Kynurenine metabolic balance is disrupted in the hippocampus following peripheral lipopolysaccharide challenge
© The Author(s). 2016
Received: 4 March 2016
Accepted: 20 May 2016
Published: 27 May 2016
Inflammation increases the risk of developing depression-related symptoms, and tryptophan metabolism is an important mediator of these behavior changes. Peripheral immune activation results in central up-regulation of pro-inflammatory cytokine expression, microglia activation, and the production of neurotoxic kynurenine metabolites. The neuroinflammatory and kynurenine metabolic response to peripheral immune activation has been largely characterized at the whole brain level. It is unknown if this metabolic response exhibits regional specificity even though the unique indoleamine 2,3-dioxygenase (IDO)-dependent depressive-like behaviors are known to be controlled by discrete brain regions. Therefore, regional characterization of neuroinflammation and kynurenine metabolism might allow for better understanding of the potential mechanisms that mediate inflammation-associated behavior changes.
Following peripheral immune challenge with lipopolysaccharide (LPS), brain tissue from behaviorally relevant regions was analyzed for changes in mRNA of neuroinflammatory targets and kynurenine pathway enzymes. The metabolic balance of the kynurenine pathway was also determined in the peripheral circulation and these brain regions.
Peripheral LPS treatment resulted in region-independent up-regulation of brain expression of pro-inflammatory cytokines and glial cellular markers indicative of a neuroinflammatory response. The expression of kynurenine pathway enzymes was also largely region-independent. While the kynurenine/tryptophan ratio was elevated significantly in both the plasma and in each brain regions evaluated, the balance of kynurenine metabolism was skewed toward production of neurotoxic metabolites in the hippocampus.
The upstream neuroinflammatory processes, such as pro-inflammatory cytokine production, glial cell activation, and kynurenine production, may be similar throughout the brain. However, it appears that the balance of downstream kynurenine metabolism is a tightly regulated brain region-dependent process.
KeywordsNeuroinflammation Kynurenine Pro-inflammatory cytokines Brain regions Microglia Indoleamine 2,3-dioxygenase Kynurenine 3-monooxygenase Hippocampus
The behavioral consequences of inflammation and pro-inflammatory cytokines have been well described, both clinically and preclinically. Chronic diseases, often characterized by prolonged immune activation, are associated with an increased risk of comorbid depression diagnosis . Approximately 50–80 % of patients receiving interferon-α (IFN-α) immunotherapy for hepatitis C or malignant melanoma develop depression-related symptoms during the course of treatment, including neurovegetative, affective, and/or cognitive impairment . Administration of endotoxin or vaccines to healthy volunteers induces transient depression-associated symptoms that parallel the appearance of pro-inflammatory cytokines [3–6], whereas direct administration of pro-inflammatory cytokines in preclinical models recapitulates inflammation-associated depressive-like behaviors [7–10]. Pharmacological or genetic inhibition of pro-inflammatory cytokine action effectively attenuates many of the inflammation-induced depressive-like behaviors in rodents, establishing that pro-inflammatory cytokines are necessary to precipitate this behavioral response [11, 12]. Clearly, pro-inflammatory cytokines are important pathogenic mediators of depression-related behavior changes resulting from immune stimulation; however, the specific mechanism by which this occurs is not clearly defined.
Depression symptomatology is heterogeneous throughout the patient population, complicating research efforts targeted at understanding the neurobiological substrates underlying the disease. Because of this, the National Institute of Mental Health (NIMH) established the Research Domain Criteria (RDoC, http://www.nimh.nih.gov/research-priorities/rdoc/index.shtml) as a construct for mental health research [29, 30]. Rather than defining patient populations by a disease diagnosis, the RDoC instead prompts investigators to focus on understanding the mechanism that underlies specific symptoms or behaviors. In healthy volunteers, endotoxin administration results in anhedonia, disruption in cognitive performance, anxiety, and mood disturbances [31, 32]. These endotoxin-associated symptoms have been associated with altered blood flow (imaged using functional magnetic resonance imaging (fMRI)) in the ventral striatum, amygdala, and dorsomedial prefrontal cortex among other regions [5, 33, 34]. Similar to human patients, lipopolysaccharide (LPS, endotoxin) challenge in mice results in similar behaviors (anhedonia, anxiety, behavioral despair, cognitive disruption) [19, 25, 26]. Determining the relevant regional neuroinflammatory response, particularly kynurenine metabolism, following peripheral immune activation with LPS will lead to a better understanding of the development of these behaviors.
While the region-specific expression of pro-inflammatory cytokines after peripheral immune challenge has been partially characterized [17, 35], the impact of peripheral LPS challenge on kynurenine pathway enzyme expression and subsequent metabolism has only been described at the whole brain level . Here, we tested the hypothesis that up-regulation of kynurenine pathway enzymes and disruption of kynurenine metabolic balance occurs in a regionally distinct manner. Brain regions associated with depressive-like behaviors were collected following peripheral immune challenge with LPS in mice. The steady-state mRNA expression of markers of glial activation, pro-inflammatory cytokines, and kynurenine pathway enzymes were measured. Tryptophan and its metabolites were also measured in each brain region. The brain regions assessed included the hippocampus, the amygdala, and the ventral striatum (nucleus accumbens). Dysfunction in these brain regions has been reported in depressed patients and is hypothesized to be a critical pathogenic mechanism underlying distinct depression-associated behaviors . Because the periphery is reported to provide much of the brain’s kynurenine , plasma metabolite levels were also measured. The data demonstrate that peripheral LPS treatment elevates the expression of pro-inflammatory cytokines and glial cell markers similarly in each region. IDO expression, but not IDO2 or tryptophan 2,3-dioxygenase (TDO), was significantly up-regulated after LPS treatment. Consistent with IDO up-regulation, the kynurenine/tryptophan ratio was elevated in each region; however, the 3-hydroxykynurenine (3-HK)/KA ratio, an indicator of a neurotoxic imbalance in kynurenine metabolism, was only increased in the hippocampus (dorsal and ventral). These data are the first that characterize the brain region-specific metabolic response of the kynurenine pathway following peripheral LPS challenge.
