PROJECT SUMMARY
Cardiovascular disease (CVD) is the leading cause of death in the United States, and obesity is one of the
highest risk factors for CVD. Our lab has shown that restricting intake of high fat diet (HFD) to the 12-hour active
period for the last 2 weeks in a 20-week diet induced obesity (DIO) model significantly reduces aortic wall
thickness and fibrosis and restores aortic endothelial function. We also found that DIO significantly increases
aortic Th17 cells, which are an inflammatory CD4+ T cell subset that are known to drive progression of
autoimmunity and organ damage. Interestingly, time restricted feeding (TRF) in the final 2 weeks of the DIO
protocol reduced aortic Th17 cells. Th17 cells are the main producer of the inflammatory cytokine, IL-17A. IL-
17A has been known to drive CVD risk factors, however, it is unclear if TRF reduces aortic damage via the IL-
17A pathway. Furthermore, we have preliminary data that TRF in DIO is associated with greater circulating
propionate and butyrate, which are two important microbial-derived short chain fatty acids (SCFA). SCFA are
important for regulating hypertension and promoting anti-inflammatory T cell subsets, however, their role in DIO
induced tissue damage is unclear. DIO is associated with decreased SCFA production, which could indicate that
propionate and butyrate are necessary for protection against DIO damage. This led us to hypothesize that
reduction of IL-17A and increased SCFA with TRF drive aortic protection and improved endothelial function in
DIO. We will use C57Bl6/J mice for our 20-week DIO model with TRF intervention in the final two weeks of
feeding. During those 2 weeks, mice will receive anti-mouse IL-17A or IgG antibody daily at Zeitgeber Time (ZT)
0, the start of the inactive period. Using these groups, we will assess aortic damage via pulse wave velocity
(PWV) and histology. We will also assess endothelial function via vascular reactivity by stimulating the aorta with
acetylcholine to assess endothelial dependent vasorelaxation. Sodium nitroprusside is used to assess
endothelial independent vasorelaxation. Furthermore, we will use our TRF intervention in DIO to assess
pathogenicity of Th17 cells in the aorta via single cell RNA sequencing. Th17 cells that upregulate the IL-23
receptor (IL-23R) are known to have greater pathogenic capabilities. In aim 2, we will use our 20-week DIO
model with C57Bl6/J mice. During the final two weeks, the diet will be supplemented with 5% butyrate and
propionate by weight in the inactive period as that is where we see increase of circulating SCFA. We will assess
how dietary SCFA affects aortic Th17 cells via flow cytometry. Flow cytometry will allow us to immunophenotype
the cells to identify if SCFA have an effect specifically on Th17 cells. We will also assess aortic damage and
function via histology and PWV measurements at the conclusion of the 20-week DIO protocol. Endothelial
function will also be assessed via vascular reactivity experiments. The main goal of this proposal is to identify
how TRF in DIO improves aortic damage and endothelial function through regulation of Th17 cell pathogenicity.
