Central interleukin-10 attenuates lipopolysaccharide-induced changes in food intake, energy expenditure and hypothalamic Fos expression

Neuropharmacology 58 (2010) 730e738

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Central interleukin-10 attenuates lipopolysaccharide-induced changes in food intake, energy expenditure and hypothalamic Fos expression Jacob H. Hollis*, Moyra Lemus, Megan J. Evetts, Brian J. Oldfield Department of Physiology, Monash University, Victoria, Australia

a r t i c l e i n f o

a b s t r a c t

Article history: Received 30 September 2009 Received in revised form 13 December 2009 Accepted 18 December 2009

Lipopolysaccharide (LPS) is often used to mimic acute infection and induces hypophagia, the selective partitioning of fat for energy, and fever. Interleukin-10 (IL-10) is an anti-inflammatory cytokine expressed in the brain which attenuates LPS-induced hypophagia; however the potential sites of interaction within the brain have not been investigated. Hypothalamic orexin (ORX) and melanin-concentrating hormone (MCH) regulate energy expenditure and food intake although the regulation of these neuropeptides through the interactions between central IL-10 and the inflammatory consequences of peripheral LPS have not been investigated. The present study in the rat investigated during the dark phase of the lightedark cycle the ability of central IL-10 (250 ng, i.c.v.) to attenuate the changes in food intake, energy substrate partitioning, and central Fos expression within the hypothalamus to peripheral LPS (100 mg/kg, i.p.); Fos expression changes specifically within ORX and MCH neurons were also investigated. Central IL-10 attenuated the peripheral LPS-induced hypophagia, reduction in motor activity, fever and reduction in respiratory exchange ratio. Central IL-10 also attenuated peripheral LPS-induced increases in Fos expression within ORX neurons and decreases in Fos expression within unidentified cells of the caudal arcuate nucleus. In contrast, both IL-10 and LPS injection independently decreased Fos expression within MCH neurons. The present study provides further insight into the interactions within the brain between the anti-inflammatory cytokine IL-10, the inflammatory consequences of LPS, and neuropeptides known to regulate energy expenditure and food intake. Ó 2010 Elsevier Ltd. All rights reserved.

Keywords: Metabolism Sickness behavior Appetite Orexin Melanin-concentrating hormone

1. Introduction Lipopolysaccharide (LPS), a cell-surface component of Gramnegative bacteria often used to mimic infection (Alexander and Rietschel, 2001), elicits a range of metabolic changes that include hypophagia, decreased motor activity, fever and a selective partitioning of fats for energy utilisation (Becskei et al., 2008; Fishkin and Winslow, 1997; Gautron et al., 2005; Hollis et al., 2006; Kelley et al., 2003; Kent et al., 1992a, 1992b, 1994; Lacosta et al., 1999; Lang et al., 1996; Larson, 2002; Laye et al., 2000; Mathias et al., 2000; Park et al., 2008; Plata-Salaman, 1995). The primary mediators of LPS, pro-inflammatory cytokines including interleukin-1b (IL-1b), alter the neural systems that regulate energy expenditure and appetite (Bret-Dibat et al., 1995; Dantzer et al., 2000; Goehler et al., 2000; Hansen et al., 1998; Konsman and Dantzer, 2001; Konsman et al., 2004; Laflamme et al., 1999, 2001; Maier et al., 1998; Reyes and Sawchenko, 2002; Schiltz and Sawchenko, 2002; Wieczorek

* Corresponding author. Tel.: þ61 (0)3 9905 8638; fax: þ61 (0)3 9905 2547. E-mail address: [email protected] (J.H. Hollis). 0028-3908/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2009.12.016

