Obesity, Fat Mass and Immune System: Role for Leptin

1Obesity, the greater public health problem in the western world, is associated with high-incident chronic autoimmune and inflammatory pathologies, such as T2DM, NAFLD, OA, and RA, thus having a huge social and economic impact. Adipose tissue, initially considered as a simple energy storage tissue, is now recognized as an active endocrine organ and a bona fide immune organ, constituted not only by adipocytes but also by fibroblasts, endothelial cells and a wide array of immune cells (adipose tissue macrophages, neutrophils, mast cells, eosinophils, T and B cells that maintains tissue homeostasis in lean individuals. The adipocyte expansion caused by positive energy balance leads to adipocyte hypoxia, apoptosis, and cell stress, ultimately resulting in the expression of chemoattractant molecules and infiltration of inflammatory cells. The obese adipose tissue is also characterized by a markedly deregulated production of adipose tissue-derived factors, i.e., adipokines, a growing family of low molecular weight, biologically active proteins with pleiotropic functions. Adipokines are crucial players not only in energy metabolism but also in inflammation and immunity, most of them being increased in obesity and contributing to the associated ‘low-grade inflammatory state’.

2Leptin was discovered in 1994 by the group of Jeffrey Friedman and is the best-characterized member of adipokine family. Encoded by LEP gene (the human homolog of murine ob gene), leptin is a 16 kDa non-glycosylated protein mainly produced by adipocytes, but also by skeletal muscle, intestine, brain, joint tissues and bone. This adipokine exerts its physiological activity through its receptor (LEPR or Ob-R), a class I cytokine receptor family from diabetes (db) gene. There are at least six LEPR isoforms that differ in the length of the cytoplasmic domain: a soluble isoform, four short isoforms, and a long isoform, which has the full intracellular domain that allows the transduction of leptin signal via JAK and STAT signaling pathways. Alternatively to canonical JAK/STAT pathway, LEPR could activate ERK 1/2, p38 MAPK, JNK, PKC, and PI3K/Akt pathways (Figure 1). This hormone, together with other regulatory molecules, has a central role in appetite and body weight homeostasis by inducing anorexigenic factors (as cocaine-amphetamine-related transcript) and suppressing orexigenic neuropeptides (as neuropeptide Y) on hypothalamus. Therefore, central leptin resistance, caused by impairment of leptin transportation, leptin signaling and leptin target neural circuits, is considered the main risk factor for the obesity pathogenesis.. Interestingly, leptin release is modulated in a circadian rhythm manner, which has been correlated with sweet taste recognition. Moreover, leptin also affects other physiological functions, namely bone metabolism, inflammation, infection and immune responses (Figure 2). Accordingly, LEPR is expressed in across the cells of innate and adaptive immune system, evoking leptin as a crucial linker of neuroendocrine and immune systems.

3This review summarizes the latest data regarding the role of leptin as a mediator of innate and adaptive immune cells activity, and its effects on obesity-associated metabolic disorders, namely T2DM and NAFLD, and autoimmune and/or inflammatory rheumatic diseases, such as OA and RA.

4The rising prevalence of obesity in western society is paralleled with a significant augment in autoimmune diseases. Accordingly, numerous association studies had demonstrated that overweight is implicated in a higher risk of developing multiple sclerosis, RA, and psoriasis. On the other hand, malnutrition/starvation has been long related to increased susceptibility to infectious diseases. These observations bring out immune response as a highly energy-dependent biological process that is dependent on an adequate food intake and metabolism. In fact, a recent article critically discuss the correlation between autoimmunity and overnutrition or metabolic pressure. At the interface of the historically distinct fields of immunology and metabolism, immunometabolism has emerged as a new research discipline. In the last years, it has been evidenced that metabolic status of immune cells directly determines their function and differentiation, thus affecting immunity and tolerance, as well as the failure of the immune response in autoimmune pathologies. In fact, innate and adaptive immune cells adapt to altered tissue microenvironment, characterized by hypoxia and nutrient competition, by reprogramming their metabolism, and failure in this metabolic reconfiguration ultimately leads to a deregulated immune response and pathology.

5Leptin, the forerunner of adipokine family, is a key sensor of energy metabolism and a cornerstone in the regulation of metabolism-immune system interplay. Malnutrition results in hypoleptinemia, while obesity leads to hyperleptinemia, both conditions affecting the immune response in an opposite manner. In particular, obese subjects demonstrated decreased levels of Treg (central players in the control of peripheral immune tolerance), which are inversely correlated with leptin levels and BMI. In malnutrition, altered T cell function and metabolism was associated with decreased leptin levels. Leptin and LepR-deficient mouse models presented augmented number and activity of Treg cells together with a resistance to autoimmune diseases, and leptin replacement rescues Treg cell levels to wild-type mice values. Accordingly, human T cell activation and production of cytokines can be induced after incubation with 10 ng/mL exogenous leptin following nutritional rehabilitation. Furthermore, the central effect of leptin on the hypothalamus is mediated, at least in part, by inhibition of hypothalamic-pituitary-adrenal axis and activation of the sympatho-adrenal axis, having the sympathetic nervous system a function in the central control of the immune system. Moreover, most immune cells express LEPR at their surface, which evokes a straight action of leptin in the modulation of the immune response.

6Human polymorphonuclear neutrophils express LEPR, but only the short-form (Ob-Ra) was been detected. Although the short-form of LEPR lacks most of the intracellular domain of the receptor, it is enough to signal through MAPK pathways, enhancing CD 11b expression and preventing apoptosis, but not through JAK-STAT pathways as long-form of LEPR. Leptin is likely to act as a survival cytokine for neutrophils. At 500 nM, leptin delayed the cleavage of Bid and Bax, mitochondrial release of cytochrome C and second mitochondria-derived activator of caspase, as well as the activation of caspase-3 and caspase-8. PI3K, NF-κB, and MAPK pathways were involved in the anti-apoptotic activity of leptin in human neutrophils in vitro. Additionally, leptin (250 ng/ml) stimulated the release of oxygen radicals, such as superoxide anion and hydrogen peroxide, by PMNs.

