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Leptin is an adipose-derived hormone with a key role in regulation of energy homeostasis and metabolism, including appetite and starvation. It also plays an important role in the regulation of immune responses and possibly in the autoimmune diseases. Leptin has pro-inflammatory effects on T cell populations, shifting the T helper balance towards a T helper 1 phenotype, by inducing release of pro-inflammatory cytokines, and by stimulating macrophage and natural killer cell function, the phenomena that happens also in multiple sclerosis. In addition, leptin or leptin receptor-deficient mice are less affected by or are protected against the development of variety of autoimmune diseases including experimental autoimmune encephalomyelitis (animal model of multiple sclerosis). Given the immunoregulatory properties of leptin it would be interesting to evaluate whether antagonising its activity could have therapeutic benefits for patients with multiple sclerosis. The link between leptin and multiple sclerosis may therefore offer a new therapeutic strategy for multiple sclerosis. This review will discuss recent findings about the role of leptin in onset, development, clinical manifestations and outcome of multiple sclerosis and applications of a possible leptin antagonist for multiple sclerosis patient and strategies for designing such an antagonist.
In 1994, leptin was discovered by Friedman and colleagues as the product encoded by the ob gene through the study of obese mice. The ob/ob or obese mouse is a mutant mouse suffering from a complex syndrome primarily characterised by excessive eating, which results in profoundly obese mice . Leptin is a protein acting as both hormone and cytokine consisting of 167 amino acids and is an Î±-helical-bundle cytokine. The structure of leptin is highly similar to other members of this large cytokine family including growth hormone, interleukins such as interleukin-6 (IL-6), IL-11, IL-12, granulocytes colony stimulating factor (G-CSF) and leukemia inhibitory factor (LIF) [4, 5]. Leptin is predominantly produced by adipocytes and its circulating level positively correlates with white adipose tissue mass. Administration of leptin to ob/ob mice increases basal metabolism and reduces food intake, leading to a marked rapid weight loss [7-9].
Leptin interacts with leptin receptor, also known as Ob-R which is encoded by the db gene in human and has a single transmembrane-spanning domain . Ob-R has also been designated as CD295 (cluster of differentiation 295) and belongs to the class I cytokine receptor superfamily . Six isoforms of leptin receptor has been identified (Ob-Ra, b, c, d, e and f): one long (Ob-Rb), four short (Ob-Ra, c, d and f), and one secreted (Ob-Re) [13, 14], which are products of alternative mRNA splicing, and differ in the length of their intracellular tails but share identical extracellular-binding domains. . Leptin binds to the ventromedial nucleus of the hypothalamus, known as the "appetite centre". Ob-Rb is present in a number of hypothalamic nuclei . The long isoform Ob-Rb has a long intracellular domain in human and is responsible for most of the known effects of leptin through its complete intracellular tail, at which the signalling of four different pathways involving JAK-STAT, MAPK, PI3K and AMPK can occur . Ob-Rb is also expressed by endothelial cells, CD34+ haematopoietic bone marrow precursors, monocytes/macrophages, T and B cells [5, 10, 17-22]. db/db mice have a deletion in the long isoform of the leptin receptor and thus resistant to leptin .
The short form (Ob-Ra) is much more widely expressed, often at higher levels compared to long form, and is expressed in different organs such as in the choroid plexus, kidney, cells of the immune system, lung and liver . The short isoforms is believed to have some signalling capabilities and also might be involved in leptin transport through the blood-brain barrier and maybe in other unknown functions .
