Neurochem Res DOI 10.1007/s11064-016-1939-4
ORIGINAL PAPER
Repeated LPS Injection Induces Distinct Changes in the Kynurenine Pathway in Mice M. K. Larsson1 • A. Faka1 • M. Bhat2 • S. Imbeault1 • M. Goiny1 • F. Orhan1 A. Oliveros3 • S. Sta˚hl4 • X. C. Liu1 • D. S. Choi3,5,6 • K. Sandberg1,3,7 • G. Engberg1 • L. Schwieler1 • S. Erhardt1
•
Received: 2 March 2016 / Revised: 19 April 2016 / Accepted: 27 April 2016 Ó Springer Science+Business Media New York 2016
Abstract The immune system has been recognized as a potential contributor to psychiatric disorders. In animals, lipopolysaccharide (LPS) is used to induce inflammation and behaviors analogous to some of the symptoms in these disorders. Recent data indicate that the kynurenine pathway contributes to LPS-induced aberrant behaviors. However, data are inconclusive regarding optimal LPS dose and treatment strategy. Here, we therefore aimed to evaluate the effects of single versus repeated administration of LPS on the kynurenine pathway. Adult C57BL6 mice were given 0.83 mg/kg LPS as a single or a repeated injection (LPS ? LPS) and sacrificed after 24, 48, 72, or 120 h. Mice receiving LPS ? LPS had significantly elevated brain
kynurenine levels at 24 and 48 h, and elevated serum kynurenine at 24, 48 and 72 h. Brain kynurenic acid and quinolinic acid were significantly increased at 24 and 48 h in mice receiving LPS ? LPS, whereas serum kynurenic acid levels were significantly decreased at 24 h. The increase of brain kynurenic acid by LPS ? LPS was likely unrelated to the higher total dose as a separate group of mice receiving 1.66 mg/kg LPS as single injection 24 h prior to sacrifice did not show increased brain kynurenic acid. Serum quinolinic acid levels were not affected by LPS ? LPS compared to vehicle. Animals given repeated injections of LPS showed a more robust induction of the kynurenine pathway in contrast to animals receiving a single injection. These results may be valuable in light of data showing the importance of the kynurenine pathway in psychiatric disorders.
M. K. Larsson and A. Faka are shared first author. & S. Erhardt
[email protected] 1
Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden
2
Protein Biomarkers, Personalized Healthcare and Biomarkers Laboratories, Innovative Medicines, AstraZeneca, Gothenburg, Sweden
3
Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic College of Medicine, Rochester, MN 55905, USA
4
Translational Science Centre, Personalized Healthcare and Biomarkers Laboratories, Innovative Medicines, Science for Life Laboratory, AstraZeneca, Stockholm, Sweden
5
Neurobiology of Disease Program, Mayo Clinic College of Medicine, Rochester, MN 55905, USA
6
Department of Psychiatry and Psychology, Mayo Clinic College of Medicine, Rochester, MN 55905, USA
7
Department of Clinical Neuroscience, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden
Keywords Kynurenic acid Lipopolysaccharide Quinolinic acid Neuroinflammation Psychiatric disorders Kynurenine pathway
Background The immune system of the brain, and in particular its critical relationship with activation of the kynurenine pathway, has emerged as a plausible causal factor underlying several psychiatric disorders as well as a putative therapeutic target. Recent studies show that patients with schizophrenia, bipolar disorder or depression show elevated brain levels of pro-inflammatory cytokines [1–4] for overview see [5–7]. Interestingly, immune activation in animals has similarly been able to recapitulate some of these phenotypes, including depressive and schizophrenia-like behaviors. Importantly, manifestation of such symptoms are prevented by clinically used and putative antidepressants [8, 9] and antipsychotic drugs [10] respectively.
