Leptin antagonism A potential novel therapeutic approach

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Objectives: This review focuses on the most recent findings about the role of leptin in onset, development, clinical manifestations and outcomes of multiple sclerosis and applications of a possible leptin antagonist for multiple sclerosis patients and strategies for designing such an antagonist. Organization: After an introduction to leptin , we will focus on its role in the immune system and autoimmunity. Afterwards we will review the literature around leptin and MS and finally will discuss the relevant potential therapeutic strategies based on the link between MS an leptin. Conclusion: Based on the available evidence strategies aimed at leptin antagonism might represent a novel therapeutic platform which deserved further attention.

Introduction

In 1994, leptin was discovered by Friedman and colleagues as a product encoded by the ob gene through the study of obese mice[1]. 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 [2]. Leptin is a protein acting as both hormone and cytokine consisting of 167 amino acids and is an ?-helical-bundle cytokine [3]. 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, granulocyte 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 [6]. Administration of leptin to ob/ob mice increases basal metabolism and reduces food intake, leading to 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 [10]. Ob-R has also been designated as CD295 (cluster of differentiation 295) [11] and belongs to the class I cytokine receptor superfamily [12]. Six isoforms of leptin receptor has been discovered (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]; these are products of alternative mRNA splicing, and differ in the length of their intracellular tails but share identical extracellular-binding domains. [15]. Leptin binds to the ventromedial nucleus of the hypothalamus, which is named the "appetite center"[16]. Ob-Rb is present in a number of hypothalamic nuclei [16]. 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 JAKSTAT, MAPK, PI3K and AMPK can occur [14]. 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 possess a deletion in the long isoform of the leptin receptor and thus are resistant to leptin [23].

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 [5]. The short isoforms are believed to have some signaling function and may also be involved in leptin transport across the blood brain barrier and possibly in other, as yet unknown functions [24].

The cytokine structure of leptin and recent evidence has indicated that it has a pleiotropic nature [25]. 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 [26]. In addition, leptin appears to be part of the complex network that coordinates immune responses to various stimuli. Leptin also balances the bodys energy status and thus adjusts the immune response to appropriate levels. Immune responses are an energy-demanding processes, and their inhibition during starvation may conserve energy necessary for survival of core body functions. Such interactions between energy homeostasis and the immune system appears to be bi-directional [27].

Leptin and the immune system

Leptin and its receptors are independently regulated gene products: the ob gene encodes for leptin, while the db gene encodes for the leptin receptor. Mice with homozygous mutations in the leptin gene are designated ob/ob and mice homozygous for mutations in the leptin receptor gene are designated as db/db. The diverse roles for leptin in mammalian physiology are clearly shown by the complex syndromes exhibited by leptin-deficient (ob/ob) mice and deficient leptin receptor mice (db/db). These mice are not only obese, but also show abnormal reproductive function, hormone levels, wound repair, bone structure, and immune function [20, 28-32]. In addition, both 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 points towards a direct effect of leptin on T lymphocytes [29], which may reflect CD4+ and CD8+ T cells express functional leptin receptor(s) [36, 37]. Leptin concentrations lowered by starvation appear to correlate with impaired immune responses in mice [38]. Since administration of leptin to ob/ob but not db/db mice prevented immune dysfunction, a central role for leptin as an immune system regulator has been proposed [29, 39].

Several authors have reviewed recent findings on leptins relationship with the immune system and autoimmune diseases [40-50]. Leptins effects 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 of the proliferation of CD4+ T cell [21]. Considering that congenital deficiency of leptin increases the frequency of infections and related mortality [51], it was hypothesized that a low concentration of serum leptin may promote 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-?), tumour necrosis factor alpha (TNF-?), and IL-18, and decreases production of the Th2 cytokines: IL-4, IL-5 and IL-10 [36, 37]. These effects are not observed in T lymphocytes from db/db mice, supporting the concept that this effect is directly mediated by leptin receptors, expressed on T lymphocytes [48].

Leptin also influences other immune cell types. Peritoneal macrophages from ob/ob mice display a lower phagocytic activity, compared to macrophages from normal mice, and when leptin was administered, the phagocytic activity was restored [52]. Furthermore, the production of granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF) [53] and the pro-inflammatory cytokines such as, TNF-?, IL-6 and IL-12 [52] by murine macrophages is enhanced after treatment with leptin. It has also been shown that leptin induces TNF-?, IL-6 and IFN-? production by resting human peripheral blood mononuclear cells (PBMCs) and enhances the release of these cytokines from stimulated PBMCs [54]. In human neutrophils, leptin appears to mediate its effects indirectly, probably involving the release of TNF-? from monocytes [55] which activates chemotaxis of lymphocytes and monocytes [56], to sites of inflammation [56, 57]. Moreover, in ob/ob mice, numbers of intraepithelial lymphocytes (IELs) are reduced and these IELs exhibit decreased IFN-? secretion, while the lamina propria mononuclear cells of these mice show increased apoptosis[58].