The cytokine structure of leptin and recent evidence has indicated that it has a pleiotropic nature . Probably the main role of leptin is to regulate body weight through the inhibition of food intake and to increase energy consumption by increased thermogenesis . Leptin appears to be part of the complex network that coordinates immune responses to various stimuli. It balances the body's energy status and thus adjusts the immune response to an appropriate level. Immune response is an energy-demanding process, and impairment of this process during starvation may save energy necessary for vital body functions. Such interaction between energy homeostasis and the immune system appears to be bi-directional .
a. Leptin and the immune system
The pleiotropic role for leptin in mammalian physiology is clearly shown by the complex syndrome exhibited by leptin-deficient ob/ob mice and leptin receptor-deficient db/db mice. Those mice are not only obese, but also have abnormalities in reproductive function, hormone levels, wound repair, bone structure, and immune function [20, 28-32]. In addition, the ob/ob and db/db mice suffer from thymic atrophy and have reduced numbers of circulating lymphocytes [33-35]. Impaired T cell immunity in these mice indicates towards a direct effect of leptin on T lymphocytes , which might be due to the fact that CD4+ and CD8+ T cells express functional leptin receptor [36, 37]. Leptin concentrations lowered by starvation appear to correlate with impaired immune responses in mice . Since administration of leptin in ob/ob but not db/db mice normalised the immune dysfunctions, a direct role for leptin on immune system has been suggested [29, 39].
Several authors have reviewed the recent findings about leptin and its relationship with immune system and autoimmune diseases [40-50]. The effects of leptin on adaptive immune responses have been more extensively investigated compared to innate immunity.In vitro studies have shown that leptin enhances proliferation of circulating blood T lymphocytes in a dose-dependent manner [36, 37]. Addition of physiological concentrations of leptin to a Mixed Lymphocytes Reaction (MLR) induces a dose-dependent increase the proliferation of CD4+ T cell . Considering that congenital deficiency of leptin increases the frequency of infection and related mortality , it was hypothesized that a low concentration of serum leptin might contribute to increased susceptibility to infection by reducing T helper cell priming and by affecting thymic function [21, 29]. Leptin appears to affect the T helper (Th) subsets, shifting the balance towards the T helper one (Th1) subtype by stimulating production of the Th1 pro-inflammatory cytokines such as, IL-2, interferon gamma (IFN-Î³), Tumour Necrosis Factor alpha (TNF-Î±), and IL-18, and decreases production of the Th2 cytokines: IL-4, IL-5 and IL-10 [36, 37]. These effects are not seen on T lymphocytes of db/db mice, supporting the concept that this effect is directly mediated through the leptin receptor, expressed on the T lymphocytes.
Leptin also exerts some effects on the immune cells. Peritoneal macrophages from ob/ob mice display a lower phagocytic activity, compared to macrophages from normal mice, and when leptin was administred , the phagocytic activity was restored . Furthermore, the production of granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF)  and the pro-inflammatory cytokines such as, TNF-Î±, IL-6 and IL-12 , by murine macrophages is enhanced after treatment with leptin. It was also shown that leptin induces production of TNF-Î±, IL-6 and IFN-Î³ from resting human peripheral blood mononuclear cells (PBMCs) and enhances release of these cytokines from stimulated PBMCs . In human neutrophils, leptin seems to mediate its effects through an indirect mechanism, probably involving the release of TNF-Î± from monocytes. This protein acts as a chemo-attractant for lymphocytes and monocytes , recruiting activated T cells to the site of inflammation [56, 57]. Moreover, in ob/ob mice, the number of intraepithelial lymphocytes is reduced and these cells show a decreased IFN-Î³ secretion, while the lamina propria mononuclear cells of these mice show increased apoptosis.
Leptin also seems to be a regulator of natural killer (NK) cells development and activation. The db/db mice show decreased numbers of NK cells in the liver, spleen, lung and peripheral blood, and in normal mice leptin administration increases the basal or induced lysis of splenocytes, but not in db/db mice .
b. Leptin and Autoimmunity
Leptin, as mentioned before, plays an important role in CD4+ T cell mediated immune responses, promoting a pro-inflammatory Th1 response. The Th1 promoting effects of leptin have been linked to an enhanced susceptibility to develop experimentally induced autoimmune disease including Experimental Autoimmune Encephalomyelitis (EAE), type 1 diabetes melitus (T1D), and Antigen-Induced Arthritis (AIA) . Accumulating evidences suggest that leptin also plays a pivotal role in the development of CD4+ T cell mediated autoimmune diseases including Crohn's disease, Rheumatoid Arthritis (RA), MS and type I diabetes melitus . ob/ob mice are resistant to the induction of several experimental models of inflammatory and autoimmune diseases, such as experimental arthritis , T cell-mediated hepatitis  and acute and chronic intestinal inflammation .