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The kynurenine pathway is the primary tryptophan catabolic route, utilizing the rate-limiting enzymes, indoleamine 2,3 dioxygenase (IDO1) and tryptophan 2,3 dioxygenase (TDO2) to convert tryptophan to formyl-kynurenine that is further metabolized to kynurenine, a central component of the pathway. In addition, certain cytokines, i.e. interleukin (IL)-1b, IL-6 and interferon (IFN)-c have proven to induce the kynurenine pathway [3, 11, 12] see [13] for review. Notably, activation of the kynurenine pathway generates at least two neuroactive compounds, i.e. kynurenic acid (KYNA) and quinolinic acid (QUIN). The former, produced in astrocytes, acts as an N-Methyl-D-aspartate (NMDA) and a7 nicotinic acetylcholine receptor (a7nAChR) antagonist. QUIN is formed in the microglia associated branch of the kynurenine pathway and acts as an agonist at NMDA receptors containing the NR1 ? NR2A and the NR1 ? NR2B subunits. Importantly, the actions of QUIN have been associated with excitotoxicity [14, 15]. Both experimental and clinical data strongly point to an involvement of the kynurenine pathway in the induction of depressive- and psychotic-like behavior [16, 17]. In particular, acute or subchronic increased brain levels of KYNA in adulthood appear to specifically induce positive psychotic symptoms as well as impairments in executive functioning [18–23] whereas elevated brain KYNA levels in adolescence or sub-chronically during late prenatal development are associated with impaired social interaction, cognitive flexibility and long-term potentiation [24–29]. Animal experiments also show that activation of the microglia branch of the kynurenine pathway is associated with depressive-like behavior, such as reduced sucrose preference and increased immobility in the forced swim test [9, 30–32]. In line with the experimental findings, elevated cerebrospinal fluid (CSF) levels and post mortem levels of KYNA have been detected in first-episode as well as in patients with chronic schizophrenia [33–39] and in euthymic bipolar disorder patients with a history of psychosis [11, 40, 41]. CSF levels of QUIN are found to be normal in patients with chronic schizophrenia [33, 34]. On the other hand, increased levels of CSF QUIN, but not KYNA have been observed in suicide attempters [42, 43]. Interestingly, CSF QUIN associated with the degree of suicidal intent and the levels had returned to normal when patients came back for a follow-up visit. Increased CSF QUIN might thus be specifically related to the pathophysiology of suicidality [28]. Systemic administration of lipopolysaccharide (LPS) has been shown to induce an activated state of microglia in the central nervous system (CNS) [44]. The subsequent inflammatory effects of LPS activate cytokines, especially IL-1b, IFN-c and IL-6 through Toll-like receptor 4 signaling [45] and generate depressive-like behavior [46]. These effects are likely induced via activation of the kynurenine pathway since IDO inhibition prevents
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depressive-like behavior in mice receiving LPS [32, 47]. In addition, studies also indicate that LPS administration induces cognitive deficits [48–50] and anxiety [51] in mice. Systemic administration of LPS alters the blood brain barrier (BBB) transport of cytokines [52]. Studies suggest that repeated administration of LPS is more effective in altering these transport systems and thus induce a greater inflammatory response compared to a single injection of the drug [53, 54]. Moreover, it is reported that the cytokine/ chemokine profiles in the brain and serum are different following repeated injections of LPS [53] and that two injections of LPS, 16 h apart, induces mRNA expression of IL-12 p40, a cytokine that plays a central role in the regulation of cell-mediated immunity [55]. Previous studies have also shown that a single injection of LPS (0.83 mg/kg) activates the kynurenine pathway and induces depression-like behavior [9, 32, 56]. Although these studies have demonstrated interactions between the neuroinflammatory response, the activation of the kynurenine pathway and the induction of depressive-like behavior, it is still poorly understood to what extent repeated injections of LPS induces the synthesis of KYNA and QUIN, respectively. The aims of the present study were to test the hypothesis that repeated injections of LPS more effectively induce the synthesis of brain KYNA.
Materials and Methods Animals A total of 136 male C57BL6/N mice bred at the Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden, were used in the study. Food and water were available ad libitum. Animals were housed in groups of 4–7 individuals on a 12 h lights on/off cycle (lights on at 06:00 h). Temperature was maintained at 25 °C and humidity between 40 and 60 %. Mice weighed between 30 and 35 g upon entering the study and were weighed on daily basis post-LPS injections. Animals decreasing more than 15 % of body weight or showing excess signs of sickness were excluded from the study and sacrificed. At experiment termination, mice were sedated with isoflurane (&4 % in air) and euthanized by decapitation. Brains were harvested and quickly frozen and stored at -80 °C. Trunk blood was also collected and stored overnight in 4 °C. The following day serum was pipetted from blood samples and centrifuged for 15 min (15009g) and supernatant transferred to a new Eppendorf tube and stored at -80 °C. All efforts were made to minimize the number of animals used. Experiments were approved by and performed in accordance with the guidelines of the Ethical Committee of Northern Stockholm, Sweden.
Neurochem Res Sacrifice
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Fig. 1 Animals were given a single or repeated injection(s) of lipopolysaccharide (LPS) 16 h apart. Animals were sacrificed 24, 48, 72, or 120 h after the first injection
were also passed through a Shimadzu SPD-10A UV–VIS spectrometer (Shimadzu Corporation), set to a wavelength of 360 nm for kynurenine determination. A second mobile phase containing 0.5 M Zinc acetate in water was delivered post-columnar by a Pharmacia P-500 (GE Healthcare, Uppsala, Sweden) at a flow rate of 10 ml/ h. Signals from the detectors were transferred to a computer for analysis with Datalys Azur (Grenoble, France). Retention times for kynurenine and KYNA were 4 and 7 min, respectively.
Drugs and Treatment
Dopamine, 5-HT and Their Metabolites
LPS (Escherichia coli serotype O111:B4, Sigma-Aldrich, lot no.: 091M4031V) was prepared fresh daily in vehicle (sterile saline) and stored at 4 °C. Animals were divided into the following groups: saline ? saline (SAL ? SAL), LPS ? saline (LPS ? SAL), and LPS ? LPS. LPS (0.83 mg/kg or 2 9 0.83 mg/kg) or saline was injected i.p. There were 16 h between the first and second injection and mice were sacrificed 24, 48, 72, or 120 h after the first injection (Fig. 1). A group of mice received saline ? LPS (SAL ? LPS) to control for the time between the last LPS injection and the time of sacrifice in the LPS ? LPS group. Some mice were injected once with 1.66 mg/kg of LPS to control for total dosage.
