Indoleamine 2, 3-Dioxygenase Promotes Aryl Hydrocarbon Receptor-Dependent Differentiation Of Regulatory B Cells in Lung Cancer.

Regulatory B cells (Breg) are IL-10 producing subsets of B cells that contribute to immunosuppression in the tumor microenvironment (TME). Breg are elevated in patients with lung cancer; however, the mechanisms underlying Breg development and their function in lung cancer have not been adequately elucidated. Herein, we report a novel role for Indoleamine 2, 3- dioxygenase (IDO), a metabolic enzyme that degrades tryptophan (Trp) and the Trp metabolite L-kynurenine (L-Kyn) in the regulation of Breg differentiation in the lung TME. Using a syngeneic mouse model of lung cancer, we report that Breg frequencies significantly increased during tumor progression in the lung TME and secondary lymphoid organs, while Breg were reduced in tumor-bearing IDO deficient mice (IDO-/-). Trp metabolite L-Kyn promoted Breg differentiation in-vitro in an aryl hydrocarbon receptor (AhR), toll-like receptor-4-myeloid differentiation primary response 88, (TLR4-MyD88) dependent manner. Importantly, using mouse models with conditional deletion of IDO in myeloid-lineage cells, we identified a significant role for immunosuppressive myeloid-derived suppressor cell (MDSC)-associated IDO in modulating in-vivo and ex-vivo differentiation of Breg. Our studies thus identify Trp metabolism as a therapeutic target to modulate regulatory B cell function during lung cancer progression.

Immunosuppressive IDO in Cancer: Mechanisms of Action, Animal Models, and Targeting Strategies.

Indoleamine 2, 3-dioxygenase 1 (IDO; IDO1; INDO) is a rate-limiting enzyme that metabolizes the essential amino acid, tryptophan, into downstream kynurenines. Canonically, the metabolic depletion of tryptophan and/or the accumulation of kynurenine is the mechanism that defines how immunosuppressive IDO inhibits immune cell effector functions and/or facilitates T cell death. Non-canonically, IDO also suppresses immunity through non-enzymic effects. Since IDO targeting compounds predominantly aim to inhibit metabolic activity as evidenced across the numerous clinical trials currently evaluating safety/efficacy in patients with cancer, in addition to the recent disappointment of IDO enzyme inhibitor therapy during the phase III ECHO-301 trial, the issue of IDO non-enzyme effects have come to the forefront of mechanistic and therapeutic consideration(s). Here, we review enzyme-dependent and -independent IDO-mediated immunosuppression as it primarily relates to glioblastoma (GBM); the most common and aggressive primary brain tumor in adults. Our group's recent discovery that IDO levels increase in the brain parenchyma during advanced age and regardless of whether GBM is present, highlights an immunosuppressive synergy between aging-increased IDO activity in cells of the central nervous system that reside outside of the brain tumor but collaborate with GBM cell IDO activity inside of the tumor. Because of their potential value for the in vivo study of IDO, we also review current transgenic animal modeling systems while highlighting three new constructs recently created by our group. This work converges on the central premise that maximal immunotherapeutic efficacy in subjects with advanced cancer requires both IDO enzyme- and non-enzyme-neutralization, which is not adequately addressed by available IDO-targeting pharmacologic approaches at this time.

Deletion of indoleamine 2,3 dioxygenase (Ido)1 but not Ido2 exacerbates disease symptoms of MOG35-55-induced experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) with pathological features of inflammation, demyelination, and neurodegeneration. Several lines of evidence suggest that the enzymes indoleamine 2,3-dioxygenase (Ido)1 and/or Ido2 influences susceptibility to autoimmune diseases. Deletion of Ido1 exacerbates experimental autoimmune encephalomyelitis (EAE) an animal model of MS. However, no data exist on the role of Ido2 in the pathogenesis of EAE. We investigated whether deletion of Ido2 affected the pathogenesis of EAE. Temporal expression of interferon gamma (Ifng), Ido1 variants, Ido2 variants, as well as genes encoding enzymes of the kynurenine pathway in the spleen and spinal cord of C57BL/6 mice with or without EAE were determined by RT-qPCR. Moreover, EAE was induced in C57BL/6, two Ido1 knockout strains (Ido1KO and Ido1TK) and one Ido2 knockout mouse strain (Ido2-/-) and disease monitored by clinical scores and weight change. Performance on the rotarod was performed on days 0, 5, 10 and 15 post induction. The extent of demyelination in the spinal cord was determined after staining with Oil red O. The development of EAE altered gene expression in both the spleen and spinal cord. Deletion of Ido1 exacerbated the clinical symptoms of EAE. In stark contrast, EAE in Ido2-/- mice did not differ clinically or histologically from control mice. These results confirm a protective role for Ido1, on the pathogenesis of MOG35-55-induced EAE in C57BL/6J mice.

