Hyunwon Yang

Obesity Increases the Production of Proinflammatory Mediators from Adipose Tissue T Cells and Compromises TCR Repertoire Diversity: Implications for Systemic Inflammation and Insulin Resistance

Emerging evidence suggests that increases in activated T cell populations in adipose tissue may contribute toward obesity-associated metabolic syndrome. The present study investigates three unanswered questions: 1) Do adipose-resident T cells (ARTs) from lean and obese mice have altered cytokine production in response to TCR ligation?; 2) Do the extralymphoid ARTs possess a unique TCR repertoire compared with lymphoid-resident T cells and whether obesity alters the TCR diversity in specific adipose depots?; and 3) Does short-term elimination of T cells in epididymal fat pad without disturbing the systemic T cell homeostasis regulate inflammation and insulin-action during obesity? We found that obesity reduced the frequency of naive ART cells in s.c. fat and increased the effector-memory populations in visceral fat. The ARTs from diet-induced obese (DIO) mice had a higher frequency of IFN-γ+, granzyme B+ cells, and upon TCR ligation, the ARTs from DIO mice produced increased levels of proinflammatory mediators. Importantly, compared with splenic T cells, ARTs exhibited markedly restricted TCR diversity, which was further compromised by obesity. Acute depletion of T cells from epididymal fat pads improved insulin action in young DIO mice but did not reverse obesity-associated feed forward cascade of chronic systemic inflammation and insulin resistance in middle-aged DIO mice. Collectively, these data establish that ARTs have a restricted TCR-Vβ repertoire, and T cells contribute toward the complex proinflammatory microenvironment of adipose tissue in obesity. Development of future long-term T cell depletion protocols specific to visceral fat may represent an additional strategy to manage obesity-associated comorbidities.

Ghrelin promotes thymopoiesis during aging

The decline in adaptive immunity, T lymphocyte output, and the contraction of the TCR repertoire with age is largely attributable to thymic involution. The loss of thymic function with age may be due to diminished numbers of progenitors and the loss of critical cytokines and hormones from the thymic microenvironment. We have previously demonstrated that the orexigenic hormone ghrelin is expressed by immune cells and regulates T cell activation and inflammation. Here we report that ghrelin and ghrelin receptor expression within the thymus diminished with progressive aging. Infusion of ghrelin into 14-month-old mice significantly improved the age-associated changes in thymic architecture and thymocyte numbers, increasing recent thymic emigrants and improving TCR diversity of peripheral T cell subsets. Ghrelin-induced thymopoiesis during aging was associated with enhanced early thymocyte progenitors and bone marrow-derived Lin(-)Sca1(+)cKit(+) cells, while ghrelin- and growth hormone secretagogue receptor-deficient (GHS-R-deficient) mice displayed enhanced age-associated thymic involution. Leptin also enhanced thymopoiesis in aged but not young mice. Our findings demonstrate what we believe to be a novel role for ghrelin and its receptor in thymic biology and suggest a possible therapeutic benefit of harnessing this pathway in the reconstitution of thymic function in immunocompromised subjects.

Obesity accelerates thymic aging

As the expanding obese population grows older, their successful immunologic aging will be critical to enhancing the health span. Obesity increases risk of infections and cancer, suggesting adverse effects on immune surveillance. Here, we report that obesity compromises the mechanisms regulating T-cell generation by inducing premature thymic involution. Diet-induced obesity reduced thymocyte counts and significantly increased apoptosis of developing T-cell populations. Obesity accelerated the age-related reduction of T-cell receptor (TCR) excision circle bearing peripheral lymphocytes, an index of recently generated T cells from thymus. Consistent with reduced thymopoiesis, dietary obesity led to reduction in peripheral naive T cells with increased frequency of effector-memory cells. Defects in thymopoiesis in obese mice were related with decrease in the lymphoid-primed multipotent progenitor (Lin-Sca1+Kit+ Flt3+) as well as common lymphoid progenitor (Lin-Sca1+CD117(lo)CD127+) pools. The TCR spectratyping analysis showed that obesity compromised V-beta TCR repertoire diversity. Furthermore, the obesity induced by melanocortin 4 receptor deficiency also constricted the T-cell repertoire diversity, recapitulating the thymic defects observed with diet-induced obesity. In middle-aged humans, progressive adiposity with or without type 2 diabetes also compromised thymic output. Collectively, these findings establish that obesity constricts T-cell diversity by accelerating age-related thymic involution.

