Leptin enhances the secretion of interleukin (IL)-18, but not IL-1β, from human monocytes via activation of caspase-1

Cytokine 65 (2014) 222–230

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Leptin enhances the secretion of interleukin (IL)-18, but not IL-1b, from human monocytes via activation of caspase-1 Paiboon Jitprasertwong 1, Katrin M. Jaedicke, Christopher J. Nile 2, Philip M. Preshaw, John J. Taylor ⇑ Centre for Oral Health Research and Institute of Cellular Medicine, Newcastle University, Framlington Place, Newcastle upon Tyne NE2 4BW, UK

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Article history: Received 24 May 2013 Received in revised form 10 September 2013 Accepted 28 October 2013 Available online 23 November 2013 Keywords: Leptin Interleukin-18 Caspase-1 Monocytes Inflammation

a b s t r a c t Circulating levels of leptin are elevated in type-2 diabetes mellitus (T2DM) and leptin plays a role in immune responses. Elevated circulating IL-18 levels are associated with clinical complications of T2DM. IL-18 regulates cytokine secretion and the function of a number of immune cells including T-cells, neutrophils and macrophages and as such has a key role in immunity and inflammation. Pro-inflammatory monocytes exhibiting elevated cytokine secretion are closely associated with inflammation in T2DM, however, little is known about the role of leptin in modifying monocyte IL-18 secretion. We therefore aimed to investigate the effect of leptin on IL-18 secretion by monocytes. We report herein that leptin increases IL-18 secretion in THP-1 and primary human monocytes but has no effect on IL-18 mRNA. Leptin and LPS signalling in monocytes occurs by overlapping but distinct pathways. Thus, in contrast to a strong stimulation by LPS, leptin has no effect on IL-1b mRNA levels or IL-1b secretion. In addition, LPS stimulates the secretion of IL-6 but leptin did not whereas both treatments up regulate IL-8 secretion from the same cells. Although leptin (and LPS) has a synergistic effect with exogenous ATP on IL-18 secretion in both THP-1 and primary monocytes, experiments involving ATP assays and pharmacological inhibition of ATP signalling failed to provide any evidence that endogenous ATP secreted by leptin-stimulated monocytes was responsible for enhancement of monocyte IL-18 secretion by leptin. Analysis of the action of caspase-1 revealed that leptin up regulates caspase-1 activity and the effect of leptin on IL-18 release is prevented by caspase-1 inhibitor (Ac-YVAD-cmk). These data suggest that leptin activates IL-18 processing rather than IL-18 transcription. In conclusion, leptin enhances IL-18 secretion via modulation of the caspase-1 inflammasome function and acts synergistically with ATP in this regard. This process may contribute to aberrant immune responses in T2DM and other conditions of hyperleptinemia. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Leptin is a 16KDa peptide hormone which is synthesised by adipocytes and has a fundamental role in the control of appetite

Abbreviations: E. coli, Escherichia coli; G-CSF, granulocyte-colony stimulating factor; JAK2, janus kinase 2; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinases; MDDC, monocyte-derived dendritic cells; NLRP3, NLR-family, pyrin containing 3; PAMPs, pathogen-activated molecular patterns; PI3 K, phosphoinositide 3-kinase; PPADS, pyrodoxal phosphor-6azo (benzene-2, 4-disulfonic acid) tetrasodium salt hydrate; STAT, signal transducer and activator of transcription; T2DM, type-2 diabetes mellitus; Th1, T helper 1; Th2, T helper 2. ⇑ Corresponding author. Address: School of Dental Sciences, Newcastle University, Framlington Place, Newcastle upon Tyne NE2 4BW, UK. E-mail addresses: [email protected] (P. Jitprasertwong), katrin.jaedicke@ncl. ac.uk (K.M. Jaedicke), [email protected] (C.J. Nile), philip.preshaw@ ncl.ac.uk (P.M. Preshaw), [email protected] (J.J. Taylor). 1 Present address: Department of Preventive Dentistry, Faculty of Dentistry, Naresuan University, Amphor Meaung, Phitsanulok 65000, Thailand. 2 Present address: Glasgow Dental School, 378 Suachiehall Street, Glasgow, Scotland G2 3JZ, UK. 1043-4666/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.cyto.2013.10.008

and metabolism [1]. Leptin also regulates hematopoiesis, immune responses, bone metabolism and wound repair [2]. Leptin levels are directly correlated with white adipose tissue mass and BMI [3]. Consequently, leptin levels are elevated in obesity and associated metabolic disorders including T2DM [1]. Also, leptin therapy is a potential approach for restoration of metabolic homeostasis in diabetes and a number of clinical trials are currently investigating this possibility [4]. The systemic effects of leptin treatment are complex and the efficacy of this treatment is influenced by endogenous leptin levels and leptin resistance. Furthermore, the potential of exogenous leptin treatment to cause side effects including altered immune function and inflammation has been highlighted [4]. Substantial clinical and experimental evidence supports a role for leptin in regulation of the immune system [5]. Indeed, leptin deficient ob/ob mice exhibit phenotypic abnormalities in macrophages and enhanced sensitivity to both LPS and TNF-a induced mortality, and exogenous leptin has a protective effect in these models [6,7]. Leptin is a member of the class 1 cytokine family

