The transcription factor nuclear factor interleukin 6 mediates pro- and anti-inflammatory responses during LPS-induced systemic inflammation in mice.

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The transcription factor nuclear factor interleukin 6 mediates pro- and anti-inflammatory responses during LPS-induced systemic inflammation in mice Jenny Schneiders a, Franziska Fuchs b, Jelena Damm a, Christiane Herden b, Rüdiger Gerstberger a, Denis Melo Soares c, Joachim Roth a, Christoph Rummel a,⇑ a

Institute of Veterinary Physiology and Biochemistry, Justus-Liebig-University Giessen, 35392 Giessen, Germany Institute of Veterinary Pathology, Justus-Liebig-University Giessen, 35392 Giessen, Germany c Laboratory of Pharmacology, Faculty of Pharmacy, Federal University of Bahia, Salvador 40110-060, Bahia, Brazil b

a r t i c l e

i n f o

Article history: Received 4 December 2014 Received in revised form 27 February 2015 Accepted 14 March 2015 Available online xxxx Keywords: Lipopolysaccharide Neuroinflammation Immune-to-brain signaling Fever NF-IL6 Cytokines Neutrophil granulocytes Hypothalamic–pituitary–adrenal-axis Tryptophan metabolism Serotonin

a b s t r a c t The transcription factor nuclear factor interleukin 6 (NF-IL6) plays a pivotal role in neuroinflammation and, as we previously suggested, hypothalamus–pituitary–adrenal-axis-activation. Here, we investigated its contribution to immune-to-brain communication and brain controlled sickness symptoms during lipopolysaccharide (LPS)-induced (50 or 2500 lg/kg i.p.) systemic inflammation in NF-IL6-deficient (KO) or wildtype mice (WT). In WT LPS induced a dose-dependent febrile response and reduction of locomotor activity. While KO developed a normal fever after low-dose LPS-injection the febrile response was almost abolished 3–7 h after a high LPS-dose. High-dose LPS-stimulation was accompanied by decreased (8 h) followed by enhanced (24 h) inflammation in KO compared to WT e.g. hypothalamic mRNA-expression including microsomal prostaglandin E synthase, inducible nitric oxide synthase and further inflammatory mediators, neutrophil recruitment to the brain as well as plasma levels of inflammatory markers such as IL-6 and IL-10. Interestingly, KO showed reduced locomotor activity even under basal conditions, but enhanced locomotor activity to novel environment stress. Hypothalamic–pituitary– adrenal-axis-activity of KO was intact, but tryptophan-metabolizing enzymes were shifted to enhanced serotonin production and reuptake. Overall, we showed for the first time that NF-IL6 plays a dual role for sickness response and immune-to-brain communication: acting pro-inflammatory at 8 h but anti-inflammatory at 24 h after onset of the inflammatory response reflecting active natural programming of inflammation. Moreover, reduced locomotor activity observed in KO might be due to altered tryptophan metabolism and serotonin reuptake suggesting some role for NF-IL6 as therapeutic target for depressive disorders. Ó 2015 Elsevier Inc. All rights reserved.

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1. Introduction

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Systemic inflammation, experimentally induced by the bacterial inflammatory compound lipopolysaccharide (LPS), leads to brain-controlled sickness symptoms including fever. Underlying immune-to-brain communication is mediated by three major signaling pathways: a humoral [I] (Roth et al., 2009; Roth and Blatteis, 2014), a cellular [II] (Rummel et al., 2010; Aguliar-Valles et al., 2014; Dantzer et al., 2008; Serrats et al., 2010) and a neural [III] one (Roth and Blatteis, 2014). (I) LPS induces a peripheral

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⇑ Corresponding author at: Institute of Veterinary Physiology and Biochemistry, Justus-Liebig-University Giessen, Frankfurter Strasse 100, D-35392 Giessen, Germany. Tel.: +49 (641) 99 38155; fax: +49 (641) 99 38159. E-mail address: [email protected] (C. Rummel).

release of proinflammatory cytokines like interleukin(IL)-6, which in addition to LPS itself act as humoral mediators on cells of the blood–brain-barrier (BBB), namely on brain endothelial cells (Eskilsson et al., 2014; Wilhelms et al., 2014; Ching et al., 2007), or brain structures with a leaky BBB such as the sensory circumventricular organs (Roth et al., 2004; Ott et al., 2010; Wuchert et al., 2008, 2009). Subsequently, hypothalamic nuclei are activated via neuronal projections or secondary mediators produced locally at the site of action (Capuron and Miller, 2011). (II) Moreover, peripheral cytokines and LPS stimulate immune cells to migrate to the brain, locally release cytokines and, thereby, contribute to brain cell activation and behavioral impairments (Aguliar-Valles et al., 2014; D’Mello et al., 2009; McColl et al., 2007). The relevance of this pathway for the manifestation of fever

http://dx.doi.org/10.1016/j.bbi.2015.03.008 0889-1591/Ó 2015 Elsevier Inc. All rights reserved.

Please cite this article in press as: Schneiders, J., et al. The transcription factor nuclear factor interleukin 6 mediates pro- and anti-inflammatory responses during LPS-induced systemic inflammation in mice. Brain Behav. Immun. (2015), http://dx.doi.org/10.1016/j.bbi.2015.03.008

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and other rapidly developing sickness symptoms has not yet been established. (III) Furthermore, LPS can activate vagal and other sensory afferent nerves, which may contribute to sickness behavior (Dantzer et al., 2008) and to the development of the early stage of fever via a rapid peripheral formation of PGE2 (Roth and Blatteis, 2014; Steiner et al., 2006). The neural pathway is not analyzed in this study. LPS induces a spatiotemporal genomic activation of brain cells. First, there is early nuclear translocation of the pivotal transcription factor nuclear factor (NF) jB, followed by signal transducer and activator of transcription (STAT)3 and, with some delay, NFIL6, which peaks at 8 h (Damm et al., 2011; Rummel et al., 2005; Nadjar et al., 2003; Gautron et al., 2002). These transcription factors in turn regulate the expression of target genes responsible for the development of the sickness response. Recently, we revealed that NF-IL6 (C/EBPb) is activated in the brain during the late phase of systemic inflammation after both, low and high dose LPS-stimulation (Damm et al., 2011; Harden et al., 2014) and we hypothesized that NF-IL6 might be involved in either the maintenance or the termination of the febrile response (Damm et al., 2011). Furthermore, we obtained evidence that NF-IL6 might be implicated in the control of hypothalamic– pituitary–adrenal-(HPA)axis-activity as it is activated in response to exposure to a novel environment, an established psychological stressor (Fuchs et al., 2013). The HPA-axis is of special interest because it acts as negative feedback mechanism on immune-tobrain communication (Haddad et al., 2002) and it influences the serotonin system (Leonard, 2005), which is the key for the development of depressive disorders (Myint and Kim, 2014; Badawy, 2013). It is also known that NF-IL6 influences the prostaglandin E2 (PGE2) synthesis by controlling one of the rate limiting enzymes, namely microsomal prostaglandin E synthase (mPGES) (Uematsu et al., 2002). In addition, it is associated with memory and learning (Taubenfeld et al., 2001), LPS-induced neuronal death (Ramji and Foka, 2002; Pan et al., 2013; Pena-Altamira et al., 2014), excitotoxic brain injury (Cortes-Canteli et al., 2008) and post-ischemic neuroinflammation and brain damage (Halterman et al., 2008; Kapadia et al., 2006). In that context, NF-IL6 is involved in several diseases that are accompanied by increased brain inflammation like, for example, Alzheimer’s disease (Strohmeyer et al., 2014). Overall, NF-IL6 seems to be important for immune-to-brain communication, brain inflammation and the development of sickness responses. The exact contribution of NF-IL6, however, still remains to be determined. Thus, the goal of the present study was to clarify for the first time the role of NF-IL6 for humoral and cellular immune-to-brain signaling pathways, for the strength of LPS-induced systemic and brain-intrinsic inflammatory responses, and especially for the manifestation of fever. Therefore, we stimulated NF-IL6-deficient mice with LPS, monitored the sickness response, analyzed critical signaling molecules including markers of inflammation, the serotonin system and conducted measures of HPA-axis responsiveness such as novel environment stress.

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2. Materials and methods

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2.1. Animals

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C/EBPb/ (KO) and C/EBPb+/+ (WT) litter mates were generated in an in-house breeding from heterozygous (C/EBPb/+) parental animals obtained from The Jackson Laboratory (Bar Harbor, ME, USA; stock number 006873). Generation of C/EBPb/ mice and their genetically background was previously described by Screpanti et al. (1995). Genotyping was performed according the protocol provided by The Jackson Laboratory (http://jaxmice.jax.

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org/protocolsdb/, stock number 006873). During the experiment 6–12 weeks old male and female animals were individually housed with constant access to water and powdered standard lab chow (Ssniff Spezialdiäten GmbH, Soest, Germany) in a climatic chamber (Weiss Umwelttechnik GmbH, Typ 100 US/+5 – + 40 DU, Germany) at an ambient temperature of 31 °C and 50% humidity at a 12:12 h light–dark cycle (lights on at 07:00). Animals were implanted with intra-abdominal radio transmitters for measurement of core body temperature (Tb) and motor activity (MiniMitter Company Inc., Sunriver, OR, USA). Implantation of transmitters was performed about 1 week before the experiments using a mixture of ketamine (approximately 4 mg/animal; Medistar Arzneimittelvertrieb GmbH, Ascheberg, Germany) and xylazine (approximately 0.2 mg/animal; CP-Pharma, Burgdorf, Germany) as anesthetic (intraperitoneally, i.p.). Meloxicam (1 mg/ kg body weight, BW; Boehringer Ingelheim Vetmedica GmbH, Ingelheim, Germany) was used for surgical analgetic treatment. An automatic data acquisition system was used (VitalView, Respironics Inc-MiniMitter, Bend, OR, USA). For habituation, mice were handled at least 3 days before experiments. All animal procedures were conducted according to the guidelines approved by the local ethics committee (ethics approval number GI 18/2 – 51/2008).

