Kynurenine metabolic balance is disrupted in the hippocampus following peripheral lipopolysaccharide challenge

Abstract

Background: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

Keywords: Brain regions; Hippocampus; Indoleamine 2,3-dioxygenase; Kynurenine; Kynurenine 3-monooxygenase; Microglia; Neuroinflammation; Pro-inflammatory cytokines.

Fig. 1

Kynurenine pathway of tryptophan metabolism. Tryptophan is metabolized to kynurenine by indoleamine 2,3-dioxygenase (IDO), IDO-like enzyme, IDO2, and tryptophan 2,3-dioxygenase (TDO). Tryptophan can also be metabolized by tryptophan hydroxylase (TrpH) and l-aromatic amino acid decarboxylase (_L -AADC) to serotonin (5-HT) which is further broken down by monoamine oxidase (MAO) to 5-hydroxyindoleacetic acid (5-HIAA). Kynurenine can be metabolized to kynurenic acid (KA) by one of four isoforms of kynurenine aminotransferase (KAT), of which KATI and KATII are most relevant to mammals. The production of KA by KATs mainly occurs in astrocytes, cells which predominantly express glial fibrillary acidic protein (GFAP). Alternately, kynurenine can be metabolized to 3-hydroxykynurenine (3-HK) by kynurenine 3-monooxygenase (KMO). 3-HK is metabolized to 3-hydroxyanthranilic acid (3-HAA) by kynureninase (KYNU) and 3-HAA to quinolinic acid (QA) by 3-hydroxyanthranilic acid dioxygenase (HAAO). This branch of the kynurenine pathway is compartmentalized in microglia, indicated by ionized calcium-binding adapter molecule 1 (Iba1) and CD11b expression. The elevation in expression of pro-inflammatory cytokines, such as interleukin-1β (IL-1β), tumor necrosis factor α (TNFα), and interleukin-6 (IL-6), during inflammatory conditions activates glial cells. Further, IDO expression is up-regulated and flux through the neurotoxic kynurenine metabolic branch increases. Underlined targets were assessed by real-time RT-PCR to determine changes in mRNA

Fig. 2

Central pro-inflammatory cytokines are elevated following peripheral LPS injections. mRNA fold changes (Δ_mRNA) of interleukin-1β (IL-1β, left panel), tumor necrosis factor α (TNFα, middle panel), and interleukin-6 (IL-6, right panel) were measured at 6 and 24 h post LPS treatment in the (a) hippocampus, (b) amygdala, and (c) striatum. Data represent sample means ± SEM, n = 8–11 samples/group. * = post hoc comparison to saline treatment group. *p < 0.05–0.01; **p < 0.01–0.001; ***p < 0.001

Fig. 3

Peripheral inflammation up-regulates central expression of glial cell markers. mRNA fold changes (Δ_mRNA) of CD11b (left panel), ionized calcium-binding adapter molecule 1 (Iba1, middle panel), and glial fibrillary acid protein (GFAP, right panel) were measured at 6 and 24 h post LPS treatment in the a hippocampus, b amygdala, and c striatum. Data represent sample means ± SEM, n = 9–11 samples/group. * = overall significance or post hoc comparison to saline treatment group. *p < 0.05–0.01; **p < 0.01–0.001; ***p < 0.001

Fig. 4

Peripheral LPS specifically increases expression of kynurenine producing enzyme, indoleamine 2,3-dioxygenase (IDO). mRNA fold changes (Δ_mRNA) of IDO were assessed at 6 and 24 h following LPS injections in the (a) hippocampus, (b) amygdala, and (c) striatum. mRNA expression of IDO2 (left panel) and tryptophan 2,3-dioygenase (TDO, right panel) were also measured at 6 and 24 h post LPS in the (d) hippocampus, (e) amygdala, and (f) striatum. Data represent sample means ± SEM, n = 8–11 samples/group. ^#,* = overall significance or post hoc comparison to saline treatment group. ^# p < 0.1–0.05; *p < 0.05–0.01; **p < 0.01–0.001; ***p < 0.001

Fig. 5

Kynurenine aminotransferases expression following peripheral LPS treatment. mRNA fold changes (Δ_mRNA) of kynurenine aminotransferase I (KAT I, left panel) and kynurenine aminotransferase II (KAT II, right panel) were measured at 6 and 24 h post LPS in the (a) hippocampus, (b) amygdala, and (c) striatum. Data represent sample means ± SEM, n = 8–11 samples/group. ^#,* = overall significance or post hoc comparison to saline treatment group. ^# p < 0.1–0.05; *p < 0.05–0.01; **p < 0.01–0.001; ***p < 0.001

Fig. 6

Peripheral inflammation alters expression of downstream kynurenine pathway enzymes. mRNA fold changes (Δ_mRNA) of kynurenine 3-monooxygenase (KMO, left panel), kynureninase (KYNU, middle panel), and 3-hydroxyanthranilic acid dioxygenase (HAAO, right panel) were measured at 6 and 24 h following LPS injections in the (a) hippocampus, (b) amygdala, and (c) striatum. Data represent sample means ± SEM, n = 9–11 samples/group. ^#,* = post hoc comparison to saline treatment group. ^# p < 0.1–0.05; *p < 0.05–0.01; **p < 0.01–0.001; ***p < 0.001

Fig. 7

Kynurenine/tryptophan ratio and hippocampal neurotoxic kynurenine metabolite ratio are up-regulated in response to peripheral LPS. The kynurenine/tryptophan ratio was measured at 24 h following LPS injections in the (a) dorsal hippocampus, (b) ventral hippocampus, (c) central amygdala, and (d) nucleus accumbens. Metabolite ratios of 3-hydroxykynurenine/kynurenic acid (3-HK/KA, neurotoxic ratio, left panel) and 5-hydroxyindoleacetic acid/serotonin (5-HIAA/5-HT, serotonin turnover, right panel) were measured 24 h post LPS in the (e) dorsal hippocampus, (f) ventral hippocampus, (g) central amygdala, and (h) nucleus accumbens. Data represent sample means ± SEM, n = 7–12 samples/group. ^# p < 0.1–0.05; *p < 0.05–0.01; **p < 0.01–0.001; ***p < 0.001