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and has structural homology to IL-6, IL-12 and G-CSF [8]. The leptin receptor is widely expressed in cells of the immune system and activates a number of independently regulated intra-cellular signalling pathways important in cytokine gene regulation; including JAK2/STAT, PI3K and MAPK [9]. Thus, in vitro, leptin influences cytokine secretion in a variety of cells including cells of the monocytes/macrophage lineage [10–14] and the levels of leptin itself are influenced by inflammatory mediators [15]. The IL-1 family of cytokines, which includes IL-18, has a number of key roles in immunity [16]. Like many cytokines, the function of IL-18 is influenced by synergistic interactions. IL-18 synergises with IL-12 (and IL-33) to regulate the development of IFN-c production by Th1 cells, NK cells and natural killer T cells [16]. Also, in the absence of IL-12, IL-18 can promote a Th2 phenotype and, with IL-23, IL-18 activates IL-17 secretion from T cells [16]. Significantly, circulating IL-18 is elevated in obesity [17], metabolic syndrome [18] and T2DM [19,20] and elevated IL-18 in T2DM is associated with the development of important clinical complications such a nephropathy [21] and atherosclerosis [22]. In common with IL-1b, IL-18 is synthesised as a pro-peptide which lacks a secretory signal and therefore requires proteolytic processing prior to secretion; this is achieved by casapse-1 [16]. However, whereas there is no constitutive expression of pro-IL-1b protein in monocytes and transcription of pro-IL-1b requires a pro-inflammatory signal [23], pro-IL-18 is constitutively expressed in monocytes, macrophages, dendritic cells and epithelial cells [16,24]. Regulation of the processing and secretion of IL-1 cytokines (including IL-1b and IL-18) are critical in the control of immune and inflammatory responses mediated by these cytokines. Caspase-1 mediated activation of pro-IL-1b and pro-IL-18 is regulated by the formation of the NLRP3 inflammasome complex which acts as the scaffold for caspase-1 activation and is, itself, activated by diverse signals such as ATP, LPS, cholesterol and molecules from dying cells such as uric acid crystals, which are all associated with tissue inflammation [25]. Therefore, understanding the signals that activate the inflammasome and caspase-1 is central to understanding inflammatory diseases and identifying novel therapeutic targets [26]. Work in our laboratory has shown that leptin provides a differentiation signal to human monocytes as evidenced by altered TLR expression [27]. Given the fact that pro-inflammatory monocytes exhibiting elevated cytokine secretion are closely associated with inflammation in T2DM [28] and the importance of IL-18 in metabolic disorders and immune responses we were therefore interested to investigate the possible role of leptin in regulation of IL-18 expression in monocytes We demonstrate that leptin upregulates IL-18 secretion in monocytes via activation of posttranscriptional pathways and this is independent of LPS signalling. The likely pathway for leptin mediated IL-18 secretion is via activation of the inflammasome determined by the fact that leptin up regulation of IL-18 secretion is mediated by caspase-1 activation and is synergistically regulated by ATP.

2. Materials and methods 2.1. Materials Unless otherwise stated, all chemical reagents and cell culture media were purchased from Sigma–Aldrich (Poole, UK) and all plasticware from Greiner Bio One (Stonehouse, UK). Vitamin D3 (1a, 25-dihydroxy-vitamin D3) was purchased from Calbiochem (Merck Chemicals, Nottingham, UK). Ultrapure LPS (TLR-4 agonist) from Escherichia coli 0111.B4 was purchased from Invivogen (Autogen Bioclear, Calne, UK). Human recombinant leptin was purchased from R&D Systems (Abingdon, UK). Analysis of endotoxin

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contamination of leptin preparations using a limulus amebocyte lysate assay (Lonza, Slough, UK) indicated that stock solutions of 100 lg/ml leptin (100x final working concentration) contained <0.06 ng/ml endotoxin. The caspase-1 inhibitor Ac-YVAD and the JAK-2 inhibitor AG490 were purchased from Calbiochem.

2.2. THP-1 monocytes cell culture and stimulation THP-1 pro-monocytes were purchased from the European Collection of Cell Cultures (Salisbury, UK) and cultured in RPMI1640 medium supplemented with 10% foetal calf serum, 2 mM lglutamine, 100 U/ml penicillin and 100 lg/ml streptomycin at 37 °C and 5% CO2. Cells were maintained at a density of 3–8  105 cells/ml. Viability was monitored using trypan blue exclusion and was always >95%. THP-1 cells have a pro-monocyte phenotype with low levels of CD14 expression, therefore prior to use in stimulation experiments we differentiated THP-1 promonocytes to a monocyte phenotype (CD14HIGH) by incubation with 0.1 lM vitamin D3 as previously described [29]. For mRNA analysis, a 6 well plate format was adopted with cells at a density of 4  106 in 4 ml of medium.