In experimental mouse model systems for human inflammatory bowel disease (Crohn's disease with acute and chronic colitis), leptin-deficient ob/ob mice showed a 72% reduction of colitis severity and a marked decrease of pro-inflammatory cytokines (IFN-Î³, TNF-Î±, IL-1Î², IL-18 and IL-6) in colon cell culture supernatants, compared to wild type mice . Administration of leptin in ob/ob mice eliminate this resistance against experimentally induced colitis. In this model, Clostridium difficile toxin A caused severe colitis but ob/ob as well as db/db mice were partially protected against the toxin A induced intestinal secretion and inflammation . In this case also leptin administration in ob/ob, but not in db/db mice reversed this effect .
Chronic idiopathic thrombocytopenic purpura (ITP) is an organ-specific autoimmune disease characterized by the production of antibodies against antigens on the membranes of platelets, resulting in enhanced destruction of the platelets by macrophages. Leptin enhances in vitro secretion of IgG anti-platelet antibodies by splenocytes and PBMCs from patients with chronic ITP. After depletion of CD4+ T cells, leptin lost this function. Further studies showed that leptin could increase platelet reactive T cells. These findings suggest that leptin may be involved in the pathogenesis of chronic ITP and might offer a potential target for the treatment of this disease .
There are data suggesting a role for leptin in the development of Rheumatoid Arthritis (RA). Injection of methylated bovine serum albumin (BSA) in the knees of mice results in the development of antigen-induced arthritis. Whereas, ob/ob and db/db mice develop less severe arthritis as compared to wild type mice, with decreased IL-1Î² and TNF-Î± in the knee synovial fluid and decreased serum levels of anti-methylated BSA antibodies. Furthermore, a decreased antigen-specific T cell proliferative response, with a lower IFN-Î³ and a higher IL-10 secretion, typical for a shift towards an anti-inflammatory Th2 phenotype is also reported . Reducing leptin levels in RA patients by fasting ameliorate the clinical signs of the disease .
In the Non-Obese Diabetic (NOD) mouse, an animal model for type 1 diabetes (an autoimmune disease, in which the pancreatic Î²-cells are destroyed by inflammatory processes), an increased serum level of leptin precedes the diabetes in susceptible females, while injection of leptin accelerates the autoimmune destruction of the pancreatic Î²-cells and increases the IFN-Î³ production in peripheral T cells. These effects indicate that leptin promotes the development of type 1 diabetes through Th1 responses . It has been found that natural leptin receptor mutants of the NOD/LtJ strain of mice (named NOD/LtJ-db5J) display reduced susceptibility to T1D .
Women have higher circulating leptin levels than men.Women are more prone to acquire autoimmune diseases , this sexual preponderance could be justified by higher average leptin concentrations in women.
c. Leptin and multiple sclerosis
MS is an autoimmune neurologic disorders which affects young adults and, although its cause is not fully understood, it appears to involve a complex interplay of genetic, environmental and immunologic factors . Disease beginning usually occurs in young adults, is more common in women. Multiple sclerosis is an example of an autoimmune disease that many cytokines and chemokines can influence its progression and severity.
It has long been known that myelin-reactive Th1 CD4+ cells can induce MS, and Th1 cytokines are elevated in the CNS inflammatory lesions of EAE . In contrast, Th2 cytokines typically associate with recovery from EAE and protection from the disease . As was mentioned before, leptin is known to promote immune response toward Th1. One of the most convincing evidences demonstrating the crucial role of lepin for induction of EAE has been presented by Matarese et al . They have clearly shown that leptin is required for development of EAE, and thus, possibly also for multiple sclerosis. Genetically leptin deficient mice (ob/ob mice) are resistant to induction of both active and adoptively transferred EAE. This protection is reversed by leptin administration and associates with a switch from Th2 to Th1 type responses and IgG1 to IgG2a isotype switch. Similarly, in susceptible wild-type C57BL/6J mice, leptin worsens EAE disease by increasing IFN-Î³ release and IgG2a production. Surge of serum leptin anticipates the onset of clinical manifestations of EAE.. Leptin gene transcription is induced attendant with the polarization toward Th1 responses which are often involved in T-cell-mediated autoimmune diseases including MS and in situ secretion of leptin near inflammatory T cells and macrophages has been observed in active EAE lesions .