All animal care and use was conducted in accord with the Guide for the Care and Use of Laboratory Animals, eighth edition (NRC), and protocols were approved by the Institutional Animal Care and Use Committee at the University of Texas Health Science Center at San Antonio (UTHSCSA). Male C57BL/6J mice were obtained from Jackson Laboratory (Bar Harbor, ME; stock# 000664) and were used at ages between 8 and 12 weeks of age (adults). Prior to use, mice were group housed in standard shoebox cages, allowed ad libitum food and water access, and general health was monitored daily by veterinary technicians or research staff.
Lipopolysaccharide (Sigma, St. Louis, MO) isolated from Escherichia coli (L-3129, serotype 0127:B8) was prepared fresh on the morning of injections and was dissolved in sterile, endotoxin-free 0.9 % saline (vehicle) and injected intraperitoneally (i.p.) at a dose of 0.5 mg/kg.
Tissue sample collection
At either 6 or 24 h following saline or LPS injections, mice were euthanized by carbon-dioxide asphyxiation. To measure mRNA expression of target genes, brain regions (amygdala, hippocampus, striatum) were grossly dissected (based on stereological coordinates in a mouse brain atlas ). Subsequent analysis of brain region metabolites was performed on microdissected brain regions harvested from serial 1-mm coronal brain sections using a brain matrix (Stoelting Co., Wood Dale, IL) (based on stereological coordinates in a mouse brain atlas ). Tissue was snap frozen in liquid nitrogen and stored at −80 °C until analyzed. At 24 h following injections, prior to perfusion, blood was also collected for separation of plasma, which was stored at −80 °C.
RNA isolation and real-time RT-PCR
Brain tissues (hippocampus, amygdala, striatum) collected at 6 and 24 h post treatment (saline, LPS) were analyzed to determine relative steady-state mRNA expression using real-time RT-PCR. Isolation of RNA was carried out according manufacturer instructions using a PureLink® RNA Mini Kit (Life Technologies, Grand Island, NY), and cDNA was created using a high-capacity cDNA RT kit (Life Technologies) following manufacturer instructions. Real-time RT-PCR was performed over 40 cycles using a CFX384™ Real-Time PCR Detection System (Bio-Rad, Hercules, CA) and Taqman® Gene Expression Assays (Life Technologies): Gapdh (GAPDH, Mm99999915_g1), Il1b (IL-1β, Mm01336189_m1), Tnf (TNFα, Mm00443258_m1), Il6 (IL-6, Mm00446190_m1), Itgam (CD11b, Mm00434455_m1), Aif1 (Iba1, Mm00479862_g1), Gfap (GFAP, Mm01253033_m1), Ido (IDO, Mm00492586_m1), Ido2 (IDO2, Mm00524206_m1), Tdo2 (TDO, Mm00451266_m1), Ccbl1 (KATI, Mm01327703_m1), Aadat (KATII, Mm00496169_m1), Kmo (KMO, Mm00505511_m1), Kynu (KYNU, Mm00551012_m1), and Haao (HAAO, Mm00517945_m1). Data are expressed as relative fold change (Target ΔmRNA) using the 2−ΔΔCt calculation method and GAPDH as the housekeeping gene as previously described .
Liquid chromatography/mass spectrometry
Microdissected brain regions (dorsal and ventral hippocampus, central amygdala, nucleus accumbens) and plasma were prepared for liquid chromatography/mass spectrometry (LC/MS) and analyzed for kynurenine metabolites as previously described . Briefly, thawed plasma samples were diluted five times with 0.2 % acetic acid and 1 mM internal standards, transferred to Amicon Ultra filters (Millipore, Billerica, MA) and centrifuged at 13,500×g for 1 h at 4 °C. Frozen brain regions were diluted 30 times with 0.2 % acetic acid and 1 mM internal standards and then homogenized at 4 °C using a Bead Ruptor 24 Homogenizer (Omni International, Kennesaw, GA) with 1.4-mm zirconium ceramic oxide beads (Omni International) and settings of (pulse duration 45 s, pulse number 2, rest interval 15 s). The supernatant was filtered in a centrifuge as the plasma. Following preparation, samples were analyzed on a Q Exactive mass spectrometer (Thermo Fisher Scientific, Waltham, MA) with on-line separation by a Dionex UltiMate 3000 HPLC system (Thermo Fisher Scientific), and the data collected was analyzed using Xcalibur 2.2 software (Thermo Fisher Scientific) in the Mass Spectrometry Core Facility at the University of Texas Health Science Center at San Antonio. Metabolite ratios were calculated, as an in vivo estimation of IDO activity, kynurenine metabolic balance, and serotonin turnover, from raw data that are part of a separate study (Parrott JM, Redus L, Morales J, Xiaoli G, O’Connor JC: Neurotoxic kynurenine metabolism is increased in the dorsal hippocampus and drives distinct depressive behaviors during inflammation, Submitted). These values were used to determine the following ratios: kynurenine/tryptophan, 3-HK/KA, and 5-HIAA/5-HT. 3-Hydroxyanthranilic acid (3-HAA) and QA were not reliably detected in brain region microdissected samples due to limited sample size.
Data were analyzed using SigmaPlot 12.0 software (Systat Software Inc., San Jose, CA) and are presented as group means + standard error of the mean (SEM). Following a single-pass Chauvenet’s test for outliers as previously described , data analysis was conducted using either a t test or a one-way analysis of variance (ANOVA). Significant main effects identified by the ANOVA were further analyzed with the Holm-Sidak method for pairwise multiple comparisons post hoc test to identify between-group differences. Significant treatment effects (p < 0.05, compared to saline) are denoted as such (*), and trends (p < 0.1–0.05) are denoted as such (#) (# p < 0.1–0.05; *p < 0.05–0.01; **p < 0.01–0.001; ***p < 0.001).
