One of the consequences of an aging immune system is the process of thymic involution. The thymus undergoes a progressive reduction in size due to profound changes in its architecture associated with thymic epithelia atrophy and decreased thymopoiesis. This decline is systemically followed by decreased numbers of circulating naive T cells and cell-mediated immune responses which may play a role in the increased tumorigenesis, autoimmunity, and infectious diseases observed within an aging host. Despite the extensive study of the pathophysiology of the aging thymus, the precise molecular mechanism involved in the involution process remains unclear. In an effort to profile molecular changes that occur within the aging thymus, microarray analysis was performed using RNA derived from thymus isolated from mice of varying ages. Using mRNA derived from the thymi of 2, 4, 6, 12 and 18 month old BALB/c mice; microarray analysis was performed using three distinct custom-made cDNA microarrays developed within our laboratory. For each array, three hybridizations using three different radiolabeled cDNAs of each age group were performed per experiment yielding approximately 6 gene replicates per RNA. After analysis, hierarchical cluster analysis was then performed to determine the degree of gene expression changes and co-expression between the different age groups using the computer program, Cluster. Our results have demonstrated that the expression of approximately 100 genes were significantly up-regulated in 12 and 18 but not in 4 and 6 month old thymus compared with 2 month old thymus. Significant upregulation in gene expression was observed upon comparing older thymi with the younger groups. Gene expression associated with G protein-coupled receptors, transcription factors, hormone receptors, T cell signaling, migration, hematopoesis, and cell cycle were found to be increased in the 12- and 18-month old thymi. We have initially selected five upregulated genes for verification and functional analysis including CCR5, LIF, TSHR, IGF-2R and BDNF. We have since verified the patterns of mRNA and protein expression for each of these genes within the thymic tissue. However, the precise role of these genes in thymic function and involution remains to be defined. The success of this project relies upon the reliability of the molecular profiling aged cells from defined aged sources, both from culture and freshly isolated aged cells. The first milestone will be the definitive characterization and selection of genes associated with thymic involution. We plan to conduct a series analysis of gene expression in the thymi and spleens of mice of varying ages, H-2 and genetic backgrounds, and known involution mouse models. Our current data would suggest that thymic involution may be strain dependent and may in part be associated with distinct genetic factors rather than simply aging. We have designed a specific murine cDNA array consisting of approximately 5,000 known mouse cDNA clones to facilitate of this analysis. We are currently analyzing the data obtained from the gene profiles of aged spleens, thymi, bone marrow, B cells, T cells and thymocytes. Comparisons are being made between genes expressed only in aged thymic cells vs. aged peripheral lymphoid organs or lymphocytes. It is unclear whether certain lymphoid organs or cellular components play a critical role in longevity and lifespan. Young and old thymic CD4 and CD8 subsets, thymic nurse cells, thymic epithelium and thymic and peripheral adiopocytes are also currently being isolated and studied with the hope to identify and distinguish candidate genes that may play a role in the thymic involution process as well as those genes associated with age-related alterations in cell signaling and activity. Overall, the major goal of this project is to produce a comprehensive gene expression profile in the thymus, spleen, and lymph nodes during the aging process to identify unique and common genes and functionally related groups of genes that are expressed in age-dependent manner in these different organ systems. We have initially focused our efforts on the thymus, as its involution is believed to be one of the most significant obstacles to overcome in addressing the immunological deficits associated with aging.
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