7There is strong evidence for an effect of leptin on neutrophil chemotaxis and infiltration. Leptin (50 ng/ml) mediated the migration of human neutrophils in vitro, through activation of p38 MAPK and Src kinases, and by indirect mechanisms via TNF-α released by monocytes, having no secretagogue properties (no detectable [Ca2+]i mobilization, oxidant production, or β2-integrin upregulation). Otherwise, leptin inhibits neutrophil chemotaxis to classical chemoattractants, like interleukin (IL)-8. Murine neutrophils with Q223R LEPR mutation had reduced chemotaxis toward leptin, while neutrophils from human volunteers and wild-type C57BL/6 mice migrated toward leptin in a dose-dependent manner, requiring JAK2/PI3K signaling. Nevertheless, one study found that physiological concentrations of leptin (1–100 ng/ml) do not affect human neutrophils, and high leptin concentrations induced survival and changes in neutrophils proteome, but no effect on chemotaxis was observed. In vivo studies clarified the effect of leptin in neutrophils. It was observed that neutrophil populations were enhanced in rats with high-fat-diet induced obesity, compared with control diet rats, and neutrophils from obese subjects displayed elevated superoxide release and chemotactic activity. Furthermore, leptin administration (50 μg) increased pulmonary neutrophilia in Escherichia coli pneumonia murine model as well as in healthy mice.

8Alike neutrophils, both human eosinophils and basophils expressed LEPR on the cell surface. In eosinophils, leptin (50 ng/ml) enhanced the release of pro-inflammatory cytokines IL-1β and IL-6, and chemokines IL-8, growth-related oncogene-α and MCP-1. It also modulated the surface expression of adhesion molecules; in particular, up-regulates ICAM-1 and CD18, and suppress ICAM-3 and L-selectin. Treatment of human eosinophils with recombinant leptin in vitro delayed apoptosis via JAK, NF-κB, and p38 MAPK signaling pathways, suggesting leptin as a survival cytokine, similar to neutrophils. Furthermore, leptin also stimulated chemokinesis and enhanced chemotactic migration of eosinophils isolated from human peripheral blood, in a dose-dependent manner, however, the underlying mechanisms remain unclear. In obese individuals, eosinophils demonstrated greater adhesion and chemotaxis toward eotaxin and RANTES (CCL5), compared with non-obese healthy volunteers.

9In human basophils, leptin treatment (10 nM) induced a strong migratory response, promoted the secretion of type 2 cytokines IL-4 and IL-13, and up-regulated the cell surface expression of CD63, which may have an exacerbating action on allergic inflammation. Moreover, leptin is a survival-enhancing factor of human basophils, as aforementioned for eosinophils and neutrophils. Although leptin was a weak effect on direct induction of basophil degranulation, it potently primed basophils for enhanced degranulation in response to aggregation of IgE or its high-affinity receptor FcεRI.

10Altogether, these findings suggest leptin as a potent activator of neutrophils, eosinophils, and basophils through its positive action in cell survival, cytokines release and chemotaxis.

11Both isoforms of LEPR are expressed in PBMCs, being lower in cells from obese individuals compared with lean subjects. Functional LEPR was also expressed in macrophages. The effect of leptin on monocytes and macrophages has been well-established since its first evidence in. Leptin promoted the proliferation of human circulating monocytes in vitro as well as its activation through induction of TNF-α and IL-6 production, and stimulation of surface markers, namely CD25, HLA-DR, CD38, CD71, CD11b, CD11c, and CD16. Moreover, leptin potentiated the stimulatory effect of LPS or PMA on human monocytes, and increased CCLs in cultured murine macrophages, being JAK2-STAT3 signaling pathway involved. Leptin (625 nM) also augmented the production of several inflammatory mediators in monocytes/macrophages, such as interleukin 1 receptor antagonist (IL-1Ra), interferon-c-inducible protein, leukotrienes, nitric oxide, and pro-inflammatory cytokines, namely TNF-α, IL-6, IL-1β, and resistin. By contrast, it was reported that 1 μg/ml leptin had no effect on IL-1β secretion but enhanced IL-18 in the THP-1 murine monocytic cell line. These apparent discrepancies could be species-specific (human vs. murine cells) and/or leptin treatment-dependent (1 μg/ml for 24 h vs. 1 μg/ml for 3 h). Additionally, recombinant leptin increased the expression of TLR2, but not TLR4, in human monocytes.

12A dose-dependent effect of leptin as a trophic factor to prevent apoptosis was found in serum-deprived human monocytes, being this effect mediated by the p42/p44 MAPK pathways. Leptin (2 nM) stimulated the oxidative burst in monocytes, and increased LPL expression through oxidative stress- and PKC-dependent pathways. Moreover, leptin promoted the phagocytosis of apoptotic cells by macrophages from lupus mice, via modulation of cAMP levels. Leptin also promoted a defensive environment against Leishmania donovani infection by induction of macrophage phagocytic activity and intracellular ROS generation. Accordingly, macrophages from knocked-out LepR Tyr 985 mice presented reduced phagocytosis and killing activity of Klebsiella pneumoniae that was associated with diminished ROS production. It has been demonstrated that leptin-mediated protein radical formation, tyrosine nitration and activation of KCs are caused by peroxynitrite formation, which exacerbated NASH in diet-induced obese mice. Concerning to chemoattractive activity, it was verified that leptin induced in vitro chemotactic responses for monocytes and macrophages, via intracellular calcium influx, JAK/STAT, MAPK and PI3K pathways. However, it has been reported that hematopoietic LEPR deficiency in mice did not change macrophage accumulation in WAT after diet-induced obesity versus wild-type mice. Likely, compensatory in vivo effects of other cytokines (like IL-1 or TNF-α) present in WAT could occur in obese individuals and which are absent in in vitro assays.

