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.