2.3. Primary monocyte isolation and culture Primary human monocytes were isolated from buffy coats obtained from the National Blood Service (Newcastle upon Tyne, UK). Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats by centrifugation over Histopaque and monocytes isolated from PBMCs using an EasySep CD14 positive selection kit (StemCell Technologies, Grenoble, France). The purity of monocyte preparations was analysed using FACS with an anti-CD14 antibody (Serotec, Oxford, UK) and was routinely >95% (data not shown). Cell culture conditions and stimulation experiments were identical to those employed for THP-1 monocytes with the exception that primary cells were not differentiated with vitamin D3.

2.4. ELISA Cytokine concentrations were determined in cell culture supernatants using ELISA. IL-1b, IL-6 and IL-8 were analysed using Duoset ELISA kits (R&D Systems) according to the manufacturer’s instructions. We developed a sandwich ELISA to detect IL-18. Briefly, 96-well microplates with high protein binding properties (Greiner Bio One) were coated overnight with 2 lg/ml monoclonal anti-human IL-18 antibody (Cat No. DO44-3, R&D Systems) in 0.1 M NaHCO2 pH 8.5. After washing with PBS/0.05% Tween-20 and blocking with 10% FCS PBS for 2 h, plates were again washed, and standards of recombinant human IL-18 (Cat No. B001-5, MBL, R&D Systems) or unknowns were added. Samples were incubated for 2 h and then washed in PBS/0.05% Tween-20 and 0.5 lg/ml detection antibody (biotinylated anti-hIL-18 antibody, Cat No. DO45-6, R&D Systems) added at a 1:1000 dilution in 10% FCS PBS and incubated for 2 h. After washing, the bound antibody was detected with 1/200 diluted avidin-HRP (Sigma–Aldrich) that was incubated for 30 min. A final wash was then performed and tetramethylbenzidine and H2O2 substrate solution (R&D Systems) was added to each well and incubated for 30 min. Colour development was stopped with 2 N H2SO4, and the resulting absorbance read at 450 nm on a FL600 Microplate reader (BioTek, Potton, UK). To correct for the background absorbance of the plate, a second reading at 550 nm was subtracted. Concentrations were determined by 4-parameter standard curve-fit. The sensitivity of this ELISA was 5.92 pg/ml.

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2.5. Real time RT-PCR

2.8. Statistical analysis

Total RNA was isolated from 4  106 cells/sample using a GenElute kit (Sigma–Aldrich). 1 lg RNA was reverse transcribed into cDNA using a High-capacity RT kit (Applied Biosystems, Warrington, UK). TaqMan Gene Expression assays (IL-18: Hs 99999040_m1, Applied Biosystems) with a Realtime PCR kit (SensiMixdT, Quantace, London) were employed for quantification of IL-18 expression. Relative fold changes between stimulations were calculated with the comparative Ct method (2 DDCt) [30], using RNA polymerase II (Hs00172187_m1) as the reference gene.

Results were expressed as means ± SD from 3 independent experiments. Standard transformations such as square root, common log or inverse were used to achieve normal distribution and equality of variance. Shapiro–Wilk testing for normal distribution and Levene testing for homogeneity of variance were performed prior to ANOVA. Parametric data were analysed with ANOVA. Non-parametric data was analysed with Kruskal–Wallis. Student’s t-test or Mann–Whitney U test was applied for post hoc analysis of parametric or non-parametric data, respectively. P-Values were corrected for multiple comparisons with the Bonferroni–Holm method. A p-value of <0.05 was considered significant. Statistical analysis of Realtime RT-PCR data was performed on DCt values [31].

2.6. Caspase-1 assay Caspase-1 activity in lysates from cultured monocytes was determined using an assay employing a colorimetric peptide substrate comprising a specific peptide (WEHD) conjugated to p-nitroalanine (R&D Systems). The assay was carried out according to the manufacturer’s instructions. Protein concentrations in the lysates were determined using a Bradford assay kit (Pierce Chemicals, Fischer Scientific, Loughbourgh, UK). Caspase-1 activity was quantified using a standard curve constructed using recombinant caspase-1 (Sigma–Aldrich) and expressed as pmol/min/mg protein. 2.7. ATP assay ATP was quantified in cell culture supernatants using a standard curve constructed using ATP (Sigma) and analysed using ATP Determination Kit according to the manufacturer’s instructions (Molecular Probes, Invitrogen, Glasgow, UK). The ecto-ATPase inhibitor ARL 67156 (Sigma) was employed in experiments analysing endogenous ATP secretion.

3. Results 3.1. Leptin upregulates IL-18 secretion in monocytes via activation of post-transcriptional pathways We investigated the role of leptin in the regulation of IL-18 synthesis by human monocytes. We have previously shown [29] that 100 ng/ml E. coli LPS stimulates IL-18 secretion in vitamin D3-differentiated THP-1 pro-monocytes and we adopted a similar protocol for the present experiments. Dose–response experiments (Fig. 1A) revealed that leptin significantly stimulated IL-18 secretion in THP-1 monocytes above controls over the range 250– 1000 ng/ml leptin after incubation for 24 h (p < 0.05) (Fig. 1A). Time-course experiments (Fig. 1B) demonstrated that 1000 ng/ ml leptin stimulated IL-18 release from THP-1 monocytes significantly above controls over an incubation period of 3–48 h. Thus,

