Time Restricted Feeding in Diet Induced Obesity Improves Aortic Damage and Endothelial Function Through Reducing Th17 Cells

项目摘要 心血管疾病（CVD）是美国的主要死亡原因，而肥胖是其中之一。 CVD的最高风险因素。我们的实验室已经表明，限制摄入高脂肪饮食（HFD）的12小时活动 在20周饮食诱导的肥胖（DIO）模型中，最后2周的时间显著降低了主动脉壁 并恢复主动脉内皮功能。我们还发现，DIO显著增加 主动脉Th 17细胞是一种炎症性CD 4 + T细胞亚群，已知其可驱动 自身免疫和器官损伤。有趣的是，在DIO的最后2周， 方案减少主动脉Th 17细胞。Th 17细胞是炎性细胞因子IL-17 A的主要产生者。白介素- 已知17 A驱动CVD风险因素，然而，尚不清楚TRF是否通过IL-17降低主动脉损伤。 17 A路此外，我们有初步数据表明，DIO中的TRF与更大的循环有关。 丙酸和丁酸，这是两种重要的微生物来源的短链脂肪酸（SCFA）。SCFA是 对于调节高血压和促进抗炎性T细胞亚群很重要，然而，它们在DIO中的作用 引起的组织损伤尚不清楚。DIO与SCFA产生减少有关，这可能表明， 丙酸盐和丁酸盐是保护免受DIO损伤所必需的。这让我们假设， IL-17 A减少和SCFA增加与TRF驱动主动脉保护和改善内皮功能， DIO。我们将使用C57 B16/J小鼠用于我们的20周DIO模型，其中在最后两周进行TRF干预。 喂食在这2周期间，小鼠将在Zeitgeber时间（ZT）每天接受抗小鼠IL-17 A或IgG抗体。 0，非活动期的开始。使用这些组，我们将通过脉搏波速度评估主动脉损伤 (PWV)和组织学。我们还将通过血管反应性评估内皮功能，通过刺激主动脉 乙酰胆碱来评估内皮依赖性血管舒张。硝普钠用于评估 内皮非依赖性血管舒张。此外，我们将利用我们的扶轮基金会在DIO的介入来评估 通过单细胞RNA测序确定主动脉中Th 17细胞的致病性。上调IL-23的Th 17细胞 已知IL-23受体（IL-23 R）具有更大的致病能力。在目标2中，我们将使用我们的20周DIO C57 B16/J小鼠模型。在最后两周期间，饮食将补充5%丁酸盐， 在非活动期，按重量计，丙酸盐的浓度增加，因为这是我们看到循环SCFA增加的地方。我们将评估 通过流式细胞术观察膳食SCFA如何影响主动脉Th 17细胞。流式细胞术可以让我们对免疫表型 以鉴定SCFA是否对Th 17细胞具有特异性作用。我们还将评估主动脉损伤， 在20周DIO方案结束时通过组织学和PWV测量功能。内皮 还将通过血管反应性实验评估功能。该提案的主要目标是确定 DIO中TRF如何通过调节Th 17细胞致病性改善主动脉损伤和内皮功能。

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.