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 aiming at leptin antagonism might represent a novel therapeutic approach which would need to receive further attention.
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 α-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, 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 [6]. Administration of leptin to ob/ob mice increases basal metabolism and reduces food intake, leading to a markedly 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], which 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 centre"[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 JAK-STAT, 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 signalling capabilities and may also be involved in leptin transport through the bloodbrain barrier and maybe in other 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. It also 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 [27].
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 (Are any differences between these two states? Please explain in two sentences what are the differences between leptin-deficient mouse and leptin-receptor deficient mouse). 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 points towards a direct effect of leptin on T lymphocytes [29], which may be due to the fact that 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 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 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-γ), tumour necrosis factor alpha (TNF-α), and IL-18, and decreases production of the Th2 cytokines: IL-4, IL-5 and IL-10 [36, 37]. These effects are not observed 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 [48].
Leptin also exerts some effects on the other 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 [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-α, IL-6 and IL-12 [52] by murine macrophages is enhanced after treatment with leptin. It was also shown that leptin induces production of TNF-α, IL-6 and IFN-γ from resting human peripheral blood mononuclear cells (PBMCs) and enhances release of these cytokines from stimulated PBMCs [54]. In human neutrophils, leptin appears to mediate its effects through an indirect mechanism, probably involving the release of TNF-α from monocytes [55]. This protein acts as a chemo-attractant for lymphocytes and monocytes [56], 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 demonstrate decreased IFN-γ 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, but not in db/db mice [59].
Leptin and Autoimmunity
Leptin, as mentioned before, plays a signficant 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 diseases such as experimental autoimmune encephalomyelitis (EAE), type 1 diabetes melitus (T1D), and antigen-induced arthritis (AIA) [60]. Accumulating evidence suggests that leptin also plays a pivotal role in the development of CD4+ T cell mediated autoimmune diseases in human including Crohn's disease, rheumatoid Arthritis (RA), multiple sclerosis and type I diabetes mellitus (T1D) [61] (here we need one reference for each disease: One for Crohn, one for RA, type 1 DM and one for multiple sclerosis). ob/ob mice are resistant to the induction of several experimental models of inflammatory and autoimmune diseases, such as experimental arthritis [62], T cell-mediated hepatitis [63] and acute and chronic intestinal inflammation [64].
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-γ, TNF-α, IL-1β, IL-18 and IL-6) in colon cell culture supernatants, compared to wild type mice [58]. Administration of leptin to ob/ob mice eliminates this resistance against experimentally induced colitis [58]. In this model, Clostridium difficile toxin A causes severe colitis but ob/ob as well as db/db mice were partially protected against the toxin A-induced intestinal inflammation [64]. In this case, also leptin administration in ob/ob, but not in db/db mice reversed this effect [64].
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[65]. Leptin enhances in vitro secretion of IgG anti-platelet antibodies by splenocytes and PBMCs from patients with chronic ITP [66]. After depletion of CD4+ T cells, leptin lost this function [67]. Further studies showed that leptin could increase platelet reactive T cells [68]. 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 [69].
There are data suggesting a role for leptin in the development of 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-1β and TNF-α 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-γ and a higher IL-10 secretion, typical for a shift towards an anti-inflammatory Th2 phenotype is also reported [62]. Reducing leptin levels in RA patients by fasting ameliorate the clinical signs of the disease [70].
In the non-obese diabetic (NOD) mouse, an animal model for type 1 diabetes (an autoimmune disease, in which the pancreatic β-cells are destroyed by the inflammatory processes), an increased serum level of leptin precedes development of diabetes in susceptible females, while injection of leptin accelerates the autoimmune destruction of the pancreatic β-cells and increases the IFN-γ production in peripheral T cells. These effects indicate that leptin promotes the development of type 1 diabetes through Th1 responses [71]. 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 [72].
Women have higher circulating leptin levels than men[73]. In general, women are more prone to develop autoimmune diseases [74] and this sexual preponderance could be justified by higher average leptin concentrations in women [49].
Leptin and multiple sclerosis
MS is an autoimmune neurodegenerative disorder which affects young adults. While the exact cause of MS remains unknown, its pathophysiology involves complex interactions among genetic, environmental and immunologic factors [75]. Relapsing-remitting MS, the most common form of MS, is more common in females[76]. MS is an example of an autoimmune disease whose progression and severity in influenced by many cytokines and chemokines.