Twenty ll of brain homogenates previously prepared for KYNA detection were neutralized and diluted by the addition of 160 ll dH2O and 20 ll NaOH. Samples were then manually injected into a reversed-phase HPLC system using a mobile phase containing 70 mM sodium acetate (pH 4.1, 20 % methanol) with 1.5 mM octanesulfonic acid and 0.01 mM Na2EDTA. The mobile phase was delivered by an HPLC pump (LC-20AD, Shimadzu Corporation) through a C18 column (4.6 9 150 mm, ZORBAX Eclipse XDB-C18, Agilent Technologies, CA, USA) at a rate of 0.68 ml/min. Samples were quantified electrochemically by sequential oxidation and reduction in a high-sensitivity analytical cell (ESA 5011; ESA Inc., Chelmsford, MA, USA) controlled by a potentiostat (Coulochem III; ESA Inc.) with an applied potential of 500 mV for detection of dopamine, DOPAC, HVA, 5-HIAA, and 5-HT. The signals from the detector were transferred to a computer for analysis (Clarity, DataApex, Prague, The Czech Republic). Retention times in minutes: dopamine (6), DOPAC (4), HVA (7), 5-HT (11.5), 5-HIAA (5.5).
High-Performance Liquid Chromatography Kynurenic Acid and Kynurenine At the day of analysis, one hemisphere of brain tissue was placed in threefold (w/v) and serum one-fold (v/v) of 0.4 M PCA (containing 0.1 % sodium metabisulfite, 0.05 % EDTA). Brains were thereafter homogenized using a disperser (Ultra-TurraxÒ, IKA, Stauffen, Germany). Brain homogenates and serum were then centrifuged at 21,0009g for 5 min and 10 volume percent of 70 % strength pure PCA was added to the supernatant. This solution was then re-centrifuged (21,0009g, 5 min), and the supernatant transferred to a new Eppendorf tube for analysis. KYNA concentration was determined with an isocratic reversed-phase high pressure liquid chromatography (HPLC) system, coupled to a fluorescence detector (FP2020 Plus, Jasco Ltd., Hachioji City, Japan). The detector was set to an excitation wavelength of 344 nm and an emission wavelength of 398 nm (18 nm bandwidth). A mobile phase consisting of 50 mM sodium acetate and 7 % acetonitrile (pH set to 6.2 using acetic acid) was pumped through a ReproSil-Pur C18 column (4 9 150 mm, Dr. Maisch GmbH, Ammerbuch, Germany). A LC-10AD VP (Shimadzu Corporation, Kyoto, Japan) delivered the mobile phase at a flow rate of 0.5 ml/min. Serum samples
Liquid Chromatography/Mass Spectrometry Quinolinic Acid and Tryptophan Brain samples (one hemisphere) was weighed, sonicated in 600 ll PBS buffer and ultracentrifuged at 4 °C, 30,0009g. The supernatant (50 ll) was added internal standard in 5 % formic acid and filtered at 30009g for 60 min at 10 °C using 10 kDa UltracelÒ-10 filter plates from Millipore. After centrifugation, 7.5 ll of the filtrate was injected using a Waters Acquity HPLC system equipped with a SymmetryShieldTM RP18 2.1 9 100 mm, 3.5 lm particle column. Mobile phase was run at 300 ll/ min and consisted of 2.1 % formic acid in MilliQ water (A phase) and 95 % acetonitrile 0.1 % formic acid (B phase). Runs started with 5 % B in A for 2 min following gradient elution, with a total run time of 10 min. Retention times for QUIN and tryptophan were 1.1 and 3.5 min, respectively. Plasma (50 ll) was prepared using solid phase extraction (OasisÒ MAX). Cartridges were equilibrated, added
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samples and internal standard in 5 % ammonium hydroxide, washed, and analytes were eluted (60 % acetonitrile, 38 % methanol and 2 % formic acid). The organic phase was evaporated using nitrogen and analytes were redissolved in 2.5 % formic acid. After solid phase extraction, 7.5 ll of the extract was injected using a Waters Acquity HPLC system equipped with an HSST3 2.1 9 100 mm, 1.8 lm particle column. The mobile phase was run at 300 ll/minute and consisted of 2.1 % formic acid in MilliQ water (A phase) and 95 % acetonitrile 0.1 % formic acid (B phase). Runs started with 2 % B in A for 2 min followed by gradient elution, with a total run time of 15 min. Retention times for QUIN and tryptophan were 1.5 and 5.9 min, respectively. Reagents and Quantification The detection was performed using a Waters Xevo TQ-S triple quadrupole mass spectrometer operating in positive ionization MS/MS configuration. The mass spectrometer was tuned for all analytes and the mass spectral transitions were set at m/z 168 [ 106 (QUIN) and 205 [ 91 (tryptophan), 172 [ 110 (13C15 3 N1QUIN) 210 [ 150 (D5-tryptophan). QUIN and tryptophan were purchased from Sigma-Aldrich,13C15 3 N1-QUIN from Synfine research Inc., formic acid, methanol and acetonitrile were purchased as MS-grade from Sigma-Aldrich. Standards of each analyte were used to establish a linear calibration curve and plotted using the ratio of analyte peak area over internal standard peak area after integration by Masslynx 4.1 software (Waters Corporation). Statistics Data are presented as mean ± SEM. p values B0.05 were considered significant. Data were analyzed using one-way or two-way ANOVA with post hoc Bonferroni to determine differences over time and treatments.