Fig. 1. Leptin upregulates IL-18 protein secretion in monocytes but has no effect on IL-18 mRNA. (a) Secretion of IL-18 from leptin-stimulated THP-1 monocytes after 24 h culture. Data are mean ± SD of 3 separate experiments (n = 6). p < 0.05 as compared to leptin-free controls. The IL-18 responses to different doses of leptin were not significantly different from one another. (b) Time-course of IL-18 secretion from THP-1 monocytes in response to leptin (1 lg/ml) or E. coli LPS (100 ng/ml). Data are mean ± SD of 3 separate experiments (n = 6). p < 0.05 as compared to leptin-free controls. (c) Secretion of IL-18 from primary monocytes in response to leptin (1 lg/ml) or E. coli LPS (100 ng/ml) after 3 h culture. Data are mean ± SD of 3 separate experiments (n = 6). p < 0.05 as compared to leptin-free controls. (d) IL-18 mRNA levels in THP-1 monocytes in response to leptin (1 lg/ml) or E. coli LPS (100 ng/ml). Data are mean ± SD of 3 separate experiments (n = 6). p < 0.05 as compared to leptin-free controls.

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IL-18 in culture supernatants was elevated from a control level of 24.8 ± 7.7 pg/ml IL-18 to 79.4 ± 43.8 pg/ml IL-18 upon stimulation with 1000 ng/ml leptin for 3 h (p < 0.05). Although maximal IL-18 stimulation with leptin was observed after 48 h, there were no significant differences between the levels of IL-18 secreted from leptin stimulated cultures between 3 and 48 h (p > 0.05, not shown on figure). In comparison to 1000 ng/ml leptin, 100 ng/ml E. coli LPS elicited a more potent IL-18 response in THP-1 monocytes. We adopted these concentrations of leptin and E. coli LPS in all subsequent experiments. Assays of cell proliferation (Cell Titer 96 assay, Promega, Southampton, UK) indicated that 1000 ng/ml leptin did not induce any change in monocyte numbers after 6 and 24 h incubation (data not shown); the leptin-induced up regulation of IL-18 secretion was not, therefore, due to a mitogenic effect. Next, we analysed IL-18 responses using primary human monocytes purified from peripheral blood mononuclear cells using CD14 antibody selection. After 3 h in culture, primary monocytes exhibited a low level of constitutive IL-18 secretion (3.8 ± 1.5 pg/ml) which increased significantly to 12.1 ± 2.8 pg/ml (p < 0.05) in the presence of leptin (Fig. 1C). Interestingly, the IL-18 responses of primary monocytes to E. coli LPS at 3 h were also significantly elevated as compared to controls (10.9 ± 3.8 pg/ml; p < 0.05). In addition, the LPS responses of primary monocytes were quantitatively similar to those elicited by leptin (p > 0.05, not shown on figure) and this is in contrast to the data derived from similar experiments with THP-1 monocytes highlighting the particular sensitivity of THP-1 cells to LPS stimulation (Fig 1B). IL-18 mRNA was constitutively expressed in THP-1 monocytes was confirmed by RT-PCR (data not shown). Real-time RT-PCR was therefore employed to determine IL-18 mRNA expression in THP-1 monocytes in response to leptin and E. coli LPS (Fig. 1D). In contrast to changes observed in IL-18 protein secretion, both leptin and E. coli LPS had no effect on constitutive IL-18 mRNA expression after 1 and 3 h incubation (Fig. 1D). After 24 h, there was a significant down-regulation of IL-18 mRNA in THP-1 monocytes incubated with E. coli LPS (p < 0.05) but no similar changes in response to leptin (Fig. 1D). These data suggest that leptin upregulates IL-18 secretion in monocytes by signalling mechanisms downstream of mRNA synthesis.

3.2. The response of monocytes to leptin and LPS activation of cytokine secretion in monocytes is distinct but inter-related LPS delivers a potent pro-inflammatory signal to monocytes and up regulation of cytokine synthesis and secretion is one of the cellular responses elicited [32]. Both LPS and leptin stimulate IL-18 secretion in monocytes and we were interested to investigate the comparative effect of these ligands on other pro-inflammatory cytokines (Fig. 2A–D). Whereas E. coli LPS significantly (p < 0.05) stimulated IL-1b synthesis in THP-1 monocytes after 3 h of incubation at both the protein and mRNA levels, leptin had no effect (Fig. 2A. and B). Similarly, E. coli LPS significantly (p < 0.05) stimulated IL-6 protein secretion in monocytes after 3 h but leptin had no such effect (Fig. 2C). However, both leptin and E. coli LPS significantly (p < 0.05) upregulated IL-8 secretion in the same cells after 3 h (Fig. 2D). Leptin binding to its receptor activates intra-cellular signalling via JAK-2 [33] so we investigated the role of these pathways in leptin-induced IL-18 release from monocytes using the JAK-2 inhibitor AG490. In these experiments AG490 significantly (p < 0.05) inhibited both LPS and leptin stimulated IL-18 release from monocytes (Fig. 2E). Inhibition of LPS-activated IL-18 release by AG490 is consistent with the known ‘crosstalk’ between TLR-4 signalling and JAK-2 activation [34]. Taken together, these data suggest that leptin and E. coli LPS activate cytokine secretion in monocytes by over-lapping but distinct pathways.

