Molecular mechanisms of estrogen receptor alpha modulating the inflammatory response in systematic lupus erythematosus

 描述(由申请人提供)：90%被诊断为系统性红斑狼疮(SLE)的人是女性，发病率最高的年龄在15至45岁，这是女性荷尔蒙最活跃的年龄段。尽管进行了大量研究，但这种性别偏见背后的机制仍然不清楚。我们的实验室先前将雌激素受体α基因敲除(ERαKO)小鼠回交到NZM2410狼疮易发背景上。我们证明，尽管血清自身抗体和肾小球免疫复合体沉积相似，但雌性NZM/ERαKO小鼠的肾脏疾病显著少于WT产仔鼠，并显著延长了存活期。ERαKO小鼠不是ERα缺失，而是表达N端截短的ERα。它们有生理缺陷，包括由于关键激活结构域(AF-1)中断而导致的不孕不育。我们发现NZM/ERαKO小鼠的树突状细胞(DC)对Toll样受体(TLRL)配体有钝性炎症反应。当这些小鼠被切除卵巢时，保护性表型就消失了。补充雌二醇后，保护作用恢复。确实，ERα缺失的小鼠不受保护，这表明在存在AF-1突变的情况下雌激素提供了保护，而不是在没有全长ERα66的情况下提供了保护。有趣的是，在ERαKO动物中表达的截短ERα在结构上类似于ERα46，ERα46是一种内源性ER DNA剪接变异体，缺乏AF-1结构域，是基因转录的负调控因子。ERα46与ERα66具有相同的DNA结合域，是ERα66的有效抑制剂。我们推测ERα46在狼疮中的表达具有保护作用。本项目的目的是确定ERα46在系统性红斑狼疮和TLR炎症中的作用，并提高我们对ERα介导的转录在炎症发生中的作用的理解。为此，坎宁安博士将(1)确定ERα46和ERα66调控转录活性影响小鼠树突状细胞固有炎症反应的体外分子机制；(2)利用表达ERαA/B截断突变体(ERαAF10)的小鼠株，在体内外确定ERα46在TLR46诱导的狼疮中的作用；(3)使用人外周血单核细胞来源的DC，确定ERα46和ERα66在调节对照组与狼疮患者树突状细胞的固有免疫反应中的作用。坎宁安博士是一名临床医生兼研究员，他的长期职业目标是成为免疫学领域的一名独立的基础和翻译研究员，专注于SLE。为了促进她向独立的过渡，她寻求进一步培训树突状细胞生物学、ChIPseq和生物信息学，以及使用患者样本中的人类细胞。坎宁安博士有一个导师团队，他们有出色的导师记录和广泛的专业知识来支持她的项目。这项K08指导拨款申请中提出的研究将极大地促进该领域的发展，通过提供对ERα介导的转录在TLR诱导的炎症环境中的理解，包括ERα结合伙伴、基因靶点以及代表潜在治疗剂的ERα46的调节。

 DESCRIPTION (provided by applicant): Ninety percent of those diagnosed with systemic lupus erythematosus (SLE) are women, with peak incidence between the ages of 15 and 45, when women are most hormonally active. Despite significant research effort, the mechanisms underlying this sex bias remain unclear. Our laboratory previously backcrossed estrogen receptor alpha knockout (ERαKO) mice onto the NZM2410 lupus prone background. We demonstrated that female NZM/ERαKO mice had significantly less renal disease and significantly prolonged survival compared to WT littermates despite similar serum autoantibodies and glomerular immune complex deposition. ERαKO mice are not ERα null, but rather express an N-terminally truncated ERα. They have physiologic deficiencies including infertility due to disruption of a critical activation domain (AF-1). We showed that dendritic cell (DCs) from NZM/ERαKO mice have a blunted inflammatory response to Toll-like receptor (TLR) ligands. When these mice were ovariectomized, the protective phenotype was lost. Upon estradiol-repletion, protection was restored. True ERα null mice are not protected, suggesting that estrogen in the presence of the AF-1 mutant confers protection, rather than the absence of the full-length ERα66. Interestingly, the truncated ERα expressed in the ERαKO animal is structurally similar to ERα46, an endogenous ERα splice variant that lacks the AF-1 domain, and is a negative regulator of gene transcription. ERα46 has an identical DNA binding domain to ERα66 and is a powerful inhibitor of ERα66. We hypothesize that ERα46 expression has a protective effect in lupus. The goal of this project is to determine the role of ERα46 in SLE and TLR-induced inflammation and to improve our understanding of ERα-mediated transcription in the setting of inflammation. In order to accomplish this, Dr. Cunningham will (1) Define the in vitro molecular mechanisms underlying ERα46- and ERα66-regulated transcriptional activity impacting the innate inflammatory response of murine DCs, (2) Determine ex vivo and in vivo the role of ERα46 in TLR-induced lupus utilizing a murine strain expressing an A/B truncation mutant of ERα (ERαAF10), and (3) Use human peripheral blood monocyte (PBMC)-derived DCs to define the role of ERα46 and ERα66 in regulation of the innate immune response in DCs from controls vs. lupus patients. Dr. Cunningham is a clinician-investigator with a long-term career goal of becoming an independent basic and translational researcher in the field of immunology with a focus on SLE. To facilitate her transition to independence, she seeks to further her training in dendritic cell biology, ChIPseq and bioinformatics, and the use of human cells from patient samples. Dr. Cunningham has a mentorship team with an outstanding mentoring track record and wide expertise to support her project. The studies proposed in this K08 mentored grant application will significantly advance the field by providing understanding of ERα-mediated transcription in the setting of TLR-induced inflammation, including ERα binding partners, gene targets, and modulation by ERα46, which represents a potential therapeutic agent.