Peripheral lipopolysaccharide induces time-dependent central pro-inflammatory cytokine expression
Markers of glial activation increase in response to peripheral LPS challenge
Peripheral LPS challenge increases central indoleamine 2,3-dioxygenase expression
Kynurenine aminotransferases I and II are impacted in a regionally dependent manner following peripheral lipopolysaccharide
Peripheral LPS induces expression changes in neurotoxic kynurenine pathway enzymes
Peripheral LPS causes a neurotoxic shift in hippocampal kynurenine metabolism
Lipopolysaccharide increases peripheral kynurenine metabolism toward the neurotoxic route
Peripheral inflammation increases flux through the kynurenine pathway of tryptophan metabolism
24 h LPS
p < 0.01
p < 0.001
p < 0.001
3-Hydroxyanthranilic acid (3-HAA)
Quinolinic acid (QA)
Kynurenic acid (KA)
p < 0.001
p < 0.001
Under the guidelines of the RDoC framework, a renewed emphasis has been placed on understanding regionally distinct structural, cellular, and molecular events involved in the pathogenesis of neuropsychiatric disease [29, 30]. Therefore, the objective of this study was to characterize the LPS-induced neuroinflammatory response in brain regions relevant to depression-related symptoms as previous literature has described these data only at the whole brain level. Up-regulation of pro-inflammatory cytokine expression (IL-1β, TNFα, and IL-6) followed the same temporal pattern in each of the three regions studied (hippocampus, amygdala, and striatum) following peripheral LPS challenge. The relative magnitude of the LPS-induced cytokine response was similar as well. Likewise, glial cell markers (CD11b, Iba1, and GFAP), in general, were elevated in a region-independent manner. Interestingly, up-regulation of astrocytic marker GFAP preceded the subsequent increase in microglial markers CD11b and Iba1. Consistent with studies demonstrating that IDO expression is induced by pro-inflammatory cytokines, IDO expression was robustly up-regulated in a region-independent manner, peaking at 6 h following LPS treatment, while IDO2 and TDO remained largely unchanged. Interestingly, the expression of neurotoxic kynurenine pathway enzymes downstream of IDO (KMO, KYNU, and HAAO) was also similar between brain regions; however, each enzyme had a unique response pattern following peripheral inflammation. KMO expression increased, KYNU expression remained unaffected, and HAAO expression decreased as an immediate response to LPS treatment. On the opposing metabolic branch, KAT enzymes (I and II) were significantly down-regulated in the hippocampus, 24 h after LPS treatment. In accord with the mRNA data, functional assessment of kynurenine metabolism showed that the kynurenine/tryptophan ratio increased approximately twofold following peripheral immune activation in the same pattern throughout the regions assessed. Interestingly, the 3-HK/KA ratio, which reflects the relative neurotoxic/neuroprotective metabolic balance, was only up-regulated in the hippocampal regions (dorsal and ventral) following LPS treatment. Together, these data demonstrate that peripheral immune challenge results in region-independent induction of neuroinflammation and kynurenine production, while downstream metabolism of kynurenine is region specific.
To characterize the neuroinflammatory response associated with peripheral immune activation, pro-inflammatory cytokine and glial cellular marker expressions were assessed. The expression of all three pro-inflammatory cytokines, IL-1β, TNFα, and IL-6 were increased 6 h after LPS injections in all three brain regions, similar to previously published data from the hippocampus, cortex, and hypothalamus [17, 44]. This early central pro-inflammatory response drives the development of LPS-induced sickness behavior (lethargy, decreased appetite, reduced social interactions, fever) [45, 46]. At 24 h post-LPS treatment, IL-1β and TNFα expression remained elevated while IL-6 returned to saline expression levels, also an effect similar to previously published results . It is important to note that under the experimental conditions of this study, 24 h after LPS treatment corresponds to when depressive-like behaviors have been assessed previously . It is possible that the culmination of IL-6 prior to IL-1β and TNFα expression is associated with the resolution of a specific behavior, such as fever , while other behaviors persist at 24 h post-LPS and IL-1β and TNFα play an important role in the development of those behaviors. It is important to note that using steady-state mRNA analysis of brain neuroinflammatory targets has both merit and limitations. In our study, mRNA targets allow for an extremely sensitive and quantitative measure of neuroinflammatory gene expression. Since many pro-inflammatory cytokines, including IL-1β, TNFα, and IL-6, are able to enter the brain via transport across the blood brain barrier [49, 50], determination of mRNA expression ensures that any changes being measured reflect the local brain expression of the targets, not the appearance/accumulation of peripherally secreted cytokines. However, it is possible that variability in translation dynamics of mRNA to protein could influence the expression of de novo synthesized neuroinflammatory targets.
Interestingly, the early up-regulation of central pro-inflammatory cytokine mRNA was coupled with a general down-regulation of microglial markers (CD11b and Iba1) and increase of astrocytic GFAP. At both time points after LPS, expression of astrocyte cell marker GFAP was up-regulated, which is consistent with a previous study demonstrating that central LPS administration increases GFAP expression . Microglia are known to be the predominant cellular producers of pro-inflammatory cytokines in the brain, and Iba1 expression and/or staining are often reported as a proxy of activation state . The incongruence in the expression patterns between the gene targets in our study suggests that expression of pro-inflammatory cytokines precede the up-regulation of microglia-specific markers. Additionally, utilization of CD11b or Iba1, in the absence of morphologic or other complementary data, appears to be an insufficient representation of microglia activation state. Moreover, the temporal difference between up-regulation of astrocyte versus microglial markers suggests the possibility of a dynamic role for these two cell types in mediating the neuroinflammatory response. This could be particularly relevant in terms of kynurenine metabolism which is a pathway compartmentalized between astrocytes and microglia in the brain [24, 53]. It is interesting to note that both CD11b and GFAP are increased in their respective cell populations by increases in nitric oxide (NO) production [54, 55], which can be generated by elevations in pro-inflammatory cytokines and kynurenine metabolite QA [56–58]. Further investigation is warranted to more precisely explore this phenomenon.
The expression of all known rate-limiting kynurenine producing enzymes, IDO, IDO2, and TDO, was assessed to better understand how tryptophan is metabolized following peripheral LPS. IDO expression was increased at both 6 and 24 h post LPS injections in each brain region assessed, consistent with two previously published studies [17, 44]. IDO2 is a more recently discovered homologue of IDO with much lower enzymatic activity compared to IDO . In the hippocampus and amygdala, IDO2 expression decreased at 24 h following LPS while otherwise remaining unchanged, which follows conflicting previous studies demonstrating that pro-inflammatory stimuli increased or had no impact on IDO2 mRNA [60, 61]. Overall, TDO expression did not increase in any brain region at either time point in response to LPS treatment, confirming previous reports demonstrating that TDO expression is up-regulated by glucocorticoids . Together, these data confirm that IDO is the only rate-limiting kynurenine pathway enzyme up-regulated in response to peripheral LPS, demonstrating increased capacity for de novo kynurenine production during neuroinflammatory conditions.