Results Based on previously published data, LPS was administered in a dose of 0.83 mg/kg per injection [9, 32, 56]. Initial experiments confirmed that this dose of LPS produced a robust and reproducible sickness behavior response accompanied with weight loss. Brain Kynurenine Significant effects of both treatment and time were observed for levels of brain kynurenine (Fig. 2a, effect of treatment: F(3,91) = 34.44, p \ 0.0001; effect of time:
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F(3,91) = 16.80, p \ 0.0001; interaction: F(9,91) = 7.106, p \ 0.0001). Both LPS ? LPS and LPS ? SAL significantly elevated brain kynurenine at 24 h post-injection (p \ 0.0001 and p = 0.02, respectively, post hoc Bonferroni). This significant increase was maintained at 48 h post-LPS in the LPS ? LPS (p \ 0.0001) condition but returned to control levels by 72 h (p = 0.39). The double dose of LPS significantly elevated kynurenine compared to a single injection at both 24 h (p \ 0.0001 SAL ? LPS and p \ 0.0001 LPS ? SAL vs. LPS ? LPS, post hoc Bonferroni) and 48 h (p = 0.003 SAL ? LPS and p = 0.004 LPS ? SAL vs. LPS ? LPS, post hoc Bonferroni). Also notable was that the high elevation of kynurenine by LPS ? LPS at 24 h significantly decreased over time (p \ 0.0001 at 48, 72 and 120 h compared to 24 h timepoint, post hoc Bonferroni). Serum Kynurenine A similar trend was seen in serum kynurenine as in brain kynurenine, however, serum kynurenine levels were elevated by the LPS treatments for a longer period of time (Fig. 2b, effect of treatment: F(3,89) = 19.83, p \ 0.0001; effect of time: F(3,89) = 36.06, p \ 0.0001; interaction: F(9,89) = 7.106, p = 0.01). Significantly elevated levels of kynurenine were seen in LPS ? LPS and LPS ? SAL conditions at 24 h (p \ 0.0001 and p = 0.002, respectively, post hoc Bonferroni) and 48 h (p \ 0.0001 and p \ 0.0001, respectively, post hoc Bonferroni) while LPS ? LPS caused serum kynurenine to be significantly elevated until 72 h post-injection (p = 0.02, post hoc Bonferroni). Furthermore, at 48 h, a significant increase in serum kynurenine was also seen in the SAL ? LPS condition (p \ 0.0001, post hoc Bonferroni). Normalization of kynurenine levels began at 72 h for the single dose condition LPS ? SAL (as seen by the lack of significant difference from the SAL ? SAL condition and a significant change from the levels at 24 h, p = 0.03 at 72 h and p \ 0.0001 at 120 h) and at 120 h for the double dose (LPS ? LPS condition, p \ 0.0001 at 120 h). In contrast to kynurenine levels in the brain where a double dose of LPS increased kynurenine levels much more than a single dose, in the periphery, single doses of LPS were able to increase kynurenine as robustly as a double dose. Brain KYNA Both treatment and time significantly contributed to the detected changes in brain KYNA levels (Fig. 2c, effect of treatment: F(3,89) = 11.65, p \ 0.0001; effect of time: F(3,89) = 7.912, p \ 0.0001; interaction: F(9,89) = 4.718, p \ 0.0001). Significantly elevated levels of KYNA were only incurred following the double dose of LPS
Neurochem Res
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Fig. 2 Kynurenine (a, b), Kynurenic acid (KYNA, c, d), Quinolinic acid (QUIN, e, f), and tryptophan (g, h) levels in mouse brain (a, c, e, g) and serum (b, d, f, h) after single (0.83 mg/kg) or repeated (2 9 0.83 mg/kg) LPS injection. Mice were sacrificed up to 120 h after the first injection. n = 5–8 in each group. Levels are mean ± SEM. Two-way ANOVA, post hoc Bonferroni. ****p \ 0.0001, ***p \ 0.001, **p \ 0.01, *p B 0.05 versus SAL ? SAL; ????p \ 0.0001, ??? p \ 0.001, ??p \ 0.01, ? p B 0.05 versus LPS ? LPS; ## p \ 0.01, #p B 0.05 versus LPS ? SAL; °°°°p \ 0.0001, °°° p \ 0.001, °°p \ 0.01, ° p B 0.05 versus 24 h
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Neurochem Res 8
treatment (p = 0.02 and p = 0.001, respectively). In general, LPS administration did not seem to have much impact on serum KYNA levels, especially in comparison to the effects seen on serum kynurenine levels. This suggests a strong homeostatic drive to control levels of this particular kynurenine pathway metabolite in the periphery.
