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

生成 Atf3 报告小鼠系以询问免疫细胞-脂肪细胞串扰

基本信息

批准号：
10320456
负责人：
Sebastien Gingras
金额：
$ 7.65万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-12-21 至 2022-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10320456
关键词：
Adipocytes Adipose tissue Anti-Inflammatory Agents Antibodies Autoimmunity Biological Assay CRISPR/Cas technology Cell physiology Cells Central obesity Communities Data Development Endocrinology Energy Metabolism Flow Cytometry Functional disorder Gene Expression Profile Gene Targeting Generations Genes Genetic Transcription Homeostasis Immune Immune system Immunology In Vitro Inflammation Interleukin-10 Malignant Neoplasms Mediating Mediator of activation protein Metabolic Methods Mus Obesity Opioid Population Process Production Proteins Publishing Regulation Regulatory T-Lymphocyte Reporter Research Testing Th2 Cells Thermogenesis Transgenic Organisms Universities Visceral activating transcription factor activating transcription factor 3 base cytokine diet-induced obesity experience macrophage novel proenkephalin programs promoter protein expression tool transcription factor transcriptome transcriptome sequencing

项目摘要

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.

项目概要肥胖期间的脂肪细胞功能障碍受到免疫细胞串扰的调节，但我们对其如何调节知之甚少这个过程是受控制的。内脏中存在的一组重要的抗炎免疫细胞脂肪组织 (VAT) 称为脂肪驻留 T 调节细胞 (aTreg)。在饮食引起的肥胖期间 (DIO)，VAT中aTreg的数量耗尽，得出aTreg有帮助的结论防止脂肪细胞功能障碍和肥胖。然而，该研究存在严重的局限性 aTregs 及其在 VAT 中的功能：1）我们不清楚 aTregs 分泌哪些细胞因子 aTregs，2）我们用于跟踪和分析 aTregs 和 aTregs 之间串扰的工具有限。 DIO 期间的脂肪细胞。为了克服这些限制，我们的团队和其他人成功解码了全球 aTreg 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 aTreg 的一个子集中特异表达。我们组已经生成了初步数据表明，aTregs 中 Atf3 表达的丧失会减少称为五肽阿片类药物脑啡肽原（Penk）。 Penk 阿片类药物会诱导米色变，这是白色脂肪细胞上调蛋白质 Ucp-1 并启动增加 ATP 产生和能量消耗。相比之下，Atf3 缺陷的 aTregs 分泌增加的白细胞介素 10 (IL-10)。我们最近发表了抑制脂肪细胞的研究由 aTreg 产生的 IL-10 介导。根据我们的发现，我们假设 aTregs 的 Atf3 表达协调了这些细胞特有的特定转录程序决定 aTregs 如何调节脂肪细胞能量稳态和肥胖。为了检验我们的假设，我们提出以下具体建议目标：目标 1：生成 Atf3 报告小鼠并验证 Atf3 表达。我们将使用 CRISPR/Cas9 基因打靶创建 Atf3 报告小鼠，其中 mScarlet-I 荧光蛋白表达将由 Atf3 启动子驱动。目标 2：确定 Atf3+ 和 Atf3- aTregs 如何发挥作用与脂肪细胞的串扰。我们的假设是 Atf3+ 和 Atf3- aTreg 子集产生不同的可溶性调节脂肪细胞色素和能量消耗的介质。完成这些目标将提供新型经过验证的工具，用于询问 Atf3+ 和 Atf3- aTreg 的功能。这个工具是必要的进一步我们自己的以 aTreg 为重点的研究和 R01 应用，因为使用 Atf3+ 和 Atf3- aTreg 进行鉴定 Atf3 抗体会导致细胞透化，无法用于下游功能测定。这报告基因线对自身免疫和癌症中的 Treg 功能具有广泛的影响。此外，Atf3 由 Th2 细胞、Tfh 细胞和巨噬细胞表达，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界提供的研究工具。