The metabolism of kynurenine is physically compartmentalized within the brain [24, 53]. Astrocytes express mainly KATs (not KMO) for conversion of kynurenine to KA [63, 64]. Microglia express KMO (not KATs) that metabolizes kynurenine along the neurotoxic branch, leading to the potential formation of several neurotoxic metabolites [65, 66]. Contemporary research suggests that the relative balance of these opposing metabolic branches, rather than simply changes in the levels of individual metabolites, constitutes the pathogenic potential of kynurenine metabolism . Though LPS treatment increased GFAP expression and presumably astrocytic activation, there was only a significant increase in KATI expression in the hippocampus at 6 h post injections. Further, at 24 h post-LPS, KATI and KATII expression was reduced in the hippocampus. A previous expression study reported that LPS treatment did not affect KATII expression in the hippocampus and cortex , in contrast with our results in the hippocampus. The data reported here, suggesting that KAT expression (I and II) is generally unchanged by LPS treatment, are consistent with previous findings that demonstrate that whole brain KA concentration levels do not increase at 24 h post-LPS treatment . Instead, that same study demonstrated that peripheral LPS-induced elevations in central kynurenine result in increases in 3-HK, 3-HAA, and QA on the neurotoxic branch, mediated through KMO in microglia . Interestingly, KMO was the only neurotoxic enzyme up-regulated following LPS treatment, while KYNU expression did not change and HAAO decreased expression following LPS treatment. In a previous study, at 4 h post-LPS, KMO expression decreased in the cortex, and at 24 h post-LPS, expression increased in both the cortex and hippocampus . The experimental conditions, such as species (rats) and LPS dose (0.25 mg/kg), are likely contributors to the differences in expression kinetics between the two studies. In our study, it is noteworthy to consider the possibility that HAAO expression is down-regulated as a compensatory response to the increased expression of KMO and the potential increase in flux of metabolites through the neurotoxic branch. Interestingly, the disconnect between expression of microglia cell markers (Iba1, CD11b) and KMO provides further evidence that the functional cellular response is not necessarily accurately reflected by measuring standard microglial markers.
To further explore the effects of LPS on kynurenine metabolic balance within behaviorally relevant brain regions, we calculated ratios that (1) reflected IDO activity and up-regulation of overall kynurenine metabolism (kynurenine/tryptophan), (2) indicated the relative balance of kynurenine metabolism toward the neurotoxic branch (3-HK/KA), and (3) assessed serotonin turnover (5-HIAA/5-HT). Twenty-four hours post LPS was chosen as the sampling time point based on established behavioral kinetics . Additionally, mRNA increases measured at 6 h post-LPS require time in order for protein translation to begin impacting metabolism relevant to behavior changes. In all of the regions assessed, the kynurenine/tryptophan ratio was elevated as predicted based on IDO expression data and previously published data [18, 19, 23, 67]. However, assessment of the 3-HK/KA ratio, indicative of metabolism down the neurotoxic kynurenine branch, revealed that peripheral LPS treatment only increased this neurotoxic ratio in the dorsal and ventral hippocampus. Relevant to this, a recent study suggested that microglia in separate regions of the brain have functionally distinct characteristics, specifically that those in the hippocampus are more pro-inflammatory in nature . As the main producers of neurotoxic kynurenine metabolites, these more immunovigilant hippocampal microglia might drive the region-specific shift toward neurotoxic metabolism observed here. Alternatively, it is possible that astrocytic production of KA may follow a region-dependent pattern, although this has yet to be directly investigated. Further, while not significant, increasing statistical power may actually reveal that the 3-HK/KA ratio decreases in the nucleus accumbens in response to peripheral LPS treatment. This will require additional future experiments. Interestingly, recent data collected from a chronic social defeat model, also associated with a peripheral immune response, demonstrated regional variation in the up-regulation of central kynurenine and 3-HK production . However, when brain region kynurenine metabolites were assessed in response to peripheral CD40 antibody treatment, which induces inflammation, both kynurenine and 3-HK were elevated in all of the regions assessed . Previous studies have demonstrated that peripheral LPS treatment increases serotonin turnover in the whole brain . In line with those data, the 5-HIAA/5-HT ratio was increased or trended to increase in all the regions assessed in this study, except the nucleus accumbens. Together, these data illustrate, for the first time, that brain kynurenine metabolism is regulated in a regionally distinct manner following peripheral immune challenge with LPS. Our recently published data support the notion that this neurotoxic shift is functionally relevant, as targeted deletion of the KMO gene protects mice from LPS-induced disruption in a hippocampal-dependent behavioral task .
As a control measure, tryptophan and kynurenine metabolites were assessed in the plasma 24 h after LPS treatment (Table 1). As predicted, LPS increased kynurenine, 3-HK, the kynurenine/tryptophan ratio, and the 3-HK/KA ratio and did not change KA. Curiously, the tryptophan concentration increased as well, contrary to previous clinical and preclinical studies demonstrating that inflammation is associated with a decrease in tryptophan or ‘tryptophan depletion’ [16, 19, 71]. Further, there were no changes in 3-HAA or QA after LPS treatment, again differing from previous studies showing LPS elevates peripheral and central QA as well as central 3-HAA [23, 71]. The current study is the first to explore kynurenine metabolic balance downstream of IDO in C57BL/6J inbred mice while previous studies were carried out in CD-1 outbred mice . The dissociation between central and peripheral downstream kynurenine metabolites has also been observed in the clinic, when cerebrospinal fluid QA was elevated without an increase in plasma QA in hepatitis C patients receiving interferon-α immunotherapy . Importantly, our data revealed that even though kynurenine/tryptophan doubles in both the peripheral circulation and in each of the brain regions measured (supporting the notion of the periphery as a primary source of these amino acids), the manner in which kynurenine metabolism proceeds is not simply a function of substrate levels. The distinct 3-HK/KA ratios, in the face of uniform increases in kynurenine/tryptophan ratios, further underscore that downstream brain region metabolism is regionally discrete and locally regulated. Understanding how local metabolic changes impact the behaviors mediated by distinct brain regions remains an important area for future study. Together, these data demonstrate that peripheral LPS up-regulates the metabolism of tryptophan and kynurenine to 3-HK, in both the periphery and the brain. However, region-dependent regulation of kynurenine metabolic balance within the brain is potentially pertinent to the pathogenesis of depression and is a therapeutically relevant finding.
