Published online before print June 25, 2008
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Laboratory of Neuroendocrine-Immunology, Pennington Biomedical Research Center, Louisiana State University System, Baton Rouge, Louisiana, USA
1 Correspondence: Laboratory of Neuroendocrine-Immunology, Pennington Biomedical Research Center, Louisiana State University System, 6400 Perkins Rd., Baton Rouge, LA 70808, USA. E-mail: Vishwa.dixit{at}pbrc.edu
ABSTRACT
Increasing evidence suggests a tight coupling of metabolic and immune systems. This cross-talk mediated by neuroendocrine peptides as well as numerous cytokines and chemokines is believed to be responsible for integrating energy balance to immune function. These neuroendocrine-immune interactions are heightened during the state of chronic positive energy balance, as seen during obesity, and negative energy balance caused by caloric restriction (CR). Emerging evidence suggests that obesity may be associated with an immunodeficient state and chronic inflammation, which contribute to an increased risk of premature death. The direct interactions between expanded leukocyte populations within the adipose tissue during obesity and an increased number of adipocytes within an aging lymphoid microenvironment may constitute an important adaptive or pathological response as a result of change in energy balance. In stark contrast to obesity, CR causes negative energy balance and robustly prolongs a healthy lifespan in all of the species studied to date. Therefore, the endogenous neuroendocrine-metabolic sensors elevated or suppressed as a result of changes in energy balance may offer an important mechanism in understanding the antiaging and potential immune-enhancing nature of CR. Ghrelin, one such sensor of negative energy balance, is reduced during obesity and increased by CR. Ghrelin also regulates immune function by reducing proinflammatory cytokines and promotes thymopoiesis during aging and thus, may be a new CR mimetic target. The identification of immune effects and molecular pathways used by such orexigenic metabolic factors could offer potentially novel approaches to enhance immunity and increase healthy lifespan.
Key Words: orexigenic • CR mimetic • anorexigenic • AMPK • NF-
B • mTOR • food intake • dietary • nutrition • immunity • ghrelin • GHS-R • inflammation • aging • leptin • IL-6 • thymus • bone marrow • TNF • macrophage • T cells
INTRODUCTION
Charles Darwin famously noted that "Evolution is how life commutes its own death sentence". Arguably, the survival and evolution of the human race are intimately linked with the drive to find and accumulate food as well as the ability to sense and successfully clear pathogens. The discrepancy between the environment, in which hunger and the immune system evolved in prehistoric times, is vastly different from the calorie-rich "obesogenic" environment in the modern world. It has been hypothesized that because of the scarce availability of food throughout most of human evolution, dominant genetic pathways were shaped in favor of increasing caloric intake over the mechanisms causing reduction in energy intake [1 ]. An increase in the availability of high-calorie foods and exposure of these dominant genetic traits [2 ] have led to an alarming increase in obesity. The rise in obesity-associated diseases has raised concerns that the steady increase in life expectancy during the past centuries may not be sustainable. Indeed, obesity has been shown to have a substantial negative effect on longevity, reducing the length of life of people who are severely obese by an estimated 5–20 years [3 ]. Data from the National Health and Nutrition Examination Survey suggest that close to 33% of the adult U.S. population is obese, and 17% of children and adolescents are overweight [4 ]. According to current predictions, the prevalence and severity of obesity and its complications will worsen [5 ]. Given the increasing trends of obesity in younger ages, a large group of individuals will carry and express the obesity-related risks for a longer period of their lifespan. How this growing number of the adult obese population will age immunologically may have significant health and societal implications.
This overview provides insights into how immune function is affected by metabolic states of chronic caloric excess seen during obesity with an emphasis on interactions between adipocytes and immune cells. I highlight how metabolic regulators sensing the state of energy balance and regulating the adipose mass may be potent immune-regulators. I begin by outlining the potential mechanisms and metabolic regulators altered during obesity with consequences for immune function. I next examine the state of chronic caloric restriction (CR) and its immune and antiaging properties. Finally, I speculate that specific neuroendocrine signaling pathways that are elevated during CR and suppressed during obesity may offer an invaluable insight into harnessing future endogenous, immune-enhancing mediators.
IS OBESITY AN IMMUNODEFICIENT STATE?