There have been a number of studies investigating the relationship between leptin and multiple sclerosis in patients. Leptin is raised high 6.5-fold in acute/active MS versus chronic silent MS . In acute phases of MS, leptin secretion and CSF production of IFN-Î³ are increased. In this condition, increased leptin secretion is present both in the serum and in the CSF of patients with MS and does not correlate with body mass index (BMI) . The increase of leptin in the CSF is higher than in the serum, suggesting possible secondary in situ synthesis of leptin in the CNS and/or an increased transport across the blood-brain-barrier following enhanced systemic production. Reports have shown increased secretion of serum leptin before relapses in patients with MS during treatment with IFN-Î², and a capacity of leptin to enhance in vitro secretion of TNF-Î±, IL-6, and IL-10 from peripheral blood mononuclear cells of patients with MS in acute phase of the disease but not in patients with stable disease. It has been reported that the secretion of leptin is increased in both serum and cerebrospinal fluid (CSF) of naive-to-treatment patients with MS, an aspect that positively correlates with the secretion of IFN-Î³ in the CSF and inversely correlates with the percentage of circulating Regulatory T cells (or Treg cells, a subset of lymphocytes formerly known as suppressor T cells, are crucial for the maintenance of immunological tolerance that is reduced in patients with MS as compared with healthy matched controls). Importantly, the number of peripheral Treg cells in patients with MS inversely correlates with the serum levels of leptin, suggesting a link between the number of Treg cells and leptin secretion . Autoreactive human myelin basic protein (hMBP)-specific T cells from patients with MS produced leptin and upregulated the expression of leptin receptor after activation [44, 81, 83]. Up-regulation of Ob-R in mononuclear cells from relapsing-remitting multiple sclerosis (RRMS) patients in relapse, but not in remission and controls have been observed. This finding suggests that Ob-R could play a role in the pathogenesis of MS by up-regulating the immune response in the acute phase of the disease .
d. Leptin antagonism
The above presented evidence suggesta an involvement of leptin in CNS inflammation in the EAE model and MS. Therefore leptin antagonism could offer a new treatment option for MS patients.
It has been shown that blocking of leptin with anti-leptin antibodies or with a soluble mouse leptin receptor chimera either before or after onset of EAE, improved clinical score, slowed disease progression, reduced disease relapses, inhibited proteolipid protein 139-151(PLP139-151) myelin peptide-specific T cell proliferation, and switched cytokine secretion toward a Th2/regulatory profile. CD4+ T cells from mice treated with leptin antagonists showed hypo-responsiveness to PLP139-151 peptide, which was indicated by accumulation of cyclin-dependent kinase inhibitor p27(p27Kip-1). Hyporesponsive state induced by leptin antagonism was associated with marked increase of extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation, confirming involvement of ERK1/2 in the improvement of EAE .
Both anti-leptin and anti-leptin receptor blocking antibodies reduced the proliferative responses of the hMBP-specific T cell lines to antigen stimulation, underlying a possibility of leptin-based intervention on this autocrine loop to block autoreactivity .
Leptin neutralization with leptin antagonists could improve clinical onset, progression, and clinical relapses of both actively induced and passively transferred EAE. This effect was associated with marked inhibition of delayed-type hypersensitivity (DTH) reaction against PLP139-151 peptide, CD4+ T cell hyporesponsiveness, and increased IL-4 and IL-10 production against myelin antigens. Foxp3 which is a selective marker for Treg cells, a cellular subpopulation known to be involved in the control of immune tolerance, was also expressed more on CD4+ T cells in leptin-neutralized mice, suggesting the induction of a regulatory phenotype. At the biochemical level, T cell hyporesponsiveness might be explained by the failure to downmodulate the anergy factor p27Kip-1 and by the increase in the tyrosine phosphorylation levels of ERK1/2 and STAT6. These finding provide a framework for leptin-based intervention in EAE and identify molecules with possible therapeutic potential for the disease . It has been also shown that leptin neutralization improves the EAE course by profoundly altering intracellular signalling of myelin-reactive T cells and increasing the number of regulatory T cells .
