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对衰老构成重大风险 人口。在与年龄相关的胸腺退化期间，功能性细胞的减少和解体 胸腺区域，沿着成脂细胞和其他不良细胞类型的增加，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，Cellular 胸腺脂肪细胞的起源仍然不清楚，一些研究者认为脂肪细胞浸润胸腺， 胸腺退化期间和其他表明他们直接从胸腺基质细胞分化。我们实验室 已经确定了胸腺中脂肪形成的潜在调节因子，激活转录因子3（ATF 3）， 其中Atf 3基因的缺失导致在2- 100 μ mol/L时，载脂胸腺基质细胞的存在增加。 当通过流式细胞术和免疫组织化学两者评估时，10个月时 Atf 3突变体和野生型对照中的成脂细胞的年龄、FACs分析显示它们相似 数量上。然而，通过免疫组织化学定性分析揭示了成脂细胞的差异， Atf 3纯合子和杂合子突变体的类型，这值得进一步研究。我们还 使用Atf 3 null/null; Foxn 1Cre/+; Rosa 26 Tom/+小鼠进行谱系追踪研究，以鉴定是否存在胸腺细胞亚群。 上皮细胞（TEC）通过使用成像流式细胞术经历脂肪生成。我们确定了四个主要的 TEC衍生的成脂细胞的种类（LipidTox+EpCAM+ Ly 51+、LipidTox+EpCAM+ Ly 51-、LipidTox+EpCAM-Ly 51-） Ly 51-和LipidTox+ EpCAM-Ly 51+细胞），其包括载有脂质的cTEC和mTEC。在冷冻的胸腺中 在部分中，我们已经鉴定了在6、7和10月龄开始表达PPARy的载有脂质的cTEC。在 此外，当我们观察10个月时，我们发现了表达FSP 1（EMT的标志物）的成脂mTEC， 老老鼠这些发现表明，尽管cTEC和mTEC都能成为脂肪细胞，但它们各自可能是脂肪细胞。 由不同的分子机制调控。我们还观察了来自PPARγ-tdTomato报告基因的组织， 由哈佛的戴安马西斯实验室设计的老鼠。使用报告小鼠，我们已经确定 成脂血管相关细胞和血管管腔内存在球状脂肪细胞 似乎正在浸润胸腺总的来说，我们已经开始描述不同类型的胸腺细胞， 更充分地研究脂肪细胞，我们开始更多地了解可能控制脂肪细胞的基因。 过程我们已经优化了技术，使用标准和成像流式细胞术研究胸腺脂肪细胞 和免疫细胞化学我们已经获得了一个新的鼠标线，PPARy-tdTomato线，我们计划 用于我们的研究目的这些数据表明，cTEC和mTEC都产生亚群， ATF 3可能是胸腺退化过程中脂肪生成的抑制因子。目标1： 将鉴定其中ATF 3起调节胸腺脂肪形成作用的细胞类型。在目标2中，我们将确定 ATF 3调节脂肪形成的机制。

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.