Maternal viral infection causes global alterations in porcine fetal microglia.

Maternal infections during pregnancy are associated with increased risk of neurodevelopmental disorders, although the precise mechanisms remain to be elucidated. Previously, we established a maternal immune activation (MIA) model using swine, which results in altered social behaviors of piglet offspring. These behavioral abnormalities occurred in the absence of microglia priming. Thus, we examined fetal microglial activity during prenatal development in response to maternal infection with live porcine reproductive and respiratory syndrome virus. Fetuses were obtained by cesarean sections performed 7 and 21 d postinoculation (dpi). MIA fetuses had reduced brain weights at 21 dpi compared to controls. Furthermore, MIA microglia increased expression of major histocompatibility complex class II that was coupled with reduced phagocytic and chemotactic activity compared to controls. High-throughput gene-expression analysis of microglial-enriched genes involved in neurodevelopment, the microglia sensome, and inflammation revealed differential regulation in primary microglia and in whole amygdala tissue. Microglia density was increased in the fetal amygdala at 7 dpi. Our data also reveal widespread sexual dimorphisms in microglial gene expression and demonstrate that the consequences of MIA are sex dependent. Overall, these results indicate that fetal microglia are significantly altered by maternal viral infection, presenting a potential mechanism through which MIA impacts prenatal brain development and function.

Herring roe oil supplementation alters microglial cell gene expression and reduces peripheral inflammation after immune activation in a neonatal piglet model.

Neonatal brain development can be disrupted by infection that results in microglial cell activation and neuroinflammation. Studies indicate that polyunsaturated fatty acids (PUFAs) and their metabolites can resolve inflammation. It is not known if dietary PUFA increases lipid metabolites in brain or reduces neuroinflammation in neonates. We hypothesized that dietary PUFAs might suppress neuroinflammation by inhibiting pro-inflammatory cytokine over-production and promoting inflammatory resolution in the periphery and brain. Piglets were obtained on postnatal day (PD) 2 and randomly assigned to herring roe oil (HRO) or control (CON) diet. HRO was included at 2 g/kg powdered diet. HRO increased DHA levels in occipital lobe and the DHA to arachidonic acid (ARA) ratio in hippocampal tissue. HRO decreased ARA metabolites in occipital lobe. HRO failed to attenuate microglial pro-inflammatory cytokine production ex vivo. HRO did not affect fever or circulating resolvin D1 levels. HRO decreased circulating neutrophils and liver inflammatory gene expression, but increased resolution marker gene expression in liver post LPS. HRO upregulated CXCL16, TGFBR1, and C1QA in microglial cells. HRO supplementation exerted beneficial effects on inflammation in the periphery, but further studies are needed to evaluate the specific effects of omega-3 supplementation on microglial cell physiology in the neonate.

Glia- and tissue-specific changes in the Kynurenine Pathway after treatment of mice with lipopolysaccharide and dexamethasone.

Behavioral symptoms associated with mood disorders have been intimately linked with immunological and psychological stress. Induction of immune and stress pathways is accompanied by increased tryptophan entry into the Kynurenine (Kyn) Pathway as governed by the rate-limiting enzymes indoleamine/tryptophan 2,3-dioxygenases (DO's: Ido1, Ido2, Tdo2). Indeed, elevated DO expression is associated with inflammation- and stress-related depression symptoms. Here we examined central (brain, astrocyte and microglia) and peripheral (lung, liver and spleen) DO expression in mice treated intraperitoneally with lipopolysaccharide (LPS) and dexamethasone (DEX) to model the response of the Kyn Pathway to inflammation and glucocorticoids. LPS-induced expression of cytokines in peripheral tissues was attenuated by DEX, confirming inflammatory and anti-inflammatory responses, respectively. Increased Kyn levels following LPS and DEX administration verified Kyn Pathway activation. Expression of multiple mRNA isoforms for each DO, which we have shown to be differentially utilized and regulated, were quantified including reference/full-length (FL) and variant (v) transcripts. LPS increased Ido1-FL in brain (∼1000-fold), a response paralleled by increased expression in both astrocytes and microglia. Central Ido1-FL was not changed by DEX; however, LPS-induced Ido1-FL was decreased by DEX in peripheral tissues. In contrast, DEX increased Ido1-v1 expression by astrocytes and microglia, but not peripheral tissues. In comparison, brain Ido2 was minimally induced by LPS or DEX. Uniquely, Ido2-v6 was LPS- and DEX-inducible in astrocytes, suggesting a unique role for astrocytes in response to inflammation and glucocorticoids. Only DEX increased central Tdo2 expression; however, peripheral Tdo2 was upregulated by either LPS or DEX. In summary, specific DO isoforms are increased by LPS and DEX, but LPS-dependent Ido1 and Ido2 induction are attenuated by DEX only in the periphery indicating that elevated DO expression and Kyn production within the brain can occur independent of the periphery. These findings demonstrate a plausible interaction between immune activation and glucocorticoids associated with depression.