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2.2. Treatment and experimental protocols

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The NF-IL6 deficient mice showed, at the same age, lower body weight than their WT littermates (at the beginning of experiments: KO 21.16 ± 0.73 g; WT 26.01 ± 0.57 g). To account for these changes LPS doses were adapted to body weight. When mice were switched from the conventional cages after at least one week of recovery of the surgery to new, experimental cages Tb and motor activity were recorded for the first 4 h (10:00–14:00). These data were used as readouts for novel environment stress (NES; n = 47). For inflammatory stress experiments, mice were i.p. injected with LPS (2500 or 50 lg/kg BW; derived from Escherichia coli, serotype 0111:B4, Sigma Chemicals, Deisenhofen, Germany) diluted in sterile pyrogen-free 0.9% phosphate buffered saline (PBS; Dulbecco’s Phosphate Buffered saline, PAA, Cölbe, Germany) at a total injection volume of 5 ml/kg BW. The dose of LPS was chosen according to previous studies showing either (50 lg/kg) a moderate febrile response (Rudaya et al., 2005) and transcription factor activation in the brain (Rummel et al., 2008) or (2.5 mg/kg) robust systemic and brain inflammatory response accompanied by a strong fever (Rudaya et al., 2005) and recruitment of neutrophil granulocytes to the brain (Aguliar-Valles et al., 2014; Rummel et al., 2010). Another previous report showed abolished fever in NFjB deficient animals to an LPS-stimulation of 50 lg/kg (Kozak et al., 2006). Here, we wanted to reveal the role for NF-IL6 for the same type of febrile response, brain as well as peripheral inflammation and underlying humoral and cellular immune-to-brain signaling pathways. Controls were injected with an equal volume of sterile pyrogen-free 0.9% PBS. All injections were performed between 9:00 and 10:30. To reduce the number of animals used for the experiments; animals received either the low dose of LPS and were treated with PBS one week later or injected with PBS and stimulated with the high dose of LPS 7 days later. Animals were randomly assigned to the treatment groups; some received only PBS, but most of the animals were treated with LPS at one of these points. No animal received LPS twice. 8 WT and 7 KO received 50 lg/kg LPS; 6 WT and 5 KO were perfused at 8 h, 8 WT and 6 KO at 24 h after 2.5 mg/kg LPS; 14 WT and 7 KO were treated with PBS. 8 or 24 h after PBS- or high-dose-LPS-treatment the animals were killed by terminal anesthesia with pentobarbital (i.p.; approximately 100–160 mg/kg, Merial GmbH, Hallbergmoos, Germany) and transcardially perfused with ice-cold 0.9% saline. Blood samples were collected via cardiac puncture with a sterile heparinized syringe

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before perfusion; afterwards brains, livers and pituitaries were quickly removed, frozen on powdered dry ice and stored at 55 °C until analysis.

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2.3. Tissue processing

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Coronal 20 lm brain sections of several brain structures including the vascular organ of the lamina terminalis (bregma 0.62 to 0.38 mm), the median preoptic nucleus (bregma 0.62 to 0.14 mm), the subfornical organ (SFO, bregma 0.1 to 0.82 mm), the paraventricular nucleus (bregma 0.58 to 1.22 mm), the arcuate nucleus (bregma 1.22 to 2.8 mm), the median eminence (bregma 1.58 to 2.3 mm) and the area postrema (bregma 7.2 to 7.76 mm) were cut using a cryostat (2800 Grigocut E, Reichert-Jung, Nußloch, Germany). Brain structures were identified using ‘‘The mouse brain in stereotaxic coordinates’’ (Paxinos and Franklin, 2001). Brain sections were thaw-mounted on poly-L-lysin-coated glass-slides and stored at 55 °C for immunohistochemistry. Further brain sections were stacked on glass-slides, the hypothalamus was dissected, divided into two pieces (left and right hemisphere) for PCR- and Western blotanalysis and stored at 55 °C for RNA-extraction or protein isolation.

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2.4. Immunohistochemistry

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Frozen brain sections were air-dried and fixed in 2% paraformaldehyde (Merck, Darmstadt, Germany) diluted in PBS for 10 min. After washing three times with PBS, sections were incubated at room temperature (RT) for 1 h using a blocking solution containing 10% normal donkey serum (NDS, Biozol, Eching, Germany) and 0.3% Triton X-100 (Sigma–Aldrich Chemie GmbH, Steinheim, Germany; no Triton X-100 for CD163-Antibody) in PBS. For immunofluorescence primary antibodies (rabbit-anti-POMC, dilution 1:2000, gift from Dr. Blähser, Institute of Anatomy and Cellbiology, JLU Gießen, Germany, (Blahser, 1988); rabbit-antiCD163, dilution 1:1000, sc-33560; goat-anti-COX2, dilution 1:2000, sc-1747; rabbit-anti-NF-IL6, dilution 1:5000, sc-150; rabbit-anti-STAT3, dilution 1:3000, sc-482, all from Santa Cruz Biotechnology Inc., Dallas, USA; rat-anti-7/4, dilution 1:500, MCA771G, ABD Serotec, Puchheim, Germany) were diluted in blocking solution and sections were incubated for 20–22 h at 4 °C (RT for 7/4-antibody). Specificity of the respective antibodies has been tested previously (Damm et al., 2011; Fuchs et al., 2013; Rummel et al., 2010, 2008, 2006). After three further washing steps, sections were incubated with the secondary antibodies diluted in blocking solution for 2 h at RT. For visualization Alexa488-conjugated anti-rabbit IgG (life technologies, Carlsbad, CA, USA; A21206) as well as Cy3-conjugated anti-rat, anti-goat and anti-rabbit IgG (Jackson Immuno Research Europe Ltd., Newmarket, UK; 712-165-150, 705-165-147 and 711-165-152) were used as secondary antibodies. After another three PBS washes cell nuclei were stained by incubation with 4.6-diamidino-2phenylindole (DAPI, 1:8000 dilution in PBS, Mobitec GmbH, Göttingen, Germany) for 10 min, followed by further three washings in PBS. Afterwards sections were cover slipped using Citifluor (Citifluor Ltd., London, UK). Sections were stored at 4 °C until analysis.

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2.5. Microscopical analyses

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Images were acquired using a light/fluorescent Olympus BX50 microscope (Olympus Optical, Hamburg, Germany) with a black and white Spot Insight camera (Diagnostic Instruments, Visitron Systems, Puchheim, Germany). Digital brain maps for overviews were adapted from the digital version of ‘‘The mouse brain in

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Table 1 Assay-ID of used applied biosystems gene expression assays. Gene

Assay-ID

CD163 COX2 CXCL1 GAPDH IDO IL-10 IL-1b IL1-ra IL-6 iNOS KMO miR155 mPGEs IjBa PACAP POMC Slc6A4 SOCS3 TNFa TPH2 Trib1 VIPR1

Mm00474091_m1 Mm00478374_m1 Mm04207460_m1 4352339E-1009032 Mm00492586_m1 Mm00439614_m1 Mm00434228_m1 Mm00446186_m1 Mm00446190_m1 Mm00440502_m1 Mm01321343_m1 Mm01716204_m1 Mm00452105_m1 Mm00477798_m1 Mm00437433_m1 Mm00435874_m1 Mm00439391_m1 Mm00545913_s1 Mm00443258_m1 Mm00557715_m1 Mm00454875_m1 Mm00449214_m1

stereotaxic coordinates’’ (Paxinos, 2001) using CorelDraw 9 (Corel Corporation, Ottawa, Canada). Microphotographs were taken in series for each staining and time point with the same exposure times using MetaMorph 7.7.5.0 software (Molecular Devices Inc., Downingtown, PA, USA). Individual images were combined to RGB color images using MetaMorph 5.05 software and optimized for contrast and brightness (staining was performed for all animals at the same time and for quantification purposes all images were processed the same way) using Adobe Photoshop 6.0 (Adobe Systems Incorporated, San Jose, CA, USA). At least two brain sections of each animal and brain structure were semiquantitatively evaluated using a 5-level scale (, no signals detectable; ±, single nuclear signal in some cases; +, low density, ++ moderate density; +++, high density of nuclear signals) and subsequently averaged for each animal.

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2.6. Real-Time PCR

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Total RNA of the collected hypothalamic sections (approximately 15 mg tissue) and of liver samples (approximately 20 mg tissue) was extracted using Trizol (Invitrogen, Carlsbad, CA) and RNA of the pituitary glands was extracted using the NucleoSpin RNA XS kit (Macherey–Nagel, Düren, Germany) according to the manufacturer’s protocols. Reverse transcription of 1 lg RNA (0.2 lg for the pituitary glands) and quantitative real-time PCR was performed using preoptimized primer/probe mixture and TaqMan universal PCR Master Mix (StepOnePlus Real-Time PCR; TaqMan Gene Expression Assay; Applied Biosystems) as described previously by Damm et al. (2013). Assay IDs for analyzed genes are shown in Table 1. Samples were run in duplicates. For normalization of cDNA quantities GAPDH was measured as a housekeeping gene. GAPDH was chosen as the best housekeeping gene out of the 12 most commonly used housekeeping genes for mouse tissue using the PrimerDesign PerfectProbe geNorm 12 gene kit mouse and the GeNorm software (catalog number ge-PP-12-mo, PrimerDesign Ltd., Southampton, UK). Sample values are calculated as x-fold difference from a control sample (WT PBS, value determined as 1) using the DDCT-method within the experiment.

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2.7. ELISA and cytokine bioassays

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Corticosterone and IL-10 plasma levels were measured in blood plasma using mouse specific enzyme-linked immunosorbent

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assays (ELISA; DRG Instruments GmbH, Marburg, Germany; EIA5683, EIA-4164) according to the manufacturer’s protocols. The detection range for corticosterone and IL-10 were, after considering the sample dilution, 1.13–415.75 ng corticosterone/ml and 10–2000 pg IL-10/ml, respectively. IL-6 plasma levels were determined by a bioassay based on the dose-dependent effect of IL-6 on the growth of B9 hybridoma cell line, TNFa plasma levels were analyzed using a bioassay based on the cytotoxic effect of TNFa on a WEHI cell line as described previously by Welsch et al. (2012). The bioassays showed detection limits of 3 IU IL-6/ ml and 6 pg TNFa/ml.