et al., 2005; Yamagata et al., 2001). Interleukin-10 (IL-10), an endogenous anti-inflammatory cytokine, inhibits many of the actions of LPS through inhibition of cytokine production and signalling (Berg et al., 1995; Moore et al., 2001; Oberholzer et al., 2002; Strle et al., 2001; Ward et al., 2001). Central administration of IL-10 attenuates some of the peripheral LPS-induced metabolic changes and hypophagia (Bluthe et al., 1999; Ledeboer et al., 2002a); however the metabolic consequences have not been fully investigated and the central sites of interaction between IL-10 and the inflammatory consequences of peripheral LPS have yet to be studied. The hypothalamus contains a range of neuropeptides that take part in the translation of immune signals into metabolic changes. The arcuate nucleus contains both orexigenic and anorexigenic neuropeptides that are important for LPS- and cytokine-induced changes in energy expenditure and appetite (Caruso et al., 2004; Felies et al., 2004; Gayle et al., 1999; Hauser et al., 1993; Huang et al., 1998, 1999; McMahon et al., 1999; Sergeyev et al., 2001; Wisse et al., 2006). In particular, peripheral LPS or IL-1b increases gene expression of POMC (Sergeyev et al., 2001) and Fos expression within POMC neurons (Scarlett et al., 2007), whereas peripheral LPS

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decreases gene expression of the classically orexigenic NPY (Gayle et al., 1997) and agouti-related peptide (Scarlett et al., 2008). The lateral hypothalamus contains the peptides orexin (ORX) and melanin-concentrating hormone (MCH) (Bittencourt et al., 1992; Elias et al., 1999, 1998) which are important regulators of energy expenditure and appetite (Akiyama et al., 2004; Alon and Friedman, 2006; Becskei et al., 2008; Burdakov et al., 2005; Chen et al., 2002; Gonzalez et al., 2008; Ludwig et al., 2001; Marsh et al., 2002; Sakurai, 2006; Segal-Lieberman et al., 2003; Shimada et al., 1998; Sutcliffe and de Lecea, 2002; Szekely, 2006; Yamanaka et al., 2003). Unlike the arcuate neuropeptides, the role of ORX and MCH neurons in LPS-induced metabolic changes are poorly understood (Becskei et al., 2008; Ford et al., 2005; Gaykema and Goehler, 2009; Park et al., 2008; Sergeyev et al., 2001). In particular, the function of ORX neurons during LPS-induced sickness is likely influenced by time of day and feeding status (Becskei et al., 2008; Burdakov et al., 2005; Ford et al., 2005; Gaykema and Goehler, 2009; Gonzalez et al., 2008; Moriguchi et al., 1999; Park et al., 2008). The interaction between central IL-10 and the inflammatory consequences of peripheral LPS have yet to be investigated in relation to hypothalamic sites regulating energy expenditure and food intake, in particular ORX and MCH neurons. The effects of LPS are more pronounced during the active or dark phase of the circadian cycle when aspects of energy expenditure, including motor activity, food intake and energy expenditure, are highest (Linthorst et al., 1997; Mathias et al., 2000; Morrow and Opp, 2005). Therefore, the present study investigated the antiinflammatory actions of central IL-10 on the peripheral LPSinduced changes in energy expenditure (motor activity, core body temperature, O2 consumption, and CO2 expiration), food intake, and Fos expression changes within hypothalamic nuclei and specifically within ORX and MCH neurons, in rats during the dark phase of the lightedark cycle. 2. Methods 2.1. Animals and housing All experimental procedures were approved by the Monash University School of Biomedical Sciences Animal Ethics Committee. Male Sprague Dawley rats (12e16 wk, 250e350 g), with free access to standard rat chow and water, were individually housed in standard cages under a 12 h-12 h reverse lightedark cycle (lights off at 09:00), a 10 h difference from the original lightedark cycle (lights off at 19:00). Rats were acclimatised for 2 weeks prior to surgical procedures, or 3 weeks prior to experimental procedures if including recovery time from surgery. The ambient temperature of the animal housing room including the metabolic cages was 22  1  C. 2.2. Surgical procedures One week prior to experimental procedures, rats were anaesthetised (1e2% isoflurane anaesthesia with oxygen) and surgically implanted with 1) a temperature recording datalogger (Subcue, Canada) into the peritoneal cavity that allows for repeated measurement of core body temperature, and 2) an 18 gauge guide cannula (Plastics1, USA) into the left lateral ventricle (1.0 mm caudal to Bregma, þ0.5 mm lateral to Bregma, 2.7 mm ventral to the surface of the brain) that allows for i.c.v. injections.