Maternal viral infection during pregnancy elicits anti-social behavior in neonatal piglet offspring independent of postnatal microglial cell activation.

Maternal infection during pregnancy increases risk for neurodevelopmental disorders and reduced stress resilience in offspring, but the mechanisms are not fully understood. We hypothesized that piglets born from gilts infected with a respiratory virus during late gestation would exhibit aberrant microglia activity, cognitive deficits and reduced sociability. Pregnant gilts were inoculated with porcine reproductive and respiratory syndrome virus (PRRSV; 5×105 TCID50 of live PRRSV) or saline at gestational day 76. Gilts infected with PRRSV exhibited fever (p<0.01) and reduced appetite (p<0.001) for 2weekspost-inoculation and were PRRSV-positive at parturition. Piglets born from infected and control gilts were weaned at postnatal day (PD) 1 and assigned to two groups. Group 1 was challenged with lipopolysaccharide (LPS, 5µg/kg body weight i.p.) or saline on PD 14 and tissues were collected. Group 2 was tested in a T-maze task to assess spatial learning and in a 3-chamber arena with unfamiliar conspecifics to assess social behavior from PD 14-27. Microglia (CD11b+ CD45low) isolated from Group 2 piglets at PD 28 were challenged ex vivo with LPS; a subset of cells was analyzed for MHCII expression. Maternal infection did not affect offspring circulating TNFα, IL-10, or cortisol levels basally or 4h post-LPS challenge. While performance in the T-maze task was not affected by maternal infection, both sociability and preference for social novelty were decreased in piglets from infected gilts. There was no effect of maternal infection on microglial MHCII expression or LPS-induced cytokine production. Taken together, these results suggest the reduced social behavior elicited by maternal infection is not due to aberrant microglia activity postnatally.

Desipramine decreases expression of human and murine indoleamine-2,3-dioxygenases.

Abundant evidence connects depression symptomology with immune system activation, stress and subsequently elevated levels of kynurenine. Anti-depressants, such as the tricyclic norepinephrine/serotonin reuptake inhibitor desipramine (Desip), were developed under the premise that increasing extracellular neurotransmitter level was the sole mechanism by which they alleviate depressive symptomologies. However, evidence suggests that anti-depressants have additional actions that contribute to their therapeutic potential. The Kynurenine Pathway produces tryptophan metabolites that modulate neurotransmitter activity. This recognition identified another putative pathway for anti-depressant targeting. Considering a recognized role of the Kynurenine Pathway in depression, we investigated the potential for Desip to alter expression of rate-limiting enzymes of this pathway: indoleamine-2,3-dioxygenases (Ido1 and Ido2). Mice were administered lipopolysaccharide (LPS) or synthetic glucocorticoid dexamethasone (Dex) with Desip to determine if Desip alters indoleamine-dioxygenase (DO) expression in vivo following a modeled immune and stress response. This work was followed by treating murine and human peripheral blood mononuclear cells (PBMCs) with interferon-gamma (IFNγ) and Desip. In vivo: Desip blocked LPS-induced Ido1 expression in hippocampi, astrocytes, microglia and PBMCs and Ido2 expression by PBMCs. Ex vivo: Desip decreased IFNγ-induced Ido1 and Ido2 expression in murine PBMCs. This effect was directly translatable to the human system as Desip decreased IDO1 and IDO2 expression by human PBMCs. These data demonstrate for the first time that an anti-depressant alters expression of Ido1 and Ido2, identifying a possible new mechanism behind anti-depressant activity. Furthermore, we propose the assessment of PBMCs for anti-depressant responsiveness using IDO expression as a biomarker.