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2.8. Isolation of proteins and Western blot

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For protein isolation collected hypothalamic sections were homogenized on ice in a solution containing 50 mM HEPES (Biomol GmbH, Hamburg, Germany), 150 mM NaCl, 4 mM EGTA, 10 mM EDTA, 1 mM dithiothreit (all from Roth GmbH, Karlsruhe, Germany), 0.2% Nonident P-40, 15 mM sodium pyrophosphate, 100 mM b-glycerophosphate, 50 mM sodium fluoride, 5 mM sodium orthovanadate (all from Sigma–Aldrich Chemie GmbH, Steinheim, Germany) and 1 tablet protease inhibitor cocktail (Roche Holding GmbH, Grenzach-Whylen, Germany). Homogenates were centrifuged for 15 min (6000g) and supernatants were stored at 80 °C. Protein amount was determined using Bradford protein assay (Sigma–Aldrich Chemie GmbH, Steinheim, Germany) and bovine serum albumin as standard. Samples were mixed with SDS-Sample buffer and incubated at 95 °C for 8 min. 40 lg of protein and Precision Plus Protein Western C Standards (Bio-Rad Laboratories Inc., Hercules, CA, USA) were then loaded on a 12% acrylamide gel and electrophoresed for 100 min at 140 V (Mini Protean Tetra Cell, Bio-Rad Laboratories Inc., Hercules, CA, USA). Afterwards, protein was transferred (15 V, 0.18 A, 65 min) to a PVDF-membrane (Merck Milipore, Darmstadt, Germany). Membranes were air-dried and then immersed using 70% ethanol and water. For immunostaining, blocking was performed in 10% non fat dry milk in TBST for 1 h at RT. After that, membranes were incubated with the first antibodies (rabbit-anti-NF-IL6, 1:500, sc-150 Santa Cruz Biotechnology Inc., Dallas, USA and rabbit-anti-b-actin, 1:5000, A2066 Sigma– Aldrich Chemie GmbH, Steinheim, Germany) diluted in TBST and 5% non fat dry milk over night (4 °C). After 3 washing steps with TBST, membranes were incubated with the secondary antibody (HRP-conjugated donkey-anti-rabbit, 1:2000, sc-2313, Santa Cruz Biotechnology Inc., Dallas, USA) and StrepTactin-HRP conjugate (1:7000, Bio-Rad Laboratories Inc., Hercules, CA, USA) diluted in TBST for 1 h at RT. After further two washing steps in TBST and one in TBS membranes were incubated with ImmunStar Western C Chemiluminescence Kit (Bio-Rad Laboratories Inc., Hercules, CA, USA) for 5 min. Chemiluminescence was detected using a Chemidoc XRS Imaging System (Bio-Rad Laboratories Inc., Hercules, CA, USA). Data are presented as ratio NF-IL6: b-Actin multiplied by 100.

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2.9. Data analysis

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Previously, differences in febrile responses have been observed for female rats depending on their estrus cycle compared to male rats (Mouihate et al., 1998; Mouihate and Pittman, 2003). However, mice data on these differences are very scarce and might depend on the strain used for experimentation and even ambient temperature. For example some differences in basal body core temperature have been reported (Sanchez-Alavez et al., 2011). In this study 4-way repeated measures ANOVA (factors gender, treatment, genotype and time) on temperature data and 3-way repeated measures ANOVA (factors: gender, treatment and

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genotype) on PCR-data and plasma IL-6, IL-10 and corticosterone revealed no statistically significant sex difference. Analysis of each gender separately revealed the same effects (almost same group size of male and female mice for both groups e.g. WT and KO), however, with low animal numbers. Thus, as there was no significant difference between the male and female groups, data of both groups were pooled for statistical analyses and in respective Figures. Cumulative motor activity (cumulative over 15 min intervals) and abdominal temperature (at 15 min intervals) were analyzed using a three-way repeated measures analysis of variance (ANOVA) with the between subjects factors genotype and treatment and the within subjects factor time. Data were divided into 2 h-intervals (1 h-intervals for NES-data) for analysis and Bonferroni-correction for multiple comparisons was performed, followed – in case of a significant interaction – by a Tukey post hoc test (Statistica 12, StatSoft Europe, Hamburg, Germany). All other data were analyzed separately for each time point using a two-way ANOVA with the factors genotype and treatment followed by a Bonferroni post hoc test (Prism 5 software, GraphPad, San Diego, CA, USA). P < 0.05 was considered statistically significant. All data are presented as means ± SEM.

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3. Results

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3.1. KO showed prolonged fever after low dose, but abolished fever between 3 and 7 h after high-dose-LPS-stimulation

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Fig. 1 shows core body temperature (Tb) of mice in response to the different stimuli. KO and WT showed a normal circadian day night rhythm with a small stress peak in response to handling and injection. Stimulation with 50 lg/kg LPS induced a fever in both, WT and KO (Fig. 1A). In WT and KO the febrile response lasted for 7 h after LPS-stimulation, while in KO a trend to a second

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Fig. 1. Prolonged fever after low dose but abolished fever between 3 and 7 h after high dose LPS-stimulation in KO, Core body temperature of WT and KO mice over 24 h after injection of PBS (A and B), 50 lg/kg LPS (A) or 2.5 mg/kg LPS (B) as well as 4 h after NES compared to baseline (C). Injection/begin of NES at time ‘‘0’’. KO showed a tendency of prolonged fever 24 h after low dose LPS-stimulation but significantly depressed fever between 3 and 7 h after high dose LPS-stimulation. ⁄ Main effect of treatment; §main effect of genotype; #WT LPS vs. WT PBS; $KO LPS vs. WT LPS. ⁄P < 0.05; ⁄⁄⁄P < 0.001.


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late febrile phase was observed in the morning of the day after injection (lasting 5 h; P = 0.072). In response to 2.5 mg/kg LPS WT developed a robust fever that persisted and abrogated the day night rhythm over the 24 h time period until the end of recording. Interestingly, fever was almost abolished from 3 to 7 h after LPSstimulation in KO (Fig. 1B; post hoc P = 0.00016 and P = 0.00659). In response to NES (Fig. 1C) both genotypes reacted in a similar way with an increase of Tb shortly after exposure to the stressor. Tb progressively declined again, reached basal levels 2 h after stimulation and even dropped below after 3 h. Overall, we found a reduced febrile response during the first 3 h to 7 h after high-dose-LPS-stimulation.

3.2. KO showed reduced locomotor activity under basal conditions, but activity was enhanced 2–4 h after NES Locomotor activity of WT (Fig. 2A) showed a normal circadian rhythm in the PBS treated control group with low activity during the day and high activity during the night. LPS-stimulation reduced locomotor activity of WT dose dependently (Fig. 2A and D). KO already presented dramatically lower locomotor activity after PBS-treatment during the night compared to WT counterpart controls (Fig. 2B and D; post hoc P < 0.0001) whereas no signs of pain or other altered physiological parameters were present. In response to NES (Fig. 2C) both genotypes first showed an increased locomotor activity (effect of treatment P < 0.0001 F1,82 = 19,05 and P = 0.008 F1,82 = 10.57 respectively). However, while the activity of WT dropped down to basal levels after 2 h, activity of KO remained on a significant higher level (post hoc P < 0.001 and P < 0.01) also showing that these mice do not present deficits in their locomotor capacities. Furthermore, KO showed an increased basal daily gain in wt compared to WT (Fig. 2E; post hoc P < 0.05) and a trend to reduced LPS-induced anorexia after the high dose of LPS (Fig. 2G). Water intake did not differ between genotypes (Fig. 2F). Overall, NF-IL6 seems to be important for regulation of locomotor activity.

3.3. Circulating IL-6 and IL-10 are lower 8 h, but higher 24 h after LPSstimulation in KO compared to WT To elucidate the influence of NF-IL6 on the humoral pathway of immune-to-brain communication we first examined the circulating humoral mediators IL-6 and IL-10 indicative for the peripheral inflammatory response (Fig. 3). KO showed increased basal plasma IL-6 levels compared to WT, which has previously been described by Screpanti et al. (1995). Plasma IL-6 and IL-10 levels were dramatically increased in WT and KO 8 h after high dose LPS-stimulation (Fig. 3A and C). The increase in circulating IL-6 and IL-10 levels of KO, however, was significantly lower than in WT (post hoc P < 0.01 and P < 0.05 respectively). 24 h after LPS-stimulation plasma IL-6 (Fig. 3B) and IL-10 (Fig. 3D) levels of WT already returned to basal levels, while KO still showed a maintained increase in LPS-induced IL-10 levels (post hoc P < 0.001). Furthermore, the ratio of IL-6:IL-10 was calculated, which provides another indication about the inflammatory status. 8 h after stimulation (Fig. 3E) WT showed a LPS-induced increase of IL-6:IL-10ratio, while the increase of this value in the KO was significantly lower (post hoc P < 0.05). 24 h after LPS-stimulation (Fig. 3F) the ratio reached low levels in both genotypes. TNFa was not detectable in all plasma samples. Lower IL-6 levels and reduction of the IL-6:IL-10-ratio in KO compared to WT at the 8 h time point was indicative for a reduced inflammatory status in KO and might potentially be related to the reduced febrile response during 3– 7 h after LPS-stimulation.

Fig. 2. Low basal but higher stress-induced locomotor-activity in KO compared to WT. Locomotor activity of WT (A) and KO (B) over 24 h after treatment with PBS, 50 lg/kg LPS or 2.5 mg/kg LPS as well as 4 h after NES compared to baseline (C). Injection/begin of NES at time ‘‘0’’. Cumulative locomotor activity of WT and KO after PBS- or LPS-treatment is illustrated in D. KO showed lower basal locomotor activity but higher activity 2–4 h after NES compared to WT. Body weight changes (E) of both genotypes was reduced 24 h after treatment with the high dose LPS, while the basal daily increase of BW was enhanced in KO compared to WT. Water intake (F) and food intake (G) were decreased after LPS-treatment (main effect of treatment), but not significantly altered in KO compared to WT after high dose LPS-stimulation. ⁄Main effect of treatment; §main effect of genotype; #2.5 mg/kg LPS vs. PBS; $50 lg/kg LPS vs. PBS; +WT PBS vs. KO PBS; & WT NES vs. KO NES;% KO NES vs. KO baseline. ⁄P < 0.05; ⁄⁄P < 0.01; ⁄⁄⁄P < 0.001.