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LPS (100 mg/kg b.w. in approximately 0.25 ml sterile saline; Serotype 0127:B8, Cat# C3129, Sigma, Australia) or vehicle, and rats (n ¼ 3 per treatment group) were returned to the metabolic cages for 48 h. 2.4. Study 2; 2 h metabolic and Fos expression measurements A separate group of rats were placed into metabolic cages 18 h prior to experimental procedures under the same conditions as previously described. Following injections (as previously described), rats (n ¼ 6 per treatment group) were returned to the metabolic cages for 2 h. Rats were then anaesthetised with sodium pentobarbitone (100 mg/kg i.p.) and perfused through the left ventricle with 100 ml of 0.05 M phosphate buffered saline (PBS) followed by 300 ml of 0.1 M phosphate buffer, pH 7.2 (PB) containing 4% paraformaldehyde. The brains were then removed from the skull and processed in preparation for sectioning. 2.5. Tissue processing and immunostaining procedures Following perfusion fixation, rat brains were further fixed in 4% paraformaldehyde for 12 h at 4  C followed by 2e3 days in phosphate buffer (PB) containing 30% sucrose at 4  C. Rat brains were cut in the coronal plane at 40 mm thickness and all sections were collected into serial sets of four and stored in wells containing cryoprotectant solution (PB containing 30% ethylene glycol and 20% glycerol) at 20  C until required for further processing. For immunostaining of Fos protein and either ORX-A or MCH, free-floating rat forebrain sections were incubated with antisera raised against Fos (rabbit anti-c-Fos antiserum, 1:12,000; Cat# PC-38 (Ab-5), Merck Biosciences, Australia) and either ORX-A (goat anti-Orexin-A antiserum, 1:8000; Cat# SC-8070, Santa Cruz Biotechnology, USA) or MCH (goat anti-Melanin-Concentrating Hormone, 1:6000; Cat# SC14509, Santa Cruz Biotechnology, USA), using standard immunostaining procedures. Sections were incubated for 12e16 h on sequential days with Fos antiserum followed by either ORX-A or MCH antiserum. Secondary reagents included biotinylated swine anti-rabbit antibody (1:200; Cat# E0353, DAKO, Australia), biotinylated horse anti-goat antibody (1:200; Cat# BA-9500, Vector, USA), and Elite ABC reagent (1:200; PK-6100, Vector, USA). Substrate color reaction for Fos used a solution containing 0.01% 3,30 -diaminobenzidine tetrahydrochloride (DAB), 0.01% NiNH3SO4, 0.01% CoCl, and 0.0015% hydrogen peroxide, with a dark black reaction product localized to the nucleus. Substrate color reaction for ORX-A and MCH used a solution containing 0.01% DAB and 0.0015% hydrogen peroxide, with a goldenebrown reaction product localized to the cytoplasm. Brain slices were then mounted on glass slides, dehydrated and cleared with histoclear, and coverslipped using DPX mounting medium (Sigma, Australia). 2.6. Cell counting and analysis An individual blind to the treatment groups performed cell counting. In order to assess Fos expression within ORX-A or MCH neurons, 7 rostrocaudal levels of the lateral hypothalamus and perifornical area (LH/PeF) between 2.30 and 3.80 mm Bregma were analysed for the numbers of Fos/ORX-A double-positive, total ORX-Apositive, Fos/MCH double-positive, and total MCH-positive neurons. In order to assess Fos expression within regions of the hypothalamus which were not positive for ORX-A, the following regions were analysed for the numbers of Fos-positive nuclei: 7 rostrocaudal levels of the LH/PeF between 2.30 and 3.80 mm Bregma, 6 rostrocaudal levels of the arcuate nucleus between 2.30 and 3.80 mm Bregma, 6 rostrocaudal levels of the ventromedial hypothalamic nucleus (VMH) between 2.30 and 3.60 mm Bregma, 4 rostrocaudal levels of the dorsomedial hypothalamic nucleus (DMH) between 2.56 and 3.60 mm Bregma, the paraventricular nucleus of the hypothalamus (PVN) at 1.80 mm Bregma, and the medial, lateral and ventromedial preoptic areas (MPO, LPO, VMPO respectively) at 0.40 mm Bregma. Cell counts of each brain region from each rostrocaudal level were summed prior to statistical analysis and also represented accordingly in figures. Correct cannulae placement within the lateral ventricle was determined post-mortem.