Obesity, which is defined as excess adiposity with a body mass index (BMI) of greater than 30, is a multisystem disorder. Analysis of data from six cohort studies revealed that in the United States alone, obesity is responsible for approximately 300,000 deaths per year [2 3 4 ]. Type 2 diabetes, cardiovascular disease (CVD), and cancers (breast, colon, esophageal, kidney, ovarian, pancreatic, and uterine) are known to cause most obesity-related mortality and morbidity [5 ]. However, non-CVD and noncancer deaths, when subgrouped as chronic and acute respiratory infections, including bronchitis, pneumonia, tuberculosis, septicemia, and other infections, contribute to substantial adult mortality in obese individuals. Emerging epidemiological evidence suggests that underweight (BMI of <20) and obese individuals (BMI of >35) have a significant increase in mortality as a result of the above-mentioned subgroup of infections, and modestly overweight subjects have significantly reduced infection-related mortality [5 ]. Apart from mortality data about infections, there is also evidence that obese individuals are more susceptible to postoperative and nosocomial infections and more likely to develop serious complications from common infections [6 ]. However, detailed and prospective studies are needed to clarify the impact of extreme obesity on adaptive as well as an innate-immune response. Thus far, available evidence supports the view that the obesity may compromise the immune-surveillance pathways. However, the cellular and molecular mechanisms responsible for dysregulated immune response during obesity remain largely unknown.
OBESTIY AND IMMUNE FUNCTION
Emerging evidence suggests that the state of chronic caloric excess seen during obesity is associated or driven by changes in metabolic factors that can also impact immune function and lifespan (Fig. 1 ). Increased circulating levels of leptin along central leptin resistance during obesity coupled with reduced concentration of ghrelin and adiponectin are associated with an increase in inflammation. Many previous studies have investigated and demonstrated abnormalities in ob/ob or a leptin-deficient model of obesity. It is currently known that ob/ob mice display markedly involuted thymus and have significant defects in T cell responsiveness [7 8 9 ] as well as a reduced ability of dendritic cells (DC) to present antigens to T cells [10 , 11 ]. Furthermore, congenital deficiency of leptin in humans leads to obesity and is associated with lymphopenia and T cell hyporesponsiveness [12 ]. However, leptin deficiency is associated with a minute fraction of clinically relevant dietary-induced obesity (DIO) prevalence [12 ]. Thus, the studies from ob/ob mouse models are more relevant to leptin deficiency and provide only a limited understanding of consequence and mechanism of obesity on immune cell function. This is especially important, as it has already been reported that leptin deficiency and not obesity mediates inflammation in a Con A model of hepatitis [13 ]. Therefore, future studies in the models of DIO may provide a clearer understanding of the impact of obesity on adaptive immunity.
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Figure 1. Obesity and CR regulate health-span by exerting reciprocal, regulatory effects on the immune system. Aberrant elevation of leptin and reduction in ghrelin during obesity may directly regulate immune cell function. On the other hand, an increase in ghrelin and reduction in anorexigenic factors caused by CR may promote immune function and promote longevity.
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and IFN-β, as well as reduced TNF- and IL-6 expression, suggesting an impaired immune response during obesity [15
]. Therefore, the findings that obesity may increase the risk of influenza could be important from a public health perspective, especially given the World Health Organization estimates of one in 10 adults worldwide being obese. In addition, the potential reduction in protective immunity in obese individuals to the emerging strains of influenza virus could potentially increase the morbidity and mortality in an influenza pandemic. In light of these findings, future investigations into mechanisms and the impact of obesity on the T cell repertoire as well as effector functions are of utmost importance. It is also important to determine if obesity impairs the regenerative capacity of the immune system, in particular, the ability of thymus and bone marrow to produce immune cells. A large body of evidence clearly suggests that obesity is associated with a chronic. proinflammatory state [16 , 17 ]. This persistent state of inflammation appears not to be responsible for tissue repair or pathogen clearance but instead, impairs insulin receptor signaling and contributes to downstream pathology of type 2 diabetes. Emerging evidence suggests that obesity compromises the innate-immune responses to the bacterium Porphyromonas gingivalis [18 ]. Compared with lean mice, the obese mice infected with P. gingivalis display increased periodontal pathology and a blunted expression of proinflammatory cytokines [18 ]. Therefore, a nonspecific, low-grade, chronic inflammation seen during obesity does not impart an advantage to the host with regards to mounting a specific, proinflammatory response needed to clear foreign pathogens. This low-grade, metabolically triggered inflammation is described as "metainflammation" [19 ] and is believed to arise as a result of chronic nutrient excess and alteration in molecular energy-sensing pathways.
