Mechanisms of Adipose Tissue Immunoregulatory T cell (Treg) Exhaustion in Obesity

项目摘要。脂肪组织(AT)调节性T细胞(Tregs)是系统代谢的主要决定因素， 在瘦小的小鼠身上，可以预防肥胖相关的炎症和并发症。特雷格是关键 瘦肉型AT的动态平衡维持，但肥胖型AT显著减少，导致炎症， 胰岛素抵抗和其他炎症驱动的并发症。我们的初步数据显示我们已经筋疲力尽 在肥胖中导致AT-Tregs的损失。肥胖者的AT和血树节数显示增加 程序性细胞死亡蛋白1(PD-1)和其他抑制性辅助受体(OX40、CTLA4)的表达受损 抑制功能，这是可逆的；降低的肝激酶B1(LKB1)，保护Treg 疲劳；以及FOXP3基因中CNS2甲基化增加，表明不稳定。这些 特征形成了我们对疲惫的工作定义。此外，人AT PD-1 HIGH的RNAseq分析 与阴性细胞相比，75%的显著变化的基因表达下调，包括参与 抑制功能，上调的基因包括细胞凋亡和细胞死亡基因。此外，在培养的 人Tregs、干扰素-γ(IFNG)刺激抑制共受体和细胞凋亡标记物的表达 并降低LKB1的表达，提示它可能参与了AT Treg的耗竭。这些发现强调了 需要研究AT Treg丰度/功能的调节机制。我们的具体目标包括： 目的1.假设：人类肥胖的AT-Tregs表现为精疲力竭，这有助于 在高脂饮食(HFD)摄入过程中人类AT-Tregs的下降。我们将：a)确定是否有 B)利用单细胞(Sc)RNA序列和全球DNA甲基化来 确定可能耗尽的人类精瘦和肥胖的亚群；以及C)确定 吡格列酮将通过防止疲惫和减轻高脂血症引起的瘦削人类AT-Treg下降 影响AT Treg转录变化。 目的2.假设：干扰素-γ、毒性脂质和/或降低的Treg PPARγ活性是 在肥胖问题上导致精疲力竭和体重下降的调停者。以确定是否 Tregs中特定通路的衰减/敲除(由体内代谢变化的证据支持) 对于AT Treg耗竭、AT Treg丰度和HFD诱导的全身性胰岛素抵抗的变化，我们将 使用几种被预测为促进Treg耗竭的小鼠模型：a)Treg特异性敲除IFNG受体 (IFNGR1)；B)丝氨酸棕榈酰转移酶2(由Sptlc2编码)的Treg特异性丢失，限速酶 神经酰胺生物合成所需的；以及C)对AT Tregs的关键支持因子PPARγ的Treg特异性消融。 综上所述，这些调查将揭示一种新的、潜在的重要机制，可以解释 与HFD和肥胖症有关的AT-Tregs显著减少。

Project Abstract. Adipose tissue (AT) regulatory T cells (Tregs) are major determinants of systemic metabolism, and in lean mice, protect against obesity-associated inflammation and complications. Tregs are key in homeostatic maintenance in lean AT, but abundance profoundly decreases in obese AT leading to inflammation, insulin resistance, and other inflammatory-driven complications. Our preliminary data suggests exhaustion contributes to the loss of AT Tregs in obesity. AT vs. blood Tregs from obese humans reveal increased expression of programmed cell death protein 1 (PD-1) and other inhibitory co-receptors (OX40, CTLA4); impaired suppressive function, which is reversible; decreased liver kinase B1 (LKB1), which protects Tregs from exhaustion; and increased methylation of the CNS2 in the FOXP3 locus suggesting instability. These characteristics form our working definition of exhaustion. Moreover, RNAseq analyses of human AT PD-1 high vs. negative cells revealed downregulation of 75% of significantly changed genes, including genes involved in suppressor function, while upregulated genes included apoptosis and cell death genes. Furthermore, in cultured human Tregs, interferon gamma (IFNG) stimulated expression of inhibitory co-receptors and apoptosis markers and decreased LKB1 expression, suggesting it may mediate AT Treg exhaustion. These findings underscore the need to investigate mechanisms regulating AT Treg abundance/function. Our Specific Aims include: Aim 1. Hypothesis: Human obese AT Tregs are phenotypically exhausted, which contributes to the decline in human AT Tregs during high fat diet (HFD) ingestion. We will: A) Determine whether there are more exhausted Tregs in obese vs. lean AT; B) Utilize single cell (sc)RNAseq and Global DNA methylation to define subpopulations of human lean and obese AT Tregs that may be exhausted; and C) Determine whether pioglitazone will attenuate the HFD-induced AT Treg decline in lean humans by preventing exhaustion and impacting AT Treg transcriptional changes. Aim 2. Hypothesis: Interferon-gamma (IFNG), toxic lipids, and/or decreased Treg PPARγ activity are mediators that contribute to exhaustion and declining Tregs in obesity. To determine whether attenuation/knockout of specific pathways in Tregs (supported by evidence of in vivo metabolic changes) leads to changes in AT Treg exhaustion, AT Treg abundance, and HFD-induced systemic insulin resistance, we will use several mouse models predicted to promote Treg exhaustion: A) Treg-specific knockout of the IFNG receptor (IFNGR1); B) Treg-specific loss of serine palmitoyl transferase 2 (encoded by Sptlc2), a rate limiting enzyme required for ceramide biosynthesis; and C) Treg specific ablation of a key supportive factor for AT Tregs, PPARγ. Taken together, these investigations will shed light on a new, potentially important mechanism explaining the striking loss of AT Tregs that occurs with HFD and obesity.