Leptin as immune mediator: Interaction between neuroendocrine and immune system

Accepted Manuscript Leptin as immune mediator: Interaction between neuroendocrine and immune system Claudio Procaccini, Claudia La Rocca, Fortunata Carbone, Veronica De Rosa, Mario Galgani, Giuseppe Matarese PII:

S0145-305X(16)30182-3

DOI:

10.1016/j.dci.2016.06.006

Reference:

DCI 2655

To appear in:

Developmental and Comparative Immunology

Received Date: 3 November 2015 Revised Date:

27 May 2016

Accepted Date: 7 June 2016

Please cite this article as: Procaccini, C., La Rocca, C., Carbone, F., De Rosa, V., Galgani, M., Matarese, G., Leptin as immune mediator: Interaction between neuroendocrine and immune system, Developmental and Comparative Immunology (2016), doi: 10.1016/j.dci.2016.06.006. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

Leptin as immune mediator: interaction between neuroendocrine and immune system

Claudio Procaccinia1, Claudia La Roccaa1, Fortunata Carbonea1, Veronica De Rosaa,b, Mario

Laboratorio di Immunologia, Istituto di Endocrinologia e Oncologia Sperimentale, Consiglio

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Galgania, Giuseppe Mataresea,c

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Nazionale delle Ricerche (IEOS-CNR), c/o Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università di Napoli “Federico II”, 80131 Napoli, Italy b

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Unità di NeuroImmunologia, Fondazione Santa Lucia, 00143 Roma, Italy

Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università di Napoli “Federico

These authors contributed equally to this work.

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II”, 80131 Napoli, Italy

Correspondence:

Prof. Giuseppe Matarese,

Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università di Napoli “Federico II”, Via S. Pansini 5, 80131 Napoli, Italy E-mail address: [email protected]

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ACCEPTED MANUSCRIPT Abstract Leptin is an adipocyte-derived hormone/cytokine that links nutritional status with neuroendocrine and immune functions. Initially described as an anti-obesity hormone, leptin has subsequently been shown to exert pleiotropic effects, being also able to influence

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haematopoiesis, thermogenesis, reproduction, angiogenesis, and more importantly immune homeostasis. As a cytokine, leptin can affect both innate and adaptive immunity, by inducing a pro-inflammatory response and thus playing a key role in the regulation of the pathogenesis

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of several autoimmune/inflammatory diseases. In this review, we discuss the most recent advances on the role of leptin as immune-modulator in mammals and we also provide an

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overview on its main functions in non-mammalian vertebrates.

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Keywords: leptin, innate immunity, adaptive immunity, non-mammalian vertebrates

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ACCEPTED MANUSCRIPT Introduction Leptin (Ob) is one of the most important hormones secreted by adipose tissue (Zhang et al., 1994) and its implication in energetic homeostasis at central level has been largely described during the last 20 years (Flier, 1995). Once secreted into the circulation, leptin travels to the brain, where it

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enters the central nervous system (CNS), presumably via the choroid plexus and circumventricular organs. In the brain, leptin acts by binding and activating the long form of leptin receptor (LepR), which is expressed primarily on specialized subsets of neurons in certain hypothalamic and

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brainstem nuclei (Elias et al., 2000; Patterson et al., 2011; Tartaglia, 1997). Research on adipose tissue has provided important insights into the intricate network that links nutrition, metabolism and

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immune homeostasis. The neuroendocrine and immune systems communicate bi-directionally through common ligands and receptors. The hypothalamo–pituitary–adrenal (HPA) axis is one of the main structures that is responsible for this communication, and HPA hormones (corticotrophinreleasing hormone, adrenocorticotrophic hormone and glucocorticoids) secreted during stress

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responses and inflammation control immune responses. Acute-phase reactants, interleukin-1 (IL-1), IL-6 and tumour-necrosis factor-α (TNF- α) can influence the secretion of HPA hormones. Leptin is a cytokine/hormone that can also influence the HPA axis. It is mainly produced by the adipose

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tissue in proportion to the body fat mass and, at lower levels, by tissues such as the stomach,

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skeletal muscle and placenta (Friedman and Halaas, 1998). In the hypothalamus, leptin regulates appetite through the inhibition of food intake, autonomic nervous system outflow, bone mass and the secretion of HPA hormones (Friedman and Halaas, 1998). Leptin works not only as a “fasting hormone” by controlling body weight through the inhibition of food intake and stimulation of energy expenditure (Friedman and Halaas, 1998), but also in the regulation of sexual-reproduction (Chehab et al., 1996), haematopoiesis (Bennett et al., 1996), angiogenesis (Sierra-Honigmann et al., 1998; Pucino et al., 2014), bone metabolism (Ducy et al., 2000) and glucose homeostasis (Berti and Gammeltoft , 1999). Confirming leptin’s pleiotropic effects in periphery, it has been previously shown, that leptin-deficient (ob/ob) and LepR-deficient (db/db) mice are not only obese but also 3

ACCEPTED MANUSCRIPT show many other immune/endocrine alterations observed during starvation (Lord et al., 1998; Lord et al., 2001; Fraser et al., 1999). Increasing body of evidence has moreover suggested that leptin is able to modulate several innate and adaptive immune responses (La Cava and Matarese, 2004).

in the regulation of immune responses (Procaccini et al., 2012a).

Leptin and regulation of innate immune responses

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Leptin and macrophages

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Indeed, LepR is expressed by several immune cells, thus suggesting a key role displayed by leptin

Several studies in animal models with genetic abnormalities in leptin or LepR revealed that leptin-

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deficiency associates with consistent impairment in macrophage functions in terms of phagocytosis and the expression of pro-inflammatory cytokines both in vivo and in vitro, and these conditions are reverted by exogenous leptin administration (Loffreda et al., 1998) (Table 1). Gabay et al. have shown that leptin dose-dependently stimulates the production of pro-inflammatory cytokines by

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monocytes, (such as TNF-α and IL-6) (Gabay et al., 2001) and enhances CC-chemokine ligand expression in cultured murine macrophages, through the activation of a Janus kinase 2 (JAK2)Signal Transducer and Activator of Transcription-3 (STAT3) pathway (Kiguchi et al., 2009). In

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human monocytes, leptin has been shown to enhance the secretion of interleukin IL-18, via activation of caspase-1 (Jitprasertwong et al., 2014) and acting synergistically with adenosine

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triphosphate, it contributes to aberrant immune responses in Type 2 diabetes and other conditions of hyperleptinemia. Moreover leptin stimulates proliferation and activation of human circulating monocytes in vitro, promoting the expression of activation markers, such as CD69, CD25, CD38, and CD71, in addition to increasing the expression of monocytes surface markers (ie. HLA-DR, CD11b, and CD11c) (Santos-Alvarez et al., 1999). In murine alveolar macrophages (AM) leptin increases leukotriene synthesis (Mancuso et al., 2004) and it plays an important role in regulation of both energy homeostasis and innate immune response against bacterial infections. Indeed Mancuso et al. have shown that ob/ob mice exhibit an increased mortality and impaired pulmonary bacterial 4

ACCEPTED MANUSCRIPT clearance after intratracheal challenge with Klebsiella pneumoniae. Mechanistically, the impaired phagocytosis and killing of K. pneumoniae in AMs from ob/ob mice was associated with reduced reactive oxygen intermediate production in vitro. Another study has also shed light on the chemoattractant properties exerted by leptin, since it has been shown that leptin is a potent

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chemoattractant for mouse monocytes and macrophages, and that leptin-mediated chemotaxis requires the presence of full-length leptin receptors on migrating cells (Gruen et al., 2007) (Table 1). Finally, leptin causes increased influx of intracellular calcium in murine macrophages, activating

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JAK/STAT, mitogen-activated protein kinase (MAPK), and phosphatidylinositol 3-kinase (PI3K) pathways, suggesting that the canonical cell motility machinery is activated upon

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macrophage exposure to leptin (Gruen et al., 2007).

Leptin and Dendritic cells

Several reports have shown that also dendritic cells (DCs), the major antigen presenting cells

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involved in T lymphocyte priming, are affected by leptin at different levels, as leptin deficiency associates with qualitative and quantitative alteration of DCs (Macia et al., 2006) (Table 1). Indeed it has been shown that DCs from ob/ob mice are less potent in stimulation of allogenic T cells in

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vitro than those derived from wild type (WT) mice. This impaired functionality is not associated with altered expression of lineage-specific phenotypic markers, but rather with the secretion of

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immunosuppressive cytokines such as transforming growth factor-β (TGF-β) (Macia et al., 2006). Moreover it has been shown that leptin deficiency impairs the maturation of dendritic cells, decreasing their production of IL-12, TNF-α, and IL-6, and sustaining DC production of TGF-β. As a consequence of this particular phenotype, DCs generated under conditions of leptin deficiency induced regulatory T cells (Treg) or T-helper 17 (Th17) cells differentiation more efficiently than DCs generated in the presence of leptin (Moraes-Vieira et al., 2014). In addition, Mattioli et al. have shown that leptin induces also functional and morphological changes in human DCs, licensing them towards Th1 priming and promoting DC survival (Mattioli et al., 2005) (Table 1), by triggering the 5

ACCEPTED MANUSCRIPT activation of nuclear factor-kappa B (NF-kappaB) and a parallel up-regulation of bcl-2, bcl-XL gene expression and Akt activation (Mattioli et al., 2009). Another paper by the same group also showed that leptin increases immature human DC migratory capability both by favoring cytoskeleton dynamics (inducing rearrangement of actin microfilaments, leading to uropod and

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ruffle formation) and by up-regulating CCR7 surface expression, thus favoring their chemotactic responsiveness (Mattioli et al., 2008).