5'-ADENOSINE MONOPHOSPHATE (AMP)-ACTIVATED PROTEIN KINASE (AMPK) AND MAMMALIAN TARGET OF RAPAMYCIN (mTOR): YIN AND YANG OF NUTRIENT SENSING AND INFLAMMATION?
Obesity is a complex, metabolic state, and disturbance in one organ and cellular interactions in a tissue microenvironment can impact the function of several organ systems [15 16 17 18 19 20 ] (Fig. 2 ). The understanding of molecular pathways responsible for the state of metainflammation in obesity is also complicated by uncertainty of the cellular origin of proinflammatory mediators. Despite these challenges, some common themes are emerging in this rapidly evolving area, which I briefly summarize for the purposes of this review.
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Figure 2. Adipose-immune interactions during obesity. The products derived from the brain and immune system, which regulate obesity and function of these systems, may also be impacted by obesity. The expansion of the adipose organ during obesity and interactions between leukocytes and adipocytes within adipose tissue are also responsible for increased inflammation. This nutrient excess-induced inflammation seen during obesity affects immune function and is responsible for ancillary disease states of obesity, leading to premature death.
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-subunit and by phosphorylation of the catalytic -subunit by upstream kinases, including LKB1 and Ca2+/calmodulin-dependent protein kinase kinase β [22
]. It has been proposed that in T cells, AMPK responds as well as anticipates the ensuing depletion of a cellular energy supply, as AMPK is also required for rapid calcium signaling in response to TCR ligation [23
].
AMPK is activated by various neuroendocrine orexigenic factors, such as ghrelin, neuropeptide Y (NPY), Agouti-related peptide (AGRP), and adipokines such as adiponectin [24
25
26
]. On the other hand, AMPK is inhibited by proinflammatory cytokines, such as TNF-, IL-6 [27
], as well as anorexigenic neuroendocrine peptides, such as leptin and agonists of melanocortin 3 and 4 receptors [28
]. Upon activation, AMPK regulates specific transcription factors and enzymes (Fig. 3
), many of which have direct effects on regulation of inflammation.
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Figure 3. Hypothetical model for a functional role of AMPK as a central metabolic sensor affecting inflammation and immune function. An increase in AMP concentrations during negative energy balance elicited by CR activates AMPK, and excess energy reserve during obesity inhibits AMPK. Activation of AMPK negatively regulates mTOR, NF-B, and JNK pathways, thus regulating inflammation. IKKβ, Inhibitor of B kinase β; ROS, reactive oxygen species; PKC, protein kinase C.
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B, leading to reduction in TNF-, IL-6, and IL-1β release from glial cells in the brain [29
]. In addition, the activation of AMPK inhibits inflammation and has protective effects in experimental autoimmune encephalomyelitis [30
], cystic fibrosis [31
], and adipose tissue [32
]. Metformin, which exerts its insulin-sensitizing action via activation of AMPK, inhibits inflammation by inhibiting the NF-B pathway [33
]. In addition, a recent study demonstrated that AMPK is a key player in reducing inflammation and in mediating the cardioprotective effects of macrophage migration inhibitory factor during ischemia-reperfusion injury [34
]. AMPK has also been demonstrated to be directly involved in mediating the anti-inflammatory properties of adiponectin [35
]. Correlative evidence suggests that ghrelin-mediated reduction in release of proinflammatory cytokines from T cells, and monocytes may also be via activation of the AMPK pathway [24
, 36
]. Myself and others [36
37
38
39
40
] have recently shown that ghrelin inhibits proinflammatory cytokines by activation of NF-B. The transcription of proinflammatory cytokines is controlled, in a large part, by activation of NF-B. The molecular mechanisms of AMPK effects on proinflammatory cytokine transcription and its potential interactions with key transcription factors such as NF-B remain to be ascertained. Considering that phosphorylation of IB is an ATP-dependent event, the progression of this catalytic step increases the AMP:ATP ratio. I speculate that increases in cellular AMP concentration then activate AMPK, which may serve as a "metabolic brake" for NF-B activation and thus, regulate the continued transcription of proinflammatory cytokines (Fig. 4
). It is feasible that during the states of chronic nutrient excess, as seen in obesity, the inhibition of the AMPK pathway may compromise the regulatory pathways leading to "leaky", low-grade inflammation.