Inhibition of Thymic Adipogenesis by Caloric Restriction Is Coupled with Reduction in Age-Related Thymic Involution

Aging of thymus is characterized by reduction in naive T cell output together with progressive replacement of lymphostromal thymic zones with adipocytes. Determining how calorie restriction (CR), a prolongevity metabolic intervention, regulates thymic aging may allow identification of relevant mechanisms to prevent immunosenescence. Using a mouse model of chronic CR, we found that a reduction in age-related thymic adipogenic mechanism is coupled with maintenance of thymic function. The CR increased cellular density in the thymic cortex and medulla and preserved the epithelial signatures. Interestingly, CR prevented the age-related increase in epithelial-mesenchymal transition (EMT) regulators, FoxC2, and fibroblast-specific protein-1 (FSP-1), together with reduction in lipid-laden thymic fibroblasts. Additionally, CR specifically blocked the age-related elevation of thymic proadipogenic master regulator, peroxisome proliferator activated receptor γ (PPARγ), and its upstream activator xanthine-oxidoreductase (XOR). Furthermore, we found that specific inhibition of PPARγ in thymic stromal cells prevented their adipogenic transformation in an XOR-dependent mechanism. Activation of PPARγ-driven adipogenesis in OP9-DL1 stromal cells compromised their ability to support T cell development. Conversely, CR-induced reduction in EMT and thymic adipogenesis were coupled with elevated thymic output. Compared with 26-mo-old ad libitum fed mice, the T cells derived from age-matched CR animals displayed greater proliferation and higher IL-2 expression. Furthermore, CR prevented the deterioration of the peripheral TCR repertoire diversity in older animals. Collectively, our findings demonstrate that reducing proadipogenic signaling in thymus via CR may promote thymopoiesis during aging.

Deficient Ghrelin Receptor-mediated Signaling Compromises Thymic Stromal Cell Microenvironment by Accelerating Thymic Adiposity

With progressive aging, adipocytes are the major cell types that constitute the bulk of thymic microenvironment. Understanding the origin of thymic adipocytes and mechanisms responsible for age-related thymic adiposity is thus germane for the design of long lasting thymic rejuvenation strategies. We have recently identified that ghrelin, an orexigenic anti-inflammatory peptide, can partially reverse age-related thymic involution. Here we demonstrate that Ghrl and ghrelin receptor (growth hormone secretagogue receptor (GHSR)) are expressed in thymic stromal cells and that their expression declines with physiological aging. Genetic ablation of ghrelin and GHSR leads to loss of thymic epithelial cells (TEC) and an increase in adipogenic fibroblasts in the thymus, suggesting potential cellular transitions. Using FoxN1Cre;R26RstopLacZ double transgenic mice, we provide qualitative evidence that thymic epithelial cells can transition to mesenchymal cells that express proadipogenic regulators in the thymus. We found that loss of functional Ghrl-GHSR interactions facilitates EMT and induces thymic adipogenesis with age. In addition, the compromised thymic stromal microenvironment due to lack of Ghrl-GHSR interactions is associated with reduced number of naive T cells. These data suggest that Ghrl may be a novel regulator of EMT and preserves thymic stromal cell microenvironment by controlling age-related adipocyte development within the thymus.