Interactions between inflammatory mediators and corticosteroids regulate transcription of genes within the Kynurenine Pathway in the mouse hippocampus.

BACKGROUND: Increased tryptophan metabolism towards the production of kynurenine via indoleamine/tryptophan-2,3-dioxygenases (DOs: Ido1, Ido2, and Tdo2) is strongly associated with the prevalence of major depressive disorder in patients and the induction of depression-like behaviors in animal models. Several studies have suggested that activation of the immune system or elevated corticosteroids drive DO expression; however, mechanisms linking cytokines, corticosteroids, and DOs to psychiatric diseases remain unclear. Various attempts have been made to correlate DO gene expression within the brain to behavior, but disparate results have been obtained. We believe that discrepancies arise as a result of the under-recognized existence of multiple mRNA transcripts for each DO. Unfortunately, there are no reports regarding how the multiple transcripts are distributed or regulated. Here, we used organotypic hippocampal slice cultures (OHSCs) to directly test the ability of inflammatory and stress mediators to differentially regulate DO transcripts. METHODS: OHSCs were treated with pro-inflammatory mediators (interferon-gamma (IFNγ), lipopolysaccharide (LPS), and polyinosine-polycytidylic acid (pI:C)) with or without corticosteroids (dexamethasone (Dex: glucocorticoid receptor (GR) agonist), aldosterone (Aldo: mineralocorticoid receptor (MR) agonist), or corticosterone (Cort: GR/MR agonist)). RESULTS: IFNγ induced Ido1-full length (FL) and Ido1-variant (v) expression, and surprisingly, Dex, Cort, and Aldo interacted with IFNγ to further elevate expression of Ido1, importantly, in a transcript dependent manner. IFNγ, LPS, and pI:C increased expression of Ido2-v1 and Ido2-v3 transcripts, whereas only IFNγ increased expression of Ido2-v2. Overall Ido2 transcripts were relatively unaffected by GR or MR activation. Naïve mouse brain expresses multiple Tdo2 transcripts. Dex and Cort induced expression of only one of the three Tdo2 transcripts (Tdo2-FL) in OHSCs. CONCLUSIONS: These results establish that multiple transcripts for all three DOs are expressed within the mouse hippocampus, under the control of distinct regulatory pathways. These data identify a previously unrecognized interaction between corticosteroid receptor activation and inflammatory signals on DO gene expression, which suggest that corticosteroids act to differentially enhance gene expression of Ido1, Ido2, and Tdo2.

Advancing the understanding of behaviors associated with Bacille Calmette Guérin infection using multivariate analysis.

Behavioral indicators in the murine Bacille Calmette Guérin (BCG) model of inflammation have been studied individually; however, the variability of the behaviors across BCG levels and the mouse-to-mouse variation within BCG-treatment group are only partially understood. The objectives of this study were: (1) to gain a comprehensive understanding of sickness and depression-like behaviors in a BCG model of inflammation using multivariate approaches, and (2) to explore behavioral differences between BCG-treatment groups and among mice within group. Adult mice were challenged with either 0mg (saline), 5mg or 10mg of BCG (BCG-treatment groups: BCG0, BCG5, or BCG10, respectively) at Day 0 of the experiment. Sickness indicators included body weight changes between Day 0 and Day 2 and between Day 2 and Day 5, and horizontal locomotor activity and vertical activity (rearing) measured at Day 6. Depression-like indicators included duration of immobility in the forced swim test and in the tail suspension test at Day 6 and sucrose consumption in the sucrose preference test at Day 7. The simultaneous consideration of complementary sickness and depression-like indicators enabled a more precise characterization of behavioral changes associated with BCG-treatment and of mouse-to-mouse variation, relative to the analysis of indicators individually. Univariate and multivariate analyses confirmed differences between BCG-treatment groups in weight change early on the trial. Significant differences between BCG-treatment groups in depression-like behaviors were still measurable after Day 5. The potential for multivariate models to account for the correlation between behavioral indicators and to augment the analytical precision relative to univariate models was demonstrated both for sickness and for depression-like indicators. Unsupervised learning approaches revealed the complementary information provided by the sickness and depression-like indicators considered. Supervised learning approaches using cross-validation confirmed subtle differences between BCG-treatment groups and among mice within group identified by the consideration of sickness and depression-like indicators. These findings support the recommendation for multivariate and multidimensional analyses of sickness and depression-like indicators to augment the systemic understanding of the behavioral changes associated with infection.