2.3. Study 1; 48 h metabolic measurements

2.7. Metabolic analysis

Rats were housed in metabolic cages (TSE Systems, Germany) 18 h prior to experimental procedures for the regular measurement of food and water intake, motor activity, O2 consumption, and CO2 production. The indirect calorimetry design consisted of 6 cages which were used for the individual housing of rats during the study and one cage which served as the reference cage for corrections of O2 and CO2 measurements. O2 consumption and CO2 production were measured every 20 min/cage for 2.86 min and recorded using TSE Systems LabMaster software v1.8.6 (TSE systems, Germany). Food and water intake and motor activity were measured continuously and the temperature recording dataloggers were programmed to record at 5 min intervals. Between 08:00 and 09:00, just before the onset of the dark phase, rats were given i.c.v. injections of rat recombinant IL-10 (R&D Systems, Australia, Cat# 547-RL/CF; 250 ng in 2 ml sterile saline containing 0.1% bovine serum albumin) or vehicle followed immediately by i.p. injections of

To estimate the relative amount of carbohydrate and fat energy expenditure, the respiratory exchange ratio (RER) was calculated as the quotient of dCO2/dO2. Food and water intake and motor activity were calculated as total food and water consumed and total motor activity from the start of injections, respectively. In study 1 (48 h metabolic measurements), only the 2 hourly data points of total food and water intake, total motor activity, RER, and core body temperature were used in the statistical analysis and represented accordingly in figures. The change in core body temperature in 2 hourly intervals for each rat was determined by subtracting the average core body temperature of each rat over the 4 h period immediately prior to injections. For study 2 (2 h metabolic and Fos expression measurements), the 2 h total food and water intake, the final 30 min motor activity sum, and the final measurement of RER and core body temperature were used in the statistical analysis.

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2.8. Statistical analysis All statistical analyses used Statistical Package for the Social Sciences (SPSS) version 14.0 (SPSS, Australia). All data are represented as the means  the standard errors of the means. For analysis of study 1 (48 h metabolic measurements), a single multifactor analysis of variance (ANOVA) with repeated measures was performed using pre-treatment (IL-10 or vehicle) and challenge (LPS or vehicle) as the between subject factors and time (12 h increments) as the within subject factor for repeated measures analysis. For study 2 (2 h metabolic and Fos expression measurements), a single multifactor analysis of variance (ANOVA) was performed using pre-treatment (IL-10 or vehicle) and challenge (LPS or vehicle) as the between subject factors and in the case of the Fos expression data brain region was used as the within subject factor for repeated measures analysis. When appropriate, post hoc pair-wise comparisons were made using Fisher's Protected Least Significance Difference tests using Bonferroni correction for multiple comparisons. In all cases, significance was accepted at P < 0.05. 2.9. Imaging and figure preparation All photographic images were captured using a Zeiss Imager.Z1 microscope using bright-field transmitted light, color digital camera, and AxioCam image capture software v4.6 (Zeiss, Australia). All graphs were made using SigmaPlot 8.0 software (Systat, Australia), and all figures were created and assembled in Adobe Illustrator CS2 12.0.1 (Adobe Systems, Australia).

