The thymus becomes significantly atrophied with age and less functional resulting in a lower production of nave T cells, a decline in the cell-mediated immune function and reduced resistance to infections. Aging has also been associated with increases in inflammation and inflammatory disease states. Several hormones including growth hormone (GH) and insulin growth factor-1 (IGF-1) have been shown to influence thymic and bone marrow development and activity in both the young and aged hosts. Our studies have revealed that a regulatory link exists between the """"""""hunger"""""""" hormone ghrelin and the satiety hormone leptin in the regulation of immune responses. We have previously reported that the ghrelin and leptin are expressed by immune cells and regulate T-cell activation and inflammation. In addition, we have reported that both ghrelin and ghrelin receptor expression within the thymus diminishes with progressive aging. Infusion of ghrelin and leptin into old mice significantly improves the age-associated changes in thymic architecture, thymocyte numbers, thymic T cell output and the peripheral TCR diversity of both CD4+ and CD8+ T cells. Similar effects were observed with leptin, GH, IGF-1 and neuropeptide Y infusion in aged mice. Ongoing studies with ghrelin, leptin and other hormones using recently derived hormone and hormone receptor transgenic and knockout mouse models are aimed at understanding the various signaling and functional pathways by which these mediators influence bone marrow and thymic activity in the context of age, inflammation and stress. This is supported by our recent findings demonstrating a role for the ghrelin-GHS-R signaling pathway in preventing DEX-induced T-cell apoptosis, more specifically on the double positive thymocyte subset, through the activation of AKT, ERK1/2, GSK and cAMP/CREB pathways resulting in the inhibition of downstream pro-apoptotic signaling cascade. We believe that these hormone-induced changes occur via distinct signaling pathways and that each hormone may mediate their effects on distinct bone marrow and thymus subpopulations. These findings suggest a novel role for metabolic hormones and their receptors in bone marrow and thymic biology. In addition to these thymic effects, we have also further examined the signaling and functional effects of ghrelin as a potent anti-inflammatory agent in rodent and human immune responses. We have identified and characterized several of the signaling pathways activated by ghrelin treatment of monocytes/macrophages and T cells and identified its role on cell growth, activation and cytokine expression. For example, in murine CD4+ T cells, we have found that ghrelin stimulation through GHS-R1a ligation activates the extracellular signal-regulated kinases (ERK1/2) and Akt signaling pathways in a dose-dependent manner. Also, the PI-3-kinase, PKC and PLC signaling pathways also play a role in the ghrelin-mediated T-cell proliferation and phosphorylation of ERK1/2, Akt, mTOR, P70S6K, S6K, AKT, TSC2, PRAS40, 4E-BP-1, eIF4G and eIF4E. The pro-proliferative effects of ghrelin were found to be GHS-R1a-specific and involve increased expression of cell cycle proteins, cyclin D1, E, CDK2 and phospho-Rb. These data suggest that ghrelin promotes the proliferation and cytokine expression by murine T cells and macrophages via the activation of the PI-3-K, PLC, PKC, Akt, ERK1/2 and mTOR signaling pathways and that this GHS-R signal to influence cell cycle progression, cytokine production and cellular activation. Furthermore, we have recently found that repeated treatment of monocytes with ghrelin facilitates their differentiation into anti-inflammatory macrophages, which produce less proinflammatory cytokines and increased levels of the inhibitory cytokine, IL-10 both in vitro and in vivo. We demonstrate that this latter effect on IL-10 expression is mediated by an augmentation in the expression ofthe transcription factor, c-musculoaponeurotic fibrosarcoma (c-Maf) in differentiating monocytes. The ghrelin-mediated IL-10 expression appears to play a significant role in regulating proinflammatory cytokine expression by activated monocytes. This is the first report demonstrating that ghrelin has a direct effect on myeloid cell differentiation, via the enhancement of c-Maf expression in monocytes, facilitating their differentiation into macrophages possessing an anti-inflammatory phenotype. In an murine multiple sclerosis model, ghrelin infusion results in the accumulation of anti-inflammatory macrophages in the spinal cord producing high levels of IL-10 and inhibiting proinflammatory cytokines and growth factors. These findings also support additional clinical studies aimed at evaluation the efficacy of ghrelin in the treatment of inflammatory conditions. These studies were contued during the current fiscal year, especially to examine the role of ghrelin in inhibiting NF-kB activation in monocuytes and macrophages stimulated with LPS.
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