ORMDL3通过内质网应激促进B细胞自噬参与系统性红斑狼疮发生的作用机制研究

Systemic lupus erythematosus (SLE) is a prototypic systemic autoimmune disease characterized by multisystem or organ damage, abnormal T- and B-cell responses and autoantibody production. Despite vigorous research, the etiopathogenesis of SLE remains unclear. Genome-wide association studies(GWAS) identified two SNPs from autophagy-related gene 5 (Atg5) which were associated with the susceptibility of SLE, suggesting the potential contribution of autophagy to the etiopathogenesis of SLE.Recent studies reported murine and human SLE T- and B-cells showed an increased number of autophagosomes and an up-regulation of autophagy, which can increase the survival and reduce apoptosis of self-reactive lymphocytes. ORM(yeast)-like protein isoform 3 (ORMDL3) has recently been identified as a candidate gene to asthma. Although many studies confirmed ORMDL3 can inhibit sarco-ER Ca2+ ATPase (SERCA) activity to involve in the ER-mediated Ca2+ homeostasis and activate the endoplasmic reticulum stress (ERS), next to up-regulate the level of autophagy, the relationship to ORMDL3, authphagy and SLE is still unknown. In our previous studies, we found mRNA expression level of ORMDL3 was increased in SLE patients than in normal controls. Furthermore, over expression of ORMDL3 can obviously increase the expression of ERS key proteins, up-regulate autophagy level and inhibit apoptosis of B cells. The results indicated that ORMDL3 may upregulate B cells autophagy through the mediation of ERS, inhibit apoptosis and promote B cell survival and then involved in the occurrence and development of SLE. To test this hypothesis, we will apply Ormdl3 knockout Fas lpr/lpr mice model to shed light on the association of ORMDL3 with B cells differentiation, authphagy changes and its effects on SLE disease progression.On the basis of our previous work, we attempt to investigate the critical role of ORMDL3 in the pathogenesis of SLE and establish theoretical foundation for novel therapeutic targets in near future.

ORMDL3作为哮喘的易感基因，还参与了多种自身免疫病的发生发展。以往研究显示SLE患者中存在B细胞的过度活化和调控失常，同时免疫细胞自噬水平异常也被证实与SLE的发生密切相关。虽然ORMDL3能够通过激活细胞内质网应激（ERS）进而增强细胞自噬水平，但ORMDL3是否能够通过该机制影响B细胞分化进而参与SLE的发生目前尚不明确。我们前期结果显示，ORMDL3在SLE患者外周血单核细胞中表达升高。过表达ORMDL3后可促进B细胞中ERS相关基因表达升高及自噬水平上调，抑制B细胞凋亡。提示ORMDL3可能通过ERS的介导上调B细胞自噬进而参与SLE的发生发展。据此，本课题拟在前期工作基础上，利用Ormdl3敲除的Fas lpr/lpr狼疮小鼠模型，从ORMDL3与B细胞分化调控、内质网应激、自噬等方面探讨ORMDL3参与SLE发病的分子机制，以期为明确SLE的发病机制奠定理论基础。

B细胞的过度活化和调控失常及免疫细胞自噬水平异常均被发现与系统性红斑狼疮（SLE）的病理发生密切相关。以往研究显示，ORMDL3能够通过激活细胞内质网应激（ERS）进而增强细胞自噬水平，但其是否能够通过该机制影响B细胞分化进而参与SLE的发生目前尚不明确。为此，本课题利用Ormdl3敲除的Pristane诱导狼疮小鼠模型多方面探讨ORMDL3参与SLE发病的分子机制。首先，通过TALEN靶向基因敲除策略得到Ormdl3基因敲除小鼠，在与同窝野生小鼠的比较后发现，敲除小鼠脾脏质量（P<0.01）、脾脏CD19+B细胞、Mature B细胞及T2B细胞比例均显著下降（P<0.05），同时脾脏淋巴细胞中Baff的表达水平（P<0.01）及血清中Baff（P<0.05）、IgM浓度（P<0.01）均显著降低。以上结果提示缺失Ormdl3可阻碍小鼠脾脏B细胞的发育成熟。为了明确ORMDL3敲除后导致小鼠脾脏B细胞数量降低的原因，我们通过体内及体外实验发现，ORMDL3能够通过上调ATF6a表达及入核从而启动ERS的发生，随后通过上调Beclin1的表达促进LC3I向LC3II的转化，最终导致细胞自噬水平的增高。与此同时，ORMDL3还能够显著抑制凋亡相关蛋白Caspase3、Caspase9及PARP的裂解后活性片段的表达。由此结合之前的实验结果我们判断ORMDL3能够发挥促自噬抑凋亡的双重作用，从而起到维持细胞内环境稳态，促进B细胞的过度存活的作用。最后，为了明确ORMDL3对SLE病程进展的影响，我们又构建了Ormdl3基因敲除的Pristane诱导狼疮小鼠模型及其对照小鼠。通过流式细胞术、尿蛋白检测等试验，我们发现在注射了Pristane后，敲除小鼠脾脏肿大程度显著低于野生小鼠（P <0.05），同时其24h尿蛋白水平也有所降低（P=0.0664）；此外小鼠肾脏H&E染色结果显示敲除小鼠在注射Pristane后肾脏损伤程度较野生小鼠轻，而流式结果显示Pristane诱导的敲除小鼠能够明显抵抗CD4+/CD8+细胞比例上升的趋势。由此我们认为Ormdl3可部分缓解Pristane诱导狼疮小鼠病程发展，初步提示ORMDL3可能在导致SLE发病过程中具有一定作用，而这一作用部分原因是源自于ORMDL3对脾脏B细胞的促自噬抑凋亡作用，从而导致B细胞的过度存活所致。