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Fig. 3 Kynurenic acid (KYNA) levels in mouse brain after single (0.83 mg/kg OR 1.66 mg/kg) or repeated (2 9 0.83 mg/kg) LPS injection. Mice were sacrificed 24 h after the first injection. n = 3–8 in each group. Levels are mean ± SEM. One-way ANOVA post hoc Bonferroni. ****p \ 0.0001
(LPS ? LPS) and remained elevated at 24 h and 48 h (p \ 0.0001 and p = 0.02, respectively). Although KYNA levels in the LPS ? LPS condition were still higher than control levels at 48 h, they were significantly lower than what was observed for the LPS ? LPS condition at 24 h (p \ 0.0001) suggesting KYNA levels were reduced rather quickly following the double dose of LPS. Furthermore, the double dose of LPS significantly elevated brain KYNA compared to a single dose at 24 h (p \ 0.0001 SAL ? LPS and p \ 0.0001 LPS ? SAL vs. LPS ? LPS, post hoc Bonferroni). This increase in brain KYNA by LPS ? LPS likely relied on the repeated injection and not the total dosage as a separate group of mice received 1.66 mg/kg LPS in a single injection 24 h prior to sacrifice and did not show a significant increase in brain KYNA when compared to saline injected animals (Fig. 3, F(3,21) = 13.67, p \ 0.0001; post hoc Bonferroni p = 0.15). Serum KYNA In contrast to brain KYNA levels, where significant effects of time post-injection, of treatment type and of an interaction between time and treatment were observed, the levels of serum KYNA show no such effect of time post-injection and modest effect of treatment and interaction of time and treatment (Fig. 2d, effect of treatment: F(3,89) = 3.376, p = 0.02; effect of time: F(3,89) = 2.134, p = 0.10; interaction: F(9,89) = 2.405, p = 0.02). Also contrary to the situation in the brain, in the periphery, KYNA was significantly reduced in the LPS ? LPS condition compared to control at 24 h (p = 0.009). Here, a time effect of the second injection was observed as the SAL ? LPS and LPS ? LPS conditions both show reduced KYNA compared to the LPS ? SAL
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As with brain kynurenine and brain KYNA, significant effects of treatment and time were seen on brain QUIN levels (Fig. 2e, effect of treatment: F(3,88) = 28.55, p = 0.0001; effect of time: F(3,88) = 41.92, p \ 0.0001; interaction: F(9,88) = 11.31, p \ 0.0001). There was a significant increase in QUIN levels at 24 h following LPS ? SAL and LPS ? LPS (p = 0.005 and p \ 0.0001, respectively). This elevation was still seen at 48 h in the LPS ? LPS condition (p \ 0.0001) although the QUIN levels had begun falling off (p \ 0.0001 at all timepoints compared to levels at 24 h). There was a strong effect of the double dose compared to the single dose conditions at both 24 h (p \ 0.0001 for both SAL ? LPS and LPS ? SAL versus LPS ? LPS) and 48 h (p = 0.04 for SAL ? LPS and p = 0.003 for LPS ? SAL versus LPS ? LPS). Serum QUIN Similar to serum KYNA levels, serum QUIN levels showed a certain robustness against changes as there was only a trend towards significance in the effect of the treatment (Fig. 2f, effect of treatment: F(3,84) = 2.530, p = 0.06; effect of time: F(3,84) = 9.568, p \ 0.0001; interaction: F(9,84) = 3.175, p = 0.002). A significant decrease in QUIN was observed at 24 h in the SAL ? LPS condition (p = 0.0006). However, QUIN levels significantly varied across time following injection in the control SAL ? SAL condition (p = 0.02 at 48 h, p = 0.0002 at 72 h and p = 0.02 at 120 h compared to levels at 24 h) with few differences in the effect of a single or double dose of LPS. Together, this pattern suggests serum levels of QUIN or serum levels of KYNA are not good indicators of brain levels of these metabolites. Brain Tryptophan Significant effects of treatment and time were observed on levels of brain tryptophan (Fig. 2g, effect of treatment: F(3,91) = 17.21, p \ 0.0001; effect of time: F(3,91) = 29.94, p \ 0.0001; interaction: F(9,91) = 3.491, p = 0.001). Brain tryptophan was significantly increased across all treatments at the 24 h timepoint (p \ 0.0001 SAL ? LPS, p = 0.05 LPS ? SAL, p \ 0.0001 LPS ? LPS). Here, we observed an effect of the second LPS injection as tryptophan was significantly increased in the LPS ? LPS condition and a
Neurochem Res
Brain QUIN:KYNA
As with brain tryptophan levels, significant effects of treatment and time were observed on levels of serum tryptophan (Fig. 2h, effect of treatment: F(3,85) = 9.089, p \ 0.0001; effect of time: F(3,85) = 13.55 p \ 0.0001; interaction: F(9,85) = 4.370, p = 0.0001). Although the SAL ? LPS and LPS ? LPS treatments significantly reduced serum tryptophan compared to LPS ? SAL (p = 0.005 and p = 0.001, respectively), these were not significantly different from control values although the double dose showed a trend towards a significant reduction (p = 0.08). Perhaps, as with brain tryptophan, the changes compared to the LPS ? SAL condition were related to the second injection of LPS. There was some variation across time, noticeably at the 120 h timepoint for SAL ? SAL and LPS ? SAL, however the only significant change versus control conditions was a decrease in the LPS ? LPS condition at 72 h following administration (p \ 0.0001). Brain QUIN:KYNA Ratio Unlike the robust changes seen in brain QUIN and brain KYNA following LPS administration, the QUIN:KYNA ratio was more resilient with a significant effect of time being apparent but not of treatment nor an interaction of treatment with time (Fig. 4, effect of treatment: F(3,87) = 1.181, p = 0.32; effect of time: F(3,87) = 7.663, p = 0.0001; interaction: F(9,87) = 1.694, p = 0.10). Although QUIN was quite elevated at 24 and 48 h in some conditions, the levels of KYNA also increased and thus no significant changes were seen in the QUIN:KYNA ratio between treatment conditions at these timepoints. An increase in the ratio was seen at 72 h in the SAL ? SAL condition compared to 24 h (p = 0.02). A significant decrease in the QUIN:KYNA ratio of the SAL ? LPS condition was also seen at 72 h (p = 0.02) and was likely due to increased KYNA levels. In general, the QUIN:KYNA ratio seems quite stable despite the administration of a double dose of LPS. This suggests there is a homeostatic balance between the neurotoxic and neuroprotective arms of the kynurenine pathway upon LPS challenge.