13Leptin treatment (50 ng/ml) of human macrophages in culture, induced ‘alternatively activated’ or M2-phenotype surface markers, but they were able to secrete M1-typical cytokines (TNF-α, IL-6, IL-1β, IL-1ra, IL-10, MCP-1, and macrophage inflammatory protein 1-alpha (MIP-1α)), suggesting a role for leptin in the phenotype of macrophages found in adipose tissue. In macrophages, leptin also triggered catecholamine-dependent increases in cAMP-histone deacetylase 4 signaling pathway, that reduced inflammation in adipose tissue. Additionally, leptin increased the expression of LEPR in M2 macrophages and stimulated IL-8 expression via p38 and ERK signaling pathways. In tumor-associated macrophages, leptin induced the expression of IL-18 via NF-kB, possible contributing to tumor progression.

14Macrophages are indirectly regulated by leptin through mast cells. In particular, leptin expression was reduced in both human and mouse mast cells from lean adipose tissue compared with obese individuals. Leptin deficiency led to the anti-inflammatory activity of mast cells and, consequently, to a shift in macrophage polarization from M1 to M2; in vitro co-cultures of mast cells with BMDM increased IL-4-mediated arginase-1 and IL-10 expression, and suppressed LPS-mediated iNOS and IL-6 expression. Furthermore, reduction of mast cells in leptin-deficient ob/ob mice exacerbated obesity and diabetes, indicating an important role of mast cells in obesity-related inflammation through its reactivity to leptin levels.

15Biologic drugs used for the treatment of psoriatic arthritis, namely adalimumab (an anti-TNF-α monoclonal antibody) and ustekinumab (a monoclonal antibody against the p40 subunit of IL-12 and IL-23), augmented LEPR expression in THP-1 human macrophages. However, only ustekinumab was able to increase the expression of leptin, suggesting a novel mechanism for this biological drug. Further mechanistic studies focused on the leptin pathway could have potential therapeutic action in common obesity-related complications of psoriasis. The establishment of LepR-deficient macrophage cell line DB-1, derived from differentiated bone marrow cells of Lepr-knockout mice, provide a powerful tool to study the role of leptin and its receptor in obesity-associated inflammation and immune system deregulation.

16The role of leptin in regulating NK cell development and activation was first verified in obese Lepr-deficient (db/db) mice, which showed decreased NK cell function. In this animal model, the population of NK cells in bone marrow was impaired through an increase in apoptotic rate, and recombinant leptin (200 ng/ml) significantly enhanced the survival of immature NK cells from wild-type mice via modulation of Bcl-2 and Bax gene expression. Furthermore, leptin administration (500 μg/kg) led to a higher activity of NK cells in lean animals. Consistently, human NK cells expressed functional long- and short-form of LEPR that influenced NK cell cytotoxicity through STAT3 activation and, consequently, transcription of genes encoding IL-2 and perforin.

17The above-mentioned results indicated that leptin signaling is required for normal NK cell immune function. However, there are some controversial findings concerning the time of leptin treatment in vitro. Short-term stimulation of human NK cells with leptin (50 nM) raised the secretion of IFNγ and cytotoxicity. By contrast, long-term exposure to leptin decreased NK cell proliferation and immune function. Obesity is partially characterized by a state of long-term, highly elevated leptin exposure, and NK cells from obese animals were significantly resistant to leptin stimulation, which could explain the functional desensitization of NK cells after long-term exposure. Accordingly, exposure of NK-92 human cell line to hyperleptinemia (similar to that observed in obese individuals) led to metabolic activation of NK-92 cells after 24 h, but there is a reduction of cell metabolism after 96 h. Furthermore, obese individuals have lower NK function compared to lean individuals and, after weight loss, the decrease of plasma leptin levels is accompanied by a restoration of IFNγ production by NK cell.

18Overall, leptin signaling seems to be necessary for normal NK cell immune function, increasing the immune activity and cell proliferation, and reducing the apoptotic rate of NK cells. Long-term exposure to hyperleptinemia, observed in obesity, has been associated with decreased NK immune activity possibly due to the development of leptin resistance. Further studies are needed to better understand the correlation between leptin levels and NK cell development and function, as well as the potential implications in obesity.

19Human DCs, both immature and mature DCs, present functional active LEPR with the capacity to signal STAT-3 phosphorylation. Leptin (10 nM) acted as an activator of human DCs, evidenced by up-regulation of IL-1β, IL-6, IL-12, TNF-α and MIP-1α production, improvement of immature DCs migration and their chemotactic responsiveness, licensing them toward Th1 priming. Moreover, leptin treatment promoted DC survival through decreased apoptosis via activation of NF-κB and PI3K-Akt signaling pathways, with a parallel increase of bcl-2 and bcl-xL gene expression.

20Lepr-deficient db/db mouse bone marrow culture displayed a reduced number of DCs, attributable to dysregulation of Bcl-2 genes and a consequent increase of apoptosis. Moreover, DCs from db/db mice possessed markedly reduced expression of co-stimulatory molecules and a Th 2-type cytokine profile, with a poor capacity to stimulate allogeneic T cell proliferation. Consistently, db/db DCs demonstrated down-regulation of PI3K/Akt and STAT-3 pathways. Lep-deficient ob/ob mice presented a reduced expression of DC maturation markers (CD40, CD80, and CD86), decreased production of inflammatory cytokines (IL-12, TNF-α, and IL-6), and augmented TGF-β production, but ob/ob mice-derived DCs were more efficient in inducing Treg or Th17 cells than wild-type animals. In DCs from ob/ob mice, leptin deficiency resulted in defective antigen presentation function toward Leishmania donovani, which was not reversed by leptin treatment. Conversely, one report verified no changes in the phenotype, activation, antigen processing or presentation of DCs from leptin-knockout mice, but these cells showed an enhanced ability to activate T cells, suggesting that leptin may dampen T-cell responsiveness in the physiological context. Diet-induced obesity in mice fed with HFD results in an elevation of serum leptin levels and splenic CD11c+ DCs, with diminished DC cell stimulatory capacity, being these effects distinct from that caused by HFD alone in obese-resistant mice.

