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.

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.

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.

Disruption of microglia histone acetylation and protein pathways in mice exhibiting inflammation-associated depression-like symptoms.

BACKGROUND: Peripheral immune challenge can elicit microglia activation and depression-related symptoms. The balance of inflammatory signals in the tryptophan pathway can skew the activity of indoleamine-pyrrole 2,3 dioxygenase (IDO1) towards the metabolization of tryptophan into kynurenine (rather than serotonin), and towards neuroprotective or neurotoxic metabolites. The proteome changes that accompany inflammation-associated depression-related behaviors are incompletely understood. METHODS: The changes in microglia protein abundance and post-translational modifications in wild type (WT) mice that exhibit depression-like symptoms after recovery from peripheral Bacille Calmette-Guerin (BCG) challenge were studied. This WT_BGG group was compared to mice that do not express depression-like symptoms after BCG challenge due to IDO1 deficiency by means of genetic knockout (BCG_KO group), and to WT Saline-treated (Sal) mice (WT_Sal group) using a mass spectrometry-based label-free approach. RESULTS: The comparison of WT_BCG relative to WT_Sal and KO_BCG mice uncovered patterns of protein abundance and acetylation among the histone families that could influence microglia signaling and transcriptional rates. Members of the histone clusters 1, 2 and 3 families were less abundant in WT_BCG relative to WT_Sal whereas members in the H2A family exhibited the opposite pattern. Irrespective of family, the majority of the histones were less abundant in WT_BCG relative to KO_BCG microglia. Homeostatic mechanisms may temper the potentially toxic effects of high histone levels after BCG challenge to levels lower than Sal. Histone acetylation was highest in WT_BCG and the similar levels observed in WT_Sal and KO_BCG. This result suggest that histone acetylation levels are similar between IDO1 deficient mice after immune challenge and unchallenged WT mice. The over-abundance of tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation proteins (14-3-3 series) in WT_BCG relative to KO_BCG is particularly interesting because these proteins activate another rate-limiting enzyme in the tryptophan pathway. The over-representation of alcoholism and systemic lupus erythematosus pathways among the proteins exhibiting differential abundance between the groups suggest that these disorders share microglia activation pathways with BCG challenge. The over-representation of phagosome pathway among proteins differentially abundant between WT_BCG and KO_BCG microglia suggest an association between IDO1 deficiency and phagocytosis. Likewise, the over-representation of the gap junction pathway among the differentially abundant proteins between KO_BCG and WT_Sal suggest a multifactorial effect of BCG and IDO1 deficiency on cell communication. CONCLUSIONS: The present study of histone acetylation and differential protein abundance furthers the understanding of the long lasting effects of peripheral immune challenges. Our findings offer insights into target proteins and mechanisms that provide clues for therapies to ameliorate inflammation-associated depression-related behaviors.

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.

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.

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.

Immunomodulatory Factors Galectin-9 and Interferon-Gamma Synergize to Induce Expression of Rate-Limiting Enzymes of the Kynurenine Pathway in the Mouse Hippocampus.

Elevated levels of circulating pro-inflammatory cytokines are associated with symptomology of several psychiatric disorders, notably major depressive disorder. Symptomology has been linked to inflammation/cytokine-dependent induction of the Kynurenine Pathway. Galectins, like pro-inflammatory cytokines, play a role in neuroinflammation and the pathogenesis of several neurological disorders but without a clearly defined mechanism of action. Their involvement in the Kynurenine Pathway has not been investigated. Thus, we searched for a link between galectins and the Kynurenine Pathway using in vivo and ex vivo models. Mice were administered LPS and pI:C to determine if galectins (Gal's) were upregulated in the brain following in vivo inflammatory challenges. We then used organotypic hippocampal slice cultures (OHSCs) to determine if Gal's, alone or with inflammatory mediators [interferon-gamma (IFNγ), tumor necrosis factor-alpha (TNFα), interleukin-1beta (IL-1ß), polyinosine-polycytidylic acid (pI:C), and dexamethasone (Dex; synthetic glucocorticoid)], would increase expression of indoleamine/tryptophan-2,3-dioxygenases (DO's: Ido1, Ido2, and Tdo2; Kynurenine Pathway rate-limiting enzymes). In vivo, hippocampal expression of cytokines (IL-1ß, TNFα, and IFNγ), Gal-3, and Gal-9 along with Ido1 and Ido2 were increased by LPS and pI:C (bacterial and viral mimetics). Of the cytokines induced in vivo, only IFNγ increased expression of two Ido1 transcripts (Ido1-FL and Ido1-v1) by OHSCs. Although ineffective alone, Gal-9 accentuated IFNγ-induced expression of only Ido1-FL. Similarly, IFNγ induced expression of several Ido2 transcripts (Ido2-v1, Ido2-v3, Ido2-v4, Ido2-v5, and Ido2-v6). Gal-9 accentuated IFNγ-induced expression of only Ido2-v1. Surprisingly, Gal-9 alone, slightly but significantly, induced expression of Tdo2 (Tdo2-v1 and Tdo2-v2, but not Tdo2-FL). These effects were specific to Gal-9 as Gal-1 and Gal-3 did not alter DO expression. These results are the first to show that brain Gal-9 is increased during LPS- and pI:C-induced neuroinflammation. Increased expression of Gal-9 may be critical for neuroinflammation-dependent induction of DO expression, either acting alone (Tdo2-v1 and Tdo2-v2) or to enhance IFNγ activity (Ido1-FL and Ido2-v1). Although these novel actions of Gal-9 are described for hippocampus, they have the potential to operate as DO-dependent immunomodulatory processes outside the brain. With the expanding implications of Kynurenine Pathway activation across multiple immune and psychiatric disorders, this synergy provides a new target for therapeutic development.