IL-4 Knock Out Mice Display Anxiety-Like Behavior.

Inflammation is a recognized antecedent and coincident factor when examining the biology of anxiety. Little is known, however, about how reductions in endogenous anti-inflammatory mediators impact anxiety. Therefore, mood- cognition- and anxiety-associated/like behaviors were examined in IL-4 knock out (KO) mice and wild-type (WT) mice. In comparison to WT mice, IL-4 KO mice demonstrated decreased burrowing and increased social exploration. No differences were seen in forced swim or saccharine preference testing. IL-4 KO mice had similar performance to WT mice in the Morris water maze and during object location and novel object recognition. In the elevated zero-maze, IL-4 KO mice, in comparison to WT mice, demonstrated anxiety-like behavior. Anxiety-like behavior in IL-4 KO mice was not observed, however, during open-field testing. Taken together, these data indicate that IL-4 KO mice display state, but not trait, anxiety suggesting that reductions in endogenous anti-inflammatory bioactives can engender subtypes of anxiety.

Interleukin-1 beta converting enzyme is necessary for development of depression-like behavior following intracerebroventricular administration of lipopolysaccharide to mice.

BACKGROUND: Interleukin-1 beta converting enzyme (ICE, caspase 1) is a cysteine protease that processes immature pro-IL-1ß into active mature IL-1ß. IL-1ß is a pro-inflammatory cytokine that mediates many of the physiological and behavioral responses to inflammation. Genetic deletion of ICE has previously been shown to prevent some negative physiologic responses to lipopolysaccharide (LPS)-induced inflammation. METHODS: Here we used a preclinical murine model to test the hypothesis that ICE is necessary for development of depression-like behaviors following intracerebroventricular (ICV) treatment with LPS. Adult male ICE knockout (ICE KO) and congenic wild-type C57BL/6 J (WT) mice were administered LPS either ICV at 100 ng/mouse or intraperitoneally (IP) at 830 µg/kg body weight or an equal volume of saline as controls. Mice were monitored up to 48 h after treatment for both sickness and depression-like behaviors. RESULTS: LPS given ICV induced a loss of body weight in both WT and ICE KO mice. This sickness response was similar between WT and ICE KO mice. As expected, LPS administered ICV increased immobility in the forced swim test (FST) and decreased sucrose preference in WT mice but no change in either of these two depression-like behaviors was observed in ICE KO mice. Expression of TNF-α and CD11b in brain was lower in ICE-KO mice at 24 h following ICV administration of LPS compared to WT mice. In contrast, when LPS was given systemically, sickness response, depression-like behaviors, and expression of these genes were similar between the two strains of mice. CONCLUSIONS: These findings indicate that ICE plays a specific role in depression-like behavior induced by a central inflammatory stimuli even though it is not required when LPS is administered systemically.

Intracerebroventricular administration of lipopolysaccharide induces indoleamine-2,3-dioxygenase-dependent depression-like behaviors.

BACKGROUND: Activation of the tryptophan degrading enzyme indoleamine-2,3-dioxygenase 1 (IDO1) is associated with the development of behavioral signs of depression. Systemic immune challenge induces IDO1 in both the periphery and the brain, leading to increased circulating and brain concentrations of kynurenines. However, whether IDO1 activity within the brain is necessary for the manifestation of depression-like behavior of mice following a central immune challenge remains to be elucidated. METHODS: We investigated the role of brain IDO1 in mediating depression-like behavior of mice in response to intracerebroventricular injection of saline or lipopolysaccharide (LPS, 10 ng). RESULTS: LPS increased the duration of immobility in the tail suspension test and decreased preference for a sucrose solution. These effects were associated with an activation of central but not peripheral IDO1, as LPS increased brain kynurenine but had no effect on plasma concentrations of kynurenine. Interestingly, genetic deletion or pharmacological inhibition of IDO1, using 1-methyl-tryptophan, abrogated the reduction in sucrose preference induced by intracerebroventricular LPS. 1-Methyl-tryptophan also blocked the LPS-induced increase in duration of immobility during the tail suspension test. CONCLUSIONS: These data indicate that activation of brain IDO1 is sufficient to induce depression-like behaviors of mice in response to central LPS.

