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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.

项目摘要肥胖期间脂肪细胞功能障碍受免疫细胞串扰调节，但我们对如何调节知之甚少。这个过程是可控的。一个关键的抗炎免疫细胞群体存在于内脏脂肪组织（VAT）中的T细胞称为脂肪驻留调节性T细胞（aT细胞）。在饮食诱导的肥胖期间 (DIO)，增值税中的增值税税率的数量变得耗尽，从而得出增值税税率是有帮助的结论防止脂肪细胞功能障碍和肥胖。然而，研究的局限性很大， aT细胞及其在VAT中的功能：1）我们对哪些细胞因子是由 2）我们有有限的工具来跟踪和分析aT和aT之间的串扰。 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地解码了全球基因表达模式。在这个过程中，我们发现，转录因子，激活转录因子3（Atf 3）在aT细胞亚群中特异性表达。我们的小组已经产生了初步数据显示，Atf 3在aTdR中表达的缺失减少了五肽阿片类药物，脑啡肽原（Penk）。Penk阿片类药物诱导beiging，这是白色脂肪细胞上调蛋白Ucp-1和启动增加ATP的生产和能量消耗。相反，Atf 3缺陷型aT 3 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生长的研究，由aTreg产生的IL-10介导。基于我们的研究结果，我们假设Atf 3的表达是由aTcR协调的。这些细胞特有的特定转录程序决定了α T细胞如何调节脂肪细胞能量平衡和肥胖。为了验证我们的假设，我们提出了以下具体的目的：目的1：制备Atf 3报告基因小鼠并验证Atf 3的表达。我们将使用 CRISPR/Cas9基因靶向创建Atf 3报告小鼠，其中mScarlet-I荧光蛋白表达将由Atf 3启动子驱动。目的2：确定Atf 3+和Atf 3-α T如何结合与脂肪细胞的相互作用。我们的假设是Atf 3+和Atf 3- aTreg亚群产生了不同的可溶性调节脂肪细胞染色和能量消耗的介质。实现这些目标将提供一种新的验证工具，用于询问Atf 3+和Atf 3-aTf 3的功能。这个工具是必要的，进一步我们自己的aTreg为重点的研究和R 01的应用，因为鉴定Atf 3+和Atf 3-aTreg使用 atf 3抗体导致不能用于下游功能测定的透化细胞。这报道细胞系对自身免疫和癌症背景下的Treg功能具有广泛的影响。此外，Atf 3由Th 2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社区提供研究工具。