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trend towards increase was seen in SAL ? LPS over the LPS ? SAL condition (p = 0.01 and p = 0.07 respectively). At 48 h, the double dose had significantly raised tryptophan over the single dose conditions (p = 0.0007 SAL ? LPS and p = 0.01 LPS ? SAL versus LPS ? LPS). The drop back to control levels of tryptophan was noticeable in the SAL ? LPS condition by 48 h following the first injection (p = 0.003 at 48 h, p = 0.0002 at 72 h and p \ 0.0001 at 120 h), while in the LPS ? LPS condition, the tryptophan elevation remained high at 48 h (p \ 0.0001) and then decreased to control levels by 72 h.
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Time after 1st injection
Fig. 4 Brain quinolinic acid (QUIN):kynurenic acid (KYNA) ratio ratio after a single (0.83 mg/kg) or a repeated (2 9 0.83 mg/kg) LPS injection. Mice were sacrificed up to 120 h after the first injection. n = 5–8 in each group. Levels are mean ± SEM. Two-way ANOVA, post hoc Bonferroni. *p B 0.05 versus SAL ? SAL; °p B 0.05 versus 24 h
Brain Kynurenine:Tryptophan Ratio Significant effects of treatment and time were seen in the brain kynurenine:tryptophan ratio (Fig. 5a, effect of treatment: F(3,91) = 40.72, p \ 0.0001; effect of time: F(3,91) = 13.76, p \ 0.0001; interaction: F(9,91) = 7.165, p \ 0.0001). At 24 h post-injection both the LPS ? SAL and LPS ? LPS conditions showed a significantly increased ratio of brain kynurenine:tryptophan (p = 0.003 and p \ 0.0001, respectively) and by 48 h, all single doses and the double dose had increased the kynurenine:tryptophan ratio over controls (p = 0.02 SAL ? LPS, p = 0.04 LPS ? SAL, p \ 0.0001 LPS ? LPS). Of note was the significant increase of the kynurenine:tryptophan levels of the double dose compared to the single dose conditions at both 24 h (p \ 0.0001 SAL ? LPS and p \ 0.0001 LPS ? SAL) and at 48 h (p = 0.03 SAL ? LPS and p = 0.02 LPS ? SAL). The kynurenine:tryptophan ratio showed a decline from its initial spike in the LPS ? LPS condition (p \ 0.0001 at 48, 72 and 120 h compared to levels at 24 h) and showed no significant changes from control as of 72 h. Serum Kynurenine:Tryptophan Ratio As with brain, significant effects of treatment and time were seen in the serum kynurenine:tryptophan ratio (Fig. 5b, effect of treatment: F(3,83) = 25.94, p \ 0.0001; effect of time: F(3,83) = 11.98, p \ 0.0001; interaction: F(9,83) = 6.756, p \ 0.0001). At 24 h, only the double dose showed an elevated serum kynurenine:tryptophan ratio (p = 0.003). At 48 h, both the single dose and double dose showed elevated
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Neurochem Res
0.01
0.06
**
**** *+ *
****
0.08
KYN/TRP
**** ** ++++ ++++
KYN/TRP
0.02
0.10
SAL+SAL SAL+LPS LPS+SAL LPS+LPS
**
0.04
0.03
Serum KYN:TRP
B
0.04
**
++++ ++++
Brain KYN:TRP
A
48 h
72 h
+ 0.02
0.00
0.00 24 h
48 h
72 h
120 h
24 h
Time after 1st injection
120 h
Time after 1st injection
Fig. 5 Brain (a) and serum (b) kynurenine (KYN):tryptophan (TRP) ratio after a single (0.83 mg/kg) or a repeated (2 9 0.83 mg/kg) LPS injection. Mice were sacrificed up to 120 h after the first injection. n = 5–8 in each group. Levels are mean ± SEM. Two-way ANOVA,
post hoc Bonferroni. ****p \ 0.0001, **p \ 0.01, *p B 0.05 versus SAL ? SAL; ????p \ 0.0001, ?p B 0.05 versus LPS ? LPS; °°°° p \ 0.0001, °°°p \ 0.001 versus 24 h
kynurenine:tryptophan levels (p = 0.01 SAL ? LPS, p = 0.05 LPS ? SAL, p = 0.002 LPS ? LPS). However, in contrast with the brain, the double dose did not significantly elevate the kynurenine:tryptophan ratio compared to the single dose at these timepoints. At 72 h, we did see a pronounced increase of the kynurenine:tryptophan ratio in the LPS ? LPS condition compared to all other conditions (p \ 0.0001 SAL ? SAL, p \ 0.0001 SAL ? LPS, p \ 0.0001 LPS ? SAL) but this was likely due to a large decrease in serum tryptophan levels at this time (Fig. 2h).