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Leptin and Neutrophils

The immunomodulatory role of leptin has been described also in neutrophils. Indeed, it has been

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shown that human neutrophils only express the short form of the leptin receptor, which is enough to signal inside the cell, enhancing the expression of CD11b and preventing neutrophils apoptosis (Bruno et al., 2005; Zarkesh-Esfahani et al., 2004). From the molecular point of view, leptin delays the cleavage of Bid and Bax, inhibits the mitochondrial release of cytochrome c as well as the

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activation of both caspase-8 and caspase-3 in these cells, protecting them from apoptosis (Bruno et al., 2005) (Table 1). In addition, leptin sustains human neutrophils functions, as it promotes their chemotaxis (Caldefie-Chezet et al., 2003), by stimulating intracellular hydrogen peroxide

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production. In this context, confirming the critical role of leptin in leukocyte recruitment, Rummel and colleagues (Rummel et al., 2010) have shown that mice intraperitoneally injected with a septic

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dose of lipopolysaccharide (LPS) showed a dramatic increase in the number of neutrophils entering the brain of WT mice. On the contrary, leptin deficiency almost completely abolished this effect, as these mice displayed a consistent reduction in the mRNA levels of IL-1β, intracellular adhesion molecule-1 and neutrophil-specific chemokines. These effects were reversed by leptin replenishment in ob/ob mice, leading to recovery of neutrophil recruitment into the brain. Confirming the key role of leptin in this process, the same authors also showed that 48 hours food deprivation in WT mice, which decreased circulating leptin levels, attenuated the LPS-induced neutrophil recruitment as did a single injection of an anti-leptin antiserum. Finally, leptin has been 6

ACCEPTED MANUSCRIPT shown to be crucial for neutrophil complement-mediated phagocytosis, as neutrophils from ob/ob mice displayed impaired phagocytosis of Klebsiella pneumoniae and downregulated CD11b expression, condition that could be restored again by exogenous leptin replacement (Moore et al.,

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2003).

Leptin and Natural Killer cells

Recent report have shown that leptin sustains the cytotoxic activity of natural killer cells (NK), by

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activating the phosphorylation of STAT3 and then increasing the transcription of IL-2 and perforin genes (Table 1). In line with this evidence, db/db mice display a reduction in both the percentage

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and total amount of NK cells in the liver, spleen, lung, and peripheral blood, when compared to their littermate controls and the same holds true for the expression of several NK activation markers, thus indicating that NK cell development is strongly influenced by leptin. In addition, exogenous leptin treatment increases the basal or enhances the IL-15-induced specific lysis of

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splenocytes in WT but not in db/db mice (Tian et al., 2002; Zhao et al., 2003). Leptin seems to have a specific effect also on invariant Natural Killer T cells (iNKT), which are essential for several aspects of immunity, as their dysfunction or deficiency has been shown to lead

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to the development of autoimmune diseases. A recent paper has demonstrated that LepR is expressed also on murine iNKT cells and that leptin suppresses iNKT cell proliferation and their

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cytokine production in vitro, working as a key inhibitor of iNKT cell function (Venken et al., 2014). Confirming this evidence, Venken et al. have also shown that LepR blockade exacerbates Concanavalin A (ConA)-induced hepatitis only in WT mice, whereas it did not exert this function in iNKT cell-deficient mice (Venken et al., 2014). Moreover it has been demonstrated that iNKT cells are enriched in human and murine adipose tissue, and that with increasing obesity, the number of iNKT cells decrease, correlating with pro-inflammatory macrophage infiltration (Lynch et al., 2012). On the contrary, iNKT cell numbers is restored in mice and humans after weight loss. In the same study, the authors showed that mice lacking iNKT cells had enhanced weight gain, larger 7

ACCEPTED MANUSCRIPT adipocyte size, and developed insulin resistance on a high-fat diet. Confirming the crosstalk between adipose tissue and iNKT, when these cells were adoptively transferred in obese mice, they were able to decrease body fat, triglyceride, leptin levels, and improved insulin sensitivity through the sustained anti-inflammatory cytokine production by adipose-derived iNKT cells. A similar

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study also showed that adoptive transfer of a relatively small number of regulatory NKT lymphocytes into ob/ob mice results in a significant reduction in hepatic fat content, a shift from macro- to micro-steatosis, and a significant improvement in glucose control. These effects were

Leptin and Eosinophils/Basophils

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inflammatory cytokines release (Elinav et al., 2006).

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secondary to a decreased peripheral and intrahepatic CD4/CD8 ratios and inhibition of pro-

Accumulating evidence reveals that leptin is able to modulate also eosinophils function. In particular, leptin delays the apoptotic rate of mature human eosinophils in vitro. More specifically,

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it inhibits the cleavage of protein Bax, as well as the mitochondrial release of cytochrome c, suggesting that it is able to interfere with the apoptotic pathways proximal to mitochondria (Conus

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et al., 2005) (Table 1). In addition, leptin upregulates the cell surface expression of adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) and CD18 but suppresses the

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expression of ICAM-3 and L-selectin. Wong et al. have shown that leptin can also modulate migratory capability of human eosinophils, as it can stimulate the their chemokinesis, inducing the release of inflammatory cytokines (including IL-1β, IL-6) and chemokines (growth-related oncogene-alpha, and monocyte chemotactic protein-1) (Wong et al., 2007). In this context, another paper has also demonstrated that high concentrations of leptin induce human eosinophil chemotaxis and rapid phosphorylation of extracellular signal-regulated kinases 1/2 (ERK1/2) and p38 mitogenactivated protein kinase but not calcium mobilization. The authors also found that pretreatment of eosinophils with physiological concentrations of leptin amplified the chemotactic responses to 8

ACCEPTED MANUSCRIPT eotaxin (Kato et al., 2011) (Table 1). Confirming the role of leptin in the crosstalk between adipose tissue and eosinophils activities, a study by Calixto and colleagues has shown that obesity enhances eosinophilic inflammation in a murine model of allergic asthma. More in details, diet-induced obesity sustains murine eosinophil trafficking from bone marrow to lung tissues, and delays their

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transit through the airway epithelium into the airway lumen. Consequently, eosinophils remain longer in lung peribronchiolar segments due to overproduction of Th1/Th2 cytokines and chemokines (Calixto et al., 2010). Finally, it has been demonstrated that increased eosinophilic

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activity (chemotaxis and adhesion) associated with high serum leptin and TNF-α levels in atopic asthmatic obese children and adolescents compared with non-obese healthy volunteers (Grotta et

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al., 2013) (Table 1).

Only few papers have described the role of leptin in basophils activity, but Suzukawa and collaborators have shown that leptin is able to sustain the migratory capability of human basophils, and enhance their survival rate, possibly through the upregulation of CD63, which is one of the

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most studied and better characterized basophil activation marker (Table 1). Confirming specificity, LepR-neutralizing antibodies treatment reverts all these effects. The same authors also showed that leptin increases and primes human basophil degranulation in response to FcεRI aggregation and

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induces Th2-type cytokines production, thus possibly exacerbating allergic inflammation

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(Suzukawa et al., 2011).

Leptin and regulation of adaptive immune responses Leptin and T cells

The effects of leptin on adaptive immune responses have been extensively investigated in last few years. This hormone plays a key role in T cell biology, by promoting murine CD4+ T-cell proliferation, cytokine secretion and migration of these immune cells to sites of inflammation (Lord et al., 1998; Lord et al., 2001; Papathanassoglou et al., 2006) (Table 2). In particular, leptin has 9

ACCEPTED MANUSCRIPT different effects on human naive (CD45RA+) and memory (CD45RO+) CD4+ T cells both of which express the long form of the LepR. Specifically leptin promotes naïve T cells proliferation by increasing their secretion of IL-2, through the activation of the mitogen-activated protein kinase (MAPK) and PI3-K pathway (Lord et al., 1998). Whereas, on memory T cells, leptin promotes the

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switch towards Th1-cell immune responses by enhancing pro-inflammatory cytokine release, such as IFN-γ and TNF-α, by sustaining the IgG2a production from B cells and by promoting delayedtype hypersensitivity (DTH) responses, a process sustained by an autocrine loop of leptin secretion

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from Th1 cells (Lord et al., 1998). Furthermore, leptin increases the expression of adhesion molecules, such as ICAM1 and very late antigen 2 (VLA2), by CD4+ T cells, possibly through the

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induction of IFN-γ thus inducing their migration to inflammatory sites (Lord et al., 1998) (Table 2). The effect of leptin on CD4+ T cell homeostasis has been recently shown by Matarese et al. in a randomized, double-blinded, placebo-controlled trial of recombinant methionyl-human leptin (metre-leptin) administration in women affected by hypothalamic amenorrhea with acquired chronic

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hypoleptinemia. In this study, the authors show that metre-leptin was able to restore both CD4+ Tcell counts and their in vitro proliferative responses in these women (Matarese et al., 2013). Accumulating evidence indicates that leptin also affects the generation, maturation and survival of

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murine thymic T cells (Howard et al., 1999); indeed, acute caloric deprivation causes a rapid

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decrease of serum leptin concentration accompanied by reduced DTH responses and thymic atrophy, which are reversible with exogenous administration of leptin (Howard et al., 1999). Other studies have also indicated that leptin promotes lymphocyte survival in human and mouse, by upregulating lymphocyte surface expression of glucose transporters, such as GLUT1 and GLUT4 (Sivitz et al., 1997), the anti-apoptotic proteins bcl-2 and bcl-XL and also by modulating autophagy (which protects T cells from apoptosis and thymocytes from glucocorticoid-induced apoptosis) (Galgani et al., 2010; Cassano et al., 2014) (Table 2). Leptin plays also a pivotal role in T-cell polarization. Indeed, the induction of cytokine-producing Th1 or Th2 cells from murine naive CD4+ T cells under polarizing conditions in vitro was generally decreased in cells from ob/ob mice as 10