The main goal of this study was to provide a better understanding of the regional neuroinflammatory response following peripheral treatment with LPS. This response involved mRNA expression (Fig. 1) of pro-inflammatory cytokines (IL-1β, TNFα, IL-6), glia cell markers (CD11b, Iba1, GFAP), and kynurenine pathway enzymes (IDO, IDO2, TDO, KATI, KATII, KMO, KYNU, HAAO) at 6 and 24 h post LPS in relevant brain regions (hippocampus, amygdala, striatum). Additionally, kynurenine metabolite ratios (kynurenine/tryptophan, 3-HK/KA, 5-HIAA/5-HT) were calculated from previously collected data at 24 h post-LPS in relevant brain regions (dorsal and ventral hippocampus, central amygdala, nucleus accumbens). In general, these data demonstrated that in response to peripheral LPS treatment, pro-inflammatory cytokines and glia cell makers increase similarly throughout the brain. Kynurenine pathway enzymes have distinct temporal expression changes following LPS, which are region-independent; however, the metabolic balance is regulated in a region-dependent manner. Further analysis of the individual metabolite concentrations and additional brain regions will advance the understanding and potential implications of brain region-specific kynurenine metabolism. The possibility that individual depression-related behaviors are driven by regionally defined disruptions in kynurenine metabolism provides a specific target for future pharmacotherapy developments.
3-HAA, 3-hyroxyanthranilic acid; 3-HK, 3-hydroxykynurenine; 5-HIAA, 5-hyrdoxyindoleacetic acid; 5-HT, serotonin; CD11b, cluster of differentiation molecule 11b; CSF, cerebrospinal fluid; fMRI, functional magnetic resonance imaging; GFAP, glial fibrillary acidic protein; HAAO, 3-hyroxyanthranilic acid dioxygenase; HPA, hypothalamic-pituitary-adrenal; IDO, indoleamine 2,3-dioxygenase; i.p., intraperitoneal; Iba1, ionized calcium-binding adapter molecule 1; IFN-α, interferon-α; IL-1β, interleukin-1β; IL-6, interleukin-6; KA, kynurenic acid; KAT, kynurenine aminotransferase; KMO, kynurenine 3-monooxygenase; KYNU, kynureninase; LC/MS, liquid chromatography/mass spectrometry; LPS, lipopolysaccharide; MADRS, Montgomery-Asberg Depression Rating Scale; NMDAR, N-methyl-d-aspartate receptor; NO, nitric oxide; QA, quinolinic acid; RDoC, Research Domain Criteria; SEM, standard error of the mean; TDO, tryptophan 2,3-dioxygenase; TNFα, tumor necrosis factor α; UTHSCSA, University of Texas Health Science Center at San Antonio; α7nAChR, α7-nicotinic acetylcholine receptor
A special thanks to Susan T. Weintraub, PhD and Xiaoli Gao, PhD at the University of Texas Health Science Center at San Antonio Mass Spectrometry Core Facilities for their assistance with the LC/MS method design, sample preparation, sample analysis, and data collection. Mass spectrometry analyses were conducted on instrumentation obtained with funding from the National Institutes of Health (1S10OD016417-01 to STW).
This research was supported by funding from the National Institute of Mental Health (R01MH090127 and P30MH089868 to JOC; 1F31MH102070-01A1 to JP), the National Center for Advancing Translational Studies (UL1TR001120 to JOC), and the Norman Hackerman Advanced Research Program (003659-0010-2012 to JOC). The content is the sole the responsibility of the authors and does not necessarily represent the views of the National Institute of Mental Health, the National Center for Advancing Translational Studies or the National Institutes of Health. R01MH090127 funded data collection, data analysis, data interpretation, and writing of the manuscript. P30MH089868, UL1TR001120, and 003659-0010-2012 supported the design of the study, data analysis, and data interpretation. 1F31MH102070-01A1 funded data analysis, data interpretation, and writing of the manuscript.
Availability of data and materials
All raw data are clearly summarized in the body or supplemental figures of the manuscript. Even though the raw data used in the analyses presented here are not traditionally databased informatics, genomics, engineering dataset, they are freely available for review upon request from the corresponding author.
JP carried out the tissue collection, organized and analyzed the data, and drafted the manuscript. LR helped to collect the samples and performed the LC/MS analysis. JOC conceived of the study, led the design and coordination of the study, and prepared the final manuscript. All authors read and approved the final manuscript.
Although unrelated to the material presented or discussed in this manuscript, Jason C. O’Connor, PhD has consulted for Lundbeck Research, USA, and Janssen Research and Development, LLC. Jennifer M. Parrott and Laney Redus declare that they have no competing interests.