21Altogether, these data demonstrated the important role of leptin in DC activation, chemoattraction, and survival, with possible implications in DC maturation and migration. Given the ability of DCs to orchestrate immune response and promote potent immunogenic responses through activation of T cell immunity, DCs-based immunotherapies to elicit immunity against cancer and infectious diseases are currently being developed. In particular, DCs can be differentiated ex vivo, exposed to antigens and induced to mature in the presence of adjuvants. Then, the mature DCs are injected into the patient and migrate to the lymph nodes to present antigens to T cells. Thus, the modulation of DCs maturation and activity by leptin is of most importance considering a potential application of leptin in immunotherapeutic approaches and as novel adjuvant immunopotentiator in vaccination protocols employing ex vivo generated autologous DCs.

22The role of leptin in adaptive immunity has first evidenced working with ob/ob and db/db mice, which showed thymus atrophy, T-cell lymphopenia, and impaired delayed-type hypersensibility. Moreover, chronic leptin administration (1 μg/g body weight) reversed immunosuppressive status and thymic atrophy of ob/ob mice (, Nature;, J. Clin. Inv.). Since then, the role of leptin in T and B cell populations have been extensively studied.

23T lymphocytes expressed the long form of LEPR (higher in peripheral CD4+ than in CD8+ T cells), with signaling capacity to activate JAK-STAT pathway. Consequently, leptin modulated cell proliferation, responsiveness, and polarization of T cells. Leptin dose-dependently promoted the proliferation of human naïve (CD45RA+) CD4+ T cells, whereas it minimally affected memory (CD45RO+) CD4+ T cells proliferation. Additionally, morbidly obese children, who were congenitally deficient in leptin, presented a decreased number of circulating CD4+ T cells, as well as impaired T cell proliferation and cytokine release, which were reversed by administration of recombinant human leptin. Moreover, leptin inhibited autophagy in human CD4+CD25- conventional T cells via mTOR pathway, which emerged as the potential link between immunity and nutritional status.

24Leptin also promoted CD4+ T cell polarization toward a Th1 response (which secretes IFNγ and IL-2) rather than Th2 response (which secretes IL-4). Accordingly, under Th2-polarizing conditions, the in vitro leptin treatment decreased IL-4-producing T cells and inhibited T cell proliferation. However, it was recently reported that in vivo leptin-deficiency attenuated allergic airway inflammation and that high leptin levels associated with obesity promoted proliferation and survival of Th2 lymphocytes, as well as the production of type 2 cytokines, altogether contributing to allergic responses. Besides that, leptin was involved in thymus morphology and functions, particularly in thymocyte differentiation of double positive CD4+CD8+ T cells into single positive CD4+ T cells.

25IL-17-producing Th cells (Th17) have a crucial role in the promotion and maintenance of inflammatory and autoimmune pathologies. Leptin was demonstrated to increase Th17 population and responsiveness in SLE, via retinoic acid-related orphan receptor (ROR)γt. In collagen-induced arthritis mouse model, articular injection of leptin (5 μg) increased the number of Th17 in the joint tissue, resulting in exacerbating joint inflammation, and consequently early onset of arthritis and increased disease severity. Leptin, in concentrations similar to that found in blood during pregnancy, promoted the differentiation of peripheral blood CD4+ cells to Th17 cells, but suppressed the formation of Treg cells in vitro. CD4+ T cell-derived leptin, but not plasma leptin, were positively correlated with the percentage of Th17 cells or RORγt levels in chronic lymphocytic thyroiditis, an organ-specific autoimmune disease. Furthermore, Lepr-deficient CD4+ T cells verified a reduced capacity for Th17 differentiation, via down-regulation of STAT3 activation.

26Leptin also regulated CD4+CD25+ Treg proliferation. Treg lymphocytes play a critical role in controlling the inappropriate immune responses characteristic of autoimmune diseases and allergy. In humans, leptin negatively affected the proliferation of Foxp3+CD4+CD25+ Treg; in vitro leptin neutralization, during anti-CD3 and anti-CD28 stimulation, led to the proliferation of the isolated human Treg cell. Obese individuals presented a reduced number of CD4+CD25+CD127-Foxp3+ Treg cells, which was correlated with body weight, BMI, and plasma leptin levels. Moreover, leptin-deficient mice presented an increased percentage of peripheral Treg, compared with wild-type mice, which is reversed after leptin administration. It was verified that leptin played an important role in Treg dysfunction in patients with pulmonary arterial hypertension. Accordingly, Lepr-deficient rats developed less severe hypoxia-induced pulmonary hypertension and were protected against decreased Treg function after exposure to hypoxia. In SLE, the disease-associated higher leptin serum levels were negatively correlated with disease severity and number of Treg cells, and fasting-induced hypoleptinaemia was related to Treg population recovery in lupus-prone mice. Leptin-deficient ob/ob mice and a mouse model of lupus with leptin deficiency demonstrated increased frequency of Tregs cells. These data evidenced the potential of anti-leptin-based approaches for immune system-dysregulated pathologies associated with reduced Treg function, such as SLE, obesity, T2DM, and metabolic syndrome.

27T cell metabolism is directly related to its function; effector T cells, such as Th1 and Th17, demand a high glycolytic metabolism to fuel proliferation and function, while Treg cells require oxidative metabolism to fuel suppressive activity. Recently, leptin was found to directly promote T-cell glycolytic metabolism and consequently induce Th17 cell differentiation, being Treg cells unchanged, in a mouse model of experimental autoimmune encephalomyelitis. Leptin regulated glucose metabolism partly by up-regulation of glucose transporter Glut1. Moreover, fasting led to decreased ability of T cells to secrete IL-2 and IFNγ, and inability to up-regulate glucose uptake and glycolytic flux, while Treg expansion was increased; leptin administration (1 μg/g body weight) rescued peripheral T cell function and metabolism in fasted mice. Likely, fasting was extensively reported to be associated with immune deficiency and increased susceptibility to infection. Thus, leptin seems to provide a key link between nutritional status and inflammatory T cell responses.