ACCEPTED MANUSCRIPT compared with WT mice (Batra et al., 2010), since they have a decreased expression of the key transcription factors for Th1 and Th2 polarization, T-bet and GATA-3, respectively (Batra et al., 2010). In addition, it has been reported that leptin can act as a negative signal for the expansion of human naturally occurring Foxp3+CD4+CD25high regulatory T cells (Treg) (De Rosa et al., 2007)

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(Table 2), a cellular subset which suppresses autoreactive response mediated by CD4+25- T cells. De Rosa et al. indeed showed that freshly isolated human Treg cells produce leptin and express high levels of LepR and in vitro neutralization with anti-leptin monoclonal antibody (mAb) following

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anti-CD3/CD28 stimulation resulted in Treg cells proliferation. Together with the finding of enhanced numbers and proliferation of Treg cells observed in ob/ob and db/db mice, these results

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suggest a potential for therapeutic interventions in immune and autoimmune diseases, by transfer of anti-leptin-expanded antigen-specific Treg cells. Another paper by the same group has recently shown that leptin activates the mammalian target of rapamycin (mTOR) pathway to control human Treg cell responsiveness (Procaccini et al., 2010). mTOR is an evolutionarily conserved 289-kDa

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serine/threonine protein kinase that is inhibited by rapamycin and integrates environmental cues from nutrients, energy and growth factors to direct cell growth, proliferation and cell differentiation (Laplante and Sabatini, 2010). More specifically, leptin has been shown to inhibit rapamycin-

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induced proliferation of Treg cells, by increasing activation of the mTOR pathway. Moreover Tregs from db/db mice exhibited a decreased mTOR activity and increased proliferation compared with

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those of WT mice (Procaccini et al., 2010), thus suggesting that the leptin-mTOR axis sets the threshold for the responsiveness of Tregs and that this pathway might integrate cellular energy status with metabolic-related signaling in Treg cells that use this information to control immune tolerance. Interestingly, leptin has been demonstrated to exert opposite effects on human conventional T cells (Tconv), where it enhances their proliferation through the activation of mTOR pathway (Procaccini et al., 2012b). More recently, in ob/ob mice, a reduced frequency of Th17 cells has been shown (Table 2); obese mice with leptin or LepR deficiency showed reduced capacity for differentiation toward a Th17 11

ACCEPTED MANUSCRIPT phenotype, a condition that is attributed to reduced activation of the STAT3 and its downstream targets (Reis et al., 2015). These effects were restored to levels comparable to those found in WT animals after administration of exogenous leptin. Indeed leptin has been shown to facilitate Th17 responses by inducing retinoic acid-related orphan receptor-gamma t (RORγt) transcription in

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murine CD4+ T cells (Yu et al., 2013) and in the same study the authors showed that leptin was able to enhance Th17 responses in (NZB x NZW)F1 lupus-prone mice, whereas its neutralization inhibited Th17 responses (Yu et al., 2013). Finally, these findings have been confirmed also in

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humans, as Wang and collegues have shown that T cell-derived leptin contributes to increased frequency of Th17 cells in female patients with Hashimoto's thyroiditis (Wang et al., 2013).

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Different reports have also demonstrated that leptin is able to regulate the secretion of several cytokines from human peripheral CD8+ cells. Specifically, leptin enhances the secretion of IL-2 and IFN-γ and inhibits the production of IL-4 and IL-10 (Rodríguez et al., 2007) (Table 2). Furthermore, in a study in nonagenarians (≥ 90 years old), leptin has been found to play a key role in the

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maintenance of naïve CD8+ T cells functions (Chen et al., 2010). Other evidence has suggested that leptin also promotes CD8+ T cells activation in chronic obstructive pulmonary disease. Indeed, ob/ob and db/db mice, in a model of cigarette smoke-induced pulmonary inflammation, showed a

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consistent decrease in T cell number and frequency (CD4+ and CD8+) as compared to their

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littermate controls (Vernooy et al., 2010) (Table 2).

Leptin and B cells

As for T cells, leptin seems to play a central role also in the modulation of B cell compartment (Table 2). Indeed leptin deficiency has been associated with a significant reduction in lymphopoiesis, as testified by 70% fewer B cells than normal controls, as well as a reduction in the absolute number of pre-B and immature B cells. Seven days of treatment with recombinant leptin promoted a two fold increase in B cells number in the bone marrow of the obese mice, in parallel with an increase in the numbers of pre-B and immature B cells (Claycombe et al., 2008). Similar 12

ACCEPTED MANUSCRIPT results have been also detected in the bone marrow of fasted mice, in which intracerebroventricular leptin injection was sufficient to prevent the alteration of B-cell development (Tanaka et al., 2011). Furthermore, at molecular level, leptin promotes murine B-cell homeostasis by inhibiting their apoptosis and by inducing cell cycle entry through the activation of B-cell CLL/lymphoma 2 (Bcl-

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2) and cyclin D1 (Lam et al., 2010) (Table 2). In this context, Agrawal et al. have also shown that leptin activates human B cells to secrete cytokines (ie. IL-6, IL-10, and TNF-α) via activation of JAK2/STAT3 and p38MAPK/ERK1/2 signaling pathways (Agrawal et al., 2011) and that leptin-

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induced proinflammatory cytokines produced by B cells may play a role in chronic inflammation associated with human aging (Gupta et al., 2013). This effect is secondary to the activation leptin-

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LepR-STAT3 activation as suggested by increase in the P-STAT3 levels in B cells from aged humans as compared to young subjects (Gupta et al., 2013). Finally it has been shown that murine B lymphocytes appear to be more susceptible to the anti-apoptotic effects of leptin and show higher

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surface expression of LepR, when compared with T cells (Papathanassoglou et al., 2006).

Role of leptin in regulating immune/inflammatory diseases Leptin expression is not only regulated by food intake, but also by several inflammatory mediators

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(Gualillo et al., 2000). In general, serum leptin levels directly correlate with insulin levels, inversely

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correlate with glucocorticoid levels and increase during acute infection, inflammation and sepsis, sustained mainly by LPS and cytokines such as TNF-α, IL-6, and IL-1β (Boden et al., 1997; Zakrzewska et al., 1997). These pro-inflammatory mediators, which upregulate leptin expression, contribute in turn to create a loop of acute phase reactants that influence each other in promoting chronic inflammation. In this context, accumulating evidence has shed light on the role of leptin in the pathogenesis of atherosclerosis. Indeed it has been shown that leptin sustains the inflammatory milieu that fosters atherosclerosis, by promoting recruitment of monocytes to the intima, causing foam cell formation, and increasing secretion of pro-inflammatory and atherogenic cytokines (Yamagishi et al., 2001; O'Rourke et al., 2002). On the contrary, ob/ob mice are resistant to 13

ACCEPTED MANUSCRIPT atherosclerosis development (Gonzalez-Navarro et al., 2007) and leptin deficiency in low-density lipoprotein receptor knockout (LDL-R-/-/ob/ob) mice associated with a consistent reduction in atherosclerotic lesions as compared with LDL-R-/- mice. This condition has been shown to be secondary to a reduced Th1 response and an increased Treg suppressive function (Taleb et al.,

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2007). The link between leptin and inflammation has been further studied in several other inflammatory diseases. Indeed leptin is also involved in the development of glomerulosclerosis, as it promotes the deposition of extracellular matrix and proteinuria (Wolf et al., 1999). Moreover, a

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role for leptin in hepatic steatosis and steatohepatitis is suggested by studies in animal models showing that this hormone may protect from hepatic steatosis, at least at the initial stages of the

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disease, but it may act as an inflammatory and fibrogenic factor, as the disease progresses. Both ob/ob mice and fa/fa Zucker rats, lacking leptin or having a defective LepR respectively, develop hepatic steatosis, together with insulin resistance and obesity (Cipriani et al., 2010; Pelleymounter et al., 1995). On the other hand, in murine cells, leptin treatment sustains hepatic inflammation and

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fibrosis, as it activates LepR on hepatic stellate cells (HSCs), increasing the production of proinflammatory, proangiogenic cytokines and growth factors, thus contributing to hepatic inflammation (Aleffi et al., 2005). At molecular level, leptin upregulates collagen type 1 expression

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(Yan et al., 2012) and stimulates the production of tissue inhibitor of metalloproteinase (TIMP)-1 (Cao et al., 2004), all changes that are involved in the development of hepatic inflammation and

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fibrosis. In humans, it is thought that the increased leptin levels could associate with leptin resistance, contributing to fat accumulation in the liver, through a reduced hepatic oxidation, an increased synthesis of free fatty acids, that cause liver inflammation through lipid peroxidation and consequent increased ROS production (Tsochatzis et al., 2006). Finally, recent experimental evidence suggests an important effect of leptin on the chondrocytes, which represent the cellular component of the cartilage. Otero et al. demonstrated that in cultured human and murine chondrocytes, type 2 nitric oxide synthase (NOS2) is synergistically activated by the combination of leptin plus IFN-γ, and NOS2 activation by IL-1 is 14

ACCEPTED MANUSCRIPT increased by leptin via a JAK2- PI3K- and MEK1/p38-mediated mechanism (Otero et al. 2003; Otero et al., 2005; Gomez et al. 2009; Gomez et al. 2011). Moreover, it has been demonstrated that leptin is able to induce also the synthesis of relevant matrix metalloproteases (MMPs) mainly involved in cartilage damage, such as MMP9 and MMP13. Confirming this evidence, small RNA

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interference against leptin inhibits MMP-13 expression, which in turn is up-regulated after leptin’s

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reactivation at epigenetic level (Iliopoulos et al. 2007, Simopoulou et al. 2007).