Leptin also appears 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, not seen in db/db mice [59].

Leptin and Autoimmunity

Leptin, plays a signficant role in CD4+ T cell-mediated immune responses, promoting a pro-inflammatory Th1 response. The Th1 enhancing properties of leptin have been shown to increase the susceptibility of mice to develop experimentally induced autoimmune diseases such as type 1 diabetes melitus (T1D), antigen-induced arthritis (AIA) and experimental autoimmune encephalomyelitis (EAE), an immune-mediated model of human multiple sclerosis [60]. Accumulating evidence suggests that leptin also plays a pivotal role in the development of CD4+ T cell mediated autoimmune diseases in human including Crohns disease[61], rheumatoid arthritis (RA)[62], multiple sclerosis [63] and type I diabetes mellitus (T1D)[64]. ob/ob mice resist induction of several experimental models of inflammatory and autoimmune diseases, such as experimental arthritis [65], T cell-mediated hepatitis [66] and acute and chronic intestinal inflammation [67].

In experimental mouse model systems of inflammatory bowel disease (Crohns disease with acute and chronic colitis), leptin-deficient ob/ob mice showed a significant (72%) lower colitis disease severity with a concurrent decrease in pro-inflammatory cytokines (IFN-?, TNF-?, IL-1?, IL-18 and IL-6) in colon cell culture supernatants, compared to wild type mice [58]. Administration of leptin to ob/ob mice eliminates resistance to experimentally induced Clostridium difficile (CD) colitis [58]. In this model, CD toxin A caused a severe colitis in wild type mice; ob/ob as well as db/db mice appeared to be partially protected against CD toxin A-induced gut inflammation [67]. In this case, leptin administration in ob/ob, but not in db/db mice reversed this effect [67] consistent with dependency on leptin receptor signaling.

The organ-specific autoimmune condition termed chronic idiopathic thrombocytopenic purpura (ITP) is characterized by production of antibodies against platelet membrane antigens which causes their enhanced disintegration by macrophages [68]. Leptin enhances in vitro secretion of IgG anti-platelet antibodies by splenocytes and PBMCs from patients with chronic ITP [69]. After depletion of CD4+ T cells, this phenomenon was no longer observed [70]. Further studies showed that leptin could increase platelet reactive T cells [71]. These findings indicate that leptin may in some way be related to the pathogenesis of chronic ITP and may represent a target for therapy [72].

There are also data supporting a role for leptin in the development of rheumatoid arthritis (RA). Injection of methylated bovine serum albumin (BSA) into the knees of mice results in the development of antigen-induced arthritis. Ob/ob and db/db mice developed less severe arthritis (compared to wild type mice), with lower IL-1? and TNF-? present in articular synovial fluid in the knee and decreased levels of circulating methylated BSA antibody. Furthermore, decreased antigen-specific T cell proliferation, lower IFN-? and a higher IL-10 secretion, indicate a shift towards an anti-inflammatory Th2 phenotype [65]. Reducing leptin levels in RA patients by fasting ameliorate the clinical signs of the disease [73].

In non-obese diabetic mice (NOD) model, used to study type 1 diabetes (an autoimmune pancreatic inflammatory disease, which destroys ?-cells), there is a pro-dromal increase in serum leptin levels prior to development diabetes (in females). Injection of leptin also accelerates autoimmune mediated lysis of ?-cells and increases IFN-? production by peripheral T cells. These events support leptin as promoting the development of type 1 diabetes through activation of Th1 responses [74]. 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 [75]. In general, women have higher circulating leptin levels than men [76] which may help explain their greater to propensity to develop autoimmune diseases [77], leads to the conclusion that sexually dimorphic leptin concentrations constitute the basis of higher rates of autoimmunity in females [49].

Leptin and multiple sclerosis

MS, an autoimmune neurodegenerative disorder most often affects in younger (and female) adults. While the exact causes of MS remain unknown, MS pathophysiology is thought to involve complex interactions among genetic, environmental and immunologic factors [78]. Relapsing-remitting MS, the most common form of MS, is more common in females[79]. MS therefore is an example of an autoimmune disease whose progression and severity depends on increased levels of many cytokines and chemokines.