the initial 12 h period even though central IL-10 injection alone reduced cumulative motor activity for the initial 12 h period compared to vehicle injected rats (pre-treatment*challenge*time [F24,192 ¼ 1.460; P < 0.05] interaction). Peripheral injection of LPS increased the initial 12 h average core body temperature only and central IL-10 injection was without effect (Fig. 1; pre-treatment*challenge [F1,8 ¼ 7.252; P < 0.05] interaction). Central injection of IL-10 attenuated the peripheral LPS-induced reduction in the initial 12 h average of RER but not for the subsequent 12 h (12e24 h post injection) period (Fig. 1; pre-treatment*challenge*time [F24,192 ¼ 2.352; P < 0.01] interaction). In study 2, neither LPS nor IL-10 altered cumulative food or water intake (data not shown) when measured 2 h after injections. Central injection of IL-10 attenuated the peripheral LPS-induced reduction in motor activity 2 h after the injections (data not shown; challenge [F1,19 ¼ 13.122; P < 0.01] effect and pre-treatment*challenge [F1,19 ¼ 4.871; P < 0.05] interaction). Central injection of IL-10 attenuated the peripheral LPS-induced increase in core body temperature 2 h after the injections (data not shown; challenge [F1,19 ¼ 12.045; P < 0.01] effect). Neither LPS nor IL-10 altered RER when measured 2 h after injections (data not shown).

3. Results 3.1. Central IL-10 attenuated LPS-induced changes in energy expenditure

3.2. Central IL-10 attenuated LPS-induced changes in Fos expression within hypothalamic systems

In study 1, central injection of IL-10 attenuated the peripheral LPS-induced reduction in cumulative food intake for the initial 12 h but not for the subsequent 12 h (12e24 h post injection) period (Fig. 1; pre-treatment*challenge [F1,8 ¼ 6.470; P < 0.05], challenge*time [F24,192 ¼ 1.949; P < 0.01], and pre-treatment*challenge*time [F24,192 ¼ 2.526; P < 0.001] interaction). For cumulative water intake, there were no differences between treatment groups (data not shown). Central injection of IL-10 also attenuated the peripheral LPS-induced reduction in cumulative motor activity for

Peripheral injection of LPS increased Fos expression within ORX neurons and in contrast decreased Fos expression within MCH neurons compared to vehicle injection (Figs. 2 and 3). Central IL-10 injection attenuated the LPS-induced increase in Fos expression within ORX neurons (challenge [F1,19 ¼ 8.794, P < 0.01] effect). Central injection of IL-10 alone decreased Fos expression within MCH neurons compared to vehicle injection, and the combined injections of LPS and IL-10 further decreased Fos expression within MCH neurons compared to both vehicle injection and IL-10

Fig. 1. Graphs representing the effects of i.c.v. injection of interleukin-10 (IL-10; 250 ng in 2 ml) or vehicle (Veh) and i.p. injection of lipopolysaccharide (LPS; 100 mg/ml/kg b.w.) or Veh on changes in the means  standard error of the means cumulative food intake (A), cumulative activity (B), average core body temperature (C) and average respiratory exchange ratio (RER; D) over a 12 h period after the injections. *P < 0.05, **P < 0.01 versus i.c.v. Veh, i.p. Veh treated rats; ##P < 0.01 versus i.c.v. Veh, i.p. LPS treated rats; þP < 0.05 versus i.c.v. IL-10, i.p. Veh treated rats.

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Fig. 2. Graphs representing the effects of i.c.v. injection of IL-10 or Veh and i.p. injection of LPS or Veh on changes in the mean  standard error of the mean numbers of Fos/Orexin-A (ORX) double-positive (A), Fos/Melanin Concentrating Hormone (MCH) double-positive (B), total sampled ORX (C) and total sampled MCH (D) neurons. **P < 0.01, ***P < 0.001 versus i.c. v. Veh, i.p. Veh treated rats. #P < 0.05 versus i.c.v. Veh, i.p. LPS treated rats. þP < 0.05 versus i.c.v. IL-10, i.p. Veh treated rats.

Fig. 3. Photomicrographs depicting the effects of i.c.v. injection of IL-10 or Veh and i.p. injection of LPS or Veh on changes in the numbers of Fos/Orexin-A (ORX) double-positive neurons. Treatment groups are: i.c.v. Veh, i.p. Veh (A); i.c.v. IL-10, i.p. Veh (B); i.c.v. Veh, i.p. LPS (C); i.c.v. IL-10, i.p. LPS (D). Arrows designate Fos/ORX double-positive neurons. The dashed square indicates the region magnified in the bottom-right corner of each panel. Abbreviation: f, fornix. Scale bar: 100 mm, high magnification inserts 25 mm.