Leptin evolution, from fishes to mammals

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Soon after the discovery and isolation of mouse Lep gene, orthologous genes were identified in humans and several other mammalian species (Zhang et al., 1994; Doyon et al., 2001). Since the identification of leptin in mammals, many other studies have been performed to identify orthologous genes in non-mammalian species with limited success. Only in 2005 the putative homolog of mammalian leptin gene was isolated in pufferfish (Takifugu rubripes) (Kurokawa et al.,

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2005). The primary amino acid sequence of pufferfish leptin is only 13% similar to that of human leptin. However in this study, the authors did not test whether the deduced protein product of the

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putative pufferfish lep gene had biological activity homologous to mammalian leptin. After the cloning of pufferfish leptin, also a frog (Xenopus) leptin ortholog has been discovered

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with a low amino acid sequence similarity to human (approximately 35%) and pufferfish leptin (approximately 13%) (Crespi and Denver, 2006). Recombinant frog leptin has been shown to activate

the

frog

LepR

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vitro

and

exerted

anorexigenic

activity

when

injected

intracerebroventricularly into juvenile frogs (Crespi and Denver, 2006). Later, a lep gene has been also identified in the tiger salamander Ambystoma tigrinum, sharing 60% identity with the frog leptin, although the overall amino acid identity with mammalian leptin was only 29% (Boswell et al., 2006).

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ACCEPTED MANUSCRIPT Recently, leptin sequences have been identified also in birds, such as peregrine falcon (Prokop et al., 2014), zebra finch (Huang et al., 2014) and rock dove (Friedman-Einat et al., 2014). More information about the avian leptin has been collected thanks to a recent paper by Seroussi et al, showing the existence of chicken leptin (Seroussi et al., 2016). The identification and

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characterization of such molecule resolved a 17-years-old controversy regarding the existence, or not, of leptin in chicken, since previous papers reported erroneous chicken leptin sequences (Taouis et al., 1998; Ashwell et al., 1999), which gave grounds to the hypothesis that the leptin gene may

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have been lost in chicken (Daković et al., 2014).

Sequences orthologous to mammalian Lep genes have been described in several fish species, based

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on genomic analysis. However, detecting the molecular phylogeny of fish lep genes is complicated by the existence of multiple, and sometimes highly divergent, copies in several species. Indeed, while all mammals (Clarke et al., 2001) and amphibians (Boswell et al., 2006) express a single ortholog of leptin, it has been shown that some fishes have two leptin orthologs (Gorissen et al.,

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2009). This phenomenon is probably ascribed to events of entire genome duplications or may have resulted from the duplication of individual genes with the consequent divergence of one of the two copies. Despite a considerable divergence observed in the leptin primary amino acid sequence

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among vertebrates, the predicted secondary and tertiary structures and the key amino acids required for leptin’s biological activity are highly conserved (Crespi and Denver, 2006), although all non-

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mammalian leptin structures have been inferred by models of crystal structure of human leptin (Londraville et al., 2014).

In non-mammalian vertebrates, sites of leptin expression may vary among the different taxa. In this context, it has been shown that in fed fish (Atlantic salmon) the highest leptin mRNA levels have been observed in the brain, white muscle, liver, and ovaries (Rønnestad et al., 2010). In the South African clawed frog (Xenopus laevis) leptin mRNAs were found in several tissues, including fat and liver, which are the two organs where leptin is primarily expressed in mammals (Zhang et

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ACCEPTED MANUSCRIPT al., 1994), but also in brain, pituitary gland, heart, gastrointestinal tract, lungs, kidney and gonads, with the highest relative expression in the brain and heart (Crespi and Denver, 2006). Concerning the identification of leptin’s biological functions in the different taxa, the majority of the studies focused on the characterization of leptin functions in non-mammalian vertebrates,

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analyzing its role as anorexigenic and adipostat molecule. These studies have demonstrated the conserved role of leptin in the regulation of body weight, through the inhibition of food intake among vertebrates. Indeed exogenous leptin administration reduces food intake in mammals and

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also in fishes (Volkoff et al., 2003), such as trout (Murashita et al., 2008) and carp (Li et al., 2010), but also in lizard (Niewiarowski et al., 2000), frog (Crespi and Denver, 2006) and chicken (Kuo et

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al., 2005). On the other hand, contrary to what has been observed in mammals, an increase was detected in leptin mRNA or circulating protein during fasting (Trombley et al., 2012) and a rapid decrease upon refeeding (Fuentes et al., 2012) in fishes, even though it has been also observed that in common carp, leptin expression increases after food intake, but not after fasting or feeding to

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satiation (Huising et al., 2006). In addition, fish model lacking LepR do not display increased fat stores (Chisada et al., 2014; Michel et al., 2016), suggesting that leptin may not act as adipostat in fishes and that its role as an adipostatic factor is likely to be a secondary role acquired during the

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evolution of mammals (Michel et al., 2016).

Moreover, it has been shown that during the early development of Xenopus and zebrafish, leptin

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exerts several additional functions that have not been described in mammals. More in details, in Xenopus, leptin stimulates lung development by promoting the thinning of the gas exchange surface and the increase of lung surfactant synthesis (Torday et al., 2009). Moreover in Xenopus tropicalis tadpole leptin is involved in tail regeneration, since it has been observed an up regulation of leptin mRNA during phases of regeneration (Love et al., 2011). In this context, leptin administration during early prometamorphosis of Xenopus laevis induces growth and development of the hind limb (Crespi and Denver, 2006). Several data also suggest a role for leptin in early developmental processes in zebrafish; indeed, leptin-a knock-down results in smaller bodies and eyes, undeveloped 17

ACCEPTED MANUSCRIPT inner ear, enlarged pericardial cavity, curved body and/or tail, larger yolk and severely disrupted dorsal brain and retinal cells (Liu et al., 2012). As for leptin, several studies have been performed to characterize LepR expression and function in the different classes of vertebrates. Most of the studies related to the LepR have been carried out on

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the long form of the receptor (LepRb), that is the only form able to activate Jak/STAT signaling pathways (Denver et al., 2011), even though it has been reported that multiple LepRs (not only the long form) are expressed throughout vertebrates (Cao et al., 2011). Indeed a truncated LepR

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isoform has been identified in the chicken (Liu et al., 2007), whereas in the Atlantic salmon five isoforms, which differ in 3′ end of the mRNA sequence, have been detected and among them, only

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the longest form conserved the specific domains necessary for the effector functions (Rønnestad et al., 2010). The site of binding of leptin to its receptor is the cytokine homology region 2 (CHR2) (Mancour et al., 2012). A comparative analysis of leptin and LepR interaction across vertebrates has revealed a highly conserved interaction between the CHR2 of LepR and leptin, with higher levels of

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variation of this interaction in fishes (Prokop et al., 2012). Indeed, as we previously mentioned, in fishes, gene duplication events probably led to the expression of multiple leptin proteins. Since the binding site for leptin to its receptor is conserved across diverse taxa, it could be used for the

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evaluation of new leptin clones. Studies performed on non-mammalian vertebrates have shown that in Atlantic salmon, sLepR (the soluble form of LepR), similarly to leptin expression, is abundantly

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expressed in the ovary, in the brain, pituitary gland, eye, gill, skin, visceral adipose tissue, belly flap, red muscle, kidney, and testis (Rønnestad et al., 2010). In zebrafish, LepR is mainly expressed in the notochord of embryos, but as the development proceeds, LepR expression in the notochord decreases, while its expression increases in several other tissues, such as the trunk muscles and gut. LepR is expressed in the hindbrain of both larval and adult brains of zebrafish, while in adult, LepR expression has been also observed in several other brain regions such as the hypothalamus (Liu et al., 2010).

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ACCEPTED MANUSCRIPT Leptin and immune system in several non-mammalian vertebrates There is increasing body of evidence suggesting that leptin may affect also immunity in nonmammalian vertebrates such as fishes, amphibians, reptiles and birds (Fig. 1). The highly divergent leptin primary sequence in lower vertebrates, such as fish, could explain the small number of

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studies that have been performed on the role of leptin on the immune system of fishes and especially of teleosts so far, because of the difficulty to find a homologous leptin. In a recent study, to investigate the effect of leptin on the rainbow trout (Oncorhynchus mykiss) immune system, a

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rainbow trout recombinant leptin (rt-lep) has been produced and its biological capacity to trigger pathways that are usually active in mammalian cells has been tested in vitro (Mariano et al., 2013).

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Rt-lep induces inhibitor of kappa B (IKBα) phosphorylation followed by its dephosphorylation, in non-adhering leucocytes, whereas in adhering leucocytes rt-lep causes IKBα dephosphorylation. In addition, rt-lep decreases c-Jun N-terminal

kinases

(JNK) activation, activates

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phosphorylation and inhibits superoxide production in both adhering and non-adhering leucocytes

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(Mariano et al., 2013). The IKBα dephosphorylation and JNK inhibition observed in rainbow trout is not in line with what has been observed in mammalian cells, where leptin induces the activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) (Tang et al., 2007) and

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increases MAPK activity (p38, JNK and ERK) when administrated together with LPS (Shen et al.,

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2005). Overall, these data indicate that, as in mammals, leptin plays pleiotropic actions in teleosts, but its effects are not always overlapping in these two classes of vertebrates. In addition, it has been reported that in zebrafish leptin signaling influences innate immune function, since leptin knockdown significantly reduces the ability to fight invading pathogens, with consequently reduced survivability and increased bacterial load in developing fishes (Dalman et al., 2013). Other evidence supporting the role of leptin-a in the regulation of fish immune system, results from the observation that leptin is also expressed in the thymus and spleen of common carp (Cyprinus carpio) (Huising et al., 2006).