Analysis of the therapeutic functions of novel melanocortin receptor agonists in MC3R- and MC4R-deficient C57BL/6J mice

Melanocortin receptor agonists act in the brain to regulate food intake and body weight and, independently of these actions, affect insulin sensitivity. These experiments investigated the function of novel non-selective melanocortin receptor agonists (BIM-22493, BIM-22511) that cross the blood-brain barrier when administered peripherally. Treatment of diet induced obese C57BL/6J (B6) mice with melanocortin agonists administered peripherally improved obesity, hyperinsulinemia (approximately 50%) and fatty liver disease. Specificity of function was determined using B6 melanocortin-3 and melanocortin-4 receptor knockout mice (MC3RKO, MC4RKO). Chow fed MC4RKO but not MC3RKO used for these tests exhibited obesity, hyperinsulinemia and severe hepatosteatosis associated with increased expression of insulin-stimulated genes involved in lipogenesis. Reduced food intake associated with acute BIM-22493 treatment, and weight loss associated with 14 days of treatment with BIM-22511, required functional MC4R but not MC3R. However, while 14 days of treatment with BIM-22511 did not affect body weight and even increased cumulative food intake in MC4RKO, a significant reduction (approximately 50%) in fasting insulin was still observed. Despite lowering insulin, chronic treatment with BIM-22511 did not improve hepatosteatosis in MC4RKO, and did not affect hepatic lipogenic gene expression. Together, these results demonstrate that peripherally administered melanocortin receptor agonists regulate body weight, liver metabolism and glucose homeostasis through independent pathways. MC4R are necessary for melanocortin agonist-induced weight loss and improvements in liver metabolism, but are not required for improvements in hyperinsulinemia. Agonists with activity at MC4R improve glucose homeostasis at least partially by causing weight loss, however other melanocortin receptors may have potential for treating aberrations in glucose homeostasis associated with obesity.

Chronic caloric restriction induces forestomach hypertrophy with enhanced ghrelin levels during aging.

Caloric restriction (CR) is the only preventive intervention that has robust pro-longevity effects in experimental models. Various circulating hormones that regulate the state of negative energy balance may drive the multi-system beneficial effects of the CR phenomenon. Ghrelin, one such stomach-derived circulating peptide hormone stimulates food intake, promotes GH release and inhibits pro-inflammatory cytokines. We have recently demonstrated that ghrelin also reverses age-related thymic involution. Here, we report that chronic CR in aging mice results in reduction in body weight, and spleen size but remarkably, leads to a significant increase in the size and weight of stomach. The increased size of stomach was largely due to increased size of fundus (forestomach) and also smaller but statistically significant enlargement of antrum. The analysis of serial stomach sections revealed that chronic CR leads to a striking hypertrophy of lamina propria, stratum basale, stratum corneum and the stratified squamous epithelium of forestomach of the aged animals. We also report for the first time that chronic CR during aging significantly increases circulating ghrelin levels as well as total ghrelin production in the stomach and reverses age-related loss of ghrelin receptor expression in pituitary. Our data suggests that long-term CR-induced increased ghrelin production from hypertrophic stomach in mice may be an adaptive survival strategy in response to sustained negative energy balance that triggers heightened state of food seeking. Taken together, these data provide new insights into the underlying mechanism behind the salutary effects of chronic caloric restriction during aging process.

Facilitation of spinal morphine analgesia in normal and morphine tolerant animals by neuropeptide SF and related peptides.