Aging leads to prolonged duration of inflammation-induced depression-like behavior caused by Bacillus Calmette-Guérin.

Geriatric depression is a costly health issue, but little is known about its physiological underpinnings. Systemic inflammation sensitizes the innate immune system of aged animals and humans, but it is unknown if chronic, low-grade infections affect the duration of depressive-like behaviors. In this report, we infected adult (4-6 months) and aged (20-24 months) Balb/c mice with an attenuated strain of Mycobacterium bovis, Bacillus Calmette-Guérin (BCG), to induce a chronic infection. We then measured depression-like behaviors that have construct, face and predictive validity for human inflammation-associated clinical depression. Exposure to BCG caused acute sickness responses in both adult and aged mice. However, sickness behavior was prolonged in aged mice, as assessed by both locomotor and rearing activity. Two measures of depression-like behavior, which were tests involving sucrose preference and tail suspension, both showed that adult mice displayed depression-like behaviors at one day and seven days after exposure to BCG. However, aged mice continued to express both of these depression-like behaviors at three weeks following infection. Infection with BCG caused an increase in tryptophan catabolism, as evidenced by a significant rise in the plasma kynurenine/tryptophan ratio that peaked at 7 days post-infection. In aged mice, greater tryptophan catabolism persisted longer and remained elevated at 21 days post-infection. This finding is consistent with the prolonged duration of depression-like behaviors in aged mice. These are the first data using a chronic infection model to establish that recovery from inflammation-induced depression-like behavior and tryptophan catabolism are prolonged in aged animals.

HIV-1 Tat activates indoleamine 2,3 dioxygenase in murine organotypic hippocampal slice cultures in a p38 mitogen-activated protein kinase-dependent manner.

BACKGROUND: We have established that activation of the tryptophan degrading enzyme indoleamine 2,3 dioxygenase (IDO) mediates the switch from cytokine-induced sickness behavior to depressive-like behavior. Because human immunodeficiency virus type 1 (HIV-1) Tat protein causes depressive-like behavior in mice, we investigated its ability to activate IDO in organotypic hippocampal slice cultures (OHSCs) derived from neonatal C57BL/6 mice. METHODS: Depressive-like behavior in C57BL/6J mice was assessed by the forced swim test. Expression of cytokines and IDO mRNA in OHSCs was measured by real-time RT-PCR and cytokine protein was measured by enzyme-linked immunosorbent assays (ELISAs). p38 MAPK phosphorylation was analyzed by western blot. RESULTS: Intracerebroventricular (i.c.v.) administration of Tat (40 ng) induced depressive-like behavior in the absence of sickness. Addition of Tat (40 ng/slice) to the medium of OHSCs induced IDO steady-state mRNA that peaked at 6 h. This effect was potentiated by pretreatment with IFNγ. Tat also induced the synthesis and release of TNFα and IL-6 protein in the supernatant of the slices and increased expression of the inducible isoform of nitric oxide synthase (iNOS) and the serotonin transporter (SERT). Tat had no effect on endogenous synthesis of IFNγ. To explore the mechanisms of Tat-induced IDO expression, slices were pretreated with the p38 mitogen-activated protein kinase (MAPK) inhibitor SB 202190 for 30 min before Tat treatment. SB 202190 significantly decreased IDO expression induced by Tat, and this effect was accompanied by a reduction of Tat-induced expression of TNFα, IL-6, iNOS and SERT. CONCLUSION: These data establish that Tat induces IDO expression via an IFNγ-independent mechanism that depends upon activation of p38 MAPK. Targeting IDO itself or the p38 MAPK signaling pathway could provide a novel therapy for comorbid depressive disorders in HIV-1-infected patients.

Rate of fall in blood glucose and recurrent hypoglycemia affect glucose dynamics and noradrenergic activation in the ventromedial hypothalamus.