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3.3. Leptin up regulation of IL-18 secretion is mediated by caspase-1 activation Proteolytic digestion of pro-IL-18 into biologically active IL-18 is a key stage in the regulation of IL-18 synthesis and secretion; this is achieved by the activity of the enzyme caspase-1 which is part of the NLRP3 inflammasome complex [25]. Pattern-associated molecular patterns (PAMPs) including LPS are one of a number of signals which can activate caspase-1 and the inflammasome [25]. We were, therefore, interested to investigate the effect of leptin on caspase-1 activity in monocytes and to determine whether or not this pathway might contribute to leptin mediated IL-18 secretion. We used a specific colorogenic peptide assay for caspase-1 (Fig. 3A) which revealed that THP-1 monocytes exhibited considerable constitutive caspase-1 activity (11.2 ± 2.3 pmol/min/mg protein) which was increased significantly (p < 0.05) in response to incubation for 3 h with both leptin (19.2 ± 4.0 pmol/min/mg protein) and E. coli LPS (18.3 ± 3.8 pmol/min/mg protein). To investigate whether caspase-1 activity was linked to IL-18 secretion in THP-1 monocytes we pre-incubated cells with the caspase-1 inhibitor Ac-YVAD for 1 h prior to stimulation with leptin and E. coli LPS for 3 h. In these experiments caspase-1 inhibition significantly (p < 0.05) abrogated both leptin- and E. coli LPS-stimulated IL-18 release after 3 h (Fig. 3B). Caspase-1 is therefore implicated as a key mediator of leptin-stimulated IL-18 secretion in monocytes. 3.4. Leptin and ATP synergistically upregulate IL-18 secretion in monocytes Extracellular ATP binds to the purinergic P2X7 receptor and activates a number of pathways leading to caspase-1 activation and post-translational IL-1 cytokine processing [35,36]. We were therefore interested to investigate possible synergism between leptin and ATP in the stimulation of IL-18 secretion from monocytes. Monocytes were stimulated with leptin or LPS for 3 h in total and ATP (6 mM) was added for the final 30 min of incubation. Our findings confirmed that exogenous ATP stimulated IL-18 secretion (Fig. 4A). Also, leptin stimulated IL-18 secretion (64.8 ± 24.2 pg/ml) was significantly enhanced (p < 0.05) by the addition of ATP (676.8 ± 218.5 pg/ml). Similarly, ATP synergistically enhanced E. coli LPS-stimulated IL-18 secretion (from 213.1 ± 62.8 pg/ml to 1507.6 ± 331.3 pg/ml; p < 0.05). Similar effects were observed in identical experiments using primary monocytes (Fig. 4B). It has been suggested that autocrine activation of monocytes by ATP secreted in response to PAMPs might be a pathway which contributes to caspase-1 activation [36]. We addressed this in 2 experiments. Firstly, we analysed ATP secretion by THP-1 monocytes. Cells were pre-incubated with ecto-ATPase inhibitor ARL 67156 for 30 min to abrogate ATPase activity in the medium, followed by 3 h co-incubation with LPS and leptin. We found that in response to leptin and E. coli LPS (Fig. 4C), ATP release was significantly stimulated over and above basal levels in the control cultures (p < 0.05). However, the levels in both leptin and E. coli LPS-stimulated cultures (16.4 ± 3.8 nM and 17.2 ± 4.7 nM) are unlikely to be sufficient to stimulate caspase-1 activity in monocytes [37]. Secondly, we pre-incubated monocytes with the P2X receptor inhibitor pyrodoxal phosphor-6-azo (benzene-2, 4-disulfonic acid) tetrasodium salt hydrate (PPADS) for 30 min prior to stimulating with leptin and E. coli LPS for 3 h (±ATP for the final 30 min of culture) (Fig. 4D). PPADS had no effect on leptin- or E. coli LPS-stimulated IL-18 secretion from monocytes but significantly (p < 0.05) inhibited the synergistic up-regulation of IL-18 by exogenous ATP and both leptin and E. coli LPS. Therefore, although leptin appears to powerfully enhance ATP-stimulated IL-18 secretion in human monocytes, we did not find any evidence to support a role for autocrine signalling by monocyte-derived ATP in this pathway.

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Fig. 2. Leptin does not upregulate IL-1b and its effect on other monocyte derived cytokines is distinct from that of LPS. (a) Secretion of IL-1b from THP-1 monocytes in response to leptin (1 lg/ml) or E. coli LPS (100 ng/ml) after 3 h culture. Data are mean ± SD of 3 separate experiments (n = 6). p < 0.05 as compared to leptin-free controls. (b) IL-1b mRNA levels in THP-1 monocytes in response to leptin (1 lg/ml) or E. coli LPS (100 ng/ml). Data are mean ± SD of 3 separate experiments (n = 6). p < 0.05 as compared to leptin-free controls. (c) Secretion of IL-6 from THP-1 monocytes in response to leptin (1 lg/ml) or E. coli LPS (100 ng/ml) after 3 h culture. Data are mean ± SD of 3 separate experiments (n = 6). p < 0.05 as compared to leptin-free controls. (d) Secretion of IL-8 from THP-1 monocytes in response to leptin (1 lg/ml) or E. coli LPS (100 ng/ml) after 3 h culture. Data are mean ± SD of 3 separate experiments (n = 6). p < 0.05 as compared to leptin-free controls. (e) Secretion of IL-18 from THP-1 monocytes pre-treated for 30 min with and without JAK-2 inhibitor AG490 (5 lM in DMSO) followed by incubation with leptin (1 lg/ml) or E. coli LPS (100 ng/ml) after 3 h culture. Data from cells cultured with AG490 alone and vehicle (DMSO) alone are also shown. Data are mean ± SD of 3 separate experiments (n = 6). p < 0.05 as compared to leptin-free controls.