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Figure 4. Hypothetical model showing the molecular mechanism of AMPK-induced reduction in proinflammatory cytokines. Orexigenic metabolic factors, such as ghrelin, up-regulated during negative energy balance, bind to their specific receptors on immune cells and signal to inhibit NF-B-mediated inflammation. The phosphorylation of IB and subsequent translocation of p50 and p65 subunits to the nucleus and NF-B activation are ATP-dependent events. An increase in intracellular AMP levels during this reaction may lead to activation of AMPK, which may directly block the NF-B-mediated transcription of proinflammatory cytokines. GHS-R, Growth hormone secretatogue receptor.
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, IL-1β, and IL-6 [49
]. This suggest that molecular sensors of energy balance are critical in regulating a proinflammatory response, and AMPK and mTOR signals may constitute a critical molecular pathway responsible for bridging immune and metabolic systems. "ADIPOIMMUNOLOGY": LEUKOCYTE-ADIPOCYTE INTERACTIONS
The crosstalk between adipose organ and immune system is not limited to direct effects of circulating adipokines and cytokines via specific receptor binding on adipocytes and leukocytes [50 , 51 ]. These adipose-immune interactions also occur within the specific tissue microenvironments of bone marrow, thymus, and adipose tissue with important functional consequences [19 , 20 , 52 , 53 ]. A growing body of literature suggests an emerging area worthy to be considered as a specialized discipline of Adipoimmunology.
Adipoimmunology may be defined as an area of research devoted to studying the interactions between adipocytes and immune cell subsets via their secreted products and cell–cell interactions within adipose and lymphoid tissue microenvironments and consequences of these interactions during physiological and disease states of obesity (insulin resistance, infection, and cancer) and/or aging (immune deficiency and autoimmunity; Fig. 2 ). For the purposes of this review, I briefly discuss and highlight adipose-immune interaction as a result of increased immune cell infiltration in the adipose tissue depots seen during obesity and increased adipocyte differentiation within the lymphoid-stromal microenvironments of the thymus and bone marrow during aging.
Adipose organ is considered to be a specialized form of connective tissue that accumulates a vast majority of adipocytes. In the past, adipose tissue was thought to be an inert organ that primarily functions to store excess energy as triglycerides in adipocytes. However, over the past few decades and especially since the discovery of leptin, adipose tissue is now known to be a bona fide endocrine organ capable of secreting many adipokines, including adiponectin, visfatin/pre-B cell colony-enhancing factor, apelin, vaspin, omentin, resistin, and hepcidin [19 , 50 , 51 ]. Many of these adipokine receptors are expressed on immune cells, and the role of adipokines on immune response and inflammation has been the subject of excellent reviews [50 , 51 ]. Thus, it is feasible that expansion of adipose tissue mass and adipose-derived products could have a significant impact on immune cell function.
Adipose resident immune (ARI) cells
The adipose organ contains various diverse cell types including macrophages, CD4 and CD8 T cells, fibroblasts, endothelial cells, and multipotent mesenchymal cells [19
, 20
] (Fig. 2)
. Obesity-induced expansion of adipose tissue mass is known to lead to increased production of various proinflammatory cytokines, such as IL-1, IL-6, TNF-, and chemokines, such as RANTES, MCP-1, IL-8, and MIP-1 [54
, 55
]. The precise cellular origin of this cytokine and chemokine production from adipose organ are incompletely understood. Although adipocytes have been shown to produce many of these proinflammatory mediators, the exact contribution of ARI cells in inflammation of obesity remains uncertain.
