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.
Adipocyte dysfunction is a major component of obesity, and crosstalk between immune cells and adipocytes critically regulates adipocyte dysfunction. We have discovered two distinct subsets of adipose tissue resident regulatory T cells (aTregs) that express or lack expression of the transcription factor Atf3 and that produce distinct soluble mediators to crosstalk with adipocytes. This proposal will generate a novel reporter line in which expression of the fluorescent protein mScarlet-I will be driven by the Atf3 gene and used to trace and interrogate the function of Atf3+ and Atf3- aTregs and their crosstalk with adipocytes to influence adipocyte dysfunction and obesity.