4. Discussion It is clear that the expression, synthesis and processing of IL-18 is distinct from that of other members of the IL-1 cytokine family [16,24,38]. We have confirmed that IL-18 is constitutively expressed in monocytes and that LPS has no effect on IL-18 mRNA expression with the possible exception of a down-regulation at longer time points [39]. Early studies of the interaction of leptin with peripheral immune cells revealed that leptin stimulated

cytokine secretion in murine macrophages [10], human peripheral blood monocytes [12,13] and MDDC [14]. Similar results have emerged from studies of tissue macrophage-like cells including microglial cells [40] and kupffer cells [41]. A number of studies demonstrate that leptin can prime myeloid cells to mount a more vigorous cytokine response to LPS challenge [6,11,14,41,42]. We show that leptin has no effect on IL-1b mRNA synthesis and IL-1b protein secretion in THP-1 monocytes. This is consistent with previous findings: thus, leptin does not upregulate IL-1b (or IL-1a)

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Fig. 3. Leptin up regulation of IL-18 secretion in monocytes in dependent on caspase-1 activity. (a) Caspase-1 activity in monocytes in response to incubation with leptin (1 lg/ml) or E. coli LPS (100 ng/ml) for 3 h. Data are mean ± SD of 3 separate experiments (n = 6). p < 0.05 as compared to leptin-free controls. (b) IL-18 secretion from THP-1 monocytes pre-treated for 1 h with and without caspase-1 inhibitor Ac-YVAD (100 lM in DMSO) followed by incubation with leptin (1 lg/ml) or E. coli LPS (100 ng/ml) for 3 h. Data from cells cultured with Ac-YVAD alone and vehicle (DMSO) alone are also shown. Data are mean ± SD of 3 separate experiments (n = 6). p < 0.05 as compared to leptin-free controls.

release in THP-1 monocytes [43], has no effect on LPS induced IL1b release from murine macrophages [11] and ob/ob mice show no differences in LPS-induced IL-1b levels in these mice compared to lean littermates suggesting that, physiologically, there is no ‘leptin brake’ on IL-1b secretion [6]. In contrast, leptin stimulates IL-1b release from human PBMCs [43,44] upregulates IL-1b mRNA synthesis and IL-1b protein secretion in human MDDCs and enhances cytokine responses to LPS in the same cells [14]. Also, although leptin up regulated IL-1b secretion form bovine PBMCs, it had no effect on the levels of IL-1b (or IL-18) mRNA [45]. It is possible that the response of myeloid immune cells to leptin signalling is dependent on species, lineage and differentiation state. Also, the influence of cellular microenvironments and the importance of temporal relationships between stimuli on responses to leptin have been discussed [46]. However, most studies have employed different cells and conditions and data have not therefore been replicated substantially. The kinetics of transcription of IL-1b have been studied in detail and it is established that LPS induces rapid up regulation of pro-IL1b mRNA synthesis in monocytes which peaks after 2–6 h whereas IL-1b itself will stimulate a more sustained up regulation of pro-IL1b mRNA [47]. Also, there are differences in the kinetics of pro-IL1b mRNA synthesis to the same pro-inflammatory stimulus in primary monocytes isolated from peripheral blood as compared to THP-1 monocytes [48]. In agreement with this concept, we have

