The importance of Treg-intrinsic cholesterol metabolism for visceral adipose tissue Treg homeostasis, phenotype, and function

项目摘要 肥胖已成为西方社会的主要问题，在2010年影响到约40%的成年人和20%的儿童。 据美国疾病控制和预防中心（CDC）称，众所周知，肥胖 可导致胰岛素抵抗、II型糖尿病和心血管疾病，称为“代谢综合征”， 肥胖是导致心脏病和中风等可预防死亡的主要危险因素。一个关键 肥胖症中导致代谢疾病的因素是内脏脂肪组织（VAT）中的慢性低度炎症。 一群调节性T细胞（TCRs），具有独特的转录特征和克隆扩增的TCR 在VAT中高度富集，并且在控制组织炎症中起重要作用。然而，在这方面， 这一人群的肥胖症明显减少，肥胖症直接促进了VAT炎症和症状， 代谢性疾病多种证据表明，胆固醇稳态（CH）的破坏是一种 促进肥胖期间VAT TdR降低的机制。首先，此前公布的数据显示， 在长期高脂饮食（HFD）喂养后，与CH相关的转录物在VAT T中减少。第二、 初步数据显示，CH的主要调节因子SREBP 2（Srebf 2）的Treg特异性缺失显著地降低了细胞的存活率。 在稳态下，降低VAT中的TdR，但不降低其他组织中的TdR，并增加HFD诱导的VAT炎症 和胰岛素抵抗。最后，先前已经识别了VAT Tcb的多个子集，其在其特征上不同。 T细胞受体（TCR）克隆扩增的程度，初步数据也表明，Treg特异性的损失， SREBP 2基于关键表面标志物改变VAT Treg区室的子集组成，并且 减少抗原诱导的VAT T细胞扩增。因此，该提案假设干扰Treg- 内源性CH通过损害TCR信号传导和改变亚群组成来减少VAT Treg增殖， TCR克隆扩增。为了测试这一点，使用含有VAT-Treg衍生的TCR的独特TCR转基因小鼠模型， 在增值税中优先累积的情况下，将使用。将该模型与体外CRISPR结合- Cas9基因消融和过继转移允许有效评估Cas9基因的影响的独特优势。 切除对VAT Treg积累感兴趣的基因。该模型与种系敲除模型结合， 在体内和体外试验，以及单细胞组学方法将允许评估细胞的重要性， CH在增值税中。这项提案的目的1将确定破坏Treg-内在CH的机制， 通过评估Srebf 2-充足和-缺陷VAT Treg增殖，细胞死亡， 和细胞募集到组织，并通过确定胆固醇生物合成的相对重要性， 用于VAT Treg积累的摄取。AIM 2将评估破坏Treg-内在CH对VAT Treg的影响 TCR信号传导和VAT Treg区室的亚组组成和克隆扩增。在一起， 这些拟议的实验将确定破坏Treg-内在CH如何影响VAT Treg积累 和表型，其长期目标是鉴定肥胖症治疗靶向的新机制。

PROJECT SUMMARY Obesity has become major problem in western society, affecting ~40% of adults and 20% of children in the US alone, according to the Center for Disease Control and Prevention (CDC). It is well known that obesity can lead to insulin resistance, type II diabetes, and cardiovascular disease, termed “metabolic syndrome”, and that obesity is a major risk factor for leading causes of preventable death such as heart attack and stroke. A key factor driving metabolic disease in obesity is chronic low-grade inflammation in the visceral adipose tissue (VAT). A population of regulatory T cells (Tregs), with a unique transcriptional signature and clonally expanded TCR repertoire, is highly enriched in the VAT and plays an important role in controlling tissue inflammation. However, this population is significantly reduced in obesity, which directly promotes VAT inflammation and symptoms of metabolic disease. Multiple lines of evidence suggest that disruptions in cholesterol homeostasis (CH) is one mechanism promoting a reduction in VAT Tregs during obesity. First, previously published data shows that transcripts associated with CH are reduced in VAT Tregs following long-term high fat diet (HFD) feeding. Second, preliminary data shows that Treg-specific loss of SREBP2 (Srebf2), the master regulator of CH, significantly reduces Tregs in the VAT, but not in other tissues, at steady state and increases HFD-induced VAT inflammation and insulin resistance. Finally, multiple subsets of VAT Tregs have been previously identified which differ in their extent of T cell receptor (TCR) clonal expansion, and preliminary data also shows that Treg-specific loss of SREBP2 alters the subset composition of the VAT Treg compartment, based on key surface markers, and reduces antigen-induced expansion of VAT Tregs. Thus, this proposal hypothesizes that disrupting Treg- intrinsic CH reduces VAT Treg proliferation by impairing TCR signaling and altering subset composition and TCR clonal expansion. To test this, a unique TCR-transgenic mouse model containing a VAT-Treg derived TCR, where Tregs preferentially accumulate in the VAT, will be utilized. Combining this model with ex vivo CRISPR- Cas9 gene ablation and adoptive transfer, allows for the unique advantage of efficiently assessing the impact of ablating genes of interest on VAT Treg accumulation. This model in conjunction with germline knockout models, in vivo and in vitro assays, and single cell -omics approaches will allow for assessing the importance of cellular CH in VAT Tregs. AIM 1 of this proposal will determine the mechanism by which disrupting Treg-intrinsic CH impacts VAT Treg accumulation by assessing Srebf2-sufficient and -deficient VAT Treg proliferation, cell death, and cell recruitment to the tissue, and by identifying the relative importance of cholesterol biosynthesis and uptake for VAT Treg accumulation. AIM 2 will assess the impact of disrupting Treg-intrinsic CH on VAT Treg TCR signaling and on the subset composition and clonal expansion of the VAT Treg compartment. Together, these proposed experiments will determine how disrupting Treg-intrinsic CH impacts VAT Treg accumulation and phenotype with a long-term goal of identifying novel mechanisms for therapeutic targeting in obesity.