Brain DOPAC:Dopamine and HVA:Dopamine Ratios
Brain 5-HIAA:5-HT Ratio Both levels of 5-HT and its metabolite 5-HIAA were quite variable across time and treatment conditions (data not shown). We thus looked at the ratio of 5-HIAA:5-HT which proved to be more stable and which therefore allows us to make more meaningful conclusions as to the effect of LPS on serotonergic turnover. Significant effects of time, treatment and an interaction between time and treatment were observed (Fig. 6a, effect of treatment: F(3,89) = 13.08, p \ 0.0001; effect of time: F(3,89) = 38.04, p \ 0.0001; interaction: F(9,89) = 5.539, p \ 0.0001). Treatment with LPS significantly elevated the 5-HIAA:5HT ratio above control levels at 24 h across all treatment conditions (p \ 0.0001 SAL ? LPS, p \ 0.0001 LPS ? SAL, p \ 0.0001 LPS ? LPS). This remained significantly elevated by the double dose at 48 h (p \ 0.0001) but not by the single doses, which were comparable to the levels in the SAL ? SAL condition. The ratio then quickly returned to normal, by 48 h for the single doses and 72 h for the double dose. Thus, the double dose of LPS more effectively increased the 5HIAA:5-HT ratio than the single doses of LPS.
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Dopaminergic turnover was investigated by calculating the DOPAC:Dopamine and the HVA:Dopamine ratios. Only an effect of time was observed on the DOPAC:Dopamine ratio (Fig. 6b, effect of treatment: F(3,90) = 1.249, p = 0.30; effect of time: F(3,90) = 38.04, p = 0.0001; interaction: F(9,90) = 0.2399, p = 0.99) with no significant post hoc differences. In contrast, significant effects of treatment and time were seen on the HVA:Dopamine ratio (Fig. 6c, effect of treatment: F(3,90) = 15.51, p \ 0.0001; effect of time: F(3,90) = 77.10, p \ 0.0001; interaction: F(9,90) = 6.457, p \ 0.0001). A significant increase in HVA:Dopamine was caused by both the single and repeated LPS injections at 24 h (p \ 0.0001 SAL ? LPS, p \ 0.0001 LPS ? SAL, p \ 0.0001 LPS ? LPS) with levels remaining significantly elevated at 48 h in the LPS ? LPS condition compared to both control (p \ 0.0001) and single dose conditions (p = 0.006 SAL ? LPS and p = 0.0003 LPS ? SAL). The HVA levels rapidly declined following this initial rise by 48 h for the single dose and 72 h for the double dose. As levels of dopamine were not significantly affected by treatment with LPS (data not shown), this selective increase in the HVA:Dopamine ratio without changes in DOPAC may suggest an increase in COMT activity following LPS administration.
Discussion In the present study, we show that repeated injections of LPS, when compared to a single injection, result in a robust induction of the brain kynurenine pathway. Furthermore,
Neurochem Res
A
B
Brain DOPAC:Dopamine
Brain 5-HIAA:5-HT
****
0.25
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DOPAC/Dopamine
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++++ ++
0.75
**** **** ****
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Fig. 6 Brain 5-HT:5-HIAA ratio (a), DOPAC:Dopamine ratio (b), and HVA:Dopamine ratio (c) after a single (0.83 mg/ kg) or a repeated (2 9 0.83 mg/ kg) LPS injection. Mice were sacrificed up to 120 h after the first injection. n = 5–8 in each group. Levels are mean ± SEM. Two-way ANOVA, post hoc Bonferroni. ****p \ 0.0001, *p B 0.05 versus SAL ? SAL; ???? p \ 0.0001, ??? p \ 0.001, ??p \ 0.01, versus LPS ? LPS; ##p \ 0.01, # p B 0.05 versus LPS ? SAL; °°°° p \ 0.0001, °°°p \ 0.001, °° p \ 0.01, °p B 0.05 versus 24 h
0.15
0.10
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0.00 24 h
48 h
72 h
120 h
Time after 1st injection
24 h
48 h
72 h
120 h
Time after 1st injection
C Brain HVA:Dopamine
0.2
*
****
++ +++
0.3
**** **** ****
HVA/Dopamine
0.4
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0.0 24 h
48 h
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Time after 1st injection
the profile of tryptophan and its metabolites following LPS exposure clearly differs between brain and serum. Repeated injections of LPS induced a prolonged increase in brain and serum kynurenine, increased brain KYNA and increased QUIN levels compared to single injections. These findings are in a line with a study investigating the brain and serum cytokine and chemokine response following a single or repeated administration of LPS [53]. In that study, it was found that repeated LPS administration (3 mg/kg per injection) produced a larger brain immune response compared to a single injection and that the cytokine/chemokine profile differed between brain and serum. Interestingly, repeated injections of LPS clearly reduced serum KYNA levels but did not affect serum QUIN levels. The fact that brain KYNA is increased while serum KYNA is decreased is interesting with regard to schizophrenia where it is shown that brain levels of KYNA are higher in patients (c.f. Introduction) and seems to be lowered in serum compared to controls [57]. Indeed, in patients with bipolar disorder we have previously found that CSF KYNA levels are increased in patients with a history of psychosis [11, 40, 41]. Our recent data do however show that the plasma levels of KYNA, from the same individuals, is not
