Traf3ip3在小鼠边缘带B细胞分化中的功能和机制研究

TRAF3 interacting protein 3 (TRAF3IP3; also known as T3JAM) is specifically expressed in immune organs and tissues. To investigate the impact of TRAF3IP3 on immunity, we generated Traf3ip3 knockout (KO) mice. Interestingly, these mice exhibited a significant reduction in the number of common lymphoid progenitors (CLPs) and inhibition of B cell development in the bone marrow. Furthermore, Traf3ip3 KO mice lacked marginal zone (MZ) B cells in the spleen. Traf3ip3 KO mice also exhibited a reduced amount of serum natural antibodies and impaired T cell-independent type II (TI-II) responses to TNP-Ficoll antigen. Additionally, our results showed that Traf3ip3 promotes autophagy via an ATG16L1-binding motif, and MZ B cells isolated from mutant mice showed a diminished level of autophagy and a high rate of apoptosis. These results suggest that TRAF3IP3 contributes to MZ B cell survival by upregulating autophagy, thereby promoting the TI-II immune response. .There are two major pathway which are involved in the development of marginal zone B cells: BAFF/FAFFR triggered NF-κB pathway and Notch2 pathway. Autophagy is an important lysosome-mediated degradation pathway for cytoplasmic components in eukaryotes. There are many molecules participate in autophagic process, which is precisely regulated by a variety of mechanisms. It is demonstrated that NF-κB and Notch pathway could be regulated by autophagy. Since we found that MZ B cell survival was dependent on Traf3ip3-regulated autophagy, we hypothesize that Traf3ip3 could regulate the NF-κB pathway and Notch2 pathway via autophagy, therefore is involved in MZ B lymphocyte development and survival.

Traf3ip3特异表达于小鼠免疫系统，我们构建了基因敲除小鼠，发现该基因敲除后小鼠中枢和外周的淋巴细胞总数及不同亚群数量均有下降；脾脏中T1 B细胞和边缘带B（MZ B）细胞数量减少尤为显著； MZ B细胞凋亡水平增高;小鼠天然抗体减少，MZ B细胞介导的TI-II的抗体免疫应答减弱。初步研究发现Traf3ip3与ATG16L1相互作用正向调控自噬，敲除后小鼠外周血白细胞和MZ B细胞自噬水平降低。NF-κB通路及NOTCH2信号通路在MZ B发育过程中起关键作用。本研究旨在探讨Traf3ip3是否可以通过调控自噬影响NF-κB及NOTCH2信号通路的活化，进而调控MZ B细胞的发育和分化。由于MZ B细胞在清除血中病原及产生TI免疫应答过程中起关键作用，与一些自身免疫性疾病的发生也有密切的关系，本研究结果将有助于了解相关疾病的发生发展机制，为其诊断治疗提供新的线索和理论依据。

Traf3ip3特异表达于小鼠免疫系统，我们构建了基因敲除小鼠，发现该基因敲除后小鼠中枢和外周的淋巴细胞总数及不同亚群数量均有下降；脾脏中T1 B细胞和边缘带B（MZ B）细胞数量减少尤为显著： MZ B细胞凋亡水平增高；小鼠天然抗体减少，MZ B细胞介导的TI-I 的抗体免疫应答减弱。本项目利用前期引进的Traf3ip3基因敲除小鼠，分选出与MZ B细胞及其前体细胞MZ P细胞，证明Traf3ip3与Atg16l1在细胞质内共定位；Traf3ip3缺陷细胞中LC3II表达明显低于野生型小鼠、自噬标记分子p62的表达高于野生型；TEM观察了敲除Traf3ip3后MZ B细胞和MZ P细胞的自噬小体减少，线粒体增多且形态异常，证明线粒体自噬发生缺陷。我们在MZ B细胞检测了Notch2信号通路中Notch2，其配体Dll1，下游靶基因Dtx1、 Hes1、Hes5 、 Hey1 等的表达情况，发现敲除细胞中Notch2信号通路活性下降；在MZ B细胞检测了TRAF3及NF-κB 通路中RelA (p65)、 RelB、 c-Rel、 NF-κB1 (p50) 及 NF-κB2 (p52)的表达水平及活化状态，检测经典及非经典NF-κB 信号通路激活分子IκB 和IKKα 的活化状态，发现敲除细胞NF-κB可抑制经典NF-κB 信号通路的激活。在体外实验中，通过与OP9-Dll1 细胞共培养过渡期B 细胞，以表达CD21 和IgM 细胞的比例来反映细胞分化状况，证实在雷帕霉素自噬诱导的条件下敲除Traf3ip3 后细胞的自噬水平和Notch2 信号通路激活水平均显著低于野生型对照组；饥饿自噬诱导条件下基因敲除组NF-κB 非经典信号通路的活性受到了抑制，同时发现Traf3ip3的表达阻断了p100与LC3在外源性和内源性系统中的相互作用。我们还发现在基因敲除的滤泡B细胞（follicular B，FO B）中Notch2 信号通路和NF-κB 通路的激活状况与MZ B细胞恰好相反，提示Traf3ip3在B细胞分化的过程中可能起着重要的枢纽作用。本项目的研究结果为深入揭示B细胞的分化机制提供了新的线索和理论依据。