Generation of an Atf3 reporter mouse line to interrogate immune cell-adipocyte crosstalk

项目总结 肥胖时脂肪细胞功能障碍是由免疫细胞串扰调节的，但我们对此知之甚少。 这个过程是受控的。内脏中存在一种关键的抗炎免疫细胞群 脂肪组织(VAT)被称为脂肪驻留T调节细胞(ATregs)。在饮食诱导的肥胖期间 (Dio)，增值税中的aTregs数量变得枯竭，从而得出aTregs是有帮助的结论 防止脂肪细胞功能障碍和肥胖。然而，这项研究存在严重的局限性。 ATregs及其在增值税中的作用：1)我们不清楚哪些细胞因子是由哪些细胞因子分泌的 ATregs，以及2)我们有有限的工具来跟踪和分析aTregs和 DIO过程中的脂肪细胞。为了克服这些限制，我们的团队和其他人成功地解码了全球 ATregs中的基因表达模式。在这个过程中，我们发现转录因子，激活 转录因子3(ATF3)在aTregs亚群中特异表达。我们的团队已经产生了 初步数据显示，在aTregs中ATF3表达的缺失减少了名为 前脑啡肽(Penk)。Penk类阿片诱导褐变，白色脂肪细胞通过这一过程上调 蛋白UCP-1，并开始增加ATP的产生和能量消耗。相反，缺乏ATF3的aTregs 分泌升高的白介素10(IL-10)。我们最近发表了抑制脂肪细胞褐变的研究报告 是由ATREG产生的IL-10介导的。 根据我们的发现，我们假设aTregs表达的ATF3协调了一个 这些细胞特有的特定转录程序，决定了aTregs如何调节 脂肪细胞能量动态平衡与肥胖。为了验证我们的假设，我们提出了以下具体建议 目的：目的1：制备ATF3报告小鼠并验证ATF3的表达。我们将使用 CRISPR/Cas9基因打靶构建携带mScarlet-I荧光蛋白的ATF3报告小鼠 表达将由ATF3启动子驱动。目标2：确定ATF3+和ATF3-aTregs 与脂肪细胞的串扰。我们的假设是ATF3+和ATF3-ATREG亚集产生不同的可溶性 调节脂肪细胞褐变和能量消耗的介体。完成这些目标将提供一个 新的验证工具，用于询问ATF3+和ATF3-aTregs的功能。此工具是执行以下操作所必需的 进一步我们自己的ATREG研究和R01应用，因为使用ATF3+和ATF3-aTreg ATF3抗体导致不能用于下游功能分析的通透性细胞。这 记者专线对Treg在自身免疫和癌症环境中的功能有广泛的影响。 此外，ATF3由Th2细胞、Tfh细胞和巨噬细胞表达，这使该报告小鼠成为有价值的 为更广泛的免疫学和内分泌学社区提供研究工具。

PROJECT SUMMARY Adipocyte dysfunction during obesity is regulated by immune cell crosstalk, yet we understand little about how this process is controlled. One critical population of anti-inflammatory immune cells present in the visceral adipose tissue (VAT) are called adipose-resident T regulatory cells (aTregs). During diet-induced obesity (DIO), the number of aTregs in the VAT becomes depleted, resulting in the conclusion that aTregs are helpful in protection from adipocyte dysfunction and obesity. However, there are critical limitations to the study of aTregs and their function in the VAT: 1) we have no clear understanding of which cytokines are secreted by aTregs, and 2) we have limited tools with which to track and analyze the crosstalk between aTregs and adipocytes during DIO. To overcome these limitations, our group and others, successfully decoded the global gene expression pattern in aTregs. In this process, we discovered that the transcription factor, activating transcription factor 3 (Atf3) is specifically expressed in a subset of aTregs. Our group has generated preliminary data showing that loss of Atf3 expression in aTregs reduces pentapeptide opioids called Proenkephalins (Penk). Penk opioids induce beiging, the process whereby white adipocytes upregulate the protein Ucp-1 and initiate increased ATP production and energy expenditure. In contrast, Atf3-deficient aTregs secrete increased Interleukin-10 (IL-10). We have recently published that suppression of adipocyte beiging is mediated by aTreg-produced IL-10. Based on our findings, we hypothesize that Atf3 expression by aTregs orchestrates a specific transcriptional program unique to these cells that determines how aTregs regulate adipocyte energy homeostasis and obesity. To test our hypothesis, we propose the following specific aims: Aim 1: Generate an Atf3 reporter mouse and validate Atf3 expression. We will use CRISPR/Cas9 gene targeting to create an Atf3 reporter mouse in which mScarlet-I fluorescent protein expression will be driven by the Atf3 promoter. Aim 2: Determine how Atf3+ and Atf3- aTregs crosstalk with adipocytes. Our hypothesis is that Atf3+ and Atf3- aTreg subsets produce distinct soluble mediators that regulate adipocyte beiging and energy expenditure. Completion of these aims will provide a novel validated tool with which to interrogate the function of Atf3+ and Atf3- aTregs. This tool is necessary to further our own aTreg-focused studies and R01 application, since identification of Atf3+ and Atf3- aTregs using Atf3 antibodies results in permeabilized cells that cannot be used in downstream functionality assays. This reporter line has wide-ranging implications for Treg function in settings of autoimmunity and cancer. Additionally, Atf3 is expressed by Th2 cells, Tfh cells and macrophages making this reporter mouse a valuable research tool for the wider immunology and endocrinology communities.