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ACCEPTED MANUSCRIPT Studies performed on amphibians have shown that LepR mRNA is widely distributed in the African clawed frog (Xenopus laevis) and it is expressed in important immune organs such as the spleen (Crespi and Denver, 2006) (Fig. 1). Also in reptiles, there are some evidence showing the involvement of leptin in the modulation of immune responses. Indeed, studies performed on tree

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lizard (Urosaurus ornatus) suggest that leptin acts as a signal of available energy for the immune cell functions (French et al., 2011). In this context, it has been observed that in reproductive but not in non-reproductive females, limiting food intake reduces fat stores and suppresses immune

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function, suggesting that a physiological resources trade-off between reproduction and immune system exists (French et al., 2007) (Fig. 1). The mechanism of regulation of the trade-off between

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reproduction and immune function remains still unclear, even though leptin might be an ideal candidate for the regulation of energy distribution among different systems. Supporting this evidence, it has been also shown that leptin treatment is able to reverse the immunosuppressive effect of food restriction in reproductive tree lizard females, suggesting that leptin could serve as an

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endocrine signal of available energy to the immune system (French et al., 2011). In birds, recombinant chicken leptin has been shown to exert a positive effect on turkeys (Meleagris gallopavo) T cell proliferation, as Concavalin A-induced proliferation positively

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correlated with leptin concentration (Lõhmus et al., 2004). Similarly, also in Asian blue quail, leptin has been shown to enhance T cell proliferation, measured as an increased response to

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phytohaemagglutinin (PHA) injection (Lõhmus et al., 2004) (Fig. 1). Another study performed on male zebra finches (Taeniopygia guttata) has shown that leptin administration attenuates testosterone-induced immunosuppression. More specifically, male zebra finches treated with testosterone, which acts as an immunosuppressive factor, and receiving daily murine leptin injections, display increased immune responses comparable with those observed in untreated birds, as testified by the enhanced wing-web swelling in response to the mitogen PHA (Alonso-Alvarez et al., 2007). The testosterone-mediated immunosuppression could be due to a relocation of energy from the immune system to sexual traits and, in this context, leptin might interact with other 20

ACCEPTED MANUSCRIPT hormones, such as testosterone itself, to regulate the trade-off between reproductive activity and immune function (Fig. 1). Although much more research needs to be performed to understand the role of leptin in the regulation of immune system function in non-mammalian vertebrates, all these data indicate that leptin, or a leptin-like protein, could regulate immune responsiveness in different

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vertebrate classes and could have a role in the physiological trade-off between immune system and other physiological systems.

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Concluding remarks and perspective

Since its discovery in 1994, leptin has attracted increasing interest from the scientific community

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because of its pleiotropic functions. Indeed during the last decade, there has been a growing understanding of how host nutritional status and metabolism can affect the immune response, trough leptin. In this context, leptin acts as a pro-inflammatory cytokine that promotes Th1 responses on one side, and inhibits Treg cell expansion on the other, setting the basis for

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exaggerated, immunoinflammatory responses to altered self or non-self and leading to autoimmunity. Recent molecular cloning and functional studies have increased our understanding of the diversity of functions and evolutionary history of leptin. Despite low primary amino acid

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sequence conservation, leptin from different species is predicted to form similar tertiary structures and to bind to the LepR, activating specific intracellular signaling pathways. At functional level,

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although many effects of this adipocytokine have been elucidated in mammals, only few data are present for non-vertebrates. Similar actions of leptin on feeding and regulation of energy balance have been discovered in non-mammalian species, although the hypothesis that leptin functions as an adiposity signal in non-mammals remains to be tested, as it has not been completely elucidated in most species so far, even though Michel and co-workers showed that leptin may not act as adipostat in zebrafish (Michel et al., 2016). For this reason, it seems premature to draw definitive conclusions about how leptin function has evolved in vertebrates or even more generally within the animal kingdom. Therefore, comparative leptin research can further elucidate the (pleiotropic) role of leptin 21

ACCEPTED MANUSCRIPT in the earliest vertebrates, and with that, shed light on the origin and evolution of the system that plays such an important role in vertebrate energy homeostasis. Future studies are also needed to identify the precise relationship among leptin, metabolic state, and immune system function in different living organisms. Moreover it will also be crucial to focus on how leptin signaling

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interferes with the intracellular cascades activated by other factors in the immune cells, since understanding its precise mechanism of action will be pivotal to the development of novel

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therapeutic approaches for autoimmunity and obesity-induced inflammatory diseases.

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ACCEPTED MANUSCRIPT Acknowledgements G.M. is supported by grants from the Fondazione Italiana Sclerosi Multipla (FISM) 2012/R/11, Italian Space Agency (ASI) n. 2014-033-R.O. and the European Foundation for the Study of

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Diabetes/Juvenile Diabetes Research Foundation (EFSD/JDRF)/Lilly Programme 2015. V.D.R. is supported by the Ministero della Salute Grant n. GR-2010-2315414, TRIDEO-AIRC Id. 17447 and the Fondazione Italiana Sclerosi Multipla (FISM) n. 2014/R/21. M.G. is supported by grant from

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Juvenile Diabetes Research Foundation (JDRF) n. 1-PNF-2015-115-5-B.

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ACCEPTED MANUSCRIPT References Agrawal, S., Gollapudi, S., Su, H., Gupta, S., 2011. Leptin activates human B cells to secrete TNFα, IL-6, and IL-10 via JAK2/STAT3 and p38MAPK/ERK1/2 signaling pathway. J. Clin. Immunol. 31, 472-478.

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Aleffi, S., Petrai, I., Bertolani, C., Parola, M., Colombatto, S. Novo, E., et al., 2005. Upregulation of proinflammatory and proangiogenic cytokines by leptin in human hepatic stellate cells. Hepatology. 42, 1339–1348. Alonso-Alvarez, C., Bertrand, S., Sorci, G., 2007. Energetic reserves, leptin and testosterone: a refinement of the immunocompetence handicap hypothesis. Biol. Lett. 3, 271-274.

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Ashwell, C.M., Czerwinski, S.M., Brocht, D.M., McMurtry, J.P., 1999. Hormonal regulation of leptin expression in broiler chickens. Am J Physiol. 276(1 Pt 2):R226-232.

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Batra, A., Okur, B., Glauben, R., Erben, U., Ihbe, J., Stroh, T., et. Al, 2010. Leptin: a critical regulator of CD4+ T-cell polarization in vitro and in vivo. Endocrinology. 151, 56-62. Bennett, B.D., Solar, G.P., Yuan, J.Q., Mathias, J., Thomas, G.R., Matthews, W., 1996. A role for leptin and its cognate receptor in hematopoiesis. Curr Biol. 6, 1170-1180. Berti, L., Gammeltoft, S., 1999. Leptin stimulates glucose uptake in C2C12 muscle cells by activation of ERK2. Mol. Cell. Endocrinol. 157, 121–130.

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Boden, G., Chen, X., Kolaczynski, J.W., Polansky, M., 1997. Effects of prolonged hyperinsulinemia on serum leptin in normal human subjects. J. Clin. Invest. 100, 1107–1113. Boswell, T., Dunn, I.C., Wilson, P.W., Joseph, N., Burt, D.W., Sharp, P.J., 2006. Identification of a non-mammalian leptin-like gene: characterization and expression in the tiger salamander (Ambystoma tigrinum). Gen. Comp. Endocrinol. 146, 157–166.

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Bruno, A., Conus, S., Schmid, I., Simon, H.U., 2005. Apoptotic pathways are inhibited by leptin receptor activation in neutrophils. J. Immunol. 174, 8090-8096.

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Caldefie-Chezet, F., Poulin, A., Vasson, M.P., 2003. Leptin regulates functional capacities of polymorphonuclear neutrophils. Free Radic. Res. 37, 809-814. Calixto, M.C., Lintomen, L., Schenka, A., Saad, M.J., Zanesco, A., Antunes, E., 2010. Obesity enhances eosinophilic inflammation in a murine model of allergic asthma. Br. J. Pharmacol. 159, 617-625. Cao, Q., Mak, K.M., Ren, C., Lieber, C.S., 2004. Leptin stimulates tissue inhibitor of metalloproteinase-1 in human hepatic stellate cells: respective roles of the JAK/STAT and JAKmediated H2O2-dependant MAPK pathways. J. Biol. Chem. 279, 4292–4304. Cao, Y.B., Xue, J.L., Wu, L.Y., Jiang, W., Hu, P.N., Zhu, J., 2011. The detection of 3 leptin receptor isoforms in crucian carp gill and the influence of fasting and hypoxia on their expression. Domest. Anim. Endocrinol. 41, 74-80.

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ACCEPTED MANUSCRIPT Cassano, S., Pucino, V., La Rocca, C., Procaccini, C., De Rosa, V., Marone, G., et al., 2014. Leptin modulates autophagy in human CD4+CD25- conventional T cells. Metabolism. 63, 1272-1279. Chehab, F., Lim, M., Lu, R., 1996. Correction of the sterility defect in homozygous obese female mice by treatment with the human recombinant leptin. Nature Genet. 12, 318–320.

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Chen, J., Li, J., Lim, F.C., Wu, Q., Douek, D.C., Scott, D.K., et al., 2010. Louisiana Healthy Aging Study. Maintenance of naïve CD8 T cells in nonagenarians by leptin, IGFBP3 and T3. Mech. Ageing Dev. 131, 29-37.

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Chisada, S., Kurokawa, T., Murashita, K., Rønnestad, I., Taniguchi, Y., Toyoda, A., et al., Sakaki 2014. Leptin receptor-deficient (knockout) medaka, Oryzias latipes, show chronical up-regulated levels of orexigenic neuropeptides, elevated food intake and stage specific effects on growth and fat allocation. Gen Comp Endocrinol. 195, 9-20.