Aging and peripheral lipopolysaccharide can modulate epigenetic regulators and decrease IL-1ß promoter DNA methylation in microglia.

In aged mice, peripheral stimulation of the innate immune system with lipopolysaccharide (LPS) causes exaggerated neuroinflammation and prolonged sickness behavior due in part to microglial dysfunction. Epigenetic changes to DNA may play a role in microglial dysfunction; therefore, we sought to determine whether aged microglia displayed DNA hypomethylation of the interleukin-1 beta (IL-1ß) promoter and altered expression of epigenetic regulators. We further examined whether the demethylating agent 5-azacytidine induced IL-1ß expression in BV2 and primary microglia similar to microglia from aged mice. Novel findings indicated that aged mice had decreased methylation of the IL-1ß gene promoter in primary microglia basally or following systemic LPS that is associated with increased IL-1ß mRNA, intracellular IL-1ß production, as well as prolonged sickness behavior. Last, 5-azacytidine increased IL-1ß gene expression and decreased DNA methylation of BV2 and primary microglial cells similar to microglia from aged mice. Taken together, these data indicate that DNA methylation promotes heightened microglial activation in the aged brain.

Differential Transcriptome Networks between IDO1-Knockout and Wild-Type Mice in Brain Microglia and Macrophages.

Microglia in the brain and macrophages in peripheral organs are cell types responsible for immune response to challenges. Indoleamine 2,3-dioxygenase 1 (IDO1) is an immunomodulatory enzyme of the tryptophan pathway that is expressed in the brain. The higher activity of IDO1 in response to immune challenge has been implicated in behavioral disorders. The impact of IDO1 depletion on the microglia transcriptome has not been studied. An investigation of the transcript networks in the brain microglia from IDO1-knockout (IDO1-KO) mice was undertaken, relative to peripheral macrophages and to wild-type (WT) mice under unchallenged conditions. Over 105 transcript isoforms were differentially expressed between WT and IDO1-KO within cell type. Within microglia, Saa3 and Irg1 were over-expressed in IDO1-KO relative to WT. Within macrophages, Csf3 and Sele were over-expressed in IDO1-KO relative to WT. Among the genes differentially expressed between strains, enriched biological processes included ion homeostasis and ensheathment of neurons within microglia, and cytokine and chemokine expression within macrophages. Over 11,110 transcript isoforms were differentially expressed between microglia and macrophages and of these, over 10,800 transcripts overlapped between strains. Enriched biological processes among the genes over- and under-expressed in microglia relative to macrophages included cell adhesion and apoptosis, respectively. Detected only in microglia or macrophages were 421 and 43 transcript isoforms, respectively. Alternative splicing between cell types based on differential transcript isoform abundance was detected in 210 genes including Phf11d, H2afy, and Abr. Across strains, networks depicted a predominance of genes under-expressed in microglia relative to macrophages that may be a precursor for the different response of both cell types to challenges. The detected transcriptome differences enhance the understanding of the role of IDO1 in the microglia transcriptome under unchallenged conditions.

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.

Microglia Transcriptome Changes in a Model of Depressive Behavior after Immune Challenge.