3.4. Hepatic expression of cytokines in KO and WT

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To obtain further data on peripheral inflammatory mediators hepatic expression of several cytokines was analyzed. 8 h after high-dose LPS-stimulation expression of pro-IL-1b (Fig. 4A), IL-10 (Fig. 4D) and IL-1ra (Fig. 4E) was increased in LPS-treated animals with no effect of genotype. Expression of IL-6 (Fig. 4B) was

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Fig. 3. IL-6 and IL-10 plasma levels were lower 8 h but higher 24 h after LPSstimulation in KO compared to WT.IL-6 and IL-10 plasma levels as readout for peripheral cytokines. 8 h after LPS-treatment (high dose) IL-6 plasma levels (A) were increased in WT and KO, but the increase in KO was significantly lower than in WT. 24 h after LPS-treatment IL-6 plasma levels (B) returned to baseline in WT and KO with a significant effect of genotype. IL-10 plasma levels showed a similar pattern at 8 h after LPS treatment (C) but levels remained significantly elevated in KO 24 h after LPS stimulation (D). IL-6:IL-10-ratio (E) showed an LPS-induced increase in WT 8 h after stimulation, which was significantly lower in KO. 24 h after LPS-Stimulation (F) the ratio reached low levels in both genotypes. §Main effect of genotype; ⁄LPS treatment vs. PBS; §KO vs. WT. ⁄P < 0.05; ⁄⁄P < 0.01; ⁄⁄⁄P < 0.001.

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increased after LPS-treatment, but the increase was significantly lower in KO than in WT. The LPS-induced increase of TNFa (Fig. 4C), however, was significantly higher in KO compared to WT. 24 h after treatment LPS induced an increase of the expression of all cytokines with no effect of genotype for pro-IL-1b (Fig. 4F), IL-6 (Fig. 4G), TNFa (Fig. 4H) and IL-1ra (Fig. 4J). The LPS-induced increase of IL-10 (Fig. 4I), however, was lower in the KO than in the WT. 3.5. Expression of inflammatory mediators is lower in the brains of KO 8 h after LPS-stimulation compared to WT To further investigate how immune-to-brain communication and induction of the sickness response in the brain is influenced by NF-IL6 the expression of inflammatory target genes was examined in the hypothalamus. Indicative for respective signaling pathways, we analyzed the expression of suppressor of cytokine signaling 3 (SOCS3, Fig. 5A) as activation marker for STAT3 (Lebel et al., 2000), the serine/threonine kinase-like protein of the tribbles family 1 (Trib1, Fig. 5B) as negative regulator of NF-IL6 (Yamamoto et al., 2007) and inhibitor of NFjB a (IjBa, Fig. 5C) as activation marker for NFjB (Laflamme and Rivest, 1999). We acknowledge

Fig. 4. Hepatic IL-6 expression is lower but TNFa expression higher 8 h while IL-10 expression is lower 24 h after LPS-stimulation in KO compared to WT. Hepatic expression of pro-IL-1b (A and F), IL-6 (B and G), TNFa (C and H), IL-10 (D and I) and IL-1ra (E and J) 8 (A–E) and 24 h (F–J) after stimulation with PBS or 2.5 mg/kg LPS. Pro-IL-1b, IL-10 and IL-1ra showed increased expression 8 h after LPS-treatment. Expression of IL-6 was increased in response to LPS in both genotypes; the increase in KO, however, was significantly lower compared to WT. Expression of TNFa, in contrast, was higher in KO in comparison to WT 8 h after LPS-stimulation. 24 h after LPS-stimulation all cytokines showed slightly increased expression in both genotypes. The increase in IL-10 expression, however, was significantly lower in KO than in WT. §Main effect genotype, §KO vs. WT, ⁄LPS vs. PBS.

that data related to the negative regulators of transcription factors have to be interpreted with some caution as they might not only reflect activation but as well inhibition of the respective pathways (Nadjar et al., 2003). SOCS3 and IjBa showed increased expression 8 h after LPS-stimulation in both genotypes; in KO, however, the increase was significantly lower than in WT (post hoc P < 0.01 and P < 0.01). On the protein level this result was confirmed for STAT3 by immunohistochemistry in the hypothalamus (see supplementary Fig. 1). Expression of the inhibitor of NF-IL6-activity


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Fig. 5. Lower hypothalamic expression of inflammatory mediators 8 h after LPS-treatment in KO in comparison to WT. Expression of inflammatory mediators in the hypothalamus 8 h after PBS- or LPS (2.5 mg/kg)-treatment. All of the examined inflammatory signaling molecules showed an increased expression 8 h after LPS-treatment in WT. In comparison to WT, the response in KO was significantly lower for suppressor of cytokine signaling (SOCS)3 (A), inhibitor of NFjB (IjB)a (C), IL-6 (D), TNFa (E) and IL-10 (F) and abolished for microsomal prostaglandin E synthase (mPGES) (K) and inducible nitric oxide synthase (iNOS) (L). §Main effect of genotype; ⁄LPS treatment vs. PBS; §KO vs. WT. ⁄P < 0.05; ⁄⁄ P < 0.01; ⁄⁄⁄P < 0.001. n.d. = not detectable; numbers on top or within the bars indicate the number of amplified samples out of the total amount of samples used in PCR when both were not identical.

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(Trib1) was increased 8 h after LPS-stimulation but not significantly affected by genotype. LPS-induced hypothalamic expression of the cytokines IL-6 (Fig. 5D), tumor necrosis factor a (TNFa, Fig. 5E) and IL-10 (Fig. 5F) were also elevated in WT and, to a significantly lower extent, in KO (post hoc P < 0.001; P < 0.001 and P < 0.01). Moreover, expression of pro-IL-1b (Fig. 5G), IL1-receptor antagonist (IL1-ra, Fig. 5H) as well as micro RNA 155 (miR155, Fig. 5I) were not different between the two genotypes. MiR155 had previously been

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shown to be a regulator of NF-IL6 activity (Liu et al., 2011; Faraoni et al., 2009) and its expression is regulated by several inflammatory cytokines, especially TNFa (O’Connell et al., 2007). During inflammation miR155 has been reported to protect the brain from tissue damage by inhibiting the expression of several matrix metalloproteinases (Faraoni et al., 2009). Thus, we wanted to investigate if there might be some alterations in the expression of these regulatory small RNAs after LPS challenge that might compensate or counter regulate strong LPS-induced NF-IL6-activity. Prostaglandin E2 (PGE2)-synthesis is an important mechanism for fever induction and development of some other components of the sickness response. Therefore, we explored the inducible forms of its two major rate limiting enzymes (Saper et al., 2012): Cyclooxygenase 2 (COX2, Fig. 5J) mRNA-expression was unchanged and COX2-immunoreactivity slightly lower in the MnPO (Fig. 7B and C) in KO compared to WT while the LPS-induced mRNA-expression of the downstream enzyme microsomal prostaglandin E synthase (mPGES, Fig. 5K) was abolished in KO (post hoc P < 0.001). These results suggest reduced PGE2-synthesis in KO compared to WT. In addition, the inducible nitric oxide synthase (iNOS, Fig. 5L) was analyzed, which plays a role in LPS-induced anorexia and lethargy (Riediger et al., 2010) and in the modulation of fever (Kozak and Kozak, 2003). Expression of iNOS was increased in WT in response to LPS but not in KO (post hoc P < 0.001). In summary, KO displayed attenuated hypothalamic expression of several inflammatory mediators including IL-6, mPGES and iNOS 8 h after LPS-injection.

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3.6. Higher expression of inflammatory mediators in the brains of KO 24 h after LPS-stimulation compared to WT

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In WT only SOCS3 and pro-IL-1b mRNA-expression (Fig. 6A and G) were still slightly but significantly increased 24 h after LPSstimulation (post hoc P < 0.01 and post hoc P < 0.01 respectively). With exception of Trib1 (Fig. 6B), IL-6 (Fig. 6D), mPGES (Fig. 6K) and iNOS (Fig. 6L) all inflammatory target genes investigated showed a significant effect of genotype with overall increased levels in KO animals (SOCS3 P < 0.0001, F(1,32) = 24.54; NF-IL6 P < 0.0001, F(1,31) = 34.03; IjBa P = 0.0143, F(1,32) = 6.712; TNFa P = 0.0009, F(1,30) = 13.74; IL-10 P = 0.0002, F(1,31) = 17.27; pro-IL-1b P = 0.0006, F(1,32) = 14.5; IL1-ra P = 0.0005, F(1,31) = 15.31; miR155, P = 0.0435 F(1,29) = 4.459; COX2 P < 0.0001, F(1,32) = 25.32). Moreover, LPS-induced mRNA levels of SOCS3 as well as of the cytokines IL-6, TNFa (Fig. 6 E), IL-10 (Fig. 6F), pro-IL-1b and IL-1ra (Fig. 6H) remained significantly elevated in LPS-treated KO when compared to PBS-treated KO controls and even in comparison to LPS-stimulated WT counterparts (post hoc P < 0.05; P < 0.001; P < 0.001; P < 0.001; P < 0.001). miR155 (Fig. 6I) was not detectable in WT but increased in KO 24 h after LPS-injection at least detectable in 2 out of 6 animals investigated (post hoc P < 0.05). In addition to the overall effect of genotype for COX2 mRNA-expression (Fig. 6J), we confirmed elevated COX2 protein levels in KO 24 h after LPS-treatment by immunohistochemistry of the MnPO (Fig. 7D and E) potentially indicating increased PGE2-synthesis at this time point. Interestingly, expression of mPGES (Fig. 6K) and iNOS (Fig. 6L) were not altered by genotype or treatment 24 h after treatment. Overall, KO showed an enhanced expression of inflammatory mediators 24 h after LPS-treatment while most hypothalamic inflammatory markers already declined to baseline levels in WT mice.