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- Moussavi S, Chatterji S, Verdes E, Tandon A, Patel V, Ustun B. Depression, chronic diseases, and decrements in health: results from the World Health Surveys. Lancet. 2007;370:851–8.View ArticlePubMedGoogle Scholar
- Capuron L, Miller AH. Cytokines and psychopathology: lessons from interferon-alpha. Biol Psychiatry. 2004;56:819–24.View ArticlePubMedGoogle Scholar
- Brydon L, Harrison NA, Walker C, Steptoe A, Critchley HD. Peripheral inflammation is associated with altered substantia nigra activity and psychomotor slowing in humans. Biol Psychiatry. 2008;63:1022–9.View ArticlePubMedPubMed CentralGoogle Scholar
- Harrison NA, Brydon L, Walker C, Gray MA, Steptoe A, Critchley HD. Inflammation causes mood changes through alterations in subgenual cingulate activity and mesolimbic connectivity. Biol Psychiatry. 2009;66:407–14.View ArticlePubMedPubMed CentralGoogle Scholar
- Eisenberger NI, Berkman ET, Inagaki TK, Rameson LT, Mashal NM, Irwin MR. Inflammation-induced anhedonia: endotoxin reduces ventral striatum responses to reward. Biol Psychiatry. 2010;68:748–54.View ArticlePubMedPubMed CentralGoogle Scholar
- Grigoleit JS, Kullmann JS, Wolf OT, Hammes F, Wegner A, Jablonowski S, Engler H, Gizewski E, Oberbeck R, Schedlowski M. Dose-dependent effects of endotoxin on neurobehavioral functions in humans. PLoS One. 2011;6:e28330.View ArticlePubMedPubMed CentralGoogle Scholar
- Felger JC, Alagbe O, Hu F, Mook D, Freeman AA, Sanchez MM, Kalin NH, Ratti E, Nemeroff CB, Miller AH. Effects of interferon-alpha on rhesus monkeys: a nonhuman primate model of cytokine-induced depression. Biol Psychiatry. 2007;62:1324–33.View ArticlePubMedPubMed CentralGoogle Scholar
- Felger JC, Mun J, Kimmel HL, Nye JA, Drake DF, Hernandez CR, Freeman AA, Rye DB, Goodman MM, Howell LL, Miller AH. Chronic interferon-alpha decreases dopamine 2 receptor binding and striatal dopamine release in association with anhedonia-like behavior in nonhuman primates. Neuropsychopharmacology. 2013;38:2179–87.View ArticlePubMedPubMed CentralGoogle Scholar
- Orsal AS, Blois SM, Bermpohl D, Schaefer M, Coquery N. Administration of interferon-alpha in mice provokes peripheral and central modulation of immune cells, accompanied by behavioral effects. Neuropsychobiology. 2008;58:211–22.View ArticlePubMedGoogle Scholar
- Hayley S, Scharf J, Anisman H. Central administration of murine interferon-alpha induces depressive-like behavioral, brain cytokine and neurochemical alterations in mice: a mini-review and original experiments. Brain Behav Immun. 2013;31:115–27.View ArticlePubMedGoogle Scholar
- Konsman JP, Veeneman J, Combe C, Poole S, Luheshi GN, Dantzer R. Central nervous action of interleukin-1 mediates activation of limbic structures and behavioural depression in response to peripheral administration of bacterial lipopolysaccharide. Eur J Neurosci. 2008;28:2499–510.View ArticlePubMedGoogle Scholar
- O'Connor JC, Andre C, Wang Y, Lawson MA, Szegedi SS, Lestage J, Castanon N, Kelley KW, Dantzer R. Interferon-gamma and tumor necrosis factor-alpha mediate the upregulation of indoleamine 2,3-dioxygenase and the induction of depressive-like behavior in mice in response to bacillus Calmette-Guerin. J Neurosci. 2009;29:4200–9.View ArticlePubMedPubMed CentralGoogle Scholar
- Raison CL, Capuron L, Miller AH. Cytokines sing the blues: inflammation and the pathogenesis of depression. Trends Immunol. 2006;27:24–31.View ArticlePubMedPubMed CentralGoogle Scholar
- Polazzi E, Contestabile A. Reciprocal interactions between microglia and neurons: from survival to neuropathology. Rev Neurosci. 2002;13:221–42.View ArticlePubMedGoogle Scholar
- Block ML, Zecca L, Hong JS. Microglia-mediated neurotoxicity: uncovering the molecular mechanisms. Nat Rev Neurosci. 2007;8:57–69.View ArticlePubMedGoogle Scholar
- Dantzer R, O'Connor JC, Lawson MA, Kelley KW. Inflammation-associated depression: from serotonin to kynurenine. Psychoneuroendocrinology. 2011;36:426–36.View ArticlePubMedPubMed CentralGoogle Scholar
- Andre C, O'Connor JC, Kelley KW, Lestage J, Dantzer R, Castanon N. Spatio-temporal differences in the profile of murine brain expression of proinflammatory cytokines and indoleamine 2,3-dioxygenase in response to peripheral lipopolysaccharide administration. J Neuroimmunol. 2008;200:90–9.View ArticlePubMedPubMed CentralGoogle Scholar
- O'Connor JC, Lawson MA, Andre C, Briley EM, Szegedi SS, Lestage J, Castanon N, Herkenham M, Dantzer R, Kelley KW. Induction of IDO by bacille Calmette-Guerin is responsible for development of murine depressive-like behavior. J Immunol. 2009;182:3202–12.View ArticlePubMedPubMed CentralGoogle Scholar
- O'Connor JC, Lawson MA, Andre C, Moreau M, Lestage J, Castanon N, Kelley KW, Dantzer R. Lipopolysaccharide-induced depressive-like behavior is mediated by indoleamine 2,3-dioxygenase activation in mice. Mol Psychiatry. 2009;14:511–22.View ArticlePubMedPubMed CentralGoogle Scholar
- Ganong AH, Cotman CW. Kynurenic acid and quinolinic acid act at N-methyl-D-aspartate receptors in the rat hippocampus. J Pharmacol Exp Ther. 1986;236:293–9.PubMedGoogle Scholar
- Hilmas C, Pereira EF, Alkondon M, Rassoulpour A, Schwarcz R, Albuquerque EX. The brain metabolite kynurenic acid inhibits alpha7 nicotinic receptor activity and increases non-alpha7 nicotinic receptor expression: physiopathological implications. J Neurosci. 2001;21:7463–73.PubMedGoogle Scholar
- Stone TW, Perkins MN. Quinolinic acid: a potent endogenous excitant at amino acid receptors in CNS. Eur J Pharmacol. 1981;72:411–2.View ArticlePubMedGoogle Scholar