Macrophages are, so far, the best-studied immune cells in the adipose tissue microenvironment. It is well known that macrophages reside within many organs and possess tissue-specific phenotype, morphologies, and functions that are "in sync" with the functions of that particular organ. Examples of such specialized macrophages include microglia in the CNS in close contact with neurons and astrocytes, Kupffer cells lining the sinusoids of liver, multinucleated osteoclasts in the periosteum of bone, mesangial cells surrounding the glomerular network in kidney, alveolar macrophages in lungs, Langerhan cells in the skin, and antigen-presenting DC in the spleen, lymph nodes, and thymus [56 ]. It is now well established that macrophages also reside in adipose tissue, and obesity is associated with increased infiltration or presence of macrophages in the fat [57 58 59 60 61 ]. It has been estimated that in lean humans, 10% of adipose cellularity is comprised of macrophages, and in obesity, these numbers can rise to 40–50% of adipose tissue [57 ]. Compared with brain, liver, bones, kidney, lungs, thymus, spleen, and lymph nodes, the adipose tissue expands dramatically in obesity and constitutes up to 35–40% of total body mass in obese individuals [57 ]. This would make the adipose tissue unique and the largest organ during any pathophysiological state leading to a chronic disease. Therefore, it is plausible that this expanded adipose tissue may harbor substantial numbers of as-yet uncharacterized immune cell populations, whose function remains to be ascertained. Considering the available evidence that tissue macrophages are specialized cells dependent on their microenvironment [56 ], it is likely that adipose tissue macrophages (ATMs) are distinct cells with potential diverse, functional subsets.
ATMs have received considerable attention lately because of their role in producing excessive amounts of proinflammatory mediators and for causing insulin resistance and type 2 diabetes during obesity [19
, 57
58
59
60
61
]. Based on mRNA analysis of F4/80+ ATMs and F4/80– non-ATM cells, it was reported that almost all of adipose tissue TNF- and significant amounts of IL-6 and inducible NO synthase are derived from ATMs [58
]. Considering that F4/80+ Kupffer cells within the liver are not increased during obesity [57
], induced metainflammation suggests that ATMs could be significant players in an obesity-induced, chronic, proinflammatory state. A recent report [60
] suggests that JNK1 expression in the hematopoietic cells is a key molecular mediator of insulin resistance, as JNK1 removal from macrophages decreases obesity-induced inflammation and protects against insulin resistance. Emerging evidence points toward significant heterogeneity among ATMs based on severity and perhaps duration of obesity [61
]. It is known that "classically activated" M1 macrophages respond to TLR stimulation, produce large amounts of proinflammatory cytokines, such as IL-12, TNF-, IL-1β, and IL-6, and generate free radicals as a result of oxidative burst [56
]. Additionally, the M2 or "alternatively activated" macrophages are generated in response to stimulation by Th2 cytokines, such as IL-4 and IL-13. The M2 macrophages are responsible for tissue repair, produce low levels of proinflammatory cytokines, and secrete high amounts of IL-10 [56
]. Polarization of macrophages toward an M2 phenotype by IL-4 is mediated via transcription factor peroxisome proliferator-activated receptor- (PPAR) [62
]. It is well known that PPAR is the master regulator in promoting adipogenesis and is a target of an insulin-sensitizing class of thiazolidinedione drugs [63
, 64
]. Accordingly, recent evidence suggests that lean mice contain mainly M2-type tissue-reparative ATMs, and insulin resistance as a result of inflammation seen during obesity is caused by M1 or classically activated ATMs [65
]. The mechanisms responsible for directional migration of bone marrow-derived macrophages or macrophage precursor cells to specific adipose depots are incompletely understood. It is, however, known that CCR2-deficient mice have reduced ATMs [66
, 67
], and MCP-1-overexpressing mice have an increased number of ATMs [68
]. Interestingly, the CCR knockout mice are protected from high-fat, diet-induced insulin resistance as a result of an increase in M2 alternatively activated ATMs [65
].
Recent studies also suggest that obesity leads to increased T cell infiltration in adipose tissue [69 , 70 ]. Thus, it is feasible that in addition to macrophages, the proinflammatory cytokines produced from adipose tissue may be derived from various immune cell subsets. Considering that on a per-cell basis, an adipocyte produces much less proinflammatory cytokines, the increased infiltration of activated macrophages or other immune cell subsets may contribute to a large fraction of adipose-tissue inflammation. It is presently unclear if the ATMs have a role in antigen processing and presentation in adipose tissue; however, given that CD4 and CD8 T cells are also present in fat, the chance that the adipose organ has a role in immune defense is a possibility.