28Altogether, these data revealed the ability of leptin to increase immune activity by modulation of T cell number and function. Leptin can promote proliferation of naive T cells, as well as Th1 and Th17 proliferation and cytokine production. Moreover, leptin decreases Treg cell proliferation. Considering the regulatory effects of leptin on Th17 and Treg populations, revoking leptin signaling might be a potential therapeutic approach for inflammation and autoimmunity.

29In contrast to macrophages and T cells, little is known about the role of leptin in the B lymphocytes development and function. B cells expressed the long form of the LEPR, suggesting a direct effect of leptin on B cell function. Accordingly, db/db and ob/ob presented a reduced number of peripheral blood and bone marrow B lymphocytes, which was recovered after leptin treatment. Conversely, db/db mice presented an increased absolute number of B cells in the peritoneal cavity, and the increase of leptin was correlated with a decrease in B cells of mice with unbalanced diets (carbohydrate-rich and fat-rich). Thus, further investigation is needed to better clarify the role of leptin in lymphopoiesis. Leptin promoted B cell homeostasis through inhibition of apoptosis and induction of cell cycle entry via Bcl-2 and cyclin D1 activation. Furthermore, leptin dose-dependently activated human peripheral blood B cells, inducing the secretion of pro-inflammatory cytokines, namely TNF-α and IL-6, and the anti-inflammatory cytokine IL-10, via JAK-STAT and p38MAPK-ERK1/2 signaling pathways. Likewise, leptin (50 ng/ml) activated and induced the production of higher amounts of TNF-α, IL-6, and IL-10 by B cells from aged subjects compared to young individuals, which is associated with leptin-mediated STAT3 phosphorylation.

30Leptin can also modulate B cell development – decrease pro-B, pre-B and immature B cells and increase mature B cells- in bone marrow of fasted mice, characterized by low serum leptin levels. Leptin administration reversed the starvation-induced lymphopenia of bone marrow B cells, indicating an important role of central leptin in the immune system. Moreover, leptin might regulate B cell activity in obesity. In particular, B cells were described to accumulate in murine VAT and to critically regulate T2DM-associated inflammation through activation of CD8+ and Th1 cells and release of pathogenic antibodies.

31In summary, leptin can increase B cell population by augmenting proliferation and reducing the apoptotic rate, activate B cell to secrete pro-, anti- and regulatory cytokines, and also modulate B cell development.

32Obesity is associated with life-threatening co-morbidities, including insulin resistance, T2DM, NAFLD and steatohepatitis (NASH). Adipokines, in particular leptin, mediate the crosstalk between adipose tissue and metabolic organs (especially liver, muscle, pancreas and central nervous system). Thus, leptin has emerged as a significant pathological component in the development of metabolic disorders.

33T2DM is the most significant obesity-associated metabolic disorder and their prevalence is increasing worldwide in parallel. Leptin has been proposed as a therapeutic target of T2DM, for its impact on food intake and body weight as well as its potential to improve insulin action. Interestingly, leptin-deficient mice and human have diabetic features, which were reversed with leptin replacement. The anti-diabetic effect of leptin is mediated by activation of IRS-PI3K pathway that improved insulin sensitivity in peripheral tissues. Activation of JAK2/IRS/PI3K/Akt signaling pathway by leptin and insulin triggers the translocation of glucose transporter type 4 (GLUT4) from cytosol to cell surface, and glucose uptake. Moreover, in the liver, leptin deficiencies decrease AMPK activity, which is also involved in glucose homeostasis regulation. Leptin has also been implicated in the regulation of insulin secretion by pancreatic β-cells, as well as in peripheral insulin resistance. However, clinical trials to evaluate the potential of leptin monotherapy in obese humans with T2DM failed to demonstrate therapeutic activity acutely or chronically, with no observation of important weight loss or metabolic improvements (insulin sensitization, amelioration of glucose and lipid metabolism). In this context, unresponsiveness to leptin – leptin resistance, caused by hyperleptinemia observed in obese humans, should be considered. Further understanding of leptin resistance mechanisms could enable new leptin targeted therapies for obesity and diabetes in specific subsets of patients. In fact, leptin therapy improved the diabetic condition in children and adults with familial leptin deficiency, and in lipoatrophic diabetes.

34Importantly, T2DM is associated with altered components of immune system, including modified levels of specific chemokines and cytokines, changed number and activation status of leukocytes populations and increased apoptosis and tissue fibrosis, all promoted by obesity-associated inflammation in adipose tissue. Given the modulator action of leptin in innate and adaptive immune system (deeply described above), it is rational to see leptin as a linker of T2DM development, not only with metabolism but also with inflammation. Indeed, in patients with newly diagnosed T2DM, leptin levels were correlated with CRP, an inflammatory marker broadly evaluated for its association with risk factors for T2DM pathology.

35NAFLD, the major cause of chronic liver illness in developed countries, comprises a wide group of pathologies primary caused by a buildup of fat in the liver that spans from simple steatosis to NASH, liver fibrosis, cirrhosis, and hepatocellular carcinoma. Given that NAFLD is increasing worldwide and it is associated with high-incident extra-hepatic complications such as obesity, T2DM, cardiovascular diseases and chronic kidney disease, great efforts have been made in the last years to unravel the mechanisms underlying the disease pathophysiology and further development of effective NAFLD therapies.