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injection (pre-treatment [F1,19 ¼ 9.062, P < 0.01] effect, challenge [F1,19 ¼ 8.794, P < 0.001] effect, and pre-treatment*challenge [F1,19 ¼ 5.930, P < 0.05] interaction). Neither IL-10 nor LPS injection altered the total number of sampled ORX-A and MCH neurons. Peripheral injection of LPS had region-dependent effects on Fos expression within different hypothalamic nuclei, and central injection of IL-10 only attenuated the LPS-induced effect within the caudal Arc (Figs. 4 and 5; challenge*brain region [F8,152 ¼ 4.487, P < 0.001] interaction). Peripheral injection of LPS decreased Fos expression within both the rostral and caudal Arc, and central injection of IL-10 attenuated the decrease only within the caudal Arc. Peripheral injection of LPS increased Fos expression within the DMH, PVN, and MPO, and in contrast decreased Fos expression within the LPO; central injection of IL-10 did not have any effect. 4. Discussion The present study provides further evidence to support the ability of central IL-10 to attenuate peripheral LPS-induced changes in food intake and energy expenditure. In particular, central IL-10 attenuates the LPS-induced selective partitioning of fats for energy, further supporting an integral role of inflammatory processes within the brain as regulators of energy substrate partitioning. The present study also identifies hypothalamic sites of interaction between central IL-10 and the inflammatory consequences of peripheral LPS. Specifically, the inhibition by central IL-10 of LPS-induced increases in Fos expression within ORX neurons, and LPS-induced decreases in Fos within the caudal Arc, provide insight

into the hypothalamic loci and neurochemical nature of the interactions between central IL-10 and the inflammatory consequences of peripheral LPS injection. Central IL-10 attenuated the LPS-induced hypophagia, reduced motor activity and selective partitioning of fats for energy, but not fever. In terms of food intake, IL-10/ mice have a prolonged hypophagic response to LPS compared to wild-type littermates (Leon et al., 1999), and peripheral IL-10 is ineffective in attenuating LPS-induced hypophagia (Harvey et al., 2006), consistent with a central action of IL-10 as shown in the present study. The demonstrated central IL-10 blockade of the LPS-induced selective partitioning of fats for energy has not been reported elsewhere but may be related to the time of day and feeding status, i.e. LPSinduced hypophagia promotes a selective partitioning of fats for energy, as fasting also promotes the selective partitioning of fats for energy (Chwalibog et al., 1998). Of course, without the addition of a pair fed control group the dependence of feeding status on the LPS-induced regulation of energy substrate partitioning cannot be determined. In contrast to the other measurements, central IL-10 was ineffective in attenuating the LPS-induced fever. Peripheral IL-10 however does attenuate LPS-induced fever (Ledeboer et al., 2002a), providing some direction as to a possible mode of action. Peripheral IL-10 inhibits the production of pro-inflammatory cytokines by immune cells such as macrophages, an important mechanism during LPS-induced fever (Harden et al., 2006; Lenczowski et al., 1999; Williams et al., 2004) that would be unlikely to occur following the central administration of IL-10. The dissociation in

Fig. 4. Graphs representing the effects of i.c.v. injection of IL-10 or Veh and i.p. injection of LPS or Veh on changes in the mean  standard error of the mean numbers of Fos-positive (ORX-negative) nuclei within the lateral hypothalamus (LH; A), rostral and caudal arcuate (rArc, B; cArc, C), ventromedial (VMH; D), dorsomedial (DMH; E) and paraventricular (PVN; F) nucleus of the hypothalamus, and the medial (MPO, G), lateral (LPO, H) and ventromedial (VMPO, I) preoptic area. *P < 0.05, **P < 0.01 versus i.c.v. Veh. i.p. Veh treated rats. # P < 0.05 versus i.c.v. Veh, i.p. LPS treated rats.