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3.7. Less recruitment of neutrophils to the brain 8 h after LPSstimulation in KO compared to WT

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The cellular communication pathway to the brain was investigated by detecting markers for neutrophil granulocytes (7/4) and

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Fig. 6. Higher hypothalamic expression of inflammatory mediators 24 h after LPStreatment in KO in comparison to WT. Expression of inflammatory mediators in the Hypothalamus 24 h after PBS- or LPS (2.5 mg/kg)-treatment. In LPS-treated WT the expression of the analyzed inflammatory mediators decreased back to or near basal levels. In KO the expression remained increased compared to the respective controls and/or was significant higher than WT counterparts for suppressor of cytokine signaling (SOCS)3 (A), inhibitor of NFjB (IjB)a (C), IL-6 (D), TNFa (E), IL-10 (F), IL-1b (G), IL1-receptor antagonist (ra) (H), micro RNA (miR)155 (I) and cyclooxygenase 2 (COX2) (J). §Main effect of genotype; ⁄LPS treatment vs. PBS; § KO vs. WT. ⁄P < 0.05; ⁄⁄P < 0.01; ⁄⁄⁄P < 0.001. n.d. = not detectable; numbers on top or within the bars indicate the number of amplified samples out of the total amount of samples used in PCR when both were not identical.

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perivascular macrophages (CD163) using immunohistochemistry. As one critical relay station in immune-to-brain-communication (Roth et al., 2004) and important entry site for immune cells to the brain (Rummel et al., 2010) we analyzed coronal sections at the level of the SFO (Fig. 8A, representative for other brain structures with an incomplete BBB). Just single or almost no neutrophil granulocytes were observed in PBS-treated animals of both genotypes 8 h after treatment (Fig. 8Ba and Bc). In WT large amounts of neutrophils were found 8 h after LPS-injection in vessels

neighboring the SFO (Fig. 8Bb) while in KO significant lower amounts of neutrophils were detected as confirmed by semiquantitative evaluation (Fig. 8C; post hoc P < 0.001). This decreased LPS-induced neutrophil recruitment in KO animals was accompanied by respective changes in the hypothalamic expression of the neutrophil specific chemokine CXCL1 (Fig. 8D; post hoc P < 0.001). Its expression was dramatically increased in LPS-treated WT but significantly less in KO 8 h after treatment. 24 h after LPS-stimulation, similar high amounts of neutrophil granulocytes were recruited into the SFO in both genotypes (Fig. 8Eb, Ed and F). Interestingly, we even observed some enhanced neutrophil recruitment specific into the fimbria of the hippocampus in KO (data not shown) representative of increased inflammation in the KO at 24 h. CXCL1-expression already declined at this time point but there was a significant effect of genotype with slightly increased levels in KO compared to WT (Fig. 8G; P = 0.0259, F(1,31) = 5.472). NF-IL6-immunoreactivity in WT peaked at 8 h and declined at 24 h. 24 h after LPS-stimulation less NF-IL6immunoreactivity was found in the tissue and similarly also in neutrophil granulocytes compared to the 8 h time point. Immunohistochemical detection of CD163 positive perivascular macrophages (PVM) showed only few macrophages in the SFO of PBS-treated WT (Fig. 8Ha). Interestingly, higher amounts of these cell types were revealed in PBS-treated KO (Fig. 8Hc). 24 h after LPS-injection, numbers of PVM significantly increased in WT and remained at already high levels in KO mice (Fig. 8Hb and Hd). The concomitant hypothalamic expression of CD163, the marker protein for PVM, was increased in WT 24 h after LPS-injection but not in KO (Fig. 8J, post hoc P < 0.05). No changes in CD163expression were observed 8 h after stimulation (data not shown). Although being used as a very specific antibody for PVM, some cross reactivity has previously been reported between CD68, a marker for activated microglia, and CD163 staining (Damm et al., 2011; Dijkstra et al., 1985; Graeber et al., 1989). Thus, some of the observed CD163 stained cells might represent a particular subset of activated microglia but remains to be further elucidated. In summary, KO showed less recruitment of neutrophil granulocytes to the brain 8 h after LPS-stimulation compared to WT, however, similar amounts of neutrophils at the 24 h time point. PVM seemed to increase in WT in response to LPS, but numbers were already elevated in KO under basal conditions.

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With regard to the altered response to inflammatory stress and NES of KO, potential changes in the HPA-axis might explain some of the present findings. During HPA-axis-stimulation, corticotropin-releasing hormone (CRH) activates the anterior lobe of the pituitary to cleave proopiomelanocortin (POMC) to adrenocorticotropic hormone (ACTH), which induces a release of corticosterone from the adrenal gland (Haddad et al., 2002; Turnbull et al., 1998). Thus, we decided to analyze the expression of POMC mRNA in the pituitary since NF-IL6 has been reported to exhibit binding sites on the POMC promotor (Abbud et al., 2004). Moreover, corticosterone plasma levels, which did not significantly differ between male and female mice as previously shown by others for mice and rats (Malisch et al., 2007; Kitraki et al., 2004) were used as important terminal readout for HPA-axis activity. 8 h after PBS-injection (evening, Fig. 9A–D) both genotypes showed rather high corticosterone levels of about 124 ng/ml, which even increased after LPS-stimulation with no difference between genotypes (Fig. 9A). Pituitary POMC-expression was not significantly altered between groups (Fig. 9B). Interestingly, hypothalamic POMC-expression was increased in WT 8 h after LPS-stimulation, but the response was abolished in KO (data not shown). Hypothalamic expression of pituitary adenylate cyclase

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Fig. 7. Lower COX2-immunoreactivity in the MnPO in KO at 8 h, but stronger COX2-immunoreactivity at 24 h after LPS-stimulation compared to WT. Immunohistochemical detection of COX2 (red) in the MnPO (A) 8 h (B) and 24 h (D) after treatment with PBS or 2.5 mg/kg LPS. (C) and (E) show the semiquantitative evaluation of the immunohistochemitry. 8 h after LPS-stimulation both genotypes showed increased COX2-signals in the MnPO. In KO, however, the amount of signals detected was lower than in WT. 24 h after LPS-stimulation the amount of COX2 was still increased in both genotypes, while KO showed more COX2-signals than WT. §Main effect of genotype; ⁄LPS treatment vs. PBS; §KO vs. WT. ⁄P < 0.05; ⁄⁄P < 0.01; ⁄⁄⁄P < 0.001. Scale bars in Ba and Da apply to all overview pictures and represent 100 lm; 5 lm for insets. In insets von Willebrandt factor as marker for blood vessels is stained in green. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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activating polypeptide (PACAP, Fig. 9C), which is a modulator of the HPA-axis (Stroth et al., 2011), was also not altered in KO compared to WT, while mRNA-expression of its receptor, vasoactive intestinal peptide receptor 1 (VIPR1, Fig. 9D), was reduced in KO (effect of genotype, post hoc P = 0.0493, F(1,27) = 4.239). 24 h after PBS-injection (morning, Fig. 9E–H) WT showed low corticosterone plasma levels (about 18 ng/ml), whereas levels of KO were as high as in the evening (Fig. 9E). Thus, plasma corticosterone showed normal evening and morning levels in WT with low levels in the morning (24 h after injection of PBS) but high levels in

the evening (8 h after PBS-treatment). In KO, in contrast, we observed high corticosterone levels at both time points (post hoc P < 0.001). Accordingly, also basal expression of POMC (Fig. 9F) in the pituitary gland was higher in KO compared to WT at the 24 h time point (post hoc P < 0.05). At this time point, PACAP-expression (Fig. 9G) was also unchanged in both genotypes, while expression of the VIPR1 (Fig. 9H) was lower in PBS-treated KO compared to WT (post hoc P < 0.05). Exemplary immunohistochemistry of the pituitary gland (Fig. 9I) showed low amounts of POMC in the anterior lobe (AL) of PBS-treated WT (Fig. 9Ia) and LPS-treated KO


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Fig. 8. Less recruitment of neutrophil granulocytes in KO 8 h after LPS-treatment compared to WT. Immunohistochemistry was performed at the level of the subfornical organ (SFO, A) to detect neutrophil granulocytes (B and E) and perivascular macrophages (H). 8 h after LPS-treatment (2.5 mg/kg) neutrophil granulocytes (red, 7/4immunoreactivity, B) migrated into the SFO of LPS-treated WT and in lower amounts into the SFO of LPS-treated KO. Semiquantitative evaluation of the immunohistochemistry is presented in (C). Open arrows indicate NF-IL6 (green)-negative neutrophil granulocytes; white arrows indicate NF-IL6-positive neutrophil granulocytes. Expression of the neutrophil specific chemokine CXCL1 in the hypothalamus (D) showed similar results with high amounts in LPS-treated WT, but lower expression in KO. 24 h after treatment (E-G) immunohistochemistry revealed similar numbers of neutrophil granulocytes after LPS-treatment in both genotypes, while CXCL1 was slightly higher in KO. CD163 was examined as marker for perivascular macrophages (indicated by asterisks). PBS treated WT showed only few macrophages (red) in the SFO, while larger amounts were found in KO. LPS-treatment increased amounts of macrophages in both genotypes (H and I). CD163 mRNA-expression was similar in PBStreated WT and KO and increased in LPS-treated WT, but not in KO (J). §Main effect of genotype; ⁄LPS treatment vs. PBS; §KO vs. WT. ⁄P < 0.05; ⁄⁄P < 0.01; ⁄⁄⁄P < 0.001. Scale bars in Ba apply to all overview pictures and represent 100 lm; 5 lm for insets. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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(Fig. 9Ic) 24 h after treatment, while LPS seemed to increase POMC in the AL of WT (Fig. 9Ib). In the intermediate lobe (InL) of WT we observed high quantities of POMC-immunoreactivity, which seemed to be slightly reduced in KO. The posterior lobe (PL) did not contain any POMC in both genotypes. In summary, investigation of HPA-axis activity primarily indicated alterations in morning corticosterone levels in KO but no

changes in LPS-induced HPA-axis activity compared to WT counterparts.