M AN U

Cipriani, S., Mencarelli, A., Palladino, G., Fiorucci, S., 2010. FXR activation reverses insulin resistance and lipid abnormalities and protects against liver steatosis in Zucker (fa/fa) obese rats. J. Lipid. Res. 51, 771–784. Clarke, I.J., Henry, B., Iqbal, J., Goding, J.W., 2001. Leptin and the regulation of food intake and the neuroendocrine axis in sheep. Clin. Exp. Pharmacol. Physiol. 28, 106–107. Review Claycombe, K., King, L.E., Fraker, P.J., 2008. A role for leptin in sustaining lymphopoiesis and myelopoiesis. Proc. Natl. Acad. Sci. USA. 105, 2017-2021.

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Conus, S., Bruno, A., Simon, H.U., 2005. Leptin is an eosinophil survival factor. J. Allergy Clin. Immunol. 116, 1228-1234. Crespi, E.J., Denver, R.J., 2006. Leptin (ob gene) of the South African clawed frog Xenopus laevis. Proc. Natl. Acad. Sci. U S A. 103, 10092–10097.

EP

Daković, N., Térézol, M., Pitel, F., Maillard, V., Elis, S., Leroux, S., et al., 2014. The loss of adipokine genes in the chicken genome and implications for insulin metabolism. Mol Biol Evol. 31, 2637-2646

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Dalman, M.R., Mustafa, A., Liu, Q., Londraville, R.L., 2013. Leptin knockdown reduces innate immune function in zebrafish. The FASEB Journal. 27, 937.20 (Meeting Abstract). De Rosa, V., Procaccini, C., Calì, G., Pirozzi, G., Fontana, S., Zappacosta, S., et al., 2007. A key role of leptin in the control of regulatory T cell proliferation. Immunity. 26, 241-255. Denver, R.J., Bonett, R.M., Boorse, G.C., 2011. Neuroendocrinology. 94, 21-38.

Evolution of leptin structure and function.

Doyon, C., Drouin, G., Trudeau, V.L., Moon, T.W., 2001. Molecular evolution of leptin. Gen. Comp. Endocrinol. 124, 188-198. Ducy, P., Amling, M., Takeda, S., Priemel, M., Schilling, A.F., Beil, F.T., et al., 2000. Leptin inhibits bone formation through a hypothalamic relay: a central control of bone mass. Cell. 100, 197-207. 25

ACCEPTED MANUSCRIPT Elias, C.F., Kelly, J.F., Lee, C.E., Ahima, R.S., Drucker, D.J., Saper, C.B., et al., 2000. Chemical characterization of leptin-activated neurons in the rat brain. J Comp Neurol. 423, 261–281. Elinav, E.1., Pappo, O., Sklair-Levy, M., Margalit, M., Shibolet, O., Gomori, M., et al., 2006. Adoptive transfer of regulatory NKT lymphocytes ameliorates non-alcoholic steatohepatitis and glucose intolerance in ob/ob mice and is associated with intrahepatic CD8 trapping. J. Pathol. 209, 121-128.

RI PT

Flier, J.S., 1995. The adipocyte: storage depot or node on the energy information superhighway? Cell. 80, 15–18.

SC

Fraser, D.A., Thoen, J., Reseland, J.E., Førre, O., Kjeldsen-Kragh, J., 1999. Decreased CD4+ lymphocyte activation and increased interleukin-4 production inperipheral blood of rheumatoid arthritis patients after acute starvation. Clin. Rheumatol. 18, 394–401.

M AN U

French, S.S., DeNardo, D.F., Moore, M.C., 2007. Trade-offs between the reproductive and immune systems: facultative responses to resources or obligate responses to reproduction? Am. Nat. 170, 7989. French, S.S., Dearing, M.D., Demas, G.E., 2011. Leptin as a physiological mediator of energetic trade-offs in ecoimmunology: implications for disease. Integr. Comp. Biol. 51, 505-513.

Friedman, J.M., Halaas, J.L., 1998. Leptin and the regulation of body weight in mammals. Nature. 395, 763–770.

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Friedman-Einat, M., Cogburn, L.A., Yosefi, S., Hen, G., Shinder, D., Shirak, A., et al., 2014. Discovery and characterization of the first genuine avian leptin gene in the rock dove (Columba livia). Endocrinology. 155, 3376-3384.

EP

Fuentes, E.N., Kling, P., Einarsdottir, I.E., Alvarez, M., Valdés, J.A., Molina, A., et al., 2012. Plasma leptin and growth hormone levels in the fine flounder (Paralichthys adspersus) increase gradually during fasting and decline rapidly after refeeding. Gen. Comp. Endocrinol. 177, 120-127.

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Gabay, C., Dreyer, M., Pellegrinelli, N., Chicheportiche, R., Meier, C.A., 2001. Leptin directly induces the secretion of interleukin 1 receptor antagonist in human monocytes. J. Clin. Endocrinol. Metab. 86, 783-791. Galgani, M., Procaccini, C., De Rosa, V., Carbone, F., Chieffi, P., La Cava, A., et al., 2010. Leptin modulates the survival of autoreactive CD4+ T cells through the nutrient/energy-sensing mammalian target of rapamycin signaling pathway. J. Immunol. 185, 7474-7479. Gomez, R., Lago, F., Gomez-Reino, J., Dieguez, C., Gualillo, O., 2009. Adipokines in the skeleton: influence on cartilage function and joint degenerative diseases. J. Mol. Endocrinol. 43, 11-8. Gómez, R., Conde, J., Scotece, M., Gómez-Reino, J.J., Lago, F., Gualillo, O., 2011. What's new in our understanding of the role of adipokines in rheumatic diseases? Nat. Rev. Rheumatol. 2, 528536.

26

ACCEPTED MANUSCRIPT Gonzalez-Navarro, H., Burks, D.J., Andres, V., 2007. Murine models to investigate the influence of diabetic metabolism on the development of atherosclerosis and restenosis. Front. Biosci. 12, 44394455. Gorissen, M., Bernier, N.J., Nabuurs, S.B., Flik, G., Huising, M.O., 2009. Two divergent leptin paralogues in zebrafish (Danio rerio) that originate early in teleostean evolution. J Endocrinol. 201, 329-339.

RI PT

Grotta, M.B., Squebola-Cola, D.M., Toro, A.A., Ribeiro, M.A., Mazon, S.B., Ribeiro, et al., 2013. Obesity increases eosinophil activity in asthmatic children and adolescents. BMC Pulm. Med. 18, 13-39.

SC

Gruen, M.L., Hao, M., Piston, D.W., Hasty, A.H., 2007. Leptin requires canonical migratory signaling pathways for induction of monocyte and macrophage chemotaxis. Am. J. Physiol. Cell. Physiol. 293, C1481-1488.

M AN U

Gualillo, O., Eiras, S., Lago, F., Dieguez, C., Casanueva, F.F., 2000. Elevated serum leptin concentrations induced by experimental acute inflammation. Life Sci. 67, 2433–2441. Gupta, S., Agrawal, S., Gollapudi, S., 2013. Increased activation and cytokine secretion in B cells stimulated with leptin in aged humans. Immun. Ageing. 23, 10-13. Howard, J.K., Lord, G.M., Matarese, G., Vendetti, S., Ghatei, M.A., Ritter, M.A., et al., 1999. Leptin protects mice from starvation-induced lymphoid atrophy and increases thymic cellularity in ob/ob mice. J. Clin. Invest. 104, 1051-1059.

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Huang, G., Li, J., Wang, H., Lan, X., Wang, Y., 2014. Discovery of a novel functional leptin protein (LEP) in zebra finches: evidence for the existence of an authentic avian leptin gene predominantly expressed in the brain and pituitary. Endocrinology. 155, 3385-3396.

EP

Huising, M.O., Geven, E.J., Kruiswijk, C.P., Nabuurs, S.B., Stolte, E.H., Spanings, F.A., et al., 2006. Increased leptin expression in common Carp (Cyprinus carpio) after food intake but not after fasting or feeding to satiation. Endocrinology. 147, 5786-5797.

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Iliopoulos, D., Malizos, K.N., Tsezou, A., 2007. Epigenetic regulation of leptin affects MMP-13 expression in osteoarthritic chondrocytes: possible molecular target for osteoarthritis therapeutic intervention. Annals of the Rheumatic Diseases. 66, 1616–1621. Jitprasertwong, P., Jaedicke, K.M., Nile, C.J., Preshaw, P.M., 2014. Taylor JJ. Leptin enhances the secretion of interleukin (IL)-18, but not IL-1β, from human monocytes via activation of caspase-1. Cytokine. 65, 222-230. Kato, H., Ueki, S., Kamada, R., Kihara, J., Yamauchi, Y., Suzuki, T., et al., 2011. Leptin has a priming effect on eotaxin-induced human eosinophil chemotaxis. Int. Arch. Allergy Immunol. 155, 335-344. Kiguchi, N., Maeda, T., Kobayashi, Y., Fukazawa, Y., Kishioka, S., 2009. Leptin enhances CCchemokine ligand expression in cultured murine macrophage. Biochem. Biophys. Res. Commun. 384, 311-315.

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ACCEPTED MANUSCRIPT Kuo, A.Y., Cline, M.A., Werner, E., Siegel, P.B., Denbow, D.M., 2005. Leptin effects on food and water intake in lines of chickens selected for high or low body weight. Physiol. Behav. 84, 459-464. Kurokawa, T., Uji, S., Suzuki, T., 2005. Identification of cDNA coding for a homologue to mammalian leptin from pufferfish, Takifugu rubripes. Peptides. 26,745–750. La Cava, A., Matarese, G., 2004. The weight of leptin in immunity. Nat. Rev. Immunol. 4, 371-379.