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Fig. 5. Photomicrographs depicting the effects of i.c.v. injection of IL-10 or Veh and i.p. injection of LPS or Veh on changes in the numbers of Fos-positive (ORX-negative) nuclei within the arcuate (AeD) and paraventricular (EeH) nucleus of the hypothalamus. Treatment groups are: i.c.v. Veh, i.p. Veh (A); i.c.v. IL-10, i.p. Veh (B); i.c.v. Veh, i.p. LPS (C); i.c.v. IL10, i.p. LPS (D). The dashed line represents the area defined as the arcuate nucleus. Abbreviation: 3V, third ventricle. Scale bar, 100 mm.

the regulation of food intake, energy substrate partitioning, and body temperature by central IL-10 may simply relate to differences in the range of inflammatory cytokines regulated by central IL-10, which subsequently regulates food intake, energy expenditure, and fever during LPS-induced sickness. Central IL-10 alone also decreased motor activity in the present study. Conversely, when IL-10 is given peripherally prior to an open

field test, an increase in motor activity is observed (Harvey et al., 2006). It is likely that these differences are related primarily to the route of administration although other factors such as time of day, feeding status, and behavioural state are influential. One additional explanation involves the differential regulation of corticosterone by central and peripheral IL-10, which increases and decreases corticosterone production respectively (Koldzic-Zivanovic et al., 2006;

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Smith et al., 1999; Stefano et al., 1998). As the corticosterone response to LPS is integral for the behavioural response (Propes and Johnson, 1997), it is possible that corticosterone could be a mediator of the independent actions of IL-10. During LPS-induced fever there is a simultaneous increase in heat production and decrease in heat loss, thus resulting in increased body temperature (Buchanan et al., 2003). We cannot determine the mechanisms whereby central IL-10 attenuated the LPS-induced selective partitioning of fats for energy without attenuating fever, as we did not directly measure heat production and loss. Although the exact mechanisms are unknown, these interactions could be dependent on the time of day or feeding status, as heat production and loss are altered during the dark phase of the lightedark cycle (Sugimoto et al., 1996) when rats are behaviourally more active (Linthorst et al., 1997; Mathias et al., 2000; Morrow and Opp, 2005; Sugimoto et al., 1996), resulting in increased body temperature. Alternatively, the ambient temperature of the room and metabolic cages, kept below the thermoneutral temperature of rodents, may have influenced the regulation of heat production and loss to account for the discrepancy between the regulation of energy substrate partitioning and body temperature. In the present study, peripheral LPS given just prior to the dark phase of the lightedark cycle increased Fos expression within ORX neurons of the LH/PeF. Other studies during the light phase show similar results (Ford et al., 2005; Gaykema and Goehler, 2009), whereas peripheral LPS decreases Fos expression within ORX neurons during states of fasting (Becskei et al., 2008), food-related arousal (Park et al., 2008), or when given during the dark phase of the lightedark cycle (Gaykema and Goehler, 2009). Furthermore, ORX neurons are activated in states of hypoglycaemia as induced by insulin, 2-deoxygluocose, or fasting, and in contrast are inhibited by hyperglycaemia (Akiyama et al., 2004; Burdakov et al., 2005; Cai et al., 2001; Diano et al., 2003; Fujiki et al., 2001; Gonzalez et al., 2008; Kurose et al., 2002; Moriguchi et al., 1999; Sakurai et al., 1998; Yamanaka et al., 2003). Although we did not determine blood glucose concentrations nor incorporate a pair fed control group, these findings support the differential regulation of ORX neurons by LPS which is likely dependent on the time of day, feeding status and state of behavioural arousal. In contrast to ORX neurons of the LH/PeF, peripheral LPS decreased Fos expression within MCH neurons. MCH promotes feeding and inhibits energy expenditure (Alon and Friedman, 2006; Ludwig et al., 2001; Marsh et al., 2002; Segal-Lieberman et al., 2003), and peripheral LPS also decreases MCH gene expression (Sergeyev et al., 2001), in support of a role of MCH in regulating food intake and energy expenditure during LPS-induced sickness. The time of day and feeding status may also be important, as in contrast to ORX neurons, MCH neurons are inhibited by hypoglycaemia (Burdakov et al., 2005). However the exact mechanisms are not known. LPS decreased Fos expression within the Arc, consistent with previous studies in which LPS was given after fasting (Becskei et al., 2008) or trained anticipation of food reward (Park et al., 2008). In contrast, LPS increases Fos expression within the Arc when given early in the light phase (Elmquist et al., 1996; Lacroix and Rivest, 1997; Sagar et al., 1995), suggesting that the effects of LPS on Fos expression within the Arc, similar to that within ORX neurons of the LH/PeF, are influenced by the time of day and feeding status. In contrast to the Arc, peripheral LPS increased Fos expression in a range of hypothalamic nuclei including the DMH, PVN, and MPO, an effect also seen when LPS is given early in the light phase (Elmquist et al., 1996; Hollis et al., 2005; Lacroix and Rivest, 1997; Sagar et al., 1995). Contrary to previous reports during the light phase in which LPS increases Fos expression within the LPO and VMPO, peripheral injection of LPS decreased Fos expression within the LPO and was without effect in the VMPO. As with the Arc, the