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To further clarify potential underlying mechanisms for depressed locomotor activity and the altered stress response of

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Fig. 9. HPA-axis is altered in KO. Corticosterone plasma levels of WT showed basally high levels in the evening (8 h after treatment, A) and low levels in the morning (24 h after treatment, E) and increased corticosterone levels 8 and 24 h after LPS-treatment (2.5 mg/kg). KO, in contrast, showed high corticosterone levels at the 8 and 24 h time points. Proopiomelanocortin (POMC)-expression in the pituitary gland (B and F) was higher basally in KO than in WT. Hypothalamic expression of the regulatory peptide pituitary adenylate cyclase-activating polypeptide (PACAP; C, G) was not altered, but its receptor VIPR1 (D, H) was basally lower in KO. Exemplary immunohistochemistry of the pituitary gland was performed to detect POMC (red, I). LPS seemed to increase POMC in the anterior lobe (AL) of WT but not in KO 24 h after stimulation. The intermediate lobe (InL) of KO showed lower amounts of POMC than the InL of WT. a–c1 show magnified views of the InL, a–c2 of the AL of the pituitary. Scale bar in Ia applies to Ia-Ic and represents 100 lm; in a1 10 lm and applies to high magnifications. §Main effect of genotype; ⁄LPS treatment vs. PBS; §KO vs. WT. ⁄P < 0.05; ⁄⁄P < 0.01; ⁄⁄⁄P < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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KO the serotonin system was also examined. Normally, serotonin is produced out of tryptophan by tryptophan-hydroxylase (TPH) 2 (Chen and Miller, 2012). Tryptophan can also be processed to 3-hydroxy-kynurenine by two enzymes, namely indolamine-2,3dioxygenase (IDO) and 3-hydroxy-kynurenine-monooxygenase (KMO), which are known to be influenced by stress or inflammation (Myint, 2012). The serotonin reuptake transporter SLC6A4 has a regulatory role on serotonin recycling and metabolism and, thus, on the development of depressive behavior (Blier and El Mansari, 2013). Under physiological conditions tryptophan-metabolism mainly takes place in the liver, while it is shifted away from the liver to other tissues including the brain during inflammation (Myint, 2012). Here, LPS induced a dramatic elevation in IDOmRNA-expression (Fig. 10A) in the hypothalamus 8 h after treatment in WT. This response was almost abolished in KO (post hoc P < 0.001). The mRNA-expression of KMO (Fig. 10B) remained unchanged. TPH2 mRNA-expression (Fig. 10C) in the hypothalamus was significantly increased after LPS-treatment in KO (post hoc P < 0.05), but not in WT. Moreover, the expression of the

serotonin reuptake transporter SLC6A4 (Fig. 10D and H) was higher in KO compared to WT (effect of genotype, P = 0.0018 F(1,28) = 11.96). Thus, at the 8 h time point serotonin-synthesis (TPH2) and its reuptake (SLC6A4) seemed to be enhanced, while 3-hydroxy-kynurenine-synthesis (IDO) appeared to be lower in KO compared to WT. 24 h after treatment mRNA-expression of IDO and TPH2 returned to basal levels in both genotypes (Fig. 10E and G), whereas KMO (Fig. 10F) and SLC6A4 (Fig. 10H) were higher in KO compared to WT (effect of genotype, P = 0.0218, F(1,32) = 5.819; and P = 0.0003, F(1,32) = 16.26, respectively). In the liver, however, IDO-expression (Fig. 10I) was significantly higher in KO 8 h after LPS-stimulation compared to WT (post hoc P < 0.01), while KMO (Fig. 10J) remained unchanged. 24 h after treatment, no differences between the two genotypes were found for IDO-expression (Fig. 10K), but KMO-expression (Fig. 10L) was lower in KO compared to WT (effect of genotype, P = 0.0379 F(1,32) = 4.692). The liver was investigated as this organ represents the main tryptophan metabolizing organ under normal


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Fig. 10. Alterations in the tryptophan metabolism in KO. Hypothalamic expression of indolamine-2,3-dioxygenase (IDO) was increased in WT in response to LPS (2.5 mg/kg) 8 h after treatment. This response was abolished in KO (A). 3-Hydroxy-kynurenine-monooxygenase (KMO)-expression (B) was not altered by LPS-treatment. Tryptophanhydroxylase 2 (TPH2)-expression (C) was increased in KO but not in WT 8 h after LPS-treatment, while expression of the serotonin transporter SLC6A4 (D) was overall higher in KO compared to WT. 24 h after treatment expression of IDO (E) and TPH2 (G) were not altered, but KMO- (F) and SLC6A4-expression (H) were higher in KO than in WT. In the liver IDO-expression (I) of KO is higher compared to WT while KMO-expression (J) was unchanged 8 h after treatment. 24 h after treatment IDO-expression in the liver (K) was not changed between the two genotypes while KMO-expression (L) is lower in KO than in WT. §Main effect of genotype; ⁄LPS treatment compared to PBS; §KO compared to WT; ⁄P < 0.05; ⁄⁄P < 0.01; ⁄⁄⁄P < 0.001. n.d. = not detectable; numbers on top of the bars indicate the amount of amplified samples out of the total amount of samples used in PCR when both were not identical.

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physiological conditions (Myint and Kim, 2014). Overall, these data indicate that the whole serotonin system is altered in absence of NF-IL6. 3.10. Liver-enriched activating protein is increased in the hypothalamus of WT 8 h after LPS-stimulation Western blot analysis of hypothalamic tissue for NF-IL6 revealed no difference of NF-IL6 expression between morning and evening samples (Fig. 11A and B). NF-IL6 is known to be found in different isoforms: Liver-enriched activating protein (LAP1, 36 kDa and LAP2, 34 kDa) and liver-enriched inhibitory protein (LIP, 20 kDa) (Zahnow, 2009). The activating isoform of NF-IL6, LAP, was increased in WT 8 h after LPS stimulation compared to PBS-treated controls (Fig. 11A and C; student’s t-test P = 0.0027). 24 h after stimulation LAP-expression was not altered by LPS (Fig. 11B and C). We were not able to detect the inhibitory isoform,

LIP, by Western blot. According to the literature, however, LIP was so far only detected in tissues like the mammary gland (Dearth et al., 2001) and the parotid gland (Reinhold and Ekstrom, 2004) or in microglial cultures (Ejarque-Ortiz et al., 2007) but it was never detected in brain tissue.

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We report for the first time that decreased brain inflammation (8 h) and fever (3–7 h) preceded enhanced inflammation and maintained fever (24 h) in NF-IL6-KO mice during septic-like LPS-induced inflammation. These data suggest a dual role for NFIL6 e.g. an pro-inflammatory (8 h) followed by an anti-inflammatory (24 h) effect during the course of inflammation, which was also reflected by a lower recruitment of neutrophil granulocytes and absence of increases in perivascular macrophages in KO compared to WT after LPS-stimulation. Moreover, we observed

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Fig. 11. NF-IL6-protein levels in WT during LPS-induced systemic inflammation. Hypothalamic NF-IL6-protein levels normalized to b-actin increased 8 h after LPS-stimulation (2.5 mg/kg, A) and decreased back to baseline 24 after injection compared to PBS-counterpart controls (B). A representative blot is shown to depict the detected size of bands (LAP = 34.89 kDa; b-actin = 42.31 kDa) compared to the standards (middle line) displayed (C). ⁄WT LPS vs. WT PBS; ⁄⁄P < 0.01.

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reduced basal but enhanced NES-induced locomotor activity in KO, which might be related to the NF-IL6-dependent alterations in the circadian rhythmicity of HPA-axis activity and to changes in the tryptophan metabolism. As it is the case for all knockout mice and with other focus even for conditional tissue specific knockouts, there are important limitations when using such genetically modified animals. Previously, we tried to use potential inhibition strategies to block NF-IL6 action only in the brain, which was only partially successful (Damm et al., 2013). Here, effects on the brain and the periphery differed quite significantly suggesting some more specific role for NF-IL6 in these tissues and cell types. Indeed, a decrease/change in peripheral inflammation will also have contributed to the observed changes in the brain. Nonetheless, the overall role for NF-IL6 during systemic inflammation was further characterized and revealed for the first time important features of this inflammatory transcription factor for the manifestation of inflammation. The presence of NF-IL6 might be needed for some feedback mechanisms either in the periphery and/or the brain. NF-IL6 was initially described as a transcription factor implicated in IL-6-expression (Akira et al., 1992; Akira et al., 1990). Indeed, the present results suggest a participation of NF-IL6 in LPS-induced IL-6-expression in the hypothalamus and the periphery. IL-6 is one of the most prominent circulating cytokines acting as a humoral mediator on the brain (Cartmell et al., 2000) via the STAT3-signaling pathway (Rummel et al., 2006; Lebel et al., 2000). Previously, we have shown its direct action on cells of sensory circumventricular organs such as the SFO (Harre et al., 1985) and brain vasculature (Rummel et al., 2005) and its involvement for fever-induction via a STAT3-linked expression of COX2 and mPGES (Rummel et al., 2006, 2011). More recently, it was reported that the pyrogenic properties of IL-6 are mediated by the binding to its receptor on brain endothelial cells and subsequent induction of prostaglandins within these cells (Eskilsson et al., 2014). We observed reduced levels of IL-6 in the blood and reduced expression of IL-6 in the liver at 8 h after LPS-stimulation accompanied by decreased COX2-immunoreactivity in the median preoptic nucleus, a pivotal brain structure in PGE2-dependant fever induction pathways (Lazarus et al., 2007), and less pronounced mPGES-mRNA expression in the hypothalamus of KO compared to WT. It is likely that this reduced formation of peripheral IL-6 also occurs earlier than at the time point investigated, so that the pyrogenic signal exerted by this cytokine could not activate their

