The decline in adaptive immunity, nave T-cell output and a contraction in the peripheral T cell receptor (TCR) repertoire with age are largely attributable to thymic involution and the loss of critical cytokines and hormones within the thymic microenvironment. To further understand the various processes at work in the involution process, we have focused our efforts on characterizing several of the cellular and molecular changes that occur within the thymus and thymocytes during aging. We have approached these efforts in several different ways: (1) Molecular profiling: We have previously examined the transcriptomes of progressively aging mouse thymi and thymocytes of different sexes and on caloric-restricted (CR) vs. ad libitum (AL) diets. Genes involved in various biological and molecular processes including transcriptional regulators, stress response, inflammation and immune function significantly changed during thymic aging. These differences depended on variables such as sex and diet. Interestingly, many changes associated with thymic aging are either muted or almost completely reversed in mice on caloric-restricted diets. These studies provide valuable insight into the molecular mechanisms associated with thymic aging and emphasize the need to account for biological variables such as sex and diet when examining the molecular pathways responsible for thymic involution. Several of these gene families are still be explored. Moreover, in the thymocyte profiling studies, genes associated with oxidative phosphorylation, T- and B- cell receptor signaling and antigen presentation were observed to significantly change with thymocyte age. Interestingly, several immunoglobulin chains were found to have increased expression in thymocytes with age. While the increased expression of immunoglobulin genes in aged thymocytes may have been attributed to the thymic B cells (which were found to be actively producing IgG and IgM antibodies), further analysis of highly-purified thymic T cells derived from aged but not young thymi demonstrated high levels of IgM on their cell surface suggesting the possible presence of auto-antibodies on the surface thymocytes with advancing age. Whether there are autoantibodies targeting proteins on thymic subsets with advancing age remains to be determined. We are currently examining several gene pathways that were elucidated by these studies and are also utilizing pro-thymic hormone infusions and various transgenic and knockout mouse models to more fully understand the role of certain genes in maintaining thymic integrity or facilitating thymic loss. We believe array analysis of the thymi and thymocytes of hormone treated mice may yield valuable data on the common molecular processes involved in thymic regeneration. We are also exploring the interventional role of various regenerative hormones on the changes observed in these systems. (2) Proteomic profiling: We have also examined the proteomic changes that occur within the thymus during the involution process. Over 25,000 peptides were identified using mass spectroscopy in these studies to examine specimens derived from young and aged thymi and thymocytes. While the majority of the proteins identified were not significantly different between the age groups, there were several hundred proteins that were differentially expressed between the young and old thymic specimens. Many of the identified proteins were associated with oxidative stress, T cell differentiation, signal transduction and adipogenesis. We are currently writing up this work for possible publication. (3) Lipomic profiling: We have also analyzed the age-related alterations in lipid metabolism in the whole thymus and in thymocytes from young, middle aged and old mice as well as in thymus of aged mice treated or not with growth hormone (GH), known to partially reverse thymic involution. Using electrospray tandem mass spectrometry, we were able to increased amounts of tryglicerides (TG), free-cholesterol and C24:1 sphingomyelin in total thymus of old mice as compared to young ones. Moreover, we verified a decrease in sulfatide C24:1 and in unknown long-chain ceramides, comparing aged with young thymi. In addition, a significant increase in 4-HNE and 8-epi-prostaglandin F2a (PGF) in thymi of older animals. By immunohistochemistry, we verified an increase in the expression of 4-HNE as well as of tumor necrosis factor-alpha and CD204 (a marker for oxidized LDL receptor) in aging thymus. In our analysis of isolated thymocytes of various ages, we found a decrease in TG, free cholesterol and in sphingomyelin species with age. Interestingly, we detected a significant amount of ceramide C24:1 in thymocytes from old mice when compared to their younger counterparts. Following GH treatment, we found a significant decrease in the amount of TG, free-cholesterol and PGF in the thymus of old mice. In addition, GH treatment increased significantly the amount of dihydroceramides in aged mice and promoted a trend to decrease 4-HNE, as compared to control. These data suggested to us that there is increased oxidative stress in the thymus as an animal ages and that such changes may be attenuated upon growth hormone treatment. (4) Molecular gene knockouts and thymic involution: We have also collaboratively analyzed changes in thymic development in a variety of murine gene knockout mice to assess their effects on thymic involution and T- and B-cell progenitor development. Several never explored mouse models have been examined and significant changes in gender-based involution or premature age-associated involution was observed. Work in these models are still underway with various collaborators at NIA. (5) GH Infusion into human subjects: A number of laboratories have demonstrated that the supplementation of cytokines, growth factors or certain hormones (e.g., GH, IgF1) can improve thymic output and integrity upon infusion into aged or immunosuppressed mice and primates Despite a number of studies focusing on the effects of GH on thymic activity in rodents, little to no data currently exists on its effects in humans. We are interested in studying whether recombinant human GH (rhGH) can in fact stimulate thymic and immune functions in human. If so, the potential implication of using GH in treating people with diminished immune capacity such as the elderly is substantial. Based on these preclinical findings, we have initiated a collaborative clinical trial examining if GH, administered in a pulsatile pattern, can elicit significant and relevant changes in the thymus and immune system. The immunology-related aims of this study are to determine if GH administration administered in a pulsatile fashion using a subcutaneous infusion pump compared to once a day administration may elicit clinical significant and relevant changes in the human immune system and thymic size/output. A number of specific immune parameters are being examined including immunophenotyping of participants T cell and other immune cells at various time points pre- and post-GH administration, examination of subsets cytokine and gene expression, T-cell recombinant excision circle analysis and functional MRI examination of thymic changes on each participant to correlate changes in peripheral immune parameters with thymic size, cellularity and adiposity changes. The overall goal of this project is to produce comprehensive molecular and biochemical profiles of the thymus during the aging process to identify unique and common changes in gene, protein and lipid expression and functionally relate such changes to the physiological and functional alterations that occur within the thymus with age. This project will be terminiated in FY14.
|Lustig, Ana; Carter, Arnell; Bertak, Dorothy et al. (2009) Transcriptome analysis of murine thymocytes reveals age-associated changes in thymic gene expression. Int J Med Sci 6:51-64|
|Redelman, Doug; Welniak, Lisbeth A; Taub, Dennis et al. (2008) Neuroendocrine hormones such as growth hormone and prolactin are integral members of the immunological cytokine network. Cell Immunol 252:111-21|