Role of regulatory T cell glucocorticoid-induced leucine zipper (GILZ) in the chronically inflamed intestine

项目摘要/摘要 糖皮质激素诱导的亮氨酸拉链(GILZ)是一种广泛表达的转录因子，其表达是 通过内源性或合成的糖皮质激素(GC)信号通过糖皮质激素受体(GR)诱导。 GILZ被认为参与了GCs在T淋巴细胞中的许多抗炎作用，例如诱导转化生长因子β 核因子κB、丝裂原活化蛋白激酶和AP-1通路的信号和抑制。CD4+T细胞中GILZ的条件性缺失 为了增强二硝基苯磺酸(DNBS)诱导的实验性结肠炎，Th1介导的一种形式 实验性炎症性肠病(IBD)。类固醇激素，包括皮质类固醇和性类固醇 激素，如17β-雌二醇(雌激素，E_2)，在受体结合中表现出相当大的混乱。E2 已被证明在人类子宫上皮细胞中具有拮抗GILZ的作用，但关于GILZ的作用知之甚少 机制(S)，通过它可能有助于GILZ的表达和/或功能在T细胞。我们的初步数据 显示GILZ在mRNA和蛋白水平上对从小鼠胸腺分离的调节性T细胞(Tregs)的强烈诱导 克罗恩病(CD)患者和SAMP/YitFC(“SAMP”)小鼠，这是一种自发的克罗恩病样疾病模型。 这些树中GILZ上调(GILZHigh)与雌激素受体β相对减少有关 (ERβ)的表达，提示Tregs中GILZ的表达可能对雌激素信号有反应。 此外，与GILZlow对应物相比，GILZHigh Tregs显示出抑制功能降低， 提示GILZ对Treg的最佳免疫抑制作用可能需要完整的ERβ信号。 几种慢性炎症性和自身免疫性疾病表现出ERβ表达和/或活性降低， 导致一种耐人寻味的可能性，即ERβ表达减弱通过干扰 正常保护性Treg GILZ介导的机制。我们的假设是Treg的保护功能- 特异性GILZ需要完整的ERβ信号，因此不能介导足够的免疫保护 ER-β耗尽的环境，如IBD肠道。该项目的目标是确定 下调ERβ特异性信号转导对IBDGILZ表达和功能影响的机制(S) 关联树。我们将利用新的工具(GILZ转基因和基因敲除小鼠和MaxCyte慢病毒 转染人和小鼠原代T细胞)以调控ERβ缺陷对照-GILZ的表达 足够的Tregs以了解完整的ERβ信号如何影响表达(Subaim 1a)和 Treg特异性GILZ的功能(Subaim 1b)。利用大量CD患者，可通过 克利夫兰消化疾病研究核心中心(DDRCC)，我们将把单细胞RNA测序应用于CD 患者粘膜Tregs(丢弃的手术样本)以识别独特的转录信号 (Subaim 1c)与ERβ低/GILZ高树相关。这些化验将提供关键数据，供将来参考 建议集中在E2信号和GILZ活性在Tregs中的交集。成功完成这项工作 该提案将为通过雌激素调节Treg GILZ的表达和功能提供机械性见解 发信号。这些研究将确定ERβ信号减弱之间的联系，许多汽车- 炎症性疾病，包括IBD和糖皮质激素信号，最终目标是发展改善 CD患者的个性化用药方法。

Project Summary/Abstract Glucocorticoid-induced leucine zipper (GILZ) is a broadly-expressed transcription factor whose expression is induced via endogenous or synthetic glucocorticoid (GC) signaling through the glucocorticoid receptor (GR). GILZ is thought to meditate many of GCs’ anti-inflammatory effects in T lymphocytes, such as induction of TGFβ signaling and inhibition of NFκB, MAPK, and AP-1 pathways. Conditional deletion of GILZ in CD4+ T cells leads to enhancement of experimental dinitrobenzene sulfonic acid (DNBS)-induced colitis, a Th1-mediated form of experimental inflammatory bowel disease (IBD). Steroid hormones, including corticosteroids and sex steroid hormones such as 17β-estradiol (estrogen, E2), demonstrate considerable promiscuity in receptor binding. E2 has been shown to antagonize GILZ in human uterine epithelial cells, but little is known regarding the mechanism(s) by which it may contribute to GILZ expression and/or function in T cells. Our preliminary data reveal a robust induction of GILZ at the mRNA and protein levels in regulatory T cells (Tregs) isolated from Crohn’s disease (CD) patients and SAMP/YitFC (“SAMP”) mice, a spontaneous model of Crohn’s-like disease. GILZ upregulation (“GILZhigh”) in these Tregs was associated with a relative decrease in estrogen receptor beta (ERβ) expression, suggesting that GILZ expression in Tregs may be responsive to estrogen signaling. Furthermore, GILZhigh Tregs exhibited reduced suppressive function compared to their GILZlow counterparts, suggesting that optimal GILZ-mediated immunosuppression in Tregs may require intact ERβ signaling. Several chronic inflammatory and autoimmune diseases exhibit reductions in ERβ expression and/or activity, leading to the intriguing possibility that diminished ERβ expression contributes to inflammation via disruption of normally-protective Treg GILZ-mediated mechanisms. Our hypothesis is that the protective functions of Treg- specific GILZ require intact ERβ signaling, and therefore fail to mediated sufficient immunoprotection in ERβ-depleted environments, such as the IBD intestine. The goal of this project is to determine the mechanism(s) by which reduced ERβ-specific signaling influences the expression and function of GILZ in IBD- associated Tregs. We will make use of novel tools (GILZ transgenic and knockout mice and MaxCyte lentiviral transfection of primary human and murine T cells) to manipulate GILZ expression in ERβ-deficient versus – sufficient Tregs in order to understand how intact ERβ signaling influences the expression (Subaim 1a) and function (Subaim 1b) of Treg-specific GILZ. Leveraging a large cohort of CD patients available through the Cleveland Digestive Disease Research Core Center (DDRCC), we will apply single-cell RNA sequencing to CD patient mucosal Tregs (discarded surgical samples) in order to identify unique transcriptional signatures (Subaim 1c) associated with ERβlow/GILZhigh Tregs. These assays will provide critical data to inform future proposals focused on the intersection of E2 signaling and GILZ activity in Tregs. Successful completion of this proposal will provide mechanistic insight into the regulation of Treg GILZ expression and function via estrogen signaling. These studies will determine the link between diminished ERβ signaling, characteristic of many auto- inflammatory diseases including IBD, and glucocorticoid signaling, with an ultimate goal of developing improved personalized medicine approaches for CD patients.