving the clinical outcome of patients with severe injury and subsequent sepsis.

免疫功能障碍是脓毒症发病的重要基础，而树突状细胞(DC)抗原呈递能力下降、数量减少是关键所在，但迄今为止DC功能障碍及凋亡调控机制尚未阐明，临床缺乏有效调控策略。我们前期研究发现，一定浓度高迁移率族蛋白(HMG)B1体外刺激可诱导DC成熟分化且与内质网应激(ERS)密切相关，本项目拟研究烧伤机体持续高水平HMGB1刺激对DC凋亡的影响以及与ERS相关凋亡通路的关系；并通过应用衣霉素诱导DC药物性ERS、Salubrinal缓解DC中ERS、抗HMGB1抗体中和烧伤后高水平HMGB1活性、调控ERS关键分子XBP-1、SEPS1、CHOP和caspase-12表达/活化等方法，分析体内外模型中ERS介导HMGB1调控DC凋亡的分子机制及其对DC免疫反应的影响。该研究将从新的角度进一步认识ERS在HMGB1介导DC免疫功能障碍中的作用及意义，为探索严重烧伤后脓毒症的免疫调控提供新思路。

严重烧创伤伤感染脓毒症过程中，细胞内稳态被打乱，引起内质网应激(endoplasmic reticulum stress, ERS)反应，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，决定细胞适应、损伤或凋亡的发生与发展，对机体免疫系统功能状态具有重要影响。我们前期研究表明，高迁移率族蛋白B1(HMGB1)介导ERS对体外培养DC的成熟分化具有重要意义。本研究在前期研究基础上进一步探讨ERS在严重烧伤小鼠脾脏DC功能障碍及免疫调控机制中的作用意义。 方法：重组HMGB1体外刺激正常小鼠脾脏DC，或复制严重烧伤小鼠模型，以及临床烧伤患者外周血DC，给予Salubrinal、PERK抑制剂、XBP-1基因沉默等策略对ERS关键分子进行调控处理后，观察DC功能状态及凋亡情况，并检测ERS相关分子表达/活化及ERS相关性细胞凋亡途径的活化情况。从细胞及整体水平、动物实验及临床检测的不同角度明确HMGB1对DC免疫状态和凋亡的影响及其与ERS相关凋亡途径间的相关性。结果：HMGB1体外刺激小鼠脾脏DC，可诱导DC发生明显凋亡，ERS相关性细胞凋亡是HMGB1诱导DC凋亡的重要机制，Salubrinal缓解ERS可减轻细胞凋亡的发生。PERK-eIF2α对应激状态下的细胞有重要保护作用，抑制PERK活化促进HMGB1刺激诱导的细胞凋亡。严重烧伤诱导小鼠脾脏DC凋亡明显，功能分化异常。同时，ERS相关分子(GRP78, CHOP, caspase-12)表达/活化水平有明显上调(P<0.05)，提示严重烧伤后ERS反应强烈，与DC凋亡具有一定相关性。Salubrinal干预可提高严重烧伤小鼠生存率，有效缓解严重烧伤后脾脏DC中ERS反应程度，减轻DC凋亡，改善DC功能状态，促进DC的成熟分化。XBP-1基因沉默使得烧伤诱导的脾脏DC凋亡率增高，WB检测结果显示XBPi使得介导ERS相关性细胞凋亡的关键分子CHOP表达及caspase-12表达/活化水平显著升高，提示此模型中DC凋亡率增加与ERS相关性细胞凋亡途径的活化有关。临床资料亦显示过度ERS是HMGB1刺激及严重烧伤诱导的DC功能障碍的重要机制。结论：ERS作为真核细胞重要内源性保护机制，对DC功能具有至关重要的调控作用，在严重烧伤致DC免疫功能障碍中具有重要意义，可能是调控DC功能状态的重要靶点。