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putative targets in the brain (endothelial cells and/or CVOs) in its full strength, resulting in attenuated fever from 3 to 7 h after LPS-stimulation. A detailed analysis of the kinetics of peripheral formation of IL-6 and other cytokines during this phase of LPS-induced systemic inflammation could strengthen this hypothesis. With regard to the early and short-lasting initiation of LPS-induced fever, however, the importance of IL-6 and other cytokines has been questioned due to a discrepancy between the time of their appearance in the blood and the rapid induction of fever. A role for rapidly generated PGE2 in the periphery has been suggested with regard to the induction of the early phase of LPS-fever (Roth and Blatteis, 2014; Steiner et al., 2006). Due to the fact, that NFIL6 is a transcription factor, whose activation occurs with some delay, we did not focus on the early period after LPS-treatment, when we prepared the experimental design for the collection of blood and tissue samples. Furthermore, we found alterations of TNFa-expression in NFIL6 deficient mice. The role of TNFa during inflammation, however, is very complex and still controversial. Previously it was proposed that peripheral TNFa may exhibit antipyretic effects (Klir et al., 1995; Kozak et al., 1995). Indeed, we found increased hepatic TNFa expression in LPS-treated KO compared to WT 8 h after treatment in line with its proposed role as a cytokine with antipyretic properties regarding the reduced fever of KO from 3 to 7 h after LPS-stimulation. In the blood, however, TNFa was not detectable at this time point. In the hypothalamus TNFa might be involved either in the induction of fever (Dinarello et al., 1986) or in the induction of hypothermia (Tollner et al., 2000) and pro- as well as anti-inflammatory effects of TNFa have been described (Aggarwal, 2014; Marchetti et al., 2004). In the hypothalamus, we found lower expression of TNFa 8 h after LPS-stimulation in KO compared to WT, but higher expression 24 h after LPS-treatment, which was indicative for a reduced LPS-induced brain-inflammation in KO 8 h after treatment, but increased inflammation within the CNS 24 h after treatment. The physiological significance of these findings, however, remains to be elucidated. The observed reduction in circulating IL-10 levels (if it was also present between 3 h and 7 h) most likely was not involved in the reduced febrile response. Indeed, we previously have shown that neutralization of endogenous circulating IL-10 even enhances the febrile response to a septic LPS-dose (Harden et al., 2014). Reduced IL-6:IL-10-ratio in KO compared to WT 8 h after LPS-treatment is also indicative for reduced inflammation in KO at that time point, although hepatic expression of other inflammatory mediators such as pro-IL-1b were unchanged in KO. With the exception of the short, early phase of LPS-fever (Roth and Blatteis, 2014; Steiner et al., 2006) the synthesis of PGE2 by brain endothelial cells via induced expression of COX-2 and mPGES is critical for the manifestation of LPS-induced fever (Wilhelms et al., 2014). The role of NF-IL6 in mPGES-expression appears to be more complex than expected. We only observed abolished LPS-induced mPGES-expression in KO 8 h after treatment (high dose). Indeed, mPGES-deficient mice show strongly impaired fever to inflammatory stimuli (Saha et al., 2005; Engblom et al., 2002) and previous results show that NF-IL6 contributes to mPGES-expression (Uematsu et al., 2002). Straccia et al. (2013) recently even revealed that NF-IL6 is specifically implicated in microglial mPGES-expression, supporting its crucial role for induction of neuroinflammation. However, although NF-IL6-deficient mice were previously reported to be cold sensitive with some deficits in thermoregulation (Carmona et al., 2005), or showed increased energy expenditure under high fat diet (Millward et al., 2007), in our experiments KO developed a normal febrile response to the low LPS-dose when kept at thermoneutral ambient temperature. Thus, other transcription factors than NF-IL6 including NFjB, STAT3 or early growth response factor


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seem to contribute to basal or low LPS-dose induced mPGESexpression, which might be sufficient for subsequent PGE2 production in presence of COX2 (Rummel et al., 2011; Barakat et al., 2009; Diaz-Munoz et al., 2010). The response of iNOS to LPS (high dose) was also abolished in KO confirming that LPS-induced expression of iNOS is regulated by NF-IL6 (Dlaska and Weiss, 1999). However, we did not show a contribution of NO to induction of LPS-induced anorexia and lethargy as previously suggested (Riediger et al., 2010). Interestingly, a very broad variety of ligands have been reported for NF-IL6 induction. These include LPS and almost all relevant proinflammatory cytokines (Ramji and Foka, 2002). Most of these findings have been revealed using cell cultures. In vivo, the respective contribution of cytokines and LPS to NF-IL6 activation has not been thoroughly investigated so far. We previously hypothesized that interferons might be important as imiquimod-induced systemic inflammation was primarily mediated by interferons and not accompanied by strong STAT3 but strong NF-IL6 activation (Damm et al., 2012). However, a recent study using another agonist of intracellular Toll-like receptors, namely ODN, did not show interferon induction but still strong NF-IL6 signals (Damm et al., 2014). After LPS-stimulation NF-IL6 signals might be induced by IL-6, however, the timing of its activation does not match circulating IL-6 levels and IL-6 deficient mice also seem to still show some NF-IL6 signals (unpublished observation of CR). In addition, we have previously used IL-10 antiserum (Harden et al., 2014) and leptin antiserum to neutralize endogenous circulating levels of these two mediators in rats after LPS-stimulation but we did not reveal a contribution of these mediators to NF-IL6 activation in the brain (Koenig et al., 2014). Moreover, leptin deficient mice seem still to show strong LPS-induced NF-IL6 signals (unpublished observation of CR). Overall, the contribution of circulating factors to central NF-IL6 activation does not seem to be very specific for any of these mediators but rather represents an integration of the extent of inflammation in the brain. This notion is further substantiated by the fact that also traumatic brain injury (Damm et al., 2013) and neuronal activity (Cortes-Canteli et al., 2008), for example induced by psychological stress, can activate NF-IL6 (Fuchs et al., 2013). Thus, as opposed to STAT3, NF-IL6 represents a broad and useful but rather unspecific activation marker that will be applicable for a variety of future research questions. We further analyzed potential alterations in the immune cell mediated immune-to-brain communication and observed lower LPS-induced neutrophil recruitment to the brain in KO compared to WT at the 8 h time point most likely due to reduced CXCL1expression, a neutrophil specific chemokine. However, a previous report showed overall reduced extravasation of neutrophils into the brain of KO in a model of ischemic brain damage (Kapadia et al., 2006). The reason for this discrepancy might pertain to differences in the inflammatory models and the timing of analyses but our results suggest a more complex regulation of immunecell-trafficking. Besides neutrophils (Aguliar-Valles et al., 2014; Rummel et al., 2010), PVM have also been reported to play an important role in immune-to-brain communication (Schiltz and Sawchenko, 2003; Serrats et al., 2010). Here, the stimulation with a high LPS-dose induced an increase in CD163-immunoreactivity and – mRNA-expression in WT but not in KO. We are not aware of a previous report showing a response like this but it might be related to some anti-inflammatory action of PVM as previously suggested by Serrats et al. (2010). 8 h after LPS-stimulation several inflammatory markers including TNFa and IL-10 were decreased in the hypothalamus and/or the circulation in line with reduced inflammation in KO compared to WT. Indeed, for several of these target genes NF-IL6 has been reported to be implicated in their expression like for IL-10 (Liu

et al., 2003). 24 h after treatment KO showed a prolonged fever in response to the low LPS-dose. Meanwhile, after high-dose-LPSstimulation, IL-6/IL-10 plasma levels and hypothalamic expression of several pro-inflammatory mediators like TNFa were increased compared to WT suggesting enhanced and prolonged inflammation. Some of these mediators including the anti-inflammatory cytokine IL-10 (Schottelius et al., 1999) and the small regulatory RNA miR155 might represent compensatory mechanisms to tone down an exaggerated inflammatory response (Faraoni et al., 2009) in KO at the 24 h time point. Collectively, these results show that KO exhibited lower brain and peripheral inflammation at 8 h, but increased inflammation 24 h after LPS-treatment compared to WT. Previously, we proposed that NF-IL6 might be involved in either the maintenance or the termination of the febrile response to LPS (Damm et al., 2013, 2011). Now, we can hypothesize that NF-IL6 might be responsible for both and we suggest for the first time a time-dependent dual role of NF-IL6, first a pro-, thereafter an anti-inflammatory action. To our knowledge, such a dual action of an inflammatory transcription factor has not been described before during the time course of inflammation possibly reflecting the natural active programming of the inflammatory process by a transcription factor. Knowing the pivotal role of these transcription factors including NFjB this observation has important implications for a variety of inflammatory processes. Active programming of inflammation has recently also been suggested for so-called small proresolving mediators (Serhan et al., 2014). However, we have to acknowledge that the complex interaction of inflammatory transcription factors makes it rather difficult to dissect out specific contributions of one of these factors to inflammatory processes. As previously reported, a so-called ‘‘crosstalk’’, namely, a direct physical interaction of these transcription factors can enhance or decrease their promotor activity. Moreover, even ‘‘new’’ binding sites may be bound only because of this interactive process within the complex transcription machinery (Agrawal et al., 2003; Rummel et al., 2006). Another study even argued in favor of a double knockout of CEBP/beta and CEBP/delta to obtain a phenotype that is not compensated with some overlapping actions of transcription factors (Akagi et al., 2010). Nonetheless, the specific spatiotemporal, timely controlled, consecutive transcription factor activation in the brain e.g. first NFjB, followed with some overlap by STAT3 and finally NF-IL6 favors the notion that these factors should have time specific actions und functions during the inflammatory response. Overall, we believe that some of the observed findings can actually be directly or indirectly attributed to NF-IL6 function, however, keeping in mind the afore mentioned important limitations. Hypothalamic expression of Trib1, the inhibitor of LPS-induced NF-IL6-activity, remained unchanged between the two genotypes. This observation suggests that Trib1 might not be involved in the functional shift of NF-IL6 during systemic inflammation. However, further analyses also on the protein level are needed to clarify this issue. Alternatively, this dual role of NF-IL6 might also be due to a switch between its different isoforms. Indeed, the inflammatory transcription factor NF-IL6 exists in different isoforms: LAP1 and LAP2 are activating isoforms, while LIP lacks the N-terminal activation domain and functions as an inhibitor (Zahnow, 2009). Thus, the activating isoform, LAP, might be responsible for the ‘‘sustained’’ maintenance, while the inhibitory isoform, LIP, might control the ‘‘late’’ termination of the inflammatory response and, perhaps, fever. Western blot analysis revealed increased LAP-expression 8 h after LPS-stimulation in the hypothalamus of WT, which supports our hypothesis. However, we could not detect hypothalamic LIP-protein. Thus, further studies are needed to clarify underlying mechanisms.


