Characterizing the Role of ATF3 in Regulating Adipogenesis During Age-Associated Thymic Involution

由年龄相关的胸腺退化引起的免疫衰老是衰老的一个重大风险。 人类人口。在与年龄相关的胸腺退化过程中，功能细胞的减少和解体 胸腺的某些区域，随着成脂细胞和其他有害细胞类型的增加，会导致较低的 胸腺产生适应性免疫所需的功能性T细胞的能力。目前，蜂窝 胸腺脂肪细胞的起源尚不清楚，一些研究人员认为脂肪细胞渗入胸腺 退化期的胸腺和其他表明它们直接从胸腺基质细胞分化出来的细胞。我们的实验室 已经确定了胸腺中一种潜在的脂肪生成调节因子--激活转录因子3(ATF3)， 其中ATF3基因缺失导致胸腺基质细胞在2- 用流式细胞仪和免疫组织化学两种方法评估小鼠的月龄。在10个月内 ATF3突变体和野生型对照的成脂细胞的年龄、流式细胞仪分析表明它们是相似的 从数量上讲。然而，免疫组织化学的定性分析揭示了成脂细胞的差异。 ATF3纯合子和杂合子突变株的类型，值得进一步研究。我们还有 使用Atf3Null/Null；Foxn1Cre/+；Rosa26Tom/+小鼠进行了血统追踪研究，以确定胸腺的一个子集 上皮细胞(TECs)通过成像流式细胞术进行脂肪生成。我们确定了四个主要的 TEC来源的成脂细胞类别(LipidTox+EpCAM+Ly51+，LipidTox+EpCAM+Ly51-，LipidTox+EpCAM- Ly51-和LipidTox+EpCAM-Ly51+细胞)，包括载脂的cTECs和mTECs。在冷冻胸腺中 部分，我们已经确定了从6、7和10个月龄开始表达PPARy的富含脂质的cTEC。在……里面 此外，当我们观察10个月时，我们鉴定出表达EMT标志物FSP1的成脂mTEC。 老老鼠。这些发现表明，虽然cTECs和mTECs都是成脂细胞，但各自可能是 受不同的分子机制调控。我们还查看了PPARγ-tdTomato记者的组织 由哈佛大学黛安·马西斯的实验室制造的老鼠。使用记者老鼠，我们已经识别出 成脂血管相关细胞和血管管腔内球形脂肪细胞的存在 似乎正在渗入胸腺。总体而言，我们已经开始描述不同类别的胸腺 脂肪细胞变得更充分，我们开始更多地了解可能控制 进程。我们已经优化了使用标准和成像流式细胞术研究胸腺脂肪细胞的技术。 和免疫细胞化学。我们已经获得了一种新的鼠标品系，PPARy-tdTomato品系，我们计划 用于我们的研究目的。这些数据表明cTECs和mTECs都产生了一个亚群 在胸腺退化过程中，ATF3可能是脂肪生成的抑制因子。在目标1中，我们 将确定ATF3调节胸腺脂肪生成的细胞类型。在目标2中，我们将确定 ATF3调控脂肪形成的机制。

Immunosenescence caused by age-associated involution of the thymus poses a significant risk to the aging human population. During age-associated thymic involution, a reduction and disorganization of the functional regions of the thymus, along with an increase in adipogenic and other undesirous cell types, results in a lower capacity of the thymus to generate functional T-cells required for adaptive immunity. Currently, the cellular origins of thymic adipocytes remain obscure, with some investigators suggesting that adipocytes infiltrate the thymus during involution and others indicating that they differentiate directly from thymic stromal cells. Our lab has identified a potential regulator of adipogenesis in the thymus, the Activating Transcription Factor 3 (ATF3), in which deletion of the Atf3 gene results in an increased presence of lipid-laden thymic stromal cells at 2- months of age in mice when assessed by both flow cytometry and immunohistochemistry. At 10-months of age, FACs analysis of adipogenic cells in Atf3 mutants and wild-type controls show that they are similar quantitatively. However, qualitative analysis by immunohistochemistry reveals differences in adipogenic cell types in Atf3 homozygous and heterozygous mutants, which warrants further investigation. We have also conducted a lineage trace study using Atf3null/null; Foxn1Cre/+; Rosa26Tom/+ mice to identify if a subset of thymic epithelial cells (TECs) undergo adipogenesis by using imaging flow cytometry. We identified four major classes of TEC-derived adipogenic cells (LipidTox+EpCAM+Ly51+, LipidTox+EpCAM+Ly51-, LipidTox+EpCAM- Ly51-, and LipidTox+EpCAM-Ly51+ cells), which include lipid-laden cTECs and mTECs. In frozen thymic sections, we have identified lipid-laden cTECs that express PPARy starting at 6, 7, and 10-months of age. In addition, we identified adipogenic mTECs that express FSP1, a marker for EMT, when we looked at 10-month old mice. These findings suggest that although both cTECs and mTECs become adipogenic, each may be regulated by different molecular mechanisms. We have also looked at tissue from a PPARγ-tdTomato reporter mouse engineered by the laboratory of Diane Mathis at Harvard. Using the reporter mice, we have identified adipogenic vascular-associated cells and the presence of globular fat cells within the lumen of the vasculature that appear to be infiltrating the thymus. Overall, we have begun to characterize the different classes of thymic adipocytes more fully, and we are beginning to understand more about the genes that may be governing the process. We have optimized techniques to study thymic adipocytes using standard and imaging flow cytometry and immunocytochemistry. We have access to a novel mouse line, the PPARy-tdTomato line, that we plan on using for our research purposes. These data suggest that both cTECs and mTECs give rise to a subpopulation of thymic adipocytes and that ATF3 is a likely repressor of adipogenesis during thymic involution. In Aim 1, we will identify the cell type in which ATF3 acts to regulate thymic adipogenesis. In Aim 2, we will identify the mechanisms by which ATF3 regulates adipogenesis.