Noradrenergic activity in the ventromedial hypothalamus (VMH) is increased and activates a sympathoadrenal response during hypoglycemia. How the rate at which hypoglycemia develops affects local glucose concentrations and norepinephrine (NE) release was evaluated by placing microdialysis probes into the VMH of male Sprague-Dawley rats receiving insulin (20 mU·kg(-1)·min(-1)) and variable glucose infusions. During a first episode of hypoglycemia, interstitial glucose concentrations in the VMH generally declined at the same rate as plasma glucose; however, the faster hypoglycemia developed, the greater the magnitude of the initial NE release in the VMH (r(2) = 0.72, P < 0.001). Following recurrent episodes of hypoglycemia, VMH glucose decreased at a slower rate than plasma glucose, and the initial NE release was attenuated at the same rates of blood glucose decline. The plasma glucose threshold for the initial NE release in VMH was similar for all groups (∼3.23 mM); however, the VMH glucose threshold was stimulated and was lower when blood glucose declined more slowly (0.86 ± 0.06 vs. 1.06 ± 0.04 mmol/l, P < 0.01). The timing of the initial increase in NE release in VMH corresponded with an increase in plasma epinephrine during the first episode of hypoglycemia but not following recurrent hypoglycemia. Although a decrease in VMH glucose concentration is required for noradrenergic activation in VMH, there does not appear to be a set glucose threshold within the VMH for activation of this response.

Intracerebroventricular administration of HIV-1 Tat induces brain cytokine and indoleamine 2,3-dioxygenase expression: a possible mechanism for AIDS comorbid depression.

Human immunodeficiency virus (HIV) remains a major public health concern despite a large education effort during the past 25 years. A persistent problem with HIV infection is the high comorbity rate of clinical depression. We previously established that increasing proinflammatory cytokines within the brain of mice induces sickness that can culminate in depressive-like behavior. Here we investigated the role of the HIV transactivator of transcription (Tat) protein in activation of brain cytokine signaling and subsequent induction of depressive-like behavior in a murine model. Adult Balb/c mice were administered a single intracerebroventricular (ICV) injection of Tat (40 ng). Social investigation of a novel juvenile was measured at 2, 4, 8 and 24 h post-treatment. Mice treated with Tat did not display signs of sickness, as measured by either decreased social investigation or loss of body weight. At 24 h post-injection, mice were subjected to the forced swim test (FST). ICV administration of Tat to Balb/c mice increased immobility in the FST at 24 h post injection. A different strain of mice, C57BL/6J, responded similarly in the FST. Furthermore, adult C57BL/6J mice injected with Tat and tested in a two-bottle 1% sucrose preference test displayed reduced preference for sucrose during the 24 h post-injection period. Subsequently, brain tissues from Tat-treated and control C57BL/6J mice were collected at 4 and 24 h post injection. CNS tissue from Tat-treated mice had increased expression of IL-1ß, TNF-α, IL-6, and IDO mRNAs at 4 h post injection. These data demonstrate that a single exposure to Tat in the brain is sufficient to induce brain cytokine signaling that culminates in depressive-like behavior. The results reveal a potential role for Tat in the development of comorbid depression in HIV-infected individuals.

Induction of IDO by bacille Calmette-Guérin is responsible for development of murine depressive-like behavior.

Chronic inflammation activates the tryptophan-degrading enzyme IDO, which is well known to impair T cell proliferation. We have previously established that bacille Calmette-Guérin (BCG), an attenuated form of Mycobacterium bovis, is associated with persistent activation of IDO in the brain and chronic depressive-like behavior, but a causative role has not been established. In these experiments we used both pharmacologic and genetic approaches to test the hypothesis that IDO activation is responsible for the development of chronic depression that follows BCG infection. BCG induced TNF-alpha, IFN-gamma, and IDO mRNA steady-state transcripts in the brain as well as the enzyme 3-hydroxyanthranilic acid oxygenase (3-HAO) that lies downstream of IDO and generates the neuroactive metabolite, quinolinic acid. Behaviors characteristic of depression were apparent 1 wk after BCG infection. Pretreatment with the competitive IDO inhibitor 1-methyltryptophan fully blocked BCG-induced depressive-like behaviors. Importantly, IDO-deficient mice were completely resistant to BCG-induced depressive-like behavior but responded normally to BCG induction of proinflammatory cytokines. These results are the first to prove that the BCG-induced persistent activation of IDO is accompanied by the induction of 3-hydroxyanthranilic acid oxygenase and that IDO is required as an initial step for the subsequent development of chronic depressive-like behavior.

