Molecular basis for age-related thymic involution and rejuvenation

描述(申请人提供)：本提案将确定维持胸腺微环境的分子变化，这种微环境的恶化被认为会导致与年龄相关的胸腺退缩。胸腺上皮细胞(TEC)是胸腺微环境的重要组成部分，它指导胸腺细胞的发育，控制关键的胸腺功能，从而产生和维持有效的感染适应性免疫，并防止自身免疫的发展。TEC的分化需要上皮细胞自主基因叉头盒N1(Foxn1)。一些先天的Foxn1突变已经提供了关于Foxn1在胚胎胸腺器官发生过程中调节TEC功能的机制的信息。然而，Foxn1在成熟胸腺和自然衰老胸腺中的功能尚不清楚。我们已经产生了一只Foxn1fx小鼠，它允许有条件地暂时删除Foxn1，并使我们能够研究Foxn1在出生后胸腺功能维持和与年龄相关的胸腺退化中的作用。我们将验证这样的假设，即Foxn1控制的TECs介导了胸腺衰老的关键变化，包括T淋巴细胞生成和预防自身免疫。为了检验这一假设，我们提出了四个具体目标。1)我们将通过评估Foxn1快速缺失后和Foxn1自发和逐渐切除过程中的TEC表型，确定对Foxn1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Foxn1是否会影响胸腺细胞表型和T细胞凋亡。我们将有条件地在中年小鼠中供应外源性Foxn1，以测试它是否延缓了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Foxn1缺失导致目标1中确定的TEC表型的机制，如增加细胞凋亡和减少Notch配体的表达。3)我们将确定衰老过程中自身免疫增强是否与Foxn1功能丧失以及由此导致的延髓TEC(MTEC)功能降低有关。在条件性Foxn1缺失的小鼠中，我们将评估mTEC的分化和与出生后胸腺中枢耐受发展相关的基因的表达，以及调节T细胞的功能。4)。我们将在建立Foxn1geo基因陷阱小鼠模型的基础上，通过从Foxn1缺失、Foxn1正常和Foxn1表达正常然后关闭的小鼠获得TECs，在转录水平上映射Foxn1调控的分子。来自TEC的RNA将与微阵列杂交，以确定Foxn1调节TEC发育的可能机制和途径。这些研究将为与年龄相关的胸腺退化和返老还童的分子基础提供重要的新信息，并为未来保护或再生胸腺功能和维持老年人T细胞免疫的研究奠定基础。与年龄相关的胸腺退化和返老还童的分子基础建议：胸腺微环境主要由胸腺上皮细胞的状态控制，而胸腺上皮细胞受Foxn1基因调控。我们建立了一种新的Foxn1基因可暂时缺失的小鼠模型，为研究出生后胸腺退化、胸腺功能不全衍生的免疫衰老以及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类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.