altered but rather found to be decreased in bipolar patients without a history of psychosis (manuscript in preparation). The underlying reason for this discrepancy between CSF and plasma is at present unknown. The proposed mechanisms by which LPS influences brain immune activation are numerous and include e.g. activation of vagal or other afferents impinging upon the brain [58, 59] effects on the BBB [60], or the circumventricular organs [61]. Although previous studies have shown that LPS exposure is associated with a disruption of the BBB, LPS has been shown to enter the brain only in trace amounts [53]. Thus, the presently observed LPS-induced activation of the kynurenine pathway, including elevation of brain kynurenine, KYNA, and QUIN, or its influence on monoamine neurotransmission, appears primarily executed peripherally. An increase in serum kynurenine per se may be sufficient to explain the activation of the brain kynurenine pathway. Indeed, kynurenine easily crosses the BBB and may be a major source of astrocytic KYNA production (see [16]). However, in the present study, a repeated injection clearly potentiated brain kynurenine in contrast to serum kynurenine. Thus, elevated serum kynurenine appears not to account solely for the marked activation of
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brain kynurenine pathway following a repeated injection of LPS. The role of a possible change in tryptophan transport across the blood BBB is also unclear. In the present study, LPS was given in a dose that has previously been shown to induce the full spectrum of the acute sickness response [62] and depressive-like behavior [32]. The molecular mechanism responsible for LPS-induced activation of the kynurenine pathway might be associated with the release of both central and peripheral proinflammatory cytokines, including IL-1b and IL-6 [53]. Further, LPS induces pro-IL-1b transcription, e.g. in microglia [63], for review see [64], thus enabling caspase-1 dependent IL-1b release via P2X7-receptor activation [65]. Indeed, IL-1b and IL-6 were recently shown to induce the kynurenine pathway [11]. In addition, this dose is associated with activation of brain microglia, [44] and a solid induction of brain IDO1 [56, 66]. Whether lower doses of LPS also induce IDO1 and at the same time produce aberrant behavior that can be reversed by a blocker of IDO1 remain to be investigated. The increase in brain QUIN following a repeated injection of LPS was found to be in the same magnitude as that of KYNA, as can be seen in the QUIN:KYNA ratio. QUIN and KYNA oppositely influence NMDA receptors and the balance between the two compounds is suggested to be of importance for the neurodegenerative effects of QUIN. Systemic inflammation evoked by LPS is shown to induce apoptosis and cell death [67, 68]. Although the mechanism behind the influence of LPS on brain monoaminergic neurotransmission is relatively unknown, our data show an increase in brain tryptophan, increased turnover of 5-HT, and increased turnover of dopamine leading to an increase in the HVA:Dopamine ratio. It has previously been suggested that the increase in brain tryptophan following LPS treatment is related to increased levels of free tryptophan via a liberation of the amino acid from albumin [69, 70]. The elevation in brain HVA is in line with a recent microdialysis studies on dopamine metabolites following a single LPS injection [71]. The increase in the HVA:Dopamine ratio but not the DOPAC:Dopamine ratio suggests increased COMT activity and indeed, microinfusion of LPS has been shown to increase COMT [72]. Further, whereas brain 5-HT or 5-HIAA levels were too variable across time and treatments to allow any reliable interpretations of the effects of LPS on these individual metabolites, their ratio (5-HIAA:5HT), which proved much more stable, was significantly increased, as in the study of O’Connor [32]. The present study emphasizes that a repeated exposure to LPS compared to a single injection of the drug is a more effective way to induce the kynurenine pathway. This study also points to huge differences in the regulation of the kynurenine pathway in the periphery versus the brain.
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Although more studies are needed, this finding might be of relevance for the pathophysiology of schizophrenia and may thus contribute to our understanding of this disorder. In addition, given the role of elevated KYNA in psychotic disorders and in cognitive deficits, repeated LPS exposure may be a valuable strategy for inducing such conditions. Future studies are needed to investigate if repeated administration can induce cognitive deficits and aberrant behavior of relevance for psychotic disorders. Acknowledgments This work was supported by grants from the Swedish Medical Research Council (2009-7053; 2013-2838), the Swedish Brain foundation, Petrus och Augusta Hedlunds Stiftelse, Torsten So¨derbergs Stiftelse, the Mayo Clinic—Karolinska Institutet Collaborative Research Grants, the AstraZeneca-Karolinska Institutet Joint Research Program in Translational Science and the Karolinska Institutet (KID). Authors’ contributions M.L., and S.E. collected and analyzed data, contributed to discussion, and wrote, reviewed, and edited the manuscript. F.O., XC.L., D.S.C. L.S., G.E., M.G., M.B., S.S., K.S. and A.O. researched data and reviewed and critically revised the manuscript. A.F. and S.I analyzed data, wrote, reviewed and edited the manuscript. S.E. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Compliance with Ethical Standards Conflict of interest None.
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