Lymphoid resident adipocytes
The direct interactions between adipocytes and immune cells also occur within the thymus and bone marrow microenvironments. It is well known that with increasing age, bone marrow and thymus undergo marked architectural and functional changes [53
]. Among the age-related alterations in the microenvironment of these lymphoid organs, the increased number of lipid-laden adipocytes within the thymus and bone marrow is one of the most obvious and under-studied phenomenon [52
].
Bone marrow adipocytes (BMA)
Adipocytes are constituents of the bone marrow stromal cell microenvironment, but their exact origin and functional relevance remain unknown. Gimble and colleagues [52
] have hypothesized four potential roles of adipocytes in the bone marrow: BMAs are "filler" cells serving to occupy excess space in marrow cavity; BMAs play a role in regulating systemic lipid metabolism and energy storage; BMAs are a local source of energy storage and reservoir required for hematopoiesis; and BMAs may be required as "support" cells required for maturation of hematopoietic lineages and regulation of osteogenesis.
With increasing age, the bone marrow is progressively replaced with adipocytes (Fig. 5 ) with a concomitant reduction in osteoblasts and increased osteoporosis [52 ]. Increased BMAs with aging are correlated with a reduction in pre-B cell number, decreased B cell generation, and Ig diversity [71 ]. Reciprocal bone marrow chimera experiments revealed that the production rates of pre-B cells are controlled primarily by bone marrow microenvironmental factors, rather than intrinsic events [71 ]. The BMAs may arise from preferential skewing of multipotent marrow mesenchymal stem cells toward a generation of adipocytes rather than osteoblasts or stromal cells. It is also unclear if adipocytes in bone marrow transdifferentiate from stromal cells themselves in response to specific cues originating from the bone marrow niches. It has been hypothesized that bone marrow adipogenesis may impair erythropoiesis, resulting in certain forms of anemia [72 ]. Considering that adipocytes are the predominant cell type in an aging bone marrow microenvironment (Fig. 5) , the cellular interactions critical for development of hematopoietic cells may be compromised.
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Figure 5. Increase in adipocytes in the bone marrow microenvironment with age. The femurs of young (2 months) and old (18 months) mice were decalcified and stained using H&E.
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The unique three-dimensional thymic meshwork is comprised of cortex and medulla, which are composed mainly of distinct developing T cell subsets and thymic stromal cells [75 ]. The cortical and medullary thymic stromal cells provide a unique environment and cell–cell contact and produce growth factors required for various aspects of T cell development [76 ]. For the thymus to produce naïve T cells, it needs to be seeded continually by lymphoid progenitors from bone marrow. These progenitors undergo T cell development based on specific cues from thymic epithelial cells (TECs) [77 ]. The cortical TECs regulate the migration and expansion of T cell progenitors, including the positive and negative selection of developing thymocytes [78 ]. The medullary TECs, along with antigen-presenting DC, are responsible for deletion of self-reactive T cells and support the late stages of T cell development [76 , 79 ]. The thymopoietic potential is compromised with increasing age as a result of multiple causes, including loss of TEC populations [80 ], defects in lymphoid progenitors [77 ], and alteration in growth factors [53 , 81 , 82 ]. In contrast to a young thymus, where a large number of thymocytes are the major contributors to the thymic environment, the situation is reversed in aging, where the adipocytes constitute the bulk of the thymic space, thus altering the thymic milieu.
It has also been shown that although early thymocyte progenitor (ETP) cells are reduced with age [83 ], the lymphoid progenitor do not have synchronized defects with age-related thymic involution [84 ]. The importance of an aged thymic microenvironment is underscored further by studies demonstrating that lymphoid progenitor cells from the young animals develop into defective, naïve T cells when introduced in an aging microenvironment [85 , 86 ].
It is conceivable that the presence of a large number of adipocytes in the thymus or bone marrow will significantly alter and influence the intrathymic milieu required to support the development of lymphoid progenitors into competent, mature lymphocytes. Interestingly, despite dramatic changes in thymic architecture with age and the large number of adipocytes, the aging human thymus retains thymopoietic potential, albeit to a limited extent. This suggests that interactions of adipocytes with lymphoid progenitors, developing T cells, and other stromal cells may have important consequences for thymic biology. The mechanisms responsible for adipose-immune interactions within specialized lymphoid organs could have important functional consequences. Forestalling the adipocyte development in the thymus or bone marrow could thus be an important strategy to preserve or prevent the decline in immune function with age.