It has long been known that myelin-reactive Th1 CD4+ cells might participate in pathogenesis of MS and Th1 cytokines are elevated in the CNS inflammatory lesions of EAE [80]. In contrast, elevation of Th2 cytokines are typically associated with recovery from EAE as well as protection from MS disease [81]. As mentioned before, leptin is known to shift immune responses towards the Th1 polarity. One of the most convincing findings demonstrating the critical role of leptin in the induction of EAE was presented by Matarese et al [82]. A surge in serum leptin levels has been shown to precede the clinical onset of EAE manifestations [83]. Genetically ob/ob mice resist disease induction in both active and adoptively transfer models of EAE. This protection is reversed by leptin administration and is associated with a switch from Th2 to Th1 type responses as well as an IgG1 to IgG2a isotype switch. Similarly, in susceptible wild-type C57BL/6J mice, leptin worsens EAE disease by increasing IFN-? release and enhancing IgG2a production. These findings suggest that leptin is both required for development of EAE, and likely also human MS.

Investigators have also examined links between leptin and MS [84-86]. Leptin is elevated up to 6.5-fold higher in acute/active MS compared to chronic, silent MS [87]. In acute phases of MS, leptin secretion and CSF production of IFN-? were increased [44]. In acute MS, leptin secretion was increased in both the serum and in the CSF of patients with MS; however this did not correlate with body mass index (BMI) [86]. The increase of leptin in the CSF was higher than that in the serum, indicating a potential secondary site of leptin synthesis within the CNS and/or the enhanced transport of leptin across the blood vascular barrier following its systemic production [85]. Increased secretion of leptin into the serum has also been prior to relapses in patients with MS (during treatment with IFN-?), and leptin has the capacity to enhance the secretion of TNF-?, IL-6, and IL-10 from peripheral blood mononuclear cells of patients with MS in vitro during the acute phase of the disease; this does not occur in patients with stable disease [88]. It has been reported that leptin secretion is increased in both serum and cerebrospinal fluid (CSF) of treatment-naive MS patients; CSF leptin is positively correlated with the secretion of IFN-? in the CSF and is inversely related to the fraction of circulating regulatory T cells. These tolerogenic cells are essential for maintenance of anergy and are comparatively reduced in patients with MS. Importantly, the number of peripheral Treg cells in MS patients inversely correlates with the serum levels of leptin, suggesting a link between the number of Treg cells and leptin secretion [86]. T cells from MS patients which are autoreactive to human myelin basic protein (hMBP)-specific produced leptin and up-regulated the expression of leptin receptor after activation [44, 63, 86]. Up-regulation of the Ob-R in mononuclear cells is seen in relapsingremitting MS (RRMS) patients during relapse, but is not seen in remission or controls [89]. This finding suggests that Ob-R may play a role in the pathogenesis of MS by up-regulating the immune response in the acute phase of the disease [89].

Leptin antagonism

These data strongly suggest a central role for leptin in thepathogenesis of CNS inflammation in both EAE and MS. Therefore, leptin antagonism may offer a new treatment option for MS patients.

It has been shown that blocking leptin signalling with either anti-leptin antibodies or with a recombinant mouse leptin receptor decoy, prior to or following the initiation of EAE, reduced evidence of clinical disease, with reduced disease progression, fewer relapses, less evidence of proteolipid protein 139-151 myelin peptide-induced T cell proliferation, and increased conversion to a Th2 cytokine secretion profile [90]. CD4+ T cells recovered from mice which had been injected with leptin blockers showed lowered responses to PLP139151 peptide, (measured as the accumulation of intracellular cyclin-dependent kinase inhibitor p27 (p27Kip-1)). Diminished responses induced by leptin blockade were associated with a reduction in extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation, suggesting that ERK1/2 activity regulates the etiology of EAE and perhaps also human MS [91].

Both anti-leptin and anti-leptin receptor blocking antibodies reduced the proliferative responses of the hMBP-specific T cell lines to antigen stimulation, indicating the possibility of using leptin-based interventions to terminate this autocrine loop and block autoreactivity [86].

Pharmacological inhibition of leptin using several classes of receptors antagonists reduces clinical initiation, progression, and subsequent relapses in both primary or passively transferred EAE [90]. These reactions were correlated with a significant inhibition of delayed-type hypersensitivity reaction against PLP139151 peptide, reduced CD4+ T cell activation, and an elevated IL-4 and IL-10 production in response to challenge with myelin antigens. Foxp3, a marker for Treg cells and a key regulator of immune tolerance, is more intensely expressed by CD4+ T cells from mice in which leptin function had been neutralized, suggesting that they had switched to the Treg phenotype. Lower T cell responsiveness might represent maintenance of p27Kip-1, (a pro-anergy factor) or reduced phosphorylation of regulatory tyrosine residues on ERK1/2 and STAT6. These finding provide a mechanistic basis allowing for clinical intervention in EAE (and possibly in human MS), which would exploit leptin signaling in the design of therapeutic agents to treat MS (and possibly other chronic inflammatory states) [90]. Leptin neutralization profoundly alters intracellular signalling of myelin-reactive T cells, increasing the number of regulatory T cells which improve the course of EAE [92].

