Molecular basis for age-related thymic involution and rejuvenation

描述（由申请人提供）：该提案将确定维持胸腺微环境的分子变化，该微环境的恶化被认为会导致与年龄相关的胸腺退化。胸腺上皮细胞（TEC）是胸腺微环境的重要组成部分，指导胸腺细胞的发育，控制胸腺的关键功能，从而产生和维持对感染的有效适应性免疫，并防止自身免疫的发展。TEC分化需要上皮细胞自主基因叉头盒N1 （Foxn1）。一些先天的Foxn1突变提供了Foxn1在胚胎胸腺器官发生过程中调控TEC功能的机制信息。然而，Foxn1在成熟胸腺和自然胸腺衰老中的作用尚不清楚。我们培育了一只Foxn1fx小鼠，它允许Foxn1暂时有条件地删除，并允许我们研究Foxn1在出生后胸腺和年龄相关胸腺退化的功能维持中的作用。我们将验证foxn1控制的TECs介导胸腺衰老的关键变化，包括t淋巴生成和自身免疫预防的假设。为了验证这一假设，我们提出了四个具体目标。1)我们将通过评估快速删除Foxn1后的TEC表型，以及自发和逐渐切除Foxn1期间的TEC表型，确定TEC衍生的t细胞生长因子，包括Notch配体的表达，来确定对Foxn1缺失最敏感的TEC亚群。我们还将确定Foxn1的去除是否会影响胸腺细胞表型和t细胞凋亡。我们将有条件地向中年小鼠提供外源性Foxn1，以测试它是否能延缓与年龄相关的胸腺功能不全。2)我们将阐明出生后胸腺条件性Foxn1缺失导致目的1中确定的TEC表型的机制，如细胞凋亡增强和Notch配体表达减少。3)我们将确定衰老过程中自身免疫的增加是否与Foxn1功能的丧失以及由此导致的髓质TEC （mTEC）功能的降低有关。在条件foxn1缺失小鼠中，我们将评估mTEC分化和出生后胸腺中枢耐受发育相关基因的表达，以及调节性t细胞功能。4)。我们将在Foxn1基因陷阱小鼠模型的基础上，通过从Foxn1缺失、Foxn1正常和Foxn1正常后关闭的小鼠中获得TECs，在转录水平上绘制Foxn1调控分子。来自TEC的RNA将被杂交到微阵列中，以确定Foxn1调控TEC发育的可能机制和途径。这些研究将提供与年龄相关的胸腺退化和返老还童的分子基础的重要新信息，并为未来在老年人中保存或再生胸腺功能和维持t细胞免疫的研究奠定基础。年龄相关性胸腺复旧与返老返老的分子基础：胸腺微环境主要受胸腺上皮细胞状态控制，由Foxn1基因调控。我们已经建立了一种新的小鼠模型，其中Foxn1基因可以被暂时删除，这为研究出生后胸腺退化、胸腺功能不足引起的免疫衰老和衰老过程中自身免疫增强的机制提供了有力的工具。这些研究将为恢复胸腺功能障碍和延长人体细胞免疫的功能寿命奠定基础。

DESCRIPTION (provided by applicant): This proposal will identify molecular changes that maintain the thymic microenvironment, deterioration of which is believed to cause age-related thymic involution. Thymic epithelial cells (TEC) constitute a major element of the thymic microenvironment that directs thymocyte development and controls key thymic functions that generate and maintain effective adaptive immunity to infection, and prevent development of autoimmunity. TEC differentiation requires the epithelial cell-autonomous gene forkhead box N1 (Foxn1). Several inborn Foxn1 mutations have provided information on mechanisms by which Foxn1 regulates TEC function during thymic organogenesis in the embryos. However, the function of Foxn1 in the mature thymus and in natural thymic aging is unclear. We have generated a Foxn1fx mouse, which permits conditional deletion of Foxn1 temporally and allows us to study the role of Foxn1 in functional maintenance of the postnatal thymus and age-related thymic involution. We will test the hypothesis that Foxn1-controlled TECs mediate critical changes of thymic aging, including T-lymphopoiesis and prevention of autoimmunity. To test this hypothesis, we propose four specific aims. 1) We will identify TEC subpopulations that are most sensitive to loss of Foxn1 by evaluating TEC phenotypes after rapid deletion of Foxn1 and during spontaneous and gradual excision of Foxn1, determine TEC-derived T-cell growth factors, including expression of Notch ligands. We will also determine if removal of Foxn1 affects thymocyte phenotypes, T-cell apoptosis. We will conditionally supply exogenous Foxn1 in the middle-aged mice to test if it delays age-related thymic insufficiency. 2) We will elucidate the mechanisms by which conditional Foxn1 deletion in the postnatal thymus causes the TEC phenotypes identified in aim 1, such as enhanced apoptosis and reduced expression of Notch ligands. 3) We will determine if the increased autoimmunity in aging is related to loss of Foxn1 function, and resulting reduced medullary TEC (mTEC) function. In the conditional Foxn1-deleted mice, we will evaluate mTEC differentiation and expression of genes associated with development of central tolerance in the postnatal thymus, as well as regulatory T-cell function. 4). We will map Foxn1-regulated molecules at transcriptional level by obtaining TECs from mice with deleted Foxn1, normal Foxn1, and normal-then-off Foxn1 expression, based on generation of a Foxn1geo gene-trap mouse model. RNA from TECs will be hybridized to microarrays to identify possible mechanisms and pathways by which Foxn1 regulates TEC development. These studies will provide critical new information on the molecular basis for age-related thymic involution and rejuvenation, and lay the groundwork for future studies to preserve or regenerate thymic function and to maintain T-cell immunity in the elderly. Molecular basis for age-related thymic involution and rejuvenation Narrative for the proposal: The thymic microenvironment is mainly controlled by the states of thymic epithelial cells, which are regulated by Foxn1 gene. We have developed a novel mouse model in which the Foxn1 gene can be temporally deleted, which provides a powerful tool to study the mechanisms of postnatal thymic involution, thymic insufficiency-derived immunosenescence, and the increased autoimmunity in aging. These studies will lay the groundwork for restoring thymic dysfunction and extending the functional lifespan of human cellular immunity.