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.

Although the tryptophan-degrading enzyme, indoleamine 2,3-dioxygenase (IDO), is a pivotal mediator of inflammation-induced depression, its mechanism of regulation has not yet been investigated in this context. Here, we demonstrate an essential role for interferon (IFN)gamma and tumor necrosis factor (TNF)alpha in the induction of IDO and depressive-like behaviors in response to chronic immune activation. Wild-type (WT) control mice and IFNgammaR(-/-) mice were inoculated with an attenuated form of Mycobacterium bovis, bacille Calmette-Guérin (BCG). Infection with BCG induced an acute episode of sickness that was similar in WT and IFNgammaR(-/-) mice. Increased immobility during the forced swim and tail suspension tests occurred in WT mice 7 d after BCG inoculation but was entirely absent in IFNgammaR(-/-) mice. In WT mice, these indices of depressive-like behavior were associated with chronic upregulation of IFNgamma, interleukin(IL)-1beta, TNFalpha, and IDO. Proinflammatory cytokine expression was elevated in BCG-infected IFNgammaR(-/-) mice as well, but upregulation of lung and brain IDO mRNA was completely abolished. This was accompanied by an attenuation of BCG-induced TNFalpha mRNA and the lack of an increase in plasma kynurenine/tryptophan ratio in the BCG-inoculated IFNgammaR(-/-) mice compared with WT controls. Pretreatment of mice with the TNFalpha antagonist, etanercept, partially blunted BCG-induced IDO activation and depressive-like behavior. In accordance with these in vivo data, IFNgamma and TNFalpha synergized to induce IDO in primary microglia. Together, these data demonstrate that IFNgamma, with TNFalpha, is necessary for induction of IDO and depressive-like behavior in mice after BCG infection.

LPS-induced indoleamine 2,3-dioxygenase is regulated in an interferon-gamma-independent manner by a JNK signaling pathway in primary murine microglia.

Inflammation-induced activation of the tryptophan catabolizing enzyme indoleamine 2,3-dioxygenase (IDO) causes depressive-like behavior in mice following acute activation of the innate immune system by lipopolysaccharide (LPS). Here we investigated the mechanism of IDO expression induced by LPS in primary cultures of microglia derived from neonatal C57BL/6J mice. LPS (10 ng/ml) induced IDO transcripts that peaked at 8h and enzymatic activity at 24h, resulting in an increase in extracellular kynurenine, the catabolic product of IDO-induced tryptophan catabolism. This IDO induction by LPS was accompanied by synthesis and secretion of the proinflammatory cytokines TNFalpha and IL-6, but without detectable IFNgamma expression. To explore the mechanism of LPS-induced IDO expression, microglia were pretreated with the c-Jun-N-terminal kinase (JNK) inhibitor SP600125 for 30 min before LPS treatment. We found that SP600125 blocked JNK phosphorylation and significantly decreased IDO expression induced by LPS, which was accompanied by a reduction of LPS-induced expression of TNFalpha and IL-6. Collectively, these data extend to microglia the property that LPS induces IDO expression via an IFNgamma-independent mechanism that depends upon activation of JNK. Inhibition of the JNK pathway may provide a new therapy for inflammatory depression.

Primary murine microglia are resistant to nitric oxide inhibition of indoleamine 2,3-dioxygenase.