A critical point about leptin is its pleiotropic nature as discussed earlier and any attempt to block the leptin signalling in vivo should be carefully planned as it may cause undesirable effects. The concern in the development of leptin-based therapeutic strategies for autoimmune diseases is that complete leptin/leptin receptor blockage also interferes with leptins hypothalamic body weight regulating role. Indeed, treatment of mice with the S120A/T121A leptin mutant which act as leptin antagonist induces significant weight gain. The weight gain of S120A/T121A treated mice implies that the mutant works centrally, and thus is actively transported over the blood-brain-barrier .
There are different approaches for designing antagonists. Blocking common important signal pathways, such as JAK-STAT, may results in detrimental effects. So far, there is no approved commercially available leptin antagonist that can be used for clinical studies. The recent development ofleptin mutants with antagonistic properties and other proteins that block leptin activity opens up new possibilities for their use in research and, eventually, therapy . A monoclonal antibody against human leptin receptor with antagonistic effect have been previously described . This antibody inhibits the pro-inflammatory activity of leptin by its ability to block peripheral immune actions of leptin and leptin-induced induction of TNF-Î± by human monocytes, and T cell proliferation . The DNA sequence of this antibody is cloned and different parts (Fab and ScFv) are produced with the same blocking effect as whole antibody. The greatest advantage of recombinant antibody (rAb) technology is that rAb can be manipulated genetically (eg; humanized conjugated with other molecules, etc) and more importantly producing bispecific molecules which bind simultaneously to at least two different molecules. Therefore they can block a specific molecule (such as leptin receptor) on a specific target tissue.
The adipose tissue and neuroendocrine system also secretes other factors which in addition to playing an important role in the regulation of food intake and metabolism also affect significantly the immune system. These mediators include adiponectin, visfatin, neuropeptide Y (NPY), and ghrelin . Ghrelin is a hormone stimulated by NPY and agouti, and is secreted mainly by the stomach and also from the small intestine, pancreas and thyroid. Ghrelin is secreted when blood levels of leptin and glucose drop, and stimulates appetite. It usually augments before meals and decreases after food intake . It stimulates the anterior pituitary gland to secrete growth hormone and is a biological antagonistic to leptin. Ghrelin has also anti-inflammatory effects toward the leptin-induced secretion of inflammatory cytokines as well as a powerful action for thymic homeostasis . It has been shown that ghrelin blocks the leptin-induced secretion of proinflammatory cytokines by human T cells , and suppresses EAE which is mediated by reducion in mRNA levels of TNF-alpha, IL-1beta, and IL-6 in the spinal cord cellular infiltrates and microglia of treated mice. The use of ghrelin also could represent a biological antagonism for leptin and thus useful in the treatment of MS.
Adequate nutrition is a prerequisite for generating appropriate immune responses against pathogens. Adversely, sufficient energy stores may be one of the factors required for long-term, detrimental immune reactions, as observed in autoimmune diseases. Thus, leptin can be considered as a link between the immune tolerance, metabolic state, and autoimmunity. Leptin as a cytokine might be responsible for determining the balance between predisposition to infections and predisposition to autoimmune diseases, by higher circulating leptin levels predisposing to autoimmune diseases, and lower circulating leptin levels to infection. As early leptin research has primarily been focussed on the effect of leptin on body weight regulation, little attention has yet been given to the development of leptin antagonists specifically designed for peripheral effects. According to the evidence presented above, leptin receptor antagonist could represent a novel therapeutic approach for autoimmune diseases, including MS. Identification of a monoclonal antibody against the human leptin receptor which blocks leptin signalling is probably a promising tool for designing a tissue specific leptin antagonist .