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

描述（由申请人提供）：Rab 7和雌激素-ER作为自身/抗体应答的B细胞内在介导物。我们在R 01 AI 079705前四年的工作表明，雌激素促进类转换和突变抗体/自身抗体的产生，如系统性狼疮中的抗dsDNA IgG;雌激素增强AID的诱导（对CSR和SHM至关重要）通过上调HoxC 4同源结构域转录因子;雌激素受体（ER）通过与我们在HoxC 4启动子中鉴定的三个保守的ER应答元件（ERE）结合来诱导HoxC 4：HoxC 4直接与AICDA/Aicda启动子结合以诱导AID表达;并且，HoxC 4和AID在狼疮B细胞中高度表达;它们的KO钝化正常小鼠中的类别转换/突变抗体，以及狼疮易感小鼠中的自身抗体/自身免疫。因此，我们对雌激素在CSR/SHM中的B细胞内在作用的重要发现与女性中抗体/自身抗体应答升高和自身免疫（特别是狼疮）发生率升高高度相关。 这种竞争性更新将检验我们的新假设，即Rab 7介导抗体/自身抗体应答，并且在该功能中由雌激素（β-雌二醇）-ER（E2-ER）调节。在B细胞中诱导后，Rab 7小GT3将激活NF-κB（以增加HoxC 4/Aicda转录）并下调microRNA（以解除HoxC 4和Aicda转录物的沉默），从而诱导HoxC 4/AID和CSR/SHM。这种Rab 7依赖性途径将被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：证明Rab 7在T-非依赖性和T-依赖性抗体应答中诱导AID和CSR中发挥B细胞内在作用;证据表明Rab 7降低Dicer，这对microRNA生物合成至关重要;在Rab 7启动子中鉴定多个ERE;在正常B细胞中雌激素上调Rab 7诱导及其在狼疮B细胞中的表达。 目的1将通过在“诱导的”B细胞中使用Rab 7抑制剂（CID 1067700）、ER拮抗剂（氟维司群）和缺失Rab 7 [Tg（Aicda-cre）Rab 7 fl/fl]或ERα [Tg（Aicda-cre）ERαfl/fl]的MRL/Faslpr/lpr小鼠，阐明Rab 7和E2-ER在自身抗体应答和狼疮免疫病理学中的作用。目的2将通过使用缺失Rab 7的B细胞，通过在这些B细胞中表达组成型活性IKK β突变体来加强NF-κB活化，以及通过使用缺失ERα的B细胞，来阐明Rab 7在HoxC 4/Aicda诱导、CSR/SHM和抗体应答中的B细胞内在功能、潜在机制（NF-κB的诱导）和E2-ER的增强作用。目的3将阐述Rab 7和雌激素下调miR-23 b、miR-26、miR-214（沉默HoxC 4 mRNA）、miR-181 b、miR-155和miR-361（沉默Aicda mRNA），并确定Rab 7在此过程中的作用：可能通过促进自噬（-样）过程作为Dicer降解的介体。通过将自噬、雌激素和microRNA这三个研究领域结合在一起来解决自身抗体产生的调节问题，我们的提案提供了一种综合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.