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Lam, Q.L., Wang, S., Ko ,O.K., Kincade, P.W., Lu, L., 2010. Leptin signaling maintains B-cell homeostasis via induction of Bcl-2 and Cyclin D1. Proc. Natl. Acad. Sci. USA. 107, 13812-13827. Laplante, M., Sabatini, D.M., 2010. mTOR signaling at a glance. J. Cell. Sci. 122, 3589-3594.

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Li, G.G., Liang, X.F., Xie, Q., Li, G., Yu, Y., Lai, K., 2010. Gene structure, recombinant expression and functional characterization of grass carp leptin. Gen. Comp. Endocrinol. 166, 117-127.

M AN U

Liu, X., Dunn, I.C., Sharp, P.J., Boswell, T., 2007. Molecular cloning and tissue distribution of a short form chicken leptin receptor mRNA. Domest. Anim. Endocrinol. 32, 155-166. Liu, Q., Chen, Y., Copeland, D., Ball, H., Duff, R.J., Rockich, B., et al., 2010. Expression of leptin receptor gene in developing and adult zebrafish. Gen. Comp. Endocrinol. 166, 346-355. Liu, Q., Dalman, M., Chen, Y., Akhter, M., Brahmandam, S., Patel, Y., et al., 2012. Knockdown of leptin A expression dramatically alters zebrafish development. Gen. Comp. Endocrinol. 178, 562572.

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Loffreda, S., Yang, S.Q., Lin, H.Z., Karp, C.L., Brengman, M.L., Wang, D.J, et al., 1998. Leptin regulates proinflammatory immune responses. FASEB. 12, 57–65. Lõhmus, M., Olin, M., Sundström, L.F., Troedsson, M.H., Molitor, T.W., El Halawani, M., 2004. Leptin increases T-cell immune response in birds. Gen. Comp. Endocrinol. 139, 245-250.

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Londraville, R.L., Macotela, Y., Duff, R.J., Easterling, M.R., Liu, Q., Crespi, E.J., 2014. Comparative endocrinology of leptin: assessing function in a phylogenetic context. Gen. Comp. Endocrinol. 1, 146-157.

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Lord, G.M., Matarese, G., Howard, J.K., Baker, R.J., Bloom, S.R., Lechler, R.I., 1998. Leptin modulates the T-cells immune response and reverses starvation-induced immunosoppression. Nature. 394, 897-901. Lord, G.M., Matarese, G., Howard, J.K., Lechler, R.I., 2001. The bioenergetics of the immune system. Science. 292, 855–856. Love, N.R., Chen, Y., Bonev, B., Gilchrist, M.J., Fairclough, L., Lea, R., et al., 2011. Genome-wide analysis of gene expression during Xenopus tropicalis tadpole tail regeneration. BMC. Dev. Biol. 11, 70. Lynch, L., Nowak, M., Varghese, B., Clark, J., Hogan, A.E., Toxavidis, V., et al., 2012. Adipose tissue invariant NKT cells protect against diet-induced obesity and metabolic disorder through regulatory cytokine production. Immunity. 37, 574-587. 28

ACCEPTED MANUSCRIPT Macia, L., Delacre, M., Abboud, G., Ouk, T.S., Delanoye, A., Verwaerde, C., et al., 2006. Impairment of dendritic cell functionality and steady-state number in obese mice. J. Immunol. 177, 5997-6006. Mancour, L.V., Daghestani, H.N., Dutta, S., Westfield, G.H., Schilling, J., Oleskie, A.N., et al., 2012. Ligand-induced architecture of the leptin receptor signaling complex. Mol Cell. 48, 655-661.

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Mancuso, P., Canetti, C., Gottschalk, A., Tithof, P.K., Peters-Golden, M., 2004. Leptin augments alveolar macrophage leukotriene synthesis by increasing phospholipase activity and enhancing group IVC iPLA2 (cPLA2gamma) protein expression. Am. J. Physiol. Lung Cell Mol. Physiol. 287, L497-502.

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Mariano, G., Stilo, R., Terrazzano, G., Coccia, E., Vito, P., Varricchio, E., et al., 2013. Effects of recombinant trout leptin in superoxide production and NF-κB/MAPK phosphorylation in blood leukocytes. Peptides. 48, 59-69.

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Matarese, G., La Rocca, C., Moon, H.S., Huh, J.Y., Brinkoetter, M.T., Chou, S., et al., 2013. Selective capacity of metreleptin administration to reconstitute CD4+ T-cell number in females with acquired hypoleptinemia. Proc. Natl. Acad. Sci. USA. 110, E818-827. Mattioli, B., Giordani, L., Quaranta, M.G., Viora, M., 2009. Leptin exerts an anti-apoptotic effect on human dendritic cells via the PI3K-Akt signaling pathway. FEBS Lett. 583, 1102-1106. Mattioli, B., Straface, E., Matarrese, P., Quaranta, M.G., Giordani, L., Malorni W, et al., 2008. Leptin as an immunological adjuvant: enhanced migratory and CD8+ T cell stimulatory capacity of human dendritic cells exposed to leptin. FASEB J. 22, 2012-2022.

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Mattioli, B., Straface, E., Quaranta, M.G., Giordani, L., Viora, M., 2005. Leptin promotes differentiation and survival of human dendritic cells and licenses them for Th1 priming. J. Immunol. 174, 6820-6828.

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Michel, M., Page-McCaw, P.S., Chen, W., Cone, R.D., 2016. Leptin signaling regulates glucose homeostasis, but not adipostasis, in the zebrafish. Proc. Natl. Acad. Sci. U S A.

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Moore, S.I., Huffnagle, G.B., Chen, G.H., White, E.S., Mancuso, P., 2003. Leptin modulates neutrophil phagocytosis of Klebsiella pneumoniae. Infect. Immun. 71, 4182-4185. Moraes-Vieira, P.M., Larocca, R.A., Bassi, E.J., Peron, J.P., Andrade-Oliveira, V., Wasinski, F., et al., 2014. Leptin deficiency impairs maturation of dendritic cells and enhances induction of regulatory T and Th17 cells. Eur. J. Immunol. 44, 794-806. Murashita, K., Uji, S., Yamamoto, T., Rønnestad, I., Kurokawa, T., 2008. Production of recombinant leptin and its effects on food intake in rainbow trout (Oncorhynchus mykiss). Comp. Biochem. Physiol. B. Biochem. Mol. Biol. 150, 377–384. Niewiarowski, P.H., Balk, M.L., Londraville, R.L., 2000. Phenotypic effects of leptin in an ectotherm: a new tool to study the evolution of life histories and endothermy? J. Exp. Biol. 203, 295-300.

29

ACCEPTED MANUSCRIPT O'Rourke, L., Gronning, L.M., Yeaman, S.J., Shepherd, P.R., 2002. Glucose-dependent regulation of cholesterol ester metabolism in macrophages by insulin and leptin. J. Bio. Chem. 277, 4255742562. Otero, M., Gomez Reino, J.J., Gualillo, O., 2003. Synergistic induction of nitric oxide synthase type II: in vitro effect of leptin and interferongamma in human chondrocytes and ATDC5 chondrogenic cells. Arthritis and Rheumatism. 48, 404–409.

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Otero, M., Lago, R., Lago, F., Reino, J.J., Gualillo, O., 2005. Signalling pathway involved in nitric oxide synthase type II activation in chondrocytes: synergistic effect of leptin with interleukin-1. Arthritis Res. Ther. 7, R581-591.

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Papathanassoglou, E., El-Haschimi, K., Li, X.C., Matarese, G., Strom, T., Mantzoros, C., 2006. Leptin receptor expression and signaling in lymphocytes: kinetics during lymphocyte activation, role in lymphocyte survival, and response to high fat diet in mice. J. Immunol. 176, 7745-7752.

M AN U

Patterson, C.M., Leshan, R.L., Jones, J.C., Myers, M.G.Jr., 2011. Molecular mapping of mouse brain regions innervated by leptin receptor-expressing cells. Brain Res. 1378, 18-28. Pelleymounter, M.A., Cullen, M.J., Baker, M.B., Hecht, R., Winters, D., Boone, T., et al., 1995. Effects of the obese gene product on body weight regulation in ob/ob mice. Science. 269, 540–543. Procaccini, C., De Rosa, V., Galgani, M., Abanni, L., Calì, G., Porcellini, A., et al., 2010. An oscillatory switch in mTOR kinase activity sets regulatory T cell responsiveness. Immunity. 33, 929-941.

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Procaccini, C., Jirillo, E., Matarese, G., 2012a. Leptin as an immunomodulator. Mol. Aspects. Med. 33, 35-45.

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Procaccini, C., De Rosa, V., Galgani, M., Carbone, F., Cassano, S., Greco, D., et al., 2012b. Leptininduced mTOR activation defines a specific molecular and transcriptional signature controlling CD4+ effector T cell responses. J. Immunol. 189, 2941-2953.

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Prokop, J.W., Duff, R.J., Ball, H.C., Copeland, D.L., Londraville, R.L., 2012. Leptin and leptin receptor: analysis of a structure to function relationship in interaction and evolution from humans to fish. Peptides. 38, 326-336. Prokop, J.W., Schmidt, C., Gasper, D., Duff, R.J., Milsted, A., Ohkubo, T., et al., 2014. Discovery of the elusive leptin in birds: identification of several 'missing links' in the evolution of leptin and its receptor. PLoS One. 24, e92751. Pucino, V., De Rosa, V., Procaccini, C., Matarese, G., 2014. Regulatory T cells, leptin and angiogenesis. Chem Immunol Allergy. 99, 155-169. Reis, B.S., Lee, K., Fanok, M.H., Mascaraque, C., Amoury, M., Cohn, L.B., et al., 2015. Leptin receptor signaling in T cells is required for Th17 differentiation. J Immunol. 194, 5253-5260. Rodríguez, L., Graniel, J., Ortiz, R., 2007. Effect of leptin on activation and cytokine synthesis in peripheral blood lymphocytes of malnourished infected children. Clin. Exp. Immunol.148, 478-485.