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Moreover, KO also showed reduced locomotor activity under basal conditions, which might be related to altered HPA-axis activity. For example, inhibition of corticotropin releasing hormone (CRH) has been shown to reduce locomotor activity (Ohata et al., 2002). Moreover, long-term application of corticosterone was previously shown to induce depressive-like behavior in mice, which might also lead to reduced locomotor activity (van Donkelaar et al., 2014). Indeed, we found increased basal POMC-expression in the pituitary and high morning corticosterone plasma levels in KO. PACAP, one of the major regulators of the HPA-axis (Hashimoto et al., 2011) was not changed in KO compared to WT. Its receptor, VIPR1, however, was reduced in KO under basal conditions. Interestingly, it was previously shown that mice deficient in another PACAP-receptor, namely VPAC2, show reduced basal locomotor activity (Hannibal et al., 2011). Furthermore, Joo et al. (2004) proposed that VIPR1 and VPAC2 have similar functions in the regulation of the master clock, which regulates circadian rhythms, and locomotor activity. Thus, reduced expression of VIPR1 might partially explain reduced locomotor activity and potentially be even responsible for the observed modifications of corticosterone plasma levels. On the other hand, the enhanced circulating morning levels of corticosterone observed in KO could also be associated with the higher basal levels of circulating IL-6 in these animals since IL-6 contributes to HPA-axis activation (Lenczowski et al., 1999). 2–4 h after NES locomotor activity of KO was increased compared to WT. As no animals were killed after NES we can only speculate about the underlying mechanisms. We previously showed that NES leads to an activation of NF-IL6 in the brain and the pituitary (Fuchs et al., 2013). Now, using NF-IL6 deficient mice, we obtained evidence to suggest that NF-IL6 is involved in the basal regulation of the HPA-axis. Interestingly, cage change stress induced increase in body core temperature and locomotor activity was previously shown to be reduced by icv injection of a CRH antagonist (Morimoto et al., 1993). Thus, we propose that the altered reaction of KO to NES might also be due to alterations in the HPA-axis-response in addition to potential involvements of other pathways in the framework of immune-to-brain communication. Finally, we revealed that expression of IDO was reduced in the hypothalamus of KO, while expression of TPH2 and the serotonin reuptake transporter SLC6A4 were increased 8 h after LPS-stimulation. Both changes suggest for the first time a contribution of NF-IL6 in decreased 3-hydroxy-kynurenine-synthesis as an alternative pathway of tryptophan metabolism and subsequent increased serotonin-synthesis and reuptake in KO. Importantly, depressive like behavior is induced by 3-hydroxy-kynurenine and other metabolites, while it is inhibited by serotonin (Dantzer and Walker, 2014; Myint, 2012). Altered expression of SLC6A4 is known to promote depressive like behavior, while inhibition of serotonin reuptake is a therapeutic strategy in depressive disorders (Jonassen and Landro, 2014; Blier and El Mansari, 2013). Thus, it appears that KO show elevation of serotonin and reduced 3-hydroxy-kynurenine but in particular enhanced serotonin-reuptake, which potentially leads to reduction of locomotor activity. Indeed, binding sites for NF-IL6 on the IDO- and SLC6A4-regulatory region have been reported (Robinson et al., 2005; Udina et al., 2013) and NF-IL6 can inhibit SLC6A4-expression during LPS-induced inflammation in macrophages (Zimmermann et al., 2014). However, in these studies no contribution to inflammation-induced IDO-expression of NF-IL6 was observed. Moreover, in contrast to the brain, liver IDO-expression was increased 8 h after LPS-treatment in KO, while KMO-expression was slightly decreased basally as well as 24 h after LPS-treatment in KO relative to WT. Alternatively, the observed changes in brain and circulating IL-6 in KO might also have contributed to IDO-expression as previously suggested (Kim et al., 2012). Indeed, Robinson et al. (2005) stimulated neuronal cell cultures and hippocampal

organotypic slice cultures with IL-6 and, thereby, increased IDOexpression most likely via the STAT3-signaling pathway.

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5. Systematic summary

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The schematic Fig. 12 conveys a summary of potential mechanisms revealed for NF-IL6-action in the present manuscript. Overall, KO do mount a normal fever to a low LPS dose opposed to what has been reported for NFjB deficient mice (Kozak et al., 2006). However, after the initiation phase (1–3 h) they show some lack (3–7 h) of fever to high grade inflammation but develop fever in the morning of the second day similar to WT. The reduced febrile response might be due to lower circulating IL-6 levels in KO compared to WT (8 h). Indeed, IL-6 induces COX2 and mPGES dependent PGE2 production in brain endothelial cells and subsequently fever (Eskilsson et al., 2014). Alternatively, enhanced TNFa production might have acted as a cryogen within the 3–7 h time period to reduce the febrile response. Our additional measurements of TNFa expression in the liver can be indicative of such a view. Even reduced recruitment of neutrophil granulocytes into the brain might be implicated as neutrophils have been reported to express for example IL-1b and convey prolonged behavioral changes during septic like inflammation (Aguliar-Valles et al., 2014). However, the role of neutrophil recruitment for fever remains to be investigated. Moreover, while basal locomotor activity is low in KO they showed more locomotor activity to NES stress, which demonstrates that these animals are capable to move normally. Interestingly, NF-IL6 deficiency leads to higher expression of SLC6A4, the serotonin reuptake transporter, indicative for a basal depressed state e.g. reduced locomotor activity of these animals due to reduced amounts of serotonin in the synaptic clefts. During the development of these animals high SLC6A4 levels might also just represent a compensatory increase in its expression because of changes in the whole serotonin system during development. This has previously been reported to occur and causes changes in the brain serotonin network (Sibille and Lewis, 2006). In addition, LPS-stimulation seems to shift the balance from the IDO kynurenin to the TPH2 serotonin pathway. Thus, acute NFIL6 inhibition might as well be of therapeutic potential to enhance

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NF-IL6 ? NF-IL6

IL-6

?

TNFα COX2 mPGES

neutrophils ?

LPS

fever

NF-IL6 HPA STAT3 NF-IL6

corcosterone inflammaon COX2

NFκB

mPGES iNOS IL-6, IL-10 immune cells

cytokines NF-IL6

?

NES NF-IL6

locomotor acvity inner clock

serotonin NF-IL6

NF-IL6

HPA / corcosterone

NF-IL6

NF-IL6

Fig. 12. Schematic overview of potential underlying mechanisms of the observed changes in KO compared to WT. NF-IL6 regulates the expression of IL-6, which is known to induce expression of COX2 and mPGES and, thus, seems to be implicated in the induction of fever. TNFa, however, can exhibit antipyretic effects in the periphery. The role of neutrophils for fever induction is yet unclear, as well as the role of immune cells for inflammation. STAT3 and NFjB, however, induce the expression of COX2, interact (cross-talk) with NF-IL6 and induce inflammation. NF-IL6 is known to regulate the expression of mPGES, IL-6, IL-10 and iNOS, which can also influence the inflammatory response. The HPA-axis, in contrast, is known to inhibit inflammation. Locomotor activity of the mice can be influenced by HPA-axis, Serotonin and the inner clock. " = activation, > = inhibition, l = two-way interaction.


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serotonin synthesis. The changes within the serotonin system in KO could also explain some of the changes seen in locomotor activity in the present study (e.g. reduced basal activity and enhanced NES activity). Moreover, NF-IL-6 deficiency seems to have some impact on basal morning levels of corticosterone (HPA-axis) and basal expression of the PACAP receptor VIPR1, which both might also be related to changes in the observed locomotor activity (see also ‘‘Discussion’’ section). For inflammation, NF-IL6 plays an important role as it is activated by various stimuli like cytokines, LPS and even psychological stress (Fuchs et al., 2013) and itself binds to various promotor binding sides of inflammatory mediators including mPGES (Uematsu et al., 2002), iNOS (Guo et al., 2003), IL-6 (Matsusaka et al., 1993) and IL-10 (Liu et al., 2003; Poli, 1998). Indeed, we observed less hypothalamic expression of these inflammatory markers at the 8 h time point in KO compared to WT after LPSstimulation. LPS-induced COX2-immunoreactivity in KO was lower than in WT most likely depending on afore mentioned cross talk of NF-IL6 to other inflammatory transcription factors such as STAT3 and NFjB. Subsequently, altered chemokine expression seems to contribute to regulation of immune cell recruitment to the brain, a hallmark of inflammation. Some modulatory role for NF-IL6 might alter inflammation also via the HPA-axis. In summary (supplementary Fig. 2A and B), we observed primarily reduced inflammation in the periphery and the brain of KO compared to WT (8 h, A) signifying some inflammatory potential of NF-IL6 function (recruitment of neutrophil granulocytes; induction of IL-6, mPGES, iNOS but also IL-10) followed by higher inflammation than in WT at the 24 h time point (B) signifying some anti-inflammatory potential (potentially inhibition NFjB, STAT3 and miR155 and subsequent target genes e.g. IL1b, TNFa, CXCL1).

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6. Conclusions

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We characterized for the first time the role of NF-IL6 for the development of LPS-induced sickness responses. We found that it influences immune-to-brain communication via the humoral and the cellular pathway and that it is involved in the maintenance and the termination of the febrile response reflecting natural programming of inflammation. We further showed that NF-IL6 may be implicated in the rhythm of HPA-axis-activity and the serotonin system and, thus, could be a potential therapeutic target for the treatment of brain inflammation and depressive disorders.

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Acknowledgments

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This study was supported by the Emmy Noether-Program of the German Research Foundation [DFG project RU 1397/2-1]. We would like to thank Ms. D. Marks, Ms. D. Ott, Ms. S. Engel and Ms. J. Murgott for excellent technical assistance and Dr. G. Eichner for excellent statistical advice. The authors have no conflicting financial interests.

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Appendix A. Supplementary data

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Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bbi.2015.03.008.

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Activation of the inflammatory transcription factor nuclear factor interleukin-6 during inflammatory and psychological stress in the brain.

Activating transcription factor 6 derepression mediates neuroprotection in Huntington disease.

Interleukin-6: an osteotropic factor?

Matricellular Protein Periostin Mediates Intestinal Inflammation through the Activation of Nuclear Factor κB Signaling.

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C. elegans nuclear receptor NHR-6 functionally interacts with the jun-1 transcription factor during spermatheca development.

Hepatocyte nuclear factor-4α mediates redox sensitivity of inducible nitric-oxide synthase gene transcription.

The Transcription Factor ATF5 Mediates a Mammalian Mitochondrial UPR.

SLUG transcription factor: a pro-survival and prognostic factor in gastrointestinal stromal tumour.

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The regulatory role of activating transcription factor 2 in inflammation.

Interleukin-6 induction by tumor necrosis factor and interleukin-1 in human fibroblasts involves activation of a nuclear factor binding to a kappa B-like sequence.

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The Arabidopsis thaliana Nuclear Factor Y Transcription Factors.

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