Molecular basis for age-related thymic involution and rejuvenation

描述（由申请人提供）：本提案将确定维持胸腺微环境的分子变化，认为胸腺微环境的恶化会导致年龄相关的胸腺退化。胸腺上皮细胞（TEC）构成胸腺微环境的主要成分，其指导胸腺细胞发育并控制产生和维持对感染的有效适应性免疫并防止自身免疫发展的关键胸腺功能。TEC分化需要上皮细胞自主基因叉头盒N1（Foxn 1）。几个先天性Foxn 1突变提供了Foxn 1调节TEC功能的机制在胚胎胸腺器官发生的信息。然而，Foxn 1在成熟胸腺和自然胸腺老化中的功能尚不清楚。我们已经产生了一个Foxn 1fx小鼠，它允许有条件地删除Foxn 1的时间，使我们能够研究Foxn 1的作用，在功能维护的出生后胸腺和年龄相关的胸腺退化。我们将检验Foxn 1控制的TEC介导胸腺衰老的关键变化的假设，包括T淋巴细胞生成和自身免疫的预防。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缺失Foxn 1后的TEC表型以及Foxn 1的自发和逐渐切除过程中的TEC表型来鉴定对Foxn 1缺失最敏感的TEC亚群，确定TEC衍生的T细胞生长因子，包括Notch配体的表达。我们还将确定去除Foxn 1是否影响胸腺细胞表型和T细胞凋亡。我们将在中年小鼠中有条件地供应外源性Foxn 1，以测试它是否延迟与年龄相关的胸腺功能不全。2)我们将阐明出生后胸腺中条件性Foxn 1缺失导致目标1中确定的TEC表型的机制，例如增强的凋亡和减少的Notch配体表达。3)我们将确定衰老中自身免疫性的增加是否与Foxn 1功能的丧失有关，并导致髓质TEC（mTEC）功能的降低。在条件Foxn 1删除小鼠，我们将评估mTEC分化和表达的基因与发展的中枢耐受性在出生后的胸腺，以及调节T细胞功能。4）。我们将通过从Foxn 1缺失、正常Foxn 1和正常后关闭Foxn 1表达的小鼠中获得TEC，在转录水平上绘制Foxn 1调控的分子，基于Foxn 1geo基因诱捕小鼠模型的生成。来自TEC的RNA将与微阵列杂交，以确定Foxn 1调节TEC发育的可能机制和途径。这些研究将提供关键的新信息的分子基础上与年龄相关的胸腺退化和振兴，并奠定了基础，为今后的研究，以保持或再生胸腺功能，并保持T细胞免疫的老年人。与年龄相关的胸腺退化和年轻化的分子基础：胸腺微环境主要由胸腺上皮细胞的状态控制，这些状态由Foxn 1基因调节。我们已经开发了一种新的小鼠模型，其中Foxn 1基因可以暂时删除，这提供了一个强大的工具来研究出生后胸腺退化，胸腺inaccurency-derived免疫衰老，并增加自身免疫老化的机制。这些研究将为恢复胸腺功能障碍和延长人类细胞免疫功能寿命奠定基础。

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.