36Hepatic inflammation and hepatocyte injury and death are hallmarks of NAFLD/NASH. Fat overload by hepatocytes causes lipotoxicity and the release of DAMPs which activated Kupfer cells (KC; specialized liver macrophages) and HSC promoting inflammation and fibrosis, respectively. KC activation plays a central role in NAFLD pathophysiology through the production of pro-inflammatory cytokines and chemokines such as TNF-α, IL-1β, IL-6, CCL2 and CCL5, that contributed to leukocyte infiltration and inflammatory necrosis of hepatocytes, and fibrogenesis. Dysbiosis of the gut microbiota may also conduct to KC activation through PAMPs, which originate in the gut and reach liver via portal circulation due to altered intestinal permeability. Other immune cells have been implicated in the NAFLD pathophysiology, although its role is less clear. NK cells were impaired in experimental NASH, while natural killer T cells (unique immune cell subtype that expresses NK cells surface markers as well as T-cell antigen receptor) are depleted in steatosis but increased later during disease progression likely leading to inflammation and fibrosis in NASH via the production of IL-4, osteopontin, and IFN-γ. Neutrophils exacerbate the ongoing inflammation through macrophage recruitment and cell damage via the release of myeloperoxidase, ROS, and elastase. The role of DCs in NASH is complex and somehow controversial. DCs rapidly infiltrate into the liver in experimental NASH exhibiting an activated immune phenotype, but its depletion exacerbates hepatic inflammation. B- and T-cells also contributed to hepatic inflammation via secretion of pro-inflammatory cytokines that stimulated KC activation.

37Considering the strong metabolic and inflammatory components of NAFLD, leptin is regarded as a key regulator of NAFLD physiopathology. Leptin seems to feature a dual activity in NAFLD experimental models by exerting an early protective anti-steatosis effect in the initial stages of the disease, and a late pro-inflammatory and pro-fibrogenic action, when the disease persists or progress. In leptin-resistant Zucker fa/fa diabetic fatty rats, the expression of SREBP-1c (master regulator of glucose metabolism, and lipid and fatty acid production) is increased in liver, and infusion of adenovirus-leptin decreased hepatic triglyceride synthesis and β-oxidation via SREBP-1c down-regulation and PPARα up-regulation, thus preventing hepatic lipid accumulation.

38Through anti-steatotic effect, leptin can ultimately lead to hepatic detrimental effects. Leptin activated HSCs, leading to up-regulation of pro-inflammatory and pro-angiogenic factors expression (like angiopoietin-1 and vascular endothelial growth factor), as well as collagen α1 and tissue inhibitor of metalloproteinase-1, ultimately acting as hepatic fibrogenesis inducer. Activated HSCs were able to secrete leptin, thus establishing a vicious cycle that further promotes liver fibrosis. Moreover, leptin was reported as potent HSCs mitogen and to prevent HSCs apoptosis, hence promoting the pathogenesis of hepatic fibrosis. Leptin (200 nM) increased the expression of TGF-β1 in KCs and sinusoidal endothelial cells, and connective tissue growth factor in KCs, being KCs-HSCs cross-talk proposed for liver fibrosis. Additionally, leptin is involved in macrophage-mediated KCs activation via induction of oxidative stress in macrophages. Compared to healthy subjects, NAFLD patients demonstrated an increased leptin-stimulated TNFα and ROS production in peripheral monocytes, as well as IFNγ production in circulating CD4+ cells (a marker of Th1 differentiation). Altogether, these data elucidated the role of leptin in NAFLD by modulation of HSCs, KCs, and inflammatory cells response.

39In ob/ob mice, the congenital absence of leptin abrogated the development of CCl4-induced hepatic fibrosis comparing to lean littermates, which is reverted by leptin treatment (100 ng/ml). Likewise, xenobiotics- or thioacetamide-induced hepatic fibrosis was prevented in Zucker fa/fa rats, being involved the activation of HSCs and expression of procollagen-I and TGF-β1. In humans, the role of leptin is controversial. Although leptin serum levels were initially related with hepatic steatosis but not with necroinflammation or fibrosis, later studies failed to demonstrate any significant association. Recombinant leptin has been successfully used in the treatment of insulin resistance and hepatic steatosis in patients with lipodystrophy and NASH. However, large-scale and well-designed prospective cohort studies are necessary to deeply elucidate the role of leptin in hepatic lipid handling, inflammation, and fibrosis along with the identification of NAFLD patients subsets that may benefit from therapies directed to leptin system.

40Leptin has been described as a key factor in the pathophysiology of rheumatic diseases due to its capability to modulate bone and cartilage metabolism and to influence innate and adaptive immune responses (Figure 4).

41Osteoarthritis, the most common joint disease, is a painful and debilitating illness characterized by progressive degeneration of articular joints. Initially seen as simply “wear and tear” disease, OA is currently considered a complex and multifactorial pathology triggered by inflammatory and metabolic imbalances that affect the entire joint structure (articular cartilage, meniscus, ligaments, bone, and synovium). Leptin levels are increased in serum, infrapatellar fat pad (IPFP), synovial tissues, and cartilage of OA patients compared to healthy individuals. Accordingly, leptin-deficient or LepR-deficient mice developed extreme obese phenotype without increased incidence of knee OA, suggesting that leptin signaling is essential to the development and progression of obesity-associated OA. Furthermore, long form of LEPR was found to be expressed in human cartilage cells – chondrocytes.

42Some initial findings suggested an anabolic role of leptin in cartilage. In particular, exogenous leptin administration (30 μg) stimulated proteoglycan and growth factors (insulin-like growth factor-1 and TGF-β) synthesis in rat knee-joint cartilage. However, most of the studies reported a catabolic role of leptin underlying OA pathogenesis. A recent study determining the gene expression profile of leptin-induced articular rat cartilage by microarray analysis, associated the up-regulation of matrix metalloproteinases (MMPs), inflammatory factors, growth factors and osteogenic genes with leptin-induced OA phenotype. Our group demonstrated that leptin (400 or 800 nM), in synergy with IFNγ or IL-1β, activated type 2 nitric oxide synthase (NOS2) via JAK2, PI3K and MAPK (MEK1 and p38) pathways, in cultured human and murine chondrocytes. Nitric oxide (NO), is a well-known pro-inflammatory mediator which lead to joint degradation through induction of chondrocyte phenotype loss, apoptosis and metalloproteinase (MMP) activity. Leptin (800 nM), alone or in combination with IL-1β, also induced the expression of COX-2 and the production of PGE2, IL-6, and IL-8 in cartilage explants of OA patients and human primary chondrocytes, revealing that leptin contributed to the pro-inflammatory environment of OA cartilage. It was also demonstrated that leptin (500 ng/ml) enhanced IL-6 production, mediated by chondrocyte-synovial fibroblast cross-talk, in OA patients. Moreover, leptin modulated the production of inflammatory mediators by immune cells. In particular, the production of IL-6, IL-8, and CCL3 were increased by leptin in CD4+ T cells from OA patients, but not from healthy subjects; thus, demonstrating new insights into the role of leptin in the immune system and OA pathophysiology.