Diverse actions of leptin discussed earlier on many organ systems and immune functions suggest that attempts to block leptin signalling in vivo should be carefully evaluated as it may cause undesirable, off-target effects. The main concern in the development of leptin-based therapeutic strategies for autoimmune diseases, like MS remains that complete leptin/leptin receptor blockage might interfere with leptins hypothalamic body mass regulating role. Indeed, treatment of mice with the S120A/T121A leptin mutant (which acts as leptin antagonist) induces significant weight gain by affecting satiety. Weight gain in S120A/T121A treated mice also indirectly implies that the mutant also functions centrally and is actively transported across the blood-brain-barrier [93].

There are different rationales for the design of leptin antagonists. Non-specific agents which block leptin signal pathways which overlap with other systems, such as JAK-STAT, may result in detrimental off-target effects. So far, there is no approved commercially available specific leptin antagonist that can be used for clinical studies with human subjects. The recent development of leptin mutant mice and proteins that interfere with leptin activity or signalling suggest the eventual possibility of leptin modulation in clinical therapy of inflammatory states [94]. A monoclonal antibody against human leptin receptors which has a leptin antagonist effect has been previously described [95]. This antibody inhibits pro-inflammatory actions of leptin by blocking peripheral immune actions of leptin and leptin-induced induction of TNF-? by human monocytes, and T cell proliferation [95]. The DNA sequence encoding this antibody has been cloned, and different forms of blocking antibody (Fab and ScFv) produced with similar blocking efficacy as the whole antibody, a first step towards a therapeutic antibody. The greatest advantage of recombinant antibody (rAb) technology is that rAbs can be manipulated genetically to yield specific properties (e.g. humanized conjugated with other molecular motifs, etc) and more importantly producing bifunctional molecules which can simultaneously bind to at least two different ligands, one of which is cell-/tissue-specific, permitting blockade of leptin receptors on a specific target tissues.

The adipose tissue and neuroendocrine system also secretes factors which like leptin also regulate caloric intake and metabolism also affect influence immune status. These mediators include adiponectin, visfatin, neuropeptide Y (NPY), and ghrelin [96]. Ghrelin, a hormone stimulated by NPY and Agouti-related peptide (AgRP), (a neuropeptide produced in the brain), is secreted mainly by the stomach and also by the small intestine, pancreas and thyroid [97]. Ghrelin is secreted when blood levels of leptin and glucose drop, and stimulates appetite. It is usually increased before meals, decreased after food intake [98] stimulates the anterior pituitary gland to secrete growth hormone, and is a biological antagonistic to leptin. Ghrelin also has suppressive effects on leptin-induced secretion of inflammatory cytokines as well as a powerful effect on thymus function [99]. In humans, ghrelin blocks leptin-induced secretion of Th1 cytokines by T cells [100], and in mice, suppresses EAE through reduction of mRNA levels of TNF-?, IL-1?, and IL-6 in spinal cord cellular infiltrates and microglia [101]. Therefore, ghrelin may also represent an endogenous antagonist of leptin, and thus find use in the treatment of MS.

Conclusions

Adequate nutrition is a prerequisite for generating appropriate immune responses against invading pathogens. Conversely, sufficient energy stores may be one of the factors required for long-term, detrimental immune reactions, like those observed in autoimmune diseases. Therefore, leptin can be considered as a link between the immune tolerance, metabolic state, and autoimmunity. Leptin, as an inflammatory cytokine, may be responsible for balancing immune responses between immunosuppression and autoimmunity, (Fig. 1) with higher circulating leptin levels predisposing individuals to autoimmune diseases, while low serum leptin reducing autoimmunity, but increase susceptibility to infection [26]. As early leptin research has primarily focused on the roles of leptin on body weight regulation, less attention has yet been given to the development of leptin antagonists specifically designed for its peripheral immune-regulating effects. Based on the available evidence in the literature, leptin receptor antagonism might represent an important and novel therapeutic approach for treating autoimmune diseases, including MS. Development of monoclonal antibodies against the leptin receptors that block leptin signalling in specific tissues or organs could be a promising future tool for many immune mediated chronic inflammatory conditions [95].

Competing interests

The authors affirm that they have no known competing interests.

Authors contributions

All authors participated in different parts of the preparation of the manuscript.

Acknowledgements

The authors would like to thank graduate office of Isfahan University for funding of this study, J.S. Alexander and A. Minagar supported by US Department of Defense Grant (MS090035).

Figure 1. Leptin contribution to immune responses in MS susceptibility. Low calorie intake leads to low levels of leptin production. Low leptin is associated with a Th2 cytokine polarization with lower levels of inflammatory cytokines, elevated levels of ghrelin and adipocytokines suppressing automimmunity. High leptin levels intensify immune responses which increase MS susceptibility including CD4/CD8 cell, monocyte, macrophage activation and proliferation and strong activation of Th1 cytokines.

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