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found that the magnitude of IL-18 secretion from THP-1 cells in response to both leptin and LPS is substantially greater than that observed from primary cells. There are a number of possible explanations for this finding: primary monocytes are heterogeneous, their differentiation state is highly context dependant and may be subject to spontaneous change or changes in response to manipulations during the purification process itself or there may be some unique properties of THP-1 cells as a result of the original leukemic transformation process. We report that whereas leptin has no effect on IL-18 mRNA expression, leptin stimulates IL-18 protein secretion in THP-1 monocytes and primary peripheral blood monocytes; to the best of our knowledge this is the first report of this effect and it appears that leptin has a differential effect on IL-18 regulation as compared to IL-1b. There was a clear dose– response effect of leptin on IL-18 secretion and the concentrations used in this in vitro study are entirely consistent with leptin levels used in numerous other studies [11,13,27]. Although the levels generally seen in the blood of healthy individuals are lower than those used in our own and equivalent studies, leptin levels are very sensitive to food intake and body mass index (BMI) [1]. Significantly, leptin levels are likely to be higher in individual tissue and organ microenvironments, for example in and around lymph nodes which have fatty tissue deposits; indeed, there is established evidence for reciprocal interactions between these two juxtaposed tissues [49]. Furthermore, circulating levels of leptin are profoundly elevated in obesity, diabetes and during leptin therapy [4,50]. Complete processing and secretion of IL-1b requires 2 types of signal: one a priming signal that involves production of pro-IL-1b and the key inflammasome component NLRP3 which is regulated at the level of transcription [51] and a second signal which regulates the activity of the assembled inflammasome and its constituents such as caspase-1 [25]. This signal may be provided by a number of endogenous and/or exogenous pro-inflammatory molecules such as PAMPs and cytokines respectively. Given that IL-18 expression is constitutive in monocytes (which our own experiments confirm) we investigated the effect of leptin on NLRP3 mRNA expression in THP-1 monocytes but found no evidence for any effect (data not shown). Thus monocytes do not depend on inflammasome priming signals as much as macrophages albeit they seemingly have the capacity for enhanced priming in inflammatory conditions [52,53]. Previously, a limited in vitro study demonstrated that leptin treatment of a bovine monocyte/macrophage preparation had no effect on IL-18 mRNA expression but the effect of leptin on IL-18 protein was not investigated [45]. Only a limited number of studies have investigated a possible functional relationship between IL-18 and leptin. Leptin deficient ob/ob mice have reduced levels of IL-18 (and TNF-a) in response to Con A and Pseudomonas aeruginosa exotoxin A treatment with consequent reduced inflammatory liver damage; exogenous leptin restored normal immune responses [54]. Using a similar mouse model, the effects of exogenous injections of IL-18 and IL-12 on the development of acute pancreatitis was found to be independent of leptin deficiency but rather directly related to obesity. Thus, in this scenario at least, there was no direct evidence for an interaction between leptin and IL-18 [55]. Both leptin and LPS induced IL-18 secretion is linked to JAK-2 signalling, consistent with the known pathways activated by their respective receptors [33,34]. In addition to a direct effect on transcriptional regulation of cytokine genes via STAT-3 activation, JAK signalling may also transcriptionally upregulate inflammasome components such as caspase-1 [56]. However, in RT-PCR experiments we found no evidence for up regulation of caspase-1 or NLRP3 mRNA by leptin in our system (data not shown). Our own data suggests a differential effect of leptin on IL-6 and IL-8 secretion from monocytes although the data from other studies

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Fig. 4. Leptin and ATP synergistically upregulate IL-18 secretion in monocytes. (a) IL-18 secretion from THP-1 monocytes in response to leptin (1 lg/ml) or E. coli LPS (100 ng/ ml) after 3 h incubation in the presence and absence of exogenous ATP (6 mM) for the final 30 min. Data are mean ± SD of 3 separate experiments (n = 6). p < 0.05 as compared to leptin-free controls. (b) IL-18 secretion from primary monocytes in response to leptin (1 lg/ml) or E. coli LPS (100 ng/ml) after 3 h incubation in the presence and absence of exogenous ATP (6 mM) for the final 30 min. Data are mean ± SD of 3 separate experiments (n = 6). p < 0.05 as compared to leptin-free controls. (c) Stimulation of endogenous ATP production from THP-1 monocytes following 30 min pre-incubation with the ecto-ATPase inhibitor ARL 67156 and then 3 h incubation with either leptin (1 lg/ml) or E. coli LPS (100 ng/ml). Data are mean ± SD of 3 separate experiments (n = 6). p < 0.05 as compared to leptin-free controls. (d) IL-18 secretion from THP-1 monocytes pre-incubated with the P2X7 receptor inhibitor PPADS (20 lM) followed by stimulation with leptin (1 lg/ml) or E. coli LPS (100 ng/ml) for 3 h in the presence and absence of exogenous ATP (6 mM). Data are mean ± SD of 3 separate experiments (n = 6). p < 0.05 as compared to leptin-free controls.

is inconsistent [10,11,13,42,57]. A number of functional IL-18 promoter polymorphisms have been identified which may encode inter-individual variation in IL-18 expression and regulation [58]. Allelic variants of the IL-18 gene have been correlated with BMI [59] and it would be of interest to investigate inter-individual variation in monocytic IL-18 responses to leptin. We have shown that leptin stimulates caspase-1 activity in monocytes and that leptin-induced IL-18 secretion is dependent on caspase-1 activity. This suggests a signalling pathway between leptin and the inflammasome in these cells. Neither a pan-caspase inhibitor (BOC-D-FMK) nor the caspase-1 inhibitor Ac-YVAD-CHO had any effect on leptin-induced IL-1 release from primary rat microglial cells as determined by ELISA analysis of culture supernatants [60]. Western blot analysis indicated that whereas ATP enhanced LPS induced mature IL-1 release from primary rat microglial cells, a similar effect of ATP was not observed in leptin treated cells [60]. A caveat to this finding is that higher doses of leptin are needed to induce IL-1 responses in microglial cell cultures [60] which suggests that these cells are relatively insensitive to leptin signalling in comparison to monocytes and macrophages [46]. Extracellular ATP increases caspase-1 activity by binding to the P2X7 ion channel receptor which facilitates potassium ion efflux and corresponding inflammasome activation and this is viewed as an important second signal required in addition to ‘priming’