Depression symptoms following immune response to a challenge have been reported after the recovery from sickness. A RNA-Seq study of the dysregulation of the microglia transcriptome in a model of inflammation-associated depressive behavior was undertaken. The transcriptome of microglia from mice at day 7 after Bacille Calmette Guérin (BCG) challenge was compared to that from unchallenged Control mice and to the transcriptome from peripheral macrophages from the same mice. Among the 562 and 3,851 genes differentially expressed between BCG-challenged and Control mice in microglia and macrophages respectively, 353 genes overlapped between these cells types. Among the most differentially expressed genes in the microglia, serum amyloid A3 (Saa3) and cell adhesion molecule 3 (Cadm3) were over-expressed and coiled-coil domain containing 162 (Ccdc162) and titin-cap (Tcap) were under-expressed in BCG-challenged relative to Control. Many of the differentially expressed genes between BCG-challenged and Control mice were associated with neurological disorders encompassing depression symptoms. Across cell types, S100 calcium binding protein A9 (S100A9), interleukin 1 beta (Il1b) and kynurenine 3-monooxygenase (Kmo) were differentially expressed between challenged and control mice. Immune response, chemotaxis, and chemokine activity were among the functional categories enriched by the differentially expressed genes. Functional categories enriched among the 9,117 genes differentially expressed between cell types included leukocyte regulation and activation, chemokine and cytokine activities, MAP kinase activity, and apoptosis. More than 200 genes exhibited alternative splicing events between cell types including WNK lysine deficient protein kinase 1 (Wnk1) and microtubule-actin crosslinking factor 1(Macf1). Network visualization revealed the capability of microglia to exhibit transcriptome dysregulation in response to immune challenge still after resolution of sickness symptoms, albeit lower than that observed in macrophages. The persistent transcriptome dysregulation in the microglia shared patterns with neurological disorders indicating that the associated persistent depressive symptoms share a common transcriptome basis.

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.

Analytical workflow profiling gene expression in murine macrophages.

Comprehensive and simultaneous analysis of all genes in a biological sample is a capability of RNA-Seq technology. Analysis of the entire transcriptome benefits from summarization of genes at the functional level. As a cellular response of interest not previously explored with RNA-Seq, peritoneal macrophages from mice under two conditions (control and immunologically challenged) were analyzed for gene expression differences. Quantification of individual transcripts modeled RNA-Seq read distribution and uncertainty (using a Beta Negative Binomial distribution), then tested for differential transcript expression (False Discovery Rate-adjusted p-value < 0.05). Enrichment of functional categories utilized the list of differentially expressed genes. A total of 2079 differentially expressed transcripts representing 1884 genes were detected. Enrichment of 92 categories from Gene Ontology Biological Processes and Molecular Functions, and KEGG pathways were grouped into 6 clusters. Clusters included defense and inflammatory response (Enrichment Score = 11.24) and ribosomal activity (Enrichment Score = 17.89). Our work provides a context to the fine detail of individual gene expression differences in murine peritoneal macrophages during immunological challenge with high throughput RNA-Seq.

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.

The saturated fatty acid, palmitic acid, induces anxiety-like behavior in mice.

OBJECTIVES: Excess fat in the diet can impact neuropsychiatric functions by negatively affecting cognition, mood and anxiety. We sought to show that the free fatty acid (FFA), palmitic acid, can cause adverse biobehaviors in mice that last beyond an acute elevation in plasma FFAs. METHODS: Mice were administered palmitic acid or vehicle as a single intraperitoneal (IP) injection. Biobehaviors were profiled 2 and 24 h after palmitic acid treatment. Quantification of dopamine (DA), norepinephrine (NE), serotonin (5-HT) and their major metabolites was performed in cortex, hippocampus and amygdala. FFA concentration was determined in plasma. Relative fold change in mRNA expression of unfolded protein response (UPR)-associated genes was determined in brain regions. RESULTS: In a dose-dependent fashion, palmitic acid rapidly reduced mouse locomotor activity by a mechanism that did not rely on TLR4, MyD88, IL-1, IL-6 or TNFα but was dependent on fatty acid chain length. Twenty-four hours after palmitic acid administration mice exhibited anxiety-like behavior without impairment in locomotion, food intake, depressive-like behavior or spatial memory. Additionally, the serotonin metabolite 5-HIAA was increased by 33% in the amygdala 24h after palmitic acid treatment. CONCLUSIONS: Palmitic acid induces anxiety-like behavior in mice while increasing amygdala-based serotonin metabolism. These effects occur at a time point when plasma FFA levels are no longer elevated.

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.

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.

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.