RelB as a master regulator of the thymic medulla: characterization of factors that control RelB expression and the role of the RelB target gene Enpp2/Atx

在之前的资助期间，我们发现胸腺上皮细胞(TEC)特异性去除了NF-kB家族成员RelB(RelbTEC-KO)的小鼠缺乏导致自身免疫的所有髓质mTEC。令人惊讶的是，与Relb缺失的小鼠相比，RelbTEC-KO的自身免疫病理在炎症浸润的严重程度和受影响的器官数量方面都非常轻微。值得注意的是，RelaTEC-KO小鼠只有部分mTEC丢失，也没有形成自身免疫。救援实验表明，RelaTEC-KO的表型主要是由于RelB的表达减少，它以剂量依赖的方式调节mTEC的数量。我们的结果表明，RelB驱动双能TEC前体对mTEC的承诺。为了研究经典的NF-kB信号通路中的其他成分是否调节mTEC中RelB的水平，我们首先关注c-Rel，它在体外与RelA协同诱导mTEC表达。此外，我们还将研究肿瘤坏死因子受体相关因子TRAF6是否位于RelB的上游。RelB在早期mTEC发育中的作用将通过新产生的Relb-CRE-P2A-Katushka报告鼠系来解决。这些小鼠使我们能够通过流式细胞术纯化RelB阳性和RelB阴性的mTECs，用于转录组分析和鉴定新的RelB靶基因。由于与RelbTEC-KO小鼠相比，RelB缺失型小鼠的自身免疫严重程度与胸腺髓质萎缩显著相关，我们将进一步研究RelB在非TEC的发育和功能中的作用，如间充质细胞和内皮细胞。特别是，我们想找出分泌酶自体趋化素的细胞起源和功能，它是由非TEC以RelB依赖的方式在胸腺表达的。

In the previous funding period, we showed that mice with a thymic epithelial cell (TEC)-specific ablation of the NF-kB family member RelB (RelbTEC-KO) lacked all medullary mTECs resulting in autoimmunity. Surprisingly, the RelbTEC-KO autoimmune pathology was very mild compared to Relb-null mice with respect to both severity of inflammatory infiltrates and number of affected organs. Notably, RelaTEC-KO mice had only a partial loss of mTECs and did not develop autoimmunity. Rescue experiments showed that the RelaTEC-KO phenotype was largely due to decreased expression of RelB, which regulated mTEC numbers in a dose-dependent manner. Our results indicate that RelB drives commitment of bipotent TEC precursors to the mTEC lineage.To investigate whether other components of the classical NF-kB signaling pathway regulate RelB levels in mTECs we will initially focus on c-Rel, which cooperates in vitro with RelA in the induction of RelB expression. In addition, we will investigate whether the TNF receptor-associated factor TRAF6 is upstream of RelB. The role of RelB in early mTEC development will be addressed using the newly generated Relb-Cre-P2A-Katushka reporter mouse line. These mice enable us to purify RelB-positive and RelB-negative mTECs by flow cytometry for transcriptome analyses and the identification of novel RelB target genes. Since the severity of autoimmunity correlated with markedly increased atrophy of the thymic medulla in RelB-null compared to RelbTEC-KO mice, we will furthermore investigate the role of RelB in development and function of non-TECs, such as mesenchymal and endothelial cells. In particular, we want to find out the cellular origin and function of the secreted enzyme autotaxin, which is expressed in the thymus by non-TECs in a RelB-dependent manner.