- Walker AK, Budac DP, Bisulco S, Lee AW, Smith RA, Beenders B, Kelley KW, Dantzer R. NMDA receptor blockade by ketamine abrogates lipopolysaccharide-induced depressive-like behavior in C57BL/6 J mice. Neuropsychopharmacology. 2013;38:1609–16.View ArticlePubMedPubMed CentralGoogle Scholar
- Parrott JM, O'Connor JC. Kynurenine 3-monooxygenase: an influential mediator of neuropathology. Front Psychiatry. 2015;6:116.PubMedPubMed CentralGoogle Scholar
- Salazar A, Gonzalez-Rivera BL, Redus L, Parrott JM, O'Connor JC. Indoleamine 2,3-dioxygenase mediates anhedonia and anxiety-like behaviors caused by peripheral lipopolysaccharide immune challenge. Horm Behav. 2012;62:202–9.View ArticlePubMedPubMed CentralGoogle Scholar
- Heisler JM, O'Connor JC. Indoleamine 2,3-dioxygenase-dependent neurotoxic kynurenine metabolism mediates inflammation-induced deficit in recognition memory. Brain Behav Immun. 2015;50:115-24.Google Scholar
- Wichers MC, Koek GH, Robaeys G, Verkerk R, Scharpe S, Maes M. IDO and interferon-alpha-induced depressive symptoms: a shift in hypothesis from tryptophan depletion to neurotoxicity. Mol Psychiatry. 2005;10:538–44.View ArticlePubMedGoogle Scholar
- Raison CL, Dantzer R, Kelley KW, Lawson MA, Woolwine BJ, Vogt G, Spivey JR, Saito K, Miller AH. CSF concentrations of brain tryptophan and kynurenines during immune stimulation with IFN-alpha: relationship to CNS immune responses and depression. Mol Psychiatry. 2010;15:393–403.View ArticlePubMedPubMed CentralGoogle Scholar
- Insel T, Cuthbert B, Garvey M, Heinssen R, Pine DS, Quinn K, Sanislow C, Wang P. Research domain criteria (RDoC): toward a new classification framework for research on mental disorders. Am J Psychiatry. 2010;167:748–51.View ArticlePubMedGoogle Scholar
- Insel TR. The NIMH Research Domain Criteria (RDoC) Project: precision medicine for psychiatry. Am J Psychiatry. 2014;171:395–7.View ArticlePubMedGoogle Scholar
- Reichenberg A, Yirmiya R, Schuld A, Kraus T, Haack M, Morag A, Pollmacher T. Cytokine-associated emotional and cognitive disturbances in humans. Arch Gen Psychiatry. 2001;58:445–52.View ArticlePubMedGoogle Scholar
- Krabbe KS, Reichenberg A, Yirmiya R, Smed A, Pedersen BK, Bruunsgaard H. Low-dose endotoxemia and human neuropsychological functions. Brain Behav Immun. 2005;19:453–60.View ArticlePubMedGoogle Scholar
- Inagaki TK, Muscatell KA, Irwin MR, Cole SW, Eisenberger NI. Inflammation selectively enhances amygdala activity to socially threatening images. Neuroimage. 2012;59:3222–6.View ArticlePubMedPubMed CentralGoogle Scholar
- Muscatell KA, Dedovic K, Slavich GM, Jarcho MR, Breen EC, Bower JE, Irwin MR, Eisenberger NI. Greater amygdala activity and dorsomedial prefrontal-amygdala coupling are associated with enhanced inflammatory responses to stress. Brain Behav Immun. 2015;43:46–53.View ArticlePubMedPubMed CentralGoogle Scholar
- Konsman JP, Kelley K, Dantzer R. Temporal and spatial relationships between lipopolysaccharide-induced expression of Fos, interleukin-1beta and inducible nitric oxide synthase in rat brain. Neuroscience. 1999;89:535–48.View ArticlePubMedGoogle Scholar
- Drevets WC, Price JL, Furey ML. Brain structural and functional abnormalities in mood disorders: implications for neurocircuitry models of depression. Brain Struct Funct. 2008;213:93–118.View ArticlePubMedPubMed CentralGoogle Scholar
- Gal EM, Sherman AD. L-kynurenine: its synthesis and possible regulatory function in brain. Neurochem Res. 1980;5:223–39.View ArticlePubMedGoogle Scholar
- Franklin KBJ, Paxinos G. The mouse brain in sterotaxic coordinates. 3rd ed. New York, NY: Academic Press; 2007.Google Scholar
- Lawson LJ, Perry VH, Dri P, Gordon S. Heterogeneity in the distribution and morphology of microglia in the normal adult mouse brain. Neuroscience. 1990;39:151–70.View ArticlePubMedGoogle Scholar
- Buttini M, Limonta S, Boddeke HW. Peripheral administration of lipopolysaccharide induces activation of microglial cells in rat brain. Neurochem Int. 1996;29:25–35.View ArticlePubMedGoogle Scholar
- Okuno E, Nakamura M, Schwarcz R. Two kynurenine aminotransferases in human brain. Brain Res. 1991;542:307–12.View ArticlePubMedGoogle Scholar
- Corona AW, Norden DM, Skendelas JP, Huang Y, O'Connor JC, Lawson M, Dantzer R, Kelley KW, Godbout JP. Indoleamine 2,3-dioxygenase inhibition attenuates lipopolysaccharide induced persistent microglial activation and depressive-like complications in fractalkine receptor (CX(3)CR1)-deficient mice. Brain Behav Immun. 2013;31:134–42.View ArticlePubMedPubMed CentralGoogle Scholar
- Dinel AL, Andre C, Aubert A, Ferreira G, Laye S, Castanon N. Lipopolysaccharide-induced brain activation of the indoleamine 2,3-dioxygenase and depressive-like behavior are impaired in a mouse model of metabolic syndrome. Psychoneuroendocrinology. 2014;40:48–59.View ArticlePubMedGoogle Scholar
- Connor TJ, Starr N, O'Sullivan JB, Harkin A. Induction of indolamine 2,3-dioxygenase and kynurenine 3-monooxygenase in rat brain following a systemic inflammatory challenge: a role for IFN-gamma? Neurosci Lett. 2008;441:29–34.View ArticlePubMedGoogle Scholar
- Bluthe RM, Pawlowski M, Suarez S, Parnet P, Pittman Q, Kelley KW, Dantzer R. Synergy between tumor necrosis factor alpha and interleukin-1 in the induction of sickness behavior in mice. Psychoneuroendocrinology. 1994;19:197–207.View ArticlePubMedGoogle Scholar
- Kozak W, Conn CA, Kluger MJ. Lipopolysaccharide induces fever and depresses locomotor activity in unrestrained mice. Am J Physiol. 1994;266:R125–35.PubMedGoogle Scholar
- Dantzer R, O'Connor JC, Freund GG, Johnson RW, Kelley KW. From inflammation to sickness and depression: when the immune system subjugates the brain. Nat Rev Neurosci. 2008;9:46–56.View ArticlePubMedPubMed CentralGoogle Scholar