effects of LPS on Fos expression within the LPO and VMPO may be influenced by time of day and feeding status, as previous studies have only been conducted early in the light phase (Elmquist et al., 1996; Hollis et al., 2005; Lacroix and Rivest, 1997; Sagar et al., 1995). Central IL-10 attenuated the LPS-induced changes in Fos expression only within ORX neurons and within the caudal Arc. LPS increases pro-inflammatory cytokine gene expression including IL1b within the hypothalamus, particularly within the Arc (Laye et al., 1994; Quan et al., 1999), and IL-1 receptor is strongly expressed within the Arc (Ericsson et al., 1995). Because the IL-10 receptor (IL10R) is expressed by both glia and astrocytes (Ledeboer et al., 2002b; Moore et al., 2001; Strle et al., 2001), and exogenous IL-10 inhibits LPS-induced pro-inflammatory cytokine expression by glia and astrocytes (Heyen et al., 2000; Molina-Holgado et al., 2001; Pousset et al., 1999; Strle et al., 2002), it is likely that the LH/PeF and particularly the Arc are hypothalamic regions in which exogenous IL-10 inhibited pro-inflammatory cytokine production and signalling. In regards to the Arc, differences in the anatomical distribution of neuropeptides may influence the observed actions of central IL10, as NPY-containing neurons are concentrated within the caudal Arc (Morris, 1989; Urban et al., 1993) whereas the distribution of POMC (a-MSH-containing) and agouti-related peptide neurons are equally concentrated along the entire neuraxis of the Arc (HaskellLuevano et al., 1999; O'Donohue et al., 1979). The lack of effect of central IL-10 on the DMH and PVN suggests that these nuclei may be activated through routes other than the production of intraparenchymal pro-inflammatory cytokines, such as intraparenchymal production of prostaglandins or activation of vagal afferents (Dallaporta et al., 2007; Dunn and Swiergiel, 2000; Konsman et al., 2000; Lacroix and Rivest, 1997; Laflamme et al., 1999; Laye et al., 1995; Maier et al., 1998; Zhang et al., 2003). In regard to the LH/PeF, the observation that IL-10 alone decreased Fos expression within MCH neurons, and did not attenuate that LPS-induced decrease in Fos expression within MCH neurons, suggests that ORX and MCH neurons are differentially regulated by both pro- and anti-inflammatory cytokines. Although the exact mechanisms are not known, the differential expression of cytokine receptors on ORX and MCH neurons may provide a plausible explanation. To the best of our knowledge this has not been studied. Taken together, these findings provide further support for the role of hypothalamic inflammatory processes in the regulation of LPS-induced changes in food intake and energy expenditure, and identify important hypothalamic nuclei including ORX neurons of the LH/PeF that are sites of interaction between central IL-10 and the inflammatory consequences of peripheral LPS.

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