Neuropeptide FF and related synthetic amidated peptides have been shown to elicit sustained anti-nociceptive responses and potently augment spinal anti-nociceptive actions of spinal morphine in tests of thermal and mechanical nociception. Recent studies have described the occurrence of another octapeptide, neuropeptide SF (NPSF) in the spinal cord and the cerebrospinal fluid and demonstrated its affinity for the NPFF receptors. This study examined the effects of NPSF and two putative precursor peptides, EFW-NPSF and NPAF, on the spinal actions of morphine in normal and opioid tolerant rats using the tailflick and pawpressure tests. In normal rats, NPSF demonstrated weak intrinsic activity but sub-effective doses of the peptide significantly increased the magnitude and duration of spinal morphine anti-nociception in both tests. A low-dose of NPSF also augmented the spinal actions of a delta receptor agonist, deltorphin. The morphine-potentiating effect of NPSF was shared by EFW-NPSF and the octadecapeptide NPAF. In animal rendered tolerant by continuous intrathecal infusion of morphine for 6 days, low dose NPSF itself elicited a significant anti-nociceptive response and potently increased morphine-induced response in both tests. In animals made tolerant by repeated injections of intrathecal morphine, administration of NPSF, EFW-NPSF, and NPAF with morphine reversed the loss of the anti-nociceptive effect and restored the agonist potency. The results demonstrate that in normal animals NPSF and related peptides exert strong potentiating effect on morphine anti-nociception at the spinal level and in tolerant animals these agents can reverse the loss of morphine potency.

Thiazolidinedione treatment and constitutive‐PPARγ activation induces ectopic adipogenesis and promotes age‐related thymic involution

Age‐related thymic involution is characterized by reduction in T cell production together with ectopic adipocyte development within the hematopoietic and thymic niches. Peroxisome proliferator‐activated receptor gamma (PPARγ) is required for adipocyte development, glucose homeostasis and is a target for several insulin‐sensitizing drugs. Our prior studies showed that age‐related elevation of PPARγ expression in thymic stromal cells is associated with thymic involution. Here, using clinically relevant pharmacological and genetic manipulations in mouse models, we provide evidence that activation of PPARγ leads to reduction in thymopoiesis. Treatment of aged mice with antihyperglycemic PPARγ‐ligand class of thiazolidinedione drug, rosiglitazone caused robust thymic expression of classical pro‐adipogenic transcripts. Rosiglitazone reduced thymic cellularity, lowered the naïve T cell number and T cell receptor excision circles (TRECs) indicative of compromised thymopoiesis. To directly investigate whether PPARγ activation induces thymic involution, we created transgenic mice with constitutive‐active PPARγ (CA‐PPARg) fusion protein in cells of adipogenic lineage. Importantly, CA‐PPARγ transgene was expressed in thymus and in fibroblast‐specific protein‐1/S100A4 (FSP1+) cells, a marker of secondary mesenchymal cells. The CAPPARγ fusion protein mimicked the liganded PPARγ receptor and the transgenic mice displayed increased ectopic thymic adipogenesis and reduced thymopoiesis. Furthermore, the reduction in thymopoiesis in CA‐PPARγ mice was associated with higher bone marrow adiposity and lower hematopoietic stem cell progenitor pool. Consistent with lower thymic output, CAPPARγ transgenic mice had restricted T cell receptor repertoire diversity. Collectively, our data suggest that activation of PPARγ accelerates thymic aging and thymus‐specific PPARγ antagonist may forestall age‐related decline in T cell diversity.

Regulation of μ binding sites after chronic administration of antibodies directed against specific anti-opiate peptides

There is some indication that anti-opiate peptides (AOP) modulate opioid receptor systems by altering mu-receptor density. To further characterize this phenomenon, we investigated the effects of continuous infusion of anti-AOP IgG on mu binding sites in the brains of rats. Specifically, male Sprague-Dawley rats received intracerebroventricular (i.c.v.) infusions for 13 days of either control (rabbit) IgG or test IgGs: anti-dynorphin A IgG, anti-dynorphin A1-8 IgG, anti-alpha-MSH IgG, or the monoclonal anti-NPFF IgG. Administration of anti-NPFF IgG or the anti-dynorphin1-8 IgG significantly increased mu labeling by 40-70% in several brain regions at the caudate level. Contrary to these findings, anti-alpha-MSH IgG decreased (19-32%) [125I]-DAMGO labeling in several thalamic nuclei. The results suggest that the density of mu-opioid receptors is regulated in part by anti-opiate peptides in the extracellular fluid of the brain.