Rab7 and estrogen-ER as B cell-intrinsic mediators of auto/antibody responses

描述(由申请人提供)：作为自身/抗体反应的B细胞固有介质的Rab7和雌激素受体。我们在R01 AI079705的前四年的工作表明，雌激素促进类别转换和突变的抗体/自身抗体的产生，例如在系统性红斑狼疮中的抗dsDNA抗体/自身抗体；雌激素通过上调HoxC4同源结构域转录因子而增强AID(对CSR和SHM至关重要)的诱导；雌激素受体(ER)通过与我们在HoxC4启动子中发现的三个保守的ER反应元件(ERE)结合来诱导HoxC4；HoxC4直接与AICDA/Aicda启动子结合以诱导AID表达；HoxC4和AID在狼疮B细胞中高表达；它们的KO使正常小鼠的类别转换/突变抗体，以及在狼疮易发小鼠的自身抗体/自身免疫。因此，我们关于雌激素在CSR/SHM中的B细胞内在作用的重要发现与女性抗体/自身抗体反应的增强和自身免疫，特别是狼疮的更高发生率高度相关。这种竞争性更新将检验我们的新假设，即Rab7介导抗体/自身抗体反应，并受雌激素(？)-雌二醇-ER(E2-ER)的调节。在B细胞中诱导后，Rab7Small GTP酶将激活NF-κB(增加HoxC4/Aicda转录)，下调microRNAs(使HoxC4和Aicda转录本从沉默中解脱)，从而诱导HoxC4/AID和CSR/SHM。这一依赖Rab7的途径将被E2-ER增强，进一步上调CSR/SHM。我们的假设得到了令人信服的初步数据的支持，包括：在T依赖和T依赖的抗体反应中，Rab7在诱导AID和CSR方面发挥着B细胞固有的作用；有证据表明，Rab7降低了对microRNA生物发生至关重要的DICER；鉴定了Rab7启动子中的多个ERE；雌激素上调了正常B细胞中Rab7的诱导及其在狼疮B细胞中的表达。目的1将通过使用Rab7抑制剂(CID 1067700)、ER拮抗剂(Fulvestrant)和mrl/Faslpr/lpr小鼠在诱导的B细胞中缺失Rab7[TG(Aicda-cre)Rab7fl/fl]或ERα[TG(Aicda-cre)ERαfl/fl]来研究Rab7和E2-ER在自身抗体反应和狼疮免疫病理中的作用。目的2将探讨Rab7在诱导HoxC4/Aicda、CSR/SHM和抗体反应中的内在功能，潜在的机制(诱导NF-κB)和E2-ER的增强作用，通过使用B细胞删除Rab7，通过在这些B细胞中表达具有结构性活性的ikk？突变体来加强NF-κB的激活，并使用B细胞删除ERα。目的3将研究Rab7和雌激素下调miR-23b、miR-26、miR-214(沉默HoxC4 mRNA)、miR-181b、miR-155和miR-361(沉默Aicda mRNA)，并确定Rab7在这一过程中的作用：可能通过促进自噬(类)过程而作为DICER降解的中介。通过将自噬、雌激素和microRNAs这三个研究领域结合起来解决自身抗体产生的调节问题，我们的建议提供了一种综合的方法来理解狼疮的复杂问题，并有助于定义新的狼疮治疗靶点。

DESCRIPTION (provided by applicant): Rab7 and estrogen-ER as B cell-intrinsic mediators of auto/antibody responses. Our work of the first four years of R01 AI079705 has shown that estrogen promotes production of class- switched and mutated antibodies/autoantibodies, such as anti-dsDNA IgG in systemic lupus; estrogen potentiates induction of AID (critical for CSR and SHM) through upregulation of the HoxC4 homeodomain transcription factor; estrogen receptors (ERs) induce HoxC4 by binding to three conserved ER responsive elements (EREs) we identified in the HoxC4 promoter; HoxC4 directly binds to the AICDA/Aicda promoter to induce AID expression; and, HoxC4 and AID are highly expressed in lupus B cells; their KO blunts class- switched/mutated antibodies in normal mice, and autoantibodies/autoimmunity in lupus-prone mice. Thus, our significant findings on the B cell-intrinsic role of estrogen in CSR/SHM are highly relevant to heightened antibody/autoantibody responses and higher incidence of autoimmunity, particularly lupus, in females. This competitive renewal will test our novel hypothesis that Rab7 mediates antibody/autoantibody responses and is modulated in this function by estrogen (ß-estradiol)-ER (E2-ER). Upon induction in B cells, the Rab7 small GTPase would activate NF-κB (to increase HoxC4/Aicda transcription) and downregulate microRNAs (to relieve HoxC4 and Aicda transcripts from silencing), thereby inducing HoxC4/AID and CSR/SHM. This Rab7-dependent pathway would be enhanced by E2-ER, further upregulating CSR/SHM. Our hypotheses are supported by compelling preliminary data, including: demonstration that Rab7 plays a B cell-intrinsic role in inducing AID and CSR in T-independent and T-dependent antibody responses; evidence that Rab7 decreases Dicer, which is crucial to microRNA biogenesis; identification of multiple EREs in Rab7 promoter; upregulation of Rab7 induction by estrogen in normal B cells and its expression in lupus B cells. Aim 1 will address the roles of Rab7 and E2-ER in autoantibody responses and lupus immunopathology by using a Rab7-inhibitor (CID 1067700), an ER-antagonist (fulvestrant), and MRL/Faslpr/lpr mice deleting Rab7 [Tg(Aicda-cre)Rab7fl/fl] or ERα [Tg(Aicda-cre)ERαfl/fl] in "induced" B cells. Aim 2 will address the B cell- intrinsic functions of Rab7 in HoxC4/Aicda induction, CSR/SHM and antibody responses, underlying mechanisms (induction of NF-κB) and potentiation by E2-ER, by using B cells deleting Rab7, enforcing NF-κB activation through expression of a constitutively active IKKß mutant in these B cells, and by using B cells deleting ERα. Aim 3 will address Rab7 and estrogen downregulation of miR-23b, miR-26, miR-214 (silencing HoxC4 mRNA), miR-181b, miR-155 and miR-361 (silencing Aicda mRNA), and define the role of Rab7 in this process: possibly as a mediator of Dicer degradation through promotion of autophagy (-like) processes. By bringing the three research fields of autophagy, estrogen and microRNAs together to address the regulation of autoantibody production, our proposal provides an integrated approach to understand the complex problem of lupus and contributes to the definition of new lupus therapeutic targets.