We report here that leptin can act as a negative signal for the proliferation of human naturally occurring Foxp3(+)CD4(+)CD25(+) regulatory T (T(reg)) cells. Freshly isolated T(reg) cells produced leptin and expressed high amounts of leptin receptor (ObR). In vitro neutralization with leptin monoclonal antibody (mAb), during anti-CD3 and anti-CD28 stimulation, resulted in T(reg) cell proliferation, which was interleukin-2 (IL-2) dependent. T(reg) cells that proliferated in the presence of leptin mAb had increased expression of Foxp3 and remained suppressive. The phenomena appeared secondary to leptin signaling via ObR and, importantly, leptin neutralization reversed the anergic state of the T(reg) cells, as indicated by downmodulation of the cyclin-dependent kinase inhibitor p27 (p27(kip1)) and the phosphorylation of the extracellular-related kinases 1 (ERK1) and ERK2. Together with the finding of enhanced proliferation of T(reg) cells observed in leptin- and ObR-deficient mice, these results suggest a potential for therapeutic interventions in immune and autoimmune diseases.

BACKGROUND-A single family has been described in which obesity results from a mutation in the leptin-receptor gene (LEPR), but the prevalence of such mutations in severe, early-onset obesity has not been systematically examined.

SUMMARY There is a discrepancy between the in vitro anergic state of CD4+CD25hiFoxP3+ regulatory T (Treg) cells and their in vivo proliferative capability. The underlying mechanism of this paradox is unknown. Here we show that the anergic state of Treg cells depends on the elevated activity of the mammalian target of rapamycin-(mTOR)-pathway induced by leptin: a transient inhibition of mTOR with rapamycin, before T-cell-receptor-(TCR)-stimulation, made Treg cells highly proliferative in the absence of exogenous interleukin-2 (IL-2). This was a dynamic and oscillatory phenomenon characterized by an early downregulation of the leptin-mTOR-pathway followed by an increase in mTOR activation necessary for Treg cell expansion to occur. These data suggest that energy metabolism, through the leptin-mTOR-axis, sets responsiveness of Treg cells that use this information to control immune tolerance and autoimmunity.

Human regulatory T cells (Treg cells) that develop from conventional T cells (Tconv cells) following suboptimal stimulation via the T cell antigen receptor (TCR) (induced Treg cells (iTreg cells)) express the transcription factor Foxp3, are suppressive, and display an active proliferative and metabolic state. Here we found that the induction and suppressive function of iTreg cells tightly depended on glycolysis, which controlled Foxp3 splicing variants containing exon 2 (Foxp3-E2) through the glycolytic enzyme enolase-1. The Foxp3-E2–related suppressive activity of iTreg cells was altered in human autoimmune diseases, including multiple sclerosis and type 1 diabetes, and was associated with impaired glycolysis and signaling via interleukin 2. This link between glycolysis and Foxp3-E2 variants via enolase-1 shows a previously unknown mechanism for controlling the induction and function of Treg cells in health and in autoimmunity.

autoimmunity ͉ tolerance ͉ metabolism ͉ hormones I t has recently been shown that leptin, a cytokine-like hormone mainly secreted by adipocytes, can play a significant role in the pathogenesis of several autoimmune diseases, such as experimental autoimmune encephalomyelitis (EAE), antigen-induced arthritis, and experimentally induced colitis (1-6). EAE is an animal model for the human demyelinating disease multiple sclerosis (MS) (7,8). EAE can be induced in susceptible strains of mice through immunization with myelin antigens or by adoptive transfer of myelinspecific T helper 1 (Th-1) cells (7,8). Genetically leptin-deficient (ob͞ob) mice are resistant to the induction and progression of EAE, and WT EAE-susceptible mice show an increase in serum leptin preceding the clinical onset of the disease that correlates with inflammatory anorexia and disease susceptibility (2, 3). In addition, pathogenic Th-1 cells and macrophages in active EAE brain lesions secrete consistent amounts of leptin (3). These data account for an involvement of leptin in CNS autoimmunity, at least in the EAE model. Despite this finding, in humans, the role of leptin in the pathogenesis of MS is not yet fully elucidated. In this study, we analyzed the secretion of leptin in the cerebrospinal fluid (CSF) and serum of naïve-to-treatment relapsing-remitting MS (RRMS) patients and leptin's interaction with the CD4 ϩ CD25 ϩ regulatory T cells (T Regs ). T Regs are known to dampen autoreactive responses mediated by CD4 ϩ CD25 Ϫ T cells and may influence the onset and progression of autoimmunity (9). In mice, depletion of T Regs is associated with autoimmunity, and defects of T Regs have been described in nonobese diabetic mice and in humans with type 1 diabetes (9, 10). Also, reduced frequency of T Regs and͞or defective suppressor function have been observed in humans with systemic lupus erythematosus, juvenile idiopathic arthritis, autoimmune polyglandular syndrome type II, and RRMS (11-15). T Regs are anergic in vitro but can expand in vivo (9). Although the mechanisms operated by T Regs in suppression are not fully delineated, the forkhead transcription factor FoxP-3 seems to be required for suppression to occur (9).Here, we report increased leptin levels in CSF and serum of naïve-to-therapy RRMS patients and an inverse correlation with T Regs frequency. These findings may be relevant in better understanding the disease pathogenesis and may have therapeutic implications. Materials and MethodsSubjects. All MS patients and controls were recruited at the Università di Napoli ''Federico II.'' For serum and CSF leptin measurement, we included in the study 126 individuals (Table 1) with MS defined according to the criteria of McDonald et al. (16) and 117 age-, gender-, and body mass index (BMI)-matched controls with other noninf lammatory neurologic disorders (NIND). All MS patients had RRMS and an expanded disability status scale score of Յ3.5 and were naïve to treatment. The inclusion criteria for RRMS patients were (i) onset of relapse within 4 w...