43Leptin can directly induce the expression of MMPs that are involved in OA-related joint destruction, like MMP-1 (also known as interstitial collagenase), MMP-3 (also known as stromelysin), and MMP-13 (also known as collagenase), via NF-κB, PKC, and MAPK pathways. MMP-2 (72 kDa type IV collagenase), MMP-9, and disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) 4 and ADAMTS5, were also increased by leptin, while fibroblast growth factor 2 and proteoglycan were down-regulated. Leptin (800 nM) can perpetuate the cartilage-degradation processes due to induction of VCAM-1, an adhesion molecule responsible for leukocyte and monocyte chemotaxis and infiltration to inflamed joints, via JAK2 and PI3K pathways in chondrocytes. SOCS-3 was pointed as a regulator of leptin-induced expression of MMP-1, -3, and -13, and pro-inflammatory mediators IL-6, NO and COX-2. Additionally, leptin increased the production of other adipokines, namely lipocalin-2, by human cultured chondrocytes.

44MicroRNAs, small single-stranded non-coding segments of RNA, are increasingly recognized as regulatory molecules involved in disease processes, including OA, inflammation, and obesity. miR-27 was found to be decreased in OA chondrocytes and to directly targeted the 3′-untranslated region of leptin. Furthermore, the injection of OA rats with miR-27 lentiviral overexpression vector resulted in decreased levels of IL-6 and -8, as well as MMP-9 and -13, thus indicating the protective action of miR-27 in OA, possibly by targeting leptin.

45Chondrogenic progenitor cells as cartilage seed cells are crucial to maintain cartilage homeostasis and replace damaged tissue. Leptin (50 ng/ml) can reduce CPCs migratory ability and their chondrogenic potential, and augment CPCs osteogenic transformation, hence changing CPC differentiation fate. CPC cell cycle arrest and senescence are also induced by leptin. Furthermore, leptin influenced the regulation of bone metabolism through induction of abnormal osteoblast function, which is associated to joint destruction in OA patients. The augmented production of leptin by OA subchondral osteoblasts is related with in vitro elevated levels of alkaline phosphatase, osteocalcin, collagen type 1, and TGF-β1, all being responsible for dysregulated osteoblast function. Additionally, bone morphogenic protein (BMP)-2 is increased in leptin-stimulated human primary chondrocytes. Leptin also suppressed bone formation in vivo, but there are some discrepancies with in vitro results.

46Taking together, this evidence indicated a key role of leptin in OA pathophysiology by influencing pro-inflammatory status, cartilage catabolic activity, as well as cartilage and bone remodeling. However, studies in a large cohort of patients are needed to better clarify the leptin significance in the development and progression of OA.

47Rheumatoid arthritis is a chronic inflammatory joint illness characterized by synovial membrane inflammation and hyperplasia (“swelling”), production of autoantibodies, namely rheumatoid factor and anti-citrullinated protein antibody – autoimmune disease, destruction of cartilage and bone (“deformity”), and systemic features including skeletal, cardiovascular, pulmonary, and psychological complications. Evidencing the crucial role of immune system in RA pathology, RA-associated synovitis comprises both innate immune cells (like monocytes, DCs, and mast cells) and adaptive immune cells (like Th1, Th17, and B cells). As described above, leptin modulates neutrophils chemotaxis, activates proliferation and phagocytosis of monocytes and/or macrophages, regulates NK cytotoxicity, induces proliferation of naïve T cells, promotes Th1 cell immune response and down-regulates Th2 cell immune response. Moreover, leptin modulates the activity of Treg cells, which are potent inhibitors of autoimmunity, thus having a potential implication in RA pathophysiology.

48Several studies have found a positive correlation between serum and synovial leptin levels and RA pathology, but there are controversial results. Differing results may be due to the relatively small sample size, inconsistency of the baseline characteristics of participants (age, race, disease duration, BMI, …), co-existence of other auto-immune diseases, employment of different methods to measure leptin levels in RA patients, or underlying patients treatments that intervene with the endocrine system. The present consensus is that leptin levels are elevated in RA patients, and serum and synovial fluid levels of leptin were associated with disease duration and parameters of RA activity, although large cohorts studies are necessary. Experimental animal models of arthritis had demonstrated the leptin action in joint inflammation. In particular, compared with control mice, leptin-deficient mice presented a less severe antigen-induced arthritis, decreased levels of TNF-α and IL-1β in knees synovium, and an impaired antigen-specific T cell proliferative response with lower IFN-γ and higher IL-10 production, which indicates a shift toward Th2 cell response. Accordingly, injection of leptin (5 μg) into the knee joint of collagen-immunized mice augmented arthritis severity, accompanied by elevated synovial hyperplasia and joint damage through enhancement of Th17 cell response. In fact, clinical trials using a humanized anti-IL-17 monoclonal antibody added to oral disease-modifying anti-rheumatic drugs, demonstrated improved signs and symptoms of RA, indicating the therapeutic potential of IL-17-directed strategies.