CR AND IMMUNE FUNCTION
The state of chronic negative balance elicited by CR is the most robust, nongenetic means of extending the mean and maximal lifespan in various experimental models [87 ]. The pleiotropic effects of CR also extend to the immune system (Fig. 1) . There is a large body of data from animal models that suggests that CR has a significant impact on various arms of the immune system. The majority of the reports suggests that CR improves many parameters of immune responses [88 ] such as responses of T cells to mitogens, NK cell activity, CTL activity, and the ability of mononuclear cells to produce proinflammatory cytokines [89 , 90 ]. Early work by Weindruch and Makinodan [91 ] suggested a prominent immune-enhancing role of CR on NK and CTL activity as a possible reason for reduced incidence of tumors in mice [92 , 93 ]. Indeed, some studies have suggested that CR in old mice can improve thymic cellularity and double the total thymocyte and double-positive T cell numbers without significantly increasing the size of the thymus [94 ]. Thus, it was suggested that CR preserves immature T cell precursors in the thymus during aging to maintain higher concentrations of circulating Th and naive T cells in peripheral blood. In old rats, CR also attenuated the age-associated increase in memory:naïve T cell ratios, which was significantly associated with a reduction in proinflammatory cytokines such as TNF and IL-6 [95 ]. A study in primates clearly demonstrated that CR enhances thymopoiesis and improves the TCR diversity with increased naïve:memory T cell ratios in the periphery [95 ]. However, it is also important to note that short-term CR in primates was reported to have minimal and opposite effects on mitogen-induced proliferation of unfractionated mononuclear cells, NK cell activity, and influenza vaccine titers [96 ].
The mechanism of CR-induced thymic effects is currently unknown. Considering that neuroendocrine factors regulate short- and long-term energy balance in the body, it is feasible that these factors might have significant effects on immune function during CR, which is a state of chronic negative energy balance and is associated with an increase in various hunger-inducing "orexigenic" peptides that sense the negative energy balance and signal to CNS with an urge to eat. It is plausible that the high circulating levels of such hormones drive the multisystem beneficial effects of CR. Indeed, it has recently been reported that CR causes an increase in orexigenic factors, such as NPY, AGRP, and ghrelin [97 ], and reduces anorexigenic hormones, such as leptin [98 ] in rodents.
Ghrelin: endogenous CR mimetic?
Among various neuroendocrine metabolic regulators altered in CR, the functions of ghrelin and leptin on the immune system have been studied to some depth. Ghrelin is a 28-aa acylated peptide secreted predominantly from the stomach as a result of post-translational processing of the 117-aa preproghrelin form [99
]. Ghrelin was originally thought to stimulate GH release from the pituitary and induce food intake by binding to GHS-Rs in the hypothalamus [99
]. Ghrelin potently inhibits proinflammatory cytokine release from T cells, monocytes, and endothelial cells via a GHS-R-specific mechanism [36
37
38
39
40
]. It has been hypothesized that ghrelin may integrate immune and metabolic systems by conveying the state of peripheral negative energy balance to immune cells [100
]. My laboratory [97
] recently reported that long-term CR in aging mice can significantly increase circulating ghrelin levels as a result of hypertrophy of forestomach cellular layers. We [82
] have also demonstrated recently that ghrelin can partially reverse age-related thymic involution. The ghrelin infusions led to a significant increase in RTEs with restoration of the TEC compartment and an increase in ETPs. Interestingly, ghrelin supplementation in old mice reduced the lipid-expressing "preadipocyte-like" cell in the thymic parenchyma, and ghrelin knockout mice had an accelerated thymic involution with an increase in number of thymic adipocytes [82
]. In addition, CR reduces adipocyte content in the thymus and increases RTEs in aging mice (V. D. Dixit, unpublished findings). Considering similar cellular mechanisms of ghrelin and CR on thymopoiesis and inflammation during aging, it is likely that up-regulation of ghrelin secretion during CR may be responsible for these effects. CR is also known to inhibit leptin secretion, and leptin has been shown to increase proinflammatory cytokines by acting directly via the long form of leptin receptors on immune cells. However, it is likely that ghrelin serves as a primary mediator of the prothymic effects of CR, as we [82
] have shown that leptin infusions in aging mice increase thymopoiesis and improve TCR diversity.