Indoleamine 2,3-dioxygenase (IDO) is an intracellular heme-containing enzyme that is activated by proinflammatory cytokines, including interferon-γ (IFNγ), and metabolizes tryptophan along the kynurenine pathway. Activation of murine macrophages induces not only IDO but also nitric oxide synthase (iNOS), and the ensuing production of nitric oxide (NO) inhibits IDO. To determine the sensitivity of primary cultures of murine microglia to NO, microglia were stimulated with recombinant murine IFNγ (1 ng/ml) and lipopolysaccharide (LPS) (10 ng/ml). This combination of IFNγ+LPS synergized to produce maximal amounts of nitrite as early as 16h. Steady-state mRNAs for both iNOS and IDO were significantly increased by IFNγ+LPS at 4h post-treatment, followed by an increase in IDO enzymatic activity at 24h. Murine microglia (>95% CD11b(+)) were pretreated with the iNOS inhibitor, L-NIL hydrochloride, at a dose (30 µM) that completely abrogated production of nitrite. L-NIL had no effect on IDO mRNA at 4h or IDO enzymatic activity at 24h following stimulation with IFNγ+LPS. These data establish that IDO regulation in murine microglia is not restrained by NO, thereby permitting the accumulation of kynurenine and its downstream metabolites in the central nervous system.

Butyrate and Dietary Soluble Fiber Improve Neuroinflammation Associated With Aging in Mice.

Aging results in chronic systemic inflammation that can alter neuroinflammation of the brain. Specifically, microglia shift to a pro-inflammatory phenotype predisposing them to hyperactivation upon stimulation by peripheral immune signals. It is proposed that certain nutrients can delay brain aging by preventing or reversing microglial hyperactivation. Butyrate, a short-chain fatty acid (SCFA) produced primarily by bacterial fermentation of fiber in the colon, has been extensively studied pharmacologically as a histone deacetylase inhibitor and serves as an attractive therapeutic candidate, as butyrate has also been shown to be anti-inflammatory and improve memory in animal models. In this study, we demonstrate that butyrate can attenuate pro-inflammatory cytokine expression in microglia in aged mice. It is still not fully understood, however, if an increase in butyrate-producing bacteria in the gut as a consequence of a diet high in soluble fiber could affect microglial activation during aging. Adult and aged mice were fed either a 1% cellulose (low fiber) or 5% inulin (high fiber) diet for 4 weeks. Findings indicate that mice fed inulin had an altered gut microbiome and increased butyrate, acetate, and total SCFA production. In addition, histological scoring of the distal colon demonstrated that aged animals on the low fiber diet had increased inflammatory infiltrate that was significantly reduced in animals consuming the high fiber diet. Furthermore, gene expression of inflammatory markers, epigenetic regulators, and the microglial sensory apparatus (i.e., the sensome) were altered by both diet and age, with aged animals exhibiting a more anti-inflammatory microglial profile on the high fiber diet. Taken together, high fiber supplementation in aging is a non-invasive strategy to increase butyrate levels, and these data suggest that an increase in butyrate through added soluble fiber such as inulin could counterbalance the age-related microbiota dysbiosis, potentially leading to neurological benefits.

Inhibition of DNA Methylation With Zebularine Alters Lipopolysaccharide-Induced Sickness Behavior and Neuroinflammation in Mice.

Activity of DNA methyltransferases (DNMTs), the enzymes that catalyze DNA methylation, is dynamically regulated in the brain. DNMT inhibitors alter DNA methylation globally in the brain and at individual neural plasticity-associated genes, but how DNMT inhibitors centrally influence lipopolysaccharide (LPS)-induced neuroinflammation is not known. We investigated whether the DMNT inhibitor, zebularine, would alter sickness behavior, DNA methylation of the Il-1ß promoter and expression of inflammatory genes in hippocampus and microglia. Contrary to our hypothesis that zebularine may exaggerate LPS-induced sickness response and neuroinflammation, adult mice treated with an intracerebroventricular (ICV) injection of zebularine prior to LPS had surprisingly faster recovery of burrowing behavior compared to mice treated with LPS. Further, genes of inflammatory markers, epigenetic regulators, and the microglial sensory apparatus (i.e., the sensome) were differentially expressed by zebularine alone or in combination with LPS. Bisulfite pyrosequencing revealed that ICV zebularine led to decreased DNA methylation of two CpG sites near the Il-1ß proximal promoter alone or in combination with LPS. Zebularine treated mice still exhibited decreased DNA methylation 48 h after treatment when LPS-induced sickness behavior as well as hippocampal and microglial gene expression were similar to control mice. Taken together, these data suggest that decreased DNA methylation, specifically of the Il-1ß promoter region, with a DNMT inhibitor in the brain disrupts molecular mechanisms of neuroinflammation.