It has long been known that myelin-reactive Th1 CD4+ cells may participate in pathogenesis of MS and Th1 cytokines are elevated in the CNS inflammatory lesions of EAE [77]. In contrast, Th2 cytokines typically are associated with recovery from EAE and protection from the disease [78]. As was mentioned before, leptin is known to promote immune response toward Th1 response. One of the most convincing evidence demonstrating the crucial role of lepin for induction of EAE has been presented by Matarese et al [79]. They have clearly shown that leptin is required for development of EAE, and thus, possibly also for MS. 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-γ release and IgG2a production. Surge of the serum leptin preceded the onset of clinical manifestations of EAE.[80]. 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 [81]. (This sentence in red does not make sense. What do you mean ,,,,induced attendant....? Please re-write this sentence)
A number of studies have investigated the relationship between leptin and MS(Please cite at least three referenes). Leptin is raised up to 6.5-fold higher in acute/active MS versus chronic silent MS [82](please re-write this sentence). In acute phases of MS, leptin secretion and CSF production of IFN-γ are increased [44]. 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) [83]. 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 of what ??[84]. Reports (please cite at least three references) have shown increased secretion of serum leptin before relapses in patients with MS during treatment with IFN-β, and a capacity of leptin to enhance in vitro secretion of TNF-α, IL-6, and IL-10 from PBMNCs of patients with MS in acute phase of the disease but not in patients with stable disease [85]. It has been reported that the secretion of leptin is increased in both serum and cerebrospinal fluid (CSF) of treatment-naive MS patients , a feature which positively correlates with the secretion of IFN-γ 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 MS patients inversely correlates with the serum levels of leptin, suggesting a link between the number of Treg cells and leptin secretion [83]. Autoreactive human myelin basic protein (hMBP)-specific T cells from MS patients 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 MS (RRMS) patients during relapse, but not in remission and controls have been observed (Please cite reference). 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 [86].
Leptin antagonism
The above-presented evidence suggests a possible role for leptin in pathogenesis of CNS inflammation in the EAE and MS. Therefore, leptin antagonism may 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[87]. 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 [88].
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 in this autocrine loop to block autoreactivity [83].
Leptin neutralization with leptin antagonists could improve clinical onset, progression, and clinical relapses of both actively induced and passively transferred EAE (Please cite reference). 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. Biochemically, T cell hyporesponsiveness may be explained by the failure to down-modulate 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 [87]. 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 [89].
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 leptin's hypothalamic body weight regulating role. Indeed, treatment of mice with the S120A/T121A leptin mutant which acts 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 [61].
There are different approaches for designing antagonists. Blocking common important signal pathways, such as JAK-STAT, may result in detrimental effects. So far, there is no approved commercially available leptin antagonist that can be used for clinical studies on human subjects. The recent development of leptin mutant mice with antagonistic properties and other proteins that block leptin activity opens up new possibilities for their use in research and, eventually, therapy [90]. A monoclonal antibody against human leptin receptor with antagonistic effect has been previously described [91]. 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-α by human monocytes, and T cell proliferation [91]. 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 [92]. Ghrelin is a hormone stimulated by NPY and agouti (What is agouti??), and is secreted mainly by the stomach and also by the small intestine, pancreas and thyroid (Rereference). Ghrelin is secreted when blood levels of leptin and glucose drop, and stimulates appetite. It is usually increased before meals, decreased after food intake [93] 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 [94]. It has been shown that ghrelin blocks the leptin-induced secretion of proinflammatory cytokines by human T cells [95], and suppresses EAE which is mediated by reduction of mRNA levels of TNF-α, IL-1β, and IL-6 in the spinal cord cellular infiltrates and microglia of treated mice (Treated mice with what???) [96]. The use of ghrelin also could represent a biological antagonism for leptin and thus useful in the treatment of MS.
Conclusions
Adequate nutrition is a prerequisite for generating appropriate immune responses against invading 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, may 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[26] (This sentence is meaningless please re-write).. As early leptin research has primarily been focussed on the effects of leptin on the regulation of body weight, little attention has yet been given to the development of leptin antagonists specifically designed for its peripheral effects. Based on the available evidence presented above, leptin receptor antagonism may serve as a novel therapeutic approach for autoimmune diseases, including MS. Identification of a monoclonal antibodies against the human leptin receptor which block leptin signalling is probably a promising tool for designing a tissue specific leptin antagonist [91].