by PAMPs at least for IL-1b processing and secretion [61,62]. Intriguingly, we have shown that leptin synergises strongly with ATP in activating IL-18 secretion from both THP-1 and primary monocytes and confirmed a synergy between ATP and LPS in amplifying monocyte IL-18 secretion [63]. This finding is consistent with the caspase-1 activating properties of both LPS and leptin and suggests these molecules are capable of providing the ‘second signal’ to potentially effect IL-18 processing and release from monocytes. The source of extracellular ATP which might stimulate IL-1 cytokine processing and release in vivo is not known. It has been suggested that TLR agonists can trigger the secretion of endogenous ATP from monocytes and that this activates the P2X7 receptor through an autocrine loop resulting in activation of processing and release of IL-1b and IL-18 [36]. We confirmed that LPS induces release of nM quantities of endogenous ATP from monocytes in agreement with previous findings [36] and demonstrated for the first time that leptin also induces similar levels of endogenous ATP release in monocytes. However, these ATP concentrations are barely above steady state extracellular concentrations and significantly below the concentrations required to activate the P2X7 receptor [37]. Also, experiments blocking the P2X7 receptor using PPADs did not reveal any evidence for a role of endogenous ATP in an autocrine pathway mediating leptin induced IL-18 release. However, there are a number of pathways which lead to significant

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release of endogenous ATP which could, in turn, activate immune signalling pathways synergistically with PAMPS and leptin; these include Ca2+ and NO signalling which may be active during immune responses [37]. Commensal bacteria are another plausible source of ATP for immunomodulation in vivo [64]. Leptin-stimulated IL-18 could be explained by a secondary effect of the up regulation of other cytokines, for example TNF-a as has been noted by others [54]. However, in our system, leptin had no direct effect on monocyte TNF-a secretion [27] To date, there is little information concerning the intracellular pathways which link leptin stimulation and IL-1 secretion. Nevertheless, leptin does increase IL-1 in the hypothalamus of db/db mice which lack ObRb/Stat-3 signalling suggesting that IL-1 is upregulated by leptin via STAT3 independent mechanisms [65]. In contrast, inhibition of STAT3 signalling attenuated leptin-stimulated IL-1 secretion in rat microglial cells [60]. Leptin activates IL-1Ra, and possibly IP-10, through MAPK and unknown transcription factors binding to a composite NF-kB/PU.1 binding site [57,66]. Interferon-a may have similar differential effects on IL-1b and IL-18 secretion with consequences for localised immune regulation and disease pathogenesis [56]. Finally, it is possible that leptin has wider effects on monocyte function beyond stimulation of IL-18; possibly through activation of capsase-1 which exerts a wide range of actions relevant to immune responses and inflammation [26,67,68]. In conclusion, positive and negative feedback loops are likely important in modifying inflammasome activity and IL-1 cytokines during inflammation. For example TNF-a enhances inflammasome responses [69] and other signals may suppress IL-1b production through an effect on IL-1b transcription e.g. IFN-a [56]. Furthermore, non-canonical pathways for independent stimulation of IL1b and IL-18 secretion mediated by the NLRP3 inflammasome have recently been characterised [70]. Monocytes are apparently more susceptible to inflammasome activation than other immune cells and our findings suggest that leptin may be important in enhancing this effect by providing a partial activation signal for IL-18 secretion which, in the context of other endogenous and exogneous signals, enhances a pro-inflammatory response representing another level of IL-1 regulation. Our recently published findings showing the effect of leptin on monocyte differentiation and TLR expression are consistent with the hypothesis [27]. These pathways may be particularly important in conditions of dysleptinemia such as T2DM and also in the context of therapy using exogenous leptin. Acknowledgements This work was supported by a scholarship (to P.J.) from the Anandamahidol Foundation, Naresuan University, Amphor Meaung, Phitsanulok 65000, Thailand. We thank Dr Xiaoqing Wei, School of Dentistry, Cardiff University for his advice with the development of the IL-18 ELISA. References [1] Mantzoros CS, Magkos F, Brinkoetter M, Sienkiewicz E, Dardeno TA, Kim SY, et al. Leptin in human physiology and pathophysiology. Am J Physiol Endocrinol Metab 2011;301:E567–84. [2] Conde J, Scotece M, Gomez R, Gomez-Reino JJ, Lago F, Gualillo O. At the crossroad between immunity and metabolism: focus on leptin. Expert Rev Clin Immunol 2010;6:801–8. [3] Maffei M, Halaas J, Ravussin E, Pratley RE, Lee GH, Zhang Y, et al. Leptin levels in human and rodent: measurement of plasma leptin and ob RNA in obese and weight-reduced subjects. Nat Med 1995;1:1155–61. [4] Coppari R, Bjorbaek C. Leptin revisited: its mechanism of action and potential for treating diabetes. Nat Rev Drug Discov 2012;11:692–708. [5] Procaccini C, Jirillo E, Matarese G. Leptin as an immunomodulator. Mol Aspects Med 2012;33:35–45.

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