30

ACCEPTED MANUSCRIPT Rønnestad, I., Nilsen, T.O., Murashita, K., Angotzi, A.R., Gamst, Moen, A.G., Stefansson, S.O., et al., 2010. Leptin and leptin receptor genes in Atlantic salmon: Cloning, phylogeny, tissue distribution and expression correlated to long-term feeding status. Gen. Comp. Endocrinol. 168, 5570. Rummel, C., Inoue, W., Poole, S., Luheshi, G.N., 2010. Leptin regulates leukocyte recruitment into the brain following systemic LPS-induced inflammation. Mol. Psychiatry.15, 523-534.

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Santos-Alvarez, J., Goberna, R., Sánchez-Margalet, V., 1999. Human leptin stimulates proliferation and activation of human circulating monocytes. Cell. Immunol. 194, 6-11.

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Seroussi E, Cinnamon Y, Yosefi S, Genin O, Smith JG, Rafati N, et al., 2016. Identification of the Long-Sought Leptin in Chicken and Duck: Expression Pattern of the Highly GC-Rich Avian leptin Fits an Autocrine/Paracrine Rather Than Endocrine Function. Endocrinology. 157, 737-751. Shen, J., Sakaida, I., Uchida, K., Terai, S., Okita, K., 2005. Leptin enhances TNF-alpha production via p38 and JNK MAPK in LPS-stimulated Kupffer cells. Life Sci.77, 1502-1515.

M AN U

Sierra-Honigmann, M.R., Nath, A.K., Murakami, C., García-Cardeña, G., Papapetropoulos, A., Sessa, W.C., et al., 1998. Biological action of leptin as an angiogenic factor. Science. 281, 16831686. Simopoulou, T., Malizos, K.N., Iliopoulos, D., Stefanou, N., Papatheodorou, L., Ioannou, M., 2007. Differential expression of leptin and leptin’s receptor isoform (Ob-Rb) mRNA between advanced and minimally affected osteoarthritic cartilage; effect on cartilage metabolism. Osteoarthritis Cartilage. 15, 872-883.

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Sivitz, W.I., Walsh, S.A., Morgan, D.A., Thomas, M.J., Haynes, W.G., 1997. Effects of leptin on insulin sensitivity in normal rats. Endocrinology. 138, 3395-3401.

EP

Suzukawa, M., Nagase, H., Ogahara, I., Han, K., Tashimo, H., Shibui, A., et al., 2011. Leptin enhances survival and induces migration, degranulation, and cytokine synthesis of human basophils. J. Immunol. 186, 5254-5260.

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Taleb, S., Herbin, O., Ait-Oufella, H., Verreth, W., Gourdy, P., Barateau, V.,et al., 2007. Defective leptin/leptin receptor signaling improves regulatory T cell immune response and protects mice from atherosclerosis. Arterioscler Thromb Vasc Biol. 27, 2691-2698. Tanaka, M., Suganami, T., Kim-Saijo, M., Toda, C., Tsuiji, M., Ochi, K., et al., 2011. Role of central leptin signaling in the starvation-induced alteration of B-cell development. J. Neurosci. 31, 8373-8380. Tang, C.H., Lu, D.Y., Yang, R.S., Tsai, H.Y., Kao, M.C., Fu, W.M., et al., 2007. Leptin-induced IL-6 production is mediated by leptin receptor, insulin receptor substrate-1, phosphatidylinositol 3kinase, Akt, NF-kappaB, and p300 pathway in microglia. J. Immunol. 179, 1292-1302. Taouis, M., Chen, J.W., Daviaud, C., Dupont, J., Derouet, M., Simon, J., 1998. Cloning the chicken leptin gene. Gene. 27, 239-242. Tartaglia, L.A., 1997. The leptin receptor. Journal of Biological Chemistry. 272, 6093–6096. 31

ACCEPTED MANUSCRIPT Tian, Z., Sun, R., Wei, H., Gao, B., 2002. Impaired natural killer (NK) cell activity in leptin receptor deficient mice: leptin as a critical regulator in NK cell development and activation. Biochem. Biophys. Res. Commun. 298, 297-302. Torday, J.S., Ihida-Stansbury, K., Rehan, V.K., 2009. Leptin stimulates Xenopus lung development: evolution in a dish. Evol. Dev. 11, 219-224.

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Trombley, S., Maugars, G., Kling, P., Björnsson, B.T., Schmitz, M., 2012. Effects of long-term restricted feeding on plasma leptin, hepatic leptin expression and leptin receptor expression in juvenile Atlantic salmon (Salmo salar L.). Gen. Comp. Endocrinol. 175, 92-99.

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Tsochatzis, E., Papatheodoridis, G.V., Archimandritis, A.J., 2006. The evolving role of leptin and adiponectin in chronic liver diseases. Am. J. Gastroenterol. 101, 2629-2640.

M AN U

Venken, K., Seeuws, S., Zabeau, L., Jacques, P., Decruy, T., Coudenys, J., et al., 2014. A bidirectional crosstalk between iNKT cells and adipocytes mediated by leptin modulates susceptibility for T cell mediated hepatitis. J. Hepatol. 60, 175-182. Vernooy, J.H., Bracke, K.R., Drummen, N.E., Pauwels, N.S., Zabeau, L., Van Suylen, R.J., et al., 2010. Leptin modulates innate and adaptive immune cell recruitment after cigarette smoke exposure in mice. J. Immunol. 184, 7169-7177. Volkoff, H., Eykelbosh, A.J., Peter, R.E., 2003. Role of leptin in the control of feeding of goldfish Carassius auratus: interactions with cholecystokinin, neuropeptide Y and orexin A, and modulation by fasting. Brain Res. 972, 90-109.

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Wang, S., Baidoo, S.E., Liu, Y., Zhu, C., Tian, J., Ma, J., et al., 2013. T cell-derived leptin contributes to increased frequency of T helper type 17 cells in female patients with Hashimoto's thyroiditis. Clin. Exp. Immunol. 171, 63-68.

EP

Wolf , G., Hamann, A., Han, D.C., Helmchen, U., Thaiss, F., Ziyadeh, F.N., et al., 1999. Leptin stimulates proliferation and TGF- beta expression in renal glomerular endothelial cells: potential role in glomerulosclerosis. Kidney Int. 56, 860-872.

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Wong, C.K., Cheung, P.F., Lam, C.W., 2007. Leptin-mediated cytokine release and migration of eosinophils: implications for immunopathophysiology of allergic inflammation. Eur. J. Immunol. 37, 2337-2348. Yamagishi, S.I., Edelstein, D., Du, X.L., Kaneda, Y., Guzmán, M., Brownlee, M., 2001. Leptin induces mitochondrial superoxide production and monocyte chemoattractant protein-1 expression in aortic endothelial cells by increasing fatty acid oxidation via protein kinase A. J. Biol. Chem. 276, 25096-25100. Yan, K., Deng, X., Zhai, X., Zhou, M., Jia, X., Luo, L., et al., 2012. p38 mitogen-activated protein kinase and liver X receptor-alpha mediate the leptin effect on sterol regulatory element binding protein-1c. expression in hepatic stellate cells Mol. Med. 18, 10–18. Yu, Y., Liu, Y., Shi, F.D., Zou, H., Matarese, G., La Cava, A., 2013. Cutting edge: Leptin-induced RORγt expression in CD4+ T cells promotes Th17 responses in systemic lupus erythematosus. J. Immunol. 190, 3054-3058. 32

ACCEPTED MANUSCRIPT Zakrzewska, K.E., Cusin, I., Sainsbury, A., Rohner-Jeanrenaud, F., Jeanrenaud, B., 1997. Glucocorticoids as counterregulatory hormones of leptin: toward an understanding of leptin resistance. Diabetes. 46, 717–719.

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Zarkesh-Esfahani, H., Pockley, A.G., Wu, Z., Hellewell, P.G., Weetman, A.P., Ross, R.J., 2004. Leptin indirectly activates human neutrophils via induction of TNF-alpha. J. Immunol. 172, 18091814. Zhang, Y., Proenca, R., Maffei, M., Barone, M., Leopold, L., Friedman, J.M., 1994. Positional cloning of the mouse obese gene and its human homologue. Nature. 372, 425–432.

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Zhao, Y., Sun, R., You, L., Gao, C., Tian, Z., 2003. Expression of leptin receptors and response to leptin stimulation of human natural killer cell lines. Biochem. Biophys. Res. Commun. 300, 247252.

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ACCEPTED MANUSCRIPT Figure Legend: Figure 1. Model of leptin effects on immune system cells and functions in non-mammalian vertebrates. In fishes, leptin decreases JNK activation, activates ERK phosphorylation and inhibits superoxide production in leukocytes. In amphibians, leptin receptor mRNA is widely expressed in

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important immune organs such as the spleen. In reptiles, leptin is able to limit food intake, and reducing fat stores, it suppresses immune function. Indeed, leptin treatment reverses the immunosuppressive effect of food restriction in reproductive tree lizard females, suggesting that

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Reproductive system Leptin receptor mRNA

Energy trade-off

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T cells proliferation

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Leptin regulates function of innate immune cells (macrophages, dendritic cells, NK cells, neutrophils, eosinophils and basophils). Leptin increases and sustains CD4+ T cells function.

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Leptin is a negative signal for Treg cells function.

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Leptin is present in a wide range of non-mammalian vertebrate species and may affect their

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immune system function.

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