- Lenczowski MJ, Bluthe RM, Roth J, Rees GS, Rushforth DA, van Dam AM, Tilders FJ, Dantzer R, Rothwell NJ, Luheshi GN. Central administration of rat IL-6 induces HPA activation and fever but not sickness behavior in rats. Am J Physiol. 1999;276:R652–8.PubMedGoogle Scholar
- Quan N, Banks WA. Brain-immune communication pathways. Brain Behav Immun. 2007;21:727–35.View ArticlePubMedGoogle Scholar
- Banks WA, Kastin AJ, Broadwell RD. Passage of cytokines across the blood-brain barrier. Neuroimmunomodulation. 1995;2:241–8.View ArticlePubMedGoogle Scholar
- Sugaya K, Chou S, Xu SJ, McKinney M. Indicators of glial activation and brain oxidative stress after intraventricular infusion of endotoxin. Brain Res Mol Brain Res. 1998;58:1–9.View ArticlePubMedGoogle Scholar
- Ito D, Imai Y, Ohsawa K, Nakajima K, Fukuuchi Y, Kohsaka S. Microglia-specific localisation of a novel calcium binding protein, Iba1. Brain Res Mol Brain Res. 1998;57:1–9.View ArticlePubMedGoogle Scholar
- Schwarcz R, Bruno JP, Muchowski PJ, Wu HQ. Kynurenines in the mammalian brain: when physiology meets pathology. Nat Rev Neurosci. 2012;13:465–77.View ArticlePubMedPubMed CentralGoogle Scholar
- Brahmachari S, Fung YK, Pahan K. Induction of glial fibrillary acidic protein expression in astrocytes by nitric oxide. J Neurosci. 2006;26:4930–9.View ArticlePubMedPubMed CentralGoogle Scholar
- Roy A, Fung YK, Liu X, Pahan K. Up-regulation of microglial CD11b expression by nitric oxide. J Biol Chem. 2006;281:14971–80.View ArticlePubMedPubMed CentralGoogle Scholar
- Rios C, Santamaria A. Quinolinic acid is a potent lipid peroxidant in rat brain homogenates. Neurochem Res. 1991;16:1139–43.View ArticlePubMedGoogle Scholar
- Santamaria A, Santamaria D, Diaz-Munoz M, Espinoza-Gonzalez V, Rios C. Effects of N omega-nitro-L-arginine and L-arginine on quinolinic acid-induced lipid peroxidation. Toxicol Lett. 1997;93:117–24.View ArticlePubMedGoogle Scholar
- Perez-Severiano F, Escalante B, Rios C. Nitric oxide synthase inhibition prevents acute quinolinate-induced striatal neurotoxicity. Neurochem Res. 1998;23:1297–302.View ArticlePubMedGoogle Scholar
- Fatokun AA, Hunt NH, Ball HJ. Indoleamine 2,3-dioxygenase 2 (IDO2) and the kynurenine pathway: characteristics and potential roles in health and disease. Amino Acids. 2013;45:1319–29.View ArticlePubMedGoogle Scholar
- Ball HJ, Sanchez-Perez A, Weiser S, Austin CJ, Astelbauer F, Miu J, McQuillan JA, Stocker R, Jermiin LS, Hunt NH. Characterization of an indoleamine 2,3-dioxygenase-like protein found in humans and mice. Gene. 2007;396:203–13.View ArticlePubMedGoogle Scholar
- Divanovic S, Sawtell NM, Trompette A, Warning JI, Dias A, Cooper AM, Yap GS, Arditi M, Shimada K, Duhadaway JB, et al. Opposing biological functions of tryptophan catabolizing enzymes during intracellular infection. J Infect Dis. 2012;205:152–61.View ArticlePubMedPubMed CentralGoogle Scholar
- Danesch U, Hashimoto S, Renkawitz R, Schutz G. Transcriptional regulation of the tryptophan oxygenase gene in rat liver by glucocorticoids. J Biol Chem. 1983;258:4750–3.PubMedGoogle Scholar
- Kiss C, Ceresoli-Borroni G, Guidetti P, Zielke CL, Zielke HR, Schwarcz R. Kynurenate production by cultured human astrocytes. J Neural Transm. 2003;110:1–14.PubMedGoogle Scholar
- Guidetti P, Hoffman GE, Melendez-Ferro M, Albuquerque EX, Schwarcz R. Astrocytic localization of kynurenine aminotransferase II in the rat brain visualized by immunocytochemistry. Glia. 2007;55:78–92.View ArticlePubMedGoogle Scholar
- Heyes MP, Achim CL, Wiley CA, Major EO, Saito K, Markey SP. Human microglia convert l-tryptophan into the neurotoxin quinolinic acid. Biochem J. 1996;320(Pt 2):595–7.View ArticlePubMedPubMed CentralGoogle Scholar
- Guillemin GJ, Smythe G, Takikawa O, Brew BJ. Expression of indoleamine 2,3-dioxygenase and production of quinolinic acid by human microglia, astrocytes, and neurons. Glia. 2005;49:15–23.View ArticlePubMedGoogle Scholar
- Lawson MA, Parrott JM, McCusker RH, Dantzer R, Kelley KW, O'Connor JC. Intracerebroventricular administration of lipopolysaccharide induces indoleamine-2,3-dioxygenase-dependent depression-like behaviors. J Neuroinflammation. 2013;10:87.View ArticlePubMedPubMed CentralGoogle Scholar
- Zeisel A, Munoz-Manchado AB, Codeluppi S, Lonnerberg P, La Manno G, Jureus A, Marques S, Munguba H, He L, Betsholtz C, et al. Brain structure. Cell types in the mouse cortex and hippocampus revealed by single-cell RNA-seq. Science. 2015;347:1138–42.View ArticlePubMedGoogle Scholar
- Fuertig R, Azzinnari D, Bergamini G, Cathomas F, Sigrist H, Seifritz E, Vavassori S, Luippold A, Hengerer B, Ceci A, Pryce CR. Mouse chronic social stress increases blood and brain kynurenine pathway activity and fear behaviour: both effects are reversed by inhibition of indoleamine 2,3-dioxygenase. Brain Behav Immun. 2016;54:59–72.View ArticlePubMedGoogle Scholar
- Cathomas F, Fuertig R, Sigrist H, Newman GN, Hoop V, Bizzozzero M, Mueller A, Luippold A, Ceci A, Hengerer B, et al. CD40-TNF activation in mice induces extended sickness behavior syndrome co-incident with but not dependent on activation of the kynurenine pathway. Brain Behav Immun. 2015;50:125–40.View ArticlePubMedGoogle Scholar
- Saito K, Markey SP, Heyes MP. Effects of immune activation on quinolinic acid and neuroactive kynurenines in the mouse. Neuroscience. 1992;51:25–39.View ArticlePubMedGoogle Scholar
- Pettitt SJ, Liang Q, Rairdan XY, Moran JL, Prosser HM, Beier DR, Lloyd KC, Bradley A, Skarnes WC. Agouti C57BL/6N embryonic stem cells for mouse genetic resources. Nat Methods. 2009;6:493–5.View ArticlePubMedPubMed CentralGoogle Scholar