Multiple Sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS) which involves a complex interaction between immune system and neural cells. Animal modeling has been critical for addressing MS pathogenesis. The three most characterized animal models of MS are (1) the experimental autoimmune/allergic encephalomyelitis (EAE); (2) the virally-induced chronic demyelinating disease, known as Theiler׳s murine encephalomyelitis virus (TMEV) infection and (3) the toxin-induced demyelination. All these models, in a complementary way, have allowed to reach a good knowledge of the pathogenesis of MS. Specifically, EAE is the model which better reflects the autoimmune pathogenesis of MS and is extremely useful to study potential experimental treatments. Furthermore, both TMEV and toxin-induced demyelination models are suitable for characterizing the role of the axonal injury/repair and the remyelination process in MS. In conclusion, animal models, despite their limitations, remain the most useful instrument for implementing the study of MS.

To elucidate whether the role of leptin in regulating neuroendocrine and immune function during short-term starvation in healthy humans is permissive, i.e., occurs only when circulating leptin levels are below a critical threshold level, we studied seven normal-weight women during a normoleptinemic-fed state and two states of relative hypoleptinemia induced by 72-h fasting during which we administered either placebo or recombinant methionyl human leptin (r-metHuLeptin) in replacement doses. Fasting for 72 h decreased leptin levels by Ϸ80% from a midphysiologic (14.7 ؎ 2.6 ng͞ml) to a low-physiologic (2.8 ؎ 0.3 ng͞ml) level. Administration of r-metHuLeptin during fasting fully restored leptin to physiologic levels (28.8 ؎ 2.0 ng͞ml) and reversed the fasting-associated decrease in overnight luteinizing hormone pulse frequency but had no effect on fasting-induced changes in thyroid-stimulating hormone pulsatility, thyroid and IGF-1 hormone levels, hypothalamic-pituitary-adrenal and renin-aldosterone activity. FSH and sex steroid levels were not altered. Shortterm reduction of leptin levels decreased the number of circulating cells of the adaptive immune response, but r-metHuLeptin did not have major effects on their number or in vitro function. Thus, changes of leptin levels within the physiologic range have no major physiologic effects in leptin-replete humans. Studies involving more severe and͞or chronic leptin deficiency are needed to precisely define the lower limit of normal leptin levels for each of leptin's physiologic targets.fasting ͉ reproductive D eficiency of the adipocyte-secreted hormone leptin (1) is associated with distinct abnormalities in energy-demanding processes such as neuroendocrine and immune function. Leptindeficient ob͞ob mice and humans with congenital complete leptin deficiency have abnormal neuroendocrine function, including hypogonadotropic hypogonadism, hypothalamic hypothyroidism, and͞or growth-hormone-axis abnormalities (2-6) and impaired cell-mediated immunity (4, 7), which are improved with leptin replacement (4,8). Similarly, starvation-induced decline of circulating leptin to very low levels in normal mice (9) and lean men (10) causes comparable neuroendocrine (9, 10) and immune defects (11, 12) that are significantly blunted or reversed with exogenous leptin.We have shown that an 80% decline of leptin levels from Ϸ2 to 0.3 ng͞ml in men mediates the fasting-induced suppression of gonadotropin and thyroid-stimulating hormone (TSH) pulsatility as well as sex steroid, insulin-like growth factor-1 (IGF-1), and thyroid hormone levels (10). Importantly, although observational studies have proposed that leptin regulates the hypothalamic-pituitarygonadal axis only when serum leptin levels fall below a ''threshold'' of Ϸ2 ng͞ml (13), the role of decreasing leptin levels to approximately, but not below, this threshold in leptin-replete humans with higher baseline leptin levels (e.g., normal-weight women) has not yet been directly studied.To elucidate whether such a threshold exists, below...

SummaryHuman CD4+CD25hiFoxp3+CD127− Treg and CD4+CD25−Foxp3− Tconv cell functions are governed by their metabolic requirements. Here we report a comprehensive comparative analysis between ex vivo human Treg and Tconv cells that comprises analyses of the proteomic networks in subcellular compartments. We identified a dominant proteomic signature at the metabolic level that primarily impacted the highly-tuned balance between glucose and fatty-acid oxidation in the two cell types. Ex vivo Treg cells were highly glycolytic while Tconv cells used predominantly fatty-acid oxidation (FAO). When cultured in vitro, Treg cells engaged both glycolysis and FAO to proliferate, while Tconv cell proliferation mainly relied on glucose metabolism. Our unbiased proteomic analysis provides a molecular picture of the impact of metabolism on ex vivo human Treg versus Tconv cell functions that might be relevant for therapeutic manipulations of these cells.