糖调节蛋白78通过结合CD14参与免疫炎症消退的功能及机制研究

Immunological responses can be distilled into four fundamental, interdependent processes: recognition, response, regulation and resolution. Appropriate immunological recognition is of the utmost importance. However, of equal importance is the regulation and, thereafter, resolution of the acute inflammatory response. Failure of the latter is an important component of chronic inflammatory diseases, autoimmune disease, obesity, and wound repairing. A wide range of soluble molecules contribute to the regulation and resolution of inflammation. Evidence has emerged that resolution-associated molecular patterns (RAMPs) which are defined as highly evolutionarily conserved, multi-functional, constitutively expressed proteins, exert immunoregulatory activity dependent upon their rapid decompartmentalization from the intracellular environment either actively, following cell stress, or passively via necrotic cell death. Our previous report showed that Grp78 could increase the frequency of regulatory B cells which produced IL-10 and highly expressed PD-L1 and FasL, resulting in suppression of T cell proliferation and induce myeloid antigen presenting cells to maintained tolerogenic signature upon LPS stimulation. For its potent immunomodulatory properties, Grp78 has been defined as one of RAMPs. However, it is not clear how Grp78 feeds regulatory and anti-inflammatory signals into immunological networks to help restore immunological homeostasis. In our study, we found that Grp78 colocalized with CD14 at plasma membrane to enhance TLR4 endocytosis. However, unfamiliar with TLR4 endocytosis mediated by LPS binding with CD14 which triggers TRIF signal transduction, this Grp78 mediated TLR4 endocytosis fails to activate TRIF signal. To elucidate how Grp78 participates in immune resolution through engagement with CD14, changes of function and activation of signal transduction mediated by CD14 and TLR4 would be investigated. Furthermore, disparities in this changes between Grp78 and LPS engagement with CD14 would be analyzed to uncover how CD14 can influence the sensitivity and the quality of the innate immune response. This study will guide future studies to treat immune resolution related disease.

探讨免疫应答的消退机制对促进持续性炎症及时消退、抑制免疫系统过度反应从而干预相关疾病的发生发展具有重要意义。我们前期研究发现消退相关分子模式糖调节蛋白78(Grp78)能结合模式识别受体CD14，但与CD14结合传统配体LPS不同，CD14结合Grp78后诱导TLR4非炎症性内吞、抑制其信号分子激活，提示Grp78参与诱导免疫炎症消退，但作用机理尚不清楚。由此推测，Grp78能否通过结合CD14干预其TLR4依赖性及非依赖性的信号转导，参与诱导炎症消退、维持免疫自稳？据此，本项目拟研究结合Grp78/LPS对CD14和TLR4的功能、信号通路激活和细胞命运的影响及差异，明确Grp78通过CD14参与诱导免疫炎症消退的功能及机制；同时，明确CD14通过结合不同配体调节TLR4介导的炎症过程的关键分子，探寻CD14在不同的环境中如何调控天然免疫反应的平衡与稳定，为相关疾病靶标药物设计提供新思路

我们前期研究发现消退相关分子模式糖调节蛋白78(GRP78)能结合模式识别受体CD14，但与CD14结合传统配体LPS不同，CD14结合GRP78后诱导TLR4非炎症性内吞、抑制其信号分子激活，提示GRP78参与诱导免疫炎症消退，但作用机理尚不清楚。因此我们提出假设，GRP78能否通过结合CD14从而干预细胞TLR4信号转导，参与诱导炎症消退、维持免疫自稳？在随后的研究中，我们一方面继续探索分泌型GRP78在多种免疫相关性疾病中诱导免疫炎症消退的能力，另一方面研究GRP78 通过结合CD14 对TLR4 介导的炎症过程的调节方式。研究结果显示分泌型GRP78能促进巨噬细胞向M2方向极化，诱导DCs形成耐受，活化STAT3促进 B细胞表达PD-L1，从而参与免疫应答的消退，缓解炎症性疾病（如小鼠溃疡性结肠炎）、自身免疫性疾病（如小鼠糖尿病），促进肿瘤的发生发展及肿瘤细胞的肝脏定植；进一步研究表明，分泌型GRP78可以通过结合CD14促进髓系来源的单核细胞LPS–TLR4 复合物内化，但与传统LPS促进TLR4以二聚体方式内化不同，GRP78的干预导致TLR4以单体方式内化，因此不能有效地诱导MyD88及TRIF信号传导通路；这种GRP78诱导的TLR4单体内吞，还通过Atg7依赖性自噬方式辅助TLR4降解；因此GRP78结合CD14后诱导TLR4非炎症性内吞、降解，从而抑制炎症因子分泌。此外，GRP78诱导的TLR4单体内吞通过自噬作用诱发髓系细胞凋亡和铁死亡，也是GRP78介导免疫应答消退的机制之一。本研究通过消退相关分子分泌型GRP78将CD14、TLR4与天然免疫细胞的命运联系在一起，明确了TLR4的单体/二聚体内化状态是CD14通过结合不同配体（GRP78、LPS）调控天然免疫反应平衡与稳定的关键分子，为后续深入研究免疫相关疾病及靶标药物设计提供了研究基础。