Although CR has been an effective strategy to prolong a healthy lifespan in experimental animals, it remains to be studied if such effects are relevant to human physiology and immune function. The ongoing Comprehensive Assessment of Long-Term Effects of Reducing Intake of Energy (CALERIE) is a multicenter parallel group, randomized control trial to test if CR will result in similar adaptive changes in humans that occur in animal models. It is well recognized that long-term adherence to strict CR diet in humans as a "panacea" for enhanced immunity and longevity is a significant challenge in the current calorie-rich environment. Therefore, studies are underway to identify and develop compounds that mimic the biology of CR [83 , 84 ]. Based on the available evidence, it is likely that synthetic ghrelin analogues or receptor agonists may serve as another class of CR mimetic agents to promote immune function during aging.
Ghrelin: an anti-inflammatory orexigenic peptide
Ghrelin has recently emerged as a potent inhibitor of proinflammatory cytokines [36
37
38
39
40
]. Acylation of ghrelin at the third serine residue is believed to be critical for its biological activity and ability to bind to the ghrelin receptors [99
]. Given high abundance of esterases in circulation, the acyl linkage is easily cleaved and is believed to be responsible for a short half-life of ghrelin [99
]. Despite this short half-life, ghrelin exerts protective effects in various inflammatory disease models including sepsis [36
, 101
, 102
], lung injury [103
], arthritis [39
], pancreatitis [104
], gastritis [105
, 106
], inflammatory bowel disease [38
], and hepatic inflammation [107
]. Interestingly, reduction in ghrelin levels is associated with increased inflammation during obesity [108
], and increased ghrelin by CR is associated with decreased inflammation [97
, 100
]. These findings are consistent with the hypothesis that ghrelin is a novel, anti-inflammatory peptide and may be a key player in coupling metabolism to immunity.
CONCLUSIONS
Regulation of energy homeostasis is a complex and tightly regulated process under control from neural as well as endocrine inputs. The development of excess adipose tissue as a result of chronic positive energy balance during obesity is associated with alterations in neuroendocrine factors regulating metabolism as well as immunity. Immune cell subsets expand within adipose tissue during obesity causing insulin resistance, and an increase in the number of adipocytes in the thymus and bone marrow with age is associated with reduced immunity. Thus, the direct interactions between immune cells and adipocytes by cell–cell contact and via their secreted products could potentially be significant during health and diseases. Compared with obesity, CR is not only on the opposite end of the metabolic and longevity spectrum but also significantly impacts immune function. It is likely that prolongevity effects of CR are a result of its salutary effects on immune-surveillance pathways. The exact cellular and molecular mediators responsible for integrating these two extreme metabolic states of obesity and CR to immune function are only beginning to be elucidated. Obesity, for example, is known to suppress ghrelin and increase circulating leptin levels, while CR enhances ghrelin and reduces leptin production. Emerging evidence suggests that apart from classical cytokines and chemokines, immune cell function is also regulated by neuroendocrine peptides such as ghrelin and leptin. The identification of immune effects and molecular pathways used by such metabolic neurohormones could offer potentially novel approaches to promote immunity and increase healthy lifespan.
ACKNOWLEDGEMENTS
This work was supported in part by the COYPU Foundation, Department of Health and Human Services grant (National Institutes of Health AG031797), and the Pilot and Feasibility Award by a Clinical Nutrition Research Unit Center Grant #1P30 DK072476 sponsored by National Institute of Diabetes and Digestive and Kidney Diseases. I thank Drs. Hyunwon Yang and Yun-Hee Youm in my laboratory for many exciting findings and discussions that have helped to shape this review. I also thank Drs. Richard Boyd, Vijay Kuchroo, Giuseppe Matarese, Janko Nikolich-Zugich, and Joost Oppenheim for thoughtful discussions about the contents of this article presented during the Pennington Symposium on "Neuroendocrine-Immune Signaling and Inflammation" in Baton Rouge.
Received January 14, 2008; revised April 23, 2008; accepted April 25, 2008.
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