49Since leptin modulates the immune system, as well as insulin resistance and metabolic disorders like metabolic syndrome and obesity, all RA-associated conditions, this adipokine represents an attractive therapeutic target for RA. Accordingly, reducing leptin levels in RA patients by fasting improved the clinical symptoms of the disease. Amidst the possible therapeutic approaches to antagonize leptin actions in RA, are leptin mutants with antagonist activity, and monoclonal antibodies against human LEPR or leptin itself. Interestingly, clinical studies evaluating the effect of drug modulators of insulin sensitivity (affected by leptin levels as described above), such as PPARγ agonists, are ongoing to provide new potential treatment to improve the inflammatory status and cardiovascular outcome in RA patients. Further understanding of leptin mechanisms would be of utmost importance for RA treatment.

50Hence, leptin can be pointed as a link between immune tolerance, metabolic function, and autoimmunity, and leptin signaling-directed strategies could provide future innovative therapies for autoimmune disorders like RA.

51Systemic lupus erythematosus is a chronic autoimmune disorder of unclear etiology characterized by hyperactive T and B cells, autoantibody production, deposition of immune complex, elevated blood levels of pro-inflammatory cytokines and multisystem organ damage, encompassing from mild manifestations (non-erosive arthritis or skin rash) to life-threatening complications (lupus nephritis, neuropsychiatric disorders, cardiovascular disorders, and metabolic syndrome). Although the pathogenesis of SLE is poorly understood, genetic, hormonal and environmental factors have been implicated in the onset of this heterogeneous disease, which predominantly affects women of childbearing age.

52Several studies suggest the implication of adipokines, namely leptin, in the pathogenesis of SLE. Although some reports found no statistical association between disease activity and leptin levels, recently, a meta-analysis of eighteen studies determined that serum/plasma leptin levels were significantly elevated in SLE patients. Furthermore, leptin has been suggested as a player that affects the cardiovascular risk in SLE patients. Accordingly, leptin and HFD induced proinflammatory high-density lipoproteins and atherosclerosis in BWF1 lupus-prone mice, and leptin levels were correlated with BMI, disease activity index (SLEDAI), as well as insulin and CRP levels, all CVD risk factors, in SLE patients.

53The role of leptin in SLE development has been investigated using leptin-deficient (ob/ob) mice treated with lupus-inducing agent. Leptin deficiency protected mice from the development of autoantibodies as well as renal disease, and elevated the levels of Treg cells, compared with wild-type controls. Moreover, in (NZBxNZW)F1 lupus-prone mice, leptin administration accelerated the development of autoantibodies and renal disease, while leptin antagonism delayed disease progression. At cellular level, leptin promoted Th1 responses in human CD4+ T cells and in lupus-prone mice via RORγ transcription, whereas leptin neutralization inhibited Th17 responses in autoimmune-prone mice. Additionally, fasting induced hypoleptinemia or leptin-deficient mice demonstrated decreased levels of Th17 and elevated levels of Treg cells. In SLE, apoptotic cells represent the major source of self antigens that promote and fuel autoimmune responses. Leptin promoted T cell survival and proliferation of autoreactive T cells in mice with an autoreactive T cell repertoire, including (NZBxNZW)F1 lupus-prone mice. Leptin also promoted phagocytosis of apoptotic cells by macrophages in lupus-prone mice, which increase the availability of apoptotic-derived antigens to T cells and subsequent development of self-antigen-reactive T cells.

54Altogether, these data support the involvement of leptin in the development of SLE. However, further investigations are needed to fully understand the role of leptin in SLE and thus, explore this adipokine as potential therapeutic target of SLE.

55Obesity and its comorbidities, such as T2DM, non-alcoholic fatty acid liver disease, OA and RA, reached epidemic proportions and are still rising in developing countries. Anti-obesity therapeutic options have provided only a limited long-term efficacy (lifestyle changes, physical activity, diet, and pharmacotherapies) or are not completely safe (bariatric surgery). Therefore, it has become increasingly relevant to disclose new clinical biomarkers and to develop innovative therapeutic strategies for obesity-associated pathologies and chronic inflammation.

56The adipose tissue-derived factor leptin has been emerged as a key regulator of nutritional state and metabolism, as well as a modulator of immune system activation and innate-adaptive frontier; thus bridging obesity with metabolic disorders (T2DM and NAFLD) and inflammatory pathologies that affect bones and joints (OA and RA). Consequently, plasma leptin concentration could be a biological marker of the inflammatory status and the onset and evolution of pathologies associated with dysregulation of the immune system, and hereafter evaluations will be essential to establish leptin as clinical biomarker. Moreover, control of bioactive leptin levels by high-affinity leptin-binding molecules, miRNAs targeting leptin, LEPRs antagonists or monoclonal humanized antibodies against LEPR are likely to be feasible therapeutic approaches. Recombinant leptin is already available for use in patients with leptin congenital deficiency while the synthetic leptin analog metreleptin has been approved for lipodystrophy treatment. Importantly, the development of antibodies that could cross-react with endogenous leptin and cause an effective leptin-deficient state responsible for the loss of efficacy and infection has become a significant concern. However, given the pleiotropic action of leptin, a systematic approach to modulate their levels and thus prevent obesity-associated disorders might be, for the moment, unavailable. Instead, strategies targeting leptin’s actions precisely and in specific immune cell subpopulations, or targeting of specific receptor isoforms, could be a potential viable option to add novel therapeutic agents against immune-metabolic pathologies. It is now clear that leptin is an important regulator of metabolic status and influence inflammatory and immune responses in several diseases. Nonetheless, leptin network is complex and a lack of a full understanding of leptin’s immunomodulatory mechanisms in almost all of the cells of immune system and its potential side effects are still problems that need to be figured out in drug discovery. Further insights into the pathophysiological role of leptin in the immune system and in obesity-associated disorders will be of great importance for the development of novel therapeutic approaches for these diseases.

57VF and JP have made a substantial contribution to acquisition and analysis of data and critically revised it. VC-C, CR-F, AM, MG-G, and RG have been involved in drafting the manuscript and revising it critically for important intellectual content. OG made a substantial contribution to conception and design of the review article, drafting the manuscript, and critically revising it. All authors approved the final version to be published.

58The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.