Handling stress impairs learning through a mechanism involving caspase-1 activation and adenosine signaling.

Acute stressors can induce fear and physiologic responses that prepare the body to protect from danger. A key component of this response is immune system readiness. In particular, inflammasome activation appears critical to linking stress to the immune system. Here, we show that a novel combination of handling procedures used regularly in mouse research impairs novel object recognition (NOR) and activates caspase-1 in the amygdala. In male mice, this handling-stress paradigm combined weighing, scruffing and sham abdominal injection once per hr. While one round of weigh/scruff/needle-stick had no impact on NOR, two rounds compromised NOR without impacting location memory or anxiety-like behaviors. Caspase-1 knockout (KO), IL-1 receptor 1 (IL-1R1) KO and IL-1 receptor antagonist (IL-RA)-administered mice were resistant to handling stress-induced loss of NOR. In addition, examination of the brain showed that handling stress increased caspase-1 activity 85% in the amygdala without impacting hippocampal caspase-1 activity. To delineate danger signals relevant to handling stress, caffeine-administered and adenosine 2A receptor (A2AR) KO mice were tested and found resistant to impaired learning and caspase-1 activation. Finally, mice treated with the ß-adrenergic receptor antagonist, propranolol, were resistant to handling stress-induced loss of NOR and caspase-1 activation. Taken together, these results indicate that handling stress-induced impairment of object learning is reliant on a pathway requiring A2AR-dependent activation of caspase-1 in the amygdala that appears contingent on ß-adrenergic receptor functionality.

Indoleamine 2,3-dioxygenase 1 deletion promotes Theiler's virus-induced seizures in C57BL/6J mice.

OBJECTIVE: Viral encephalitis increases the risk for developing seizures and epilepsy. Indoleamine 2,3-dioxygenase 1 (Ido1) is induced by inflammatory cytokines and functions to metabolize tryptophan to kynurenine. Kynurenine can be further metabolized to produce kynurenic acid and the N-methyl-d-aspartate receptor agonist quinolinic acid (QuinA). In the present study, we sought to determine the role of Ido1 in promoting seizures in an animal model of viral encephalitis. METHODS: C57BL/6J and Ido1 knockout mice (Ido1-KO) were infected with Theiler's murine encephalomyelitis virus (TMEV). Quantitative real-time polymerase chain reaction was used to evaluate hippocampal expression of proinflammatory cytokines, Ido1, and viral RNA. Body weights and seizure scores were recorded daily. Elevated zero maze was used to assess differences in behavior, and hippocampal pathology was determined by immunohistochemistry. RESULTS: Infected C57BL/6J mice up-regulated proinflammatory cytokines, Ido1, and genes encoding the enzymatic cascade responsible for QuinA production in the kynurenine pathway prior to the onset of seizures. Seizure incidence was elevated in Ido1-KO compared to C57BL/6J mice. Infection increased locomotor activity in Ido1-KO compared to C57BL/6J mice. Furthermore, the occurrence of seizures was associated with hyperexcitability. Neither expression of proinflammatory cytokines nor viral RNA was altered as a result of genotype. Immunohistochemical analysis revealed increased hippocampal pathology in Ido1-KO mice. SIGNIFICANCE: Our findings suggest that Ido1 deletion promotes seizures and neuropathogenesis during acute TMEV encephalitis.

Behavioral response to fiber feedingis cohort-dependent and associated with gut microbiota composition in mice.

Recent data has supported a role for the gut microbiota in improving cognition and shaping behavior. Here, we assessed whether pectin, a soluble, fermentable fiber, could enhance learning and memory in mice. Two cohorts of young male C57Bl/6 J mice, C1 (n = 20) and C2 (n = 20), were obtained from Jackson Laboratory and randomized to semi-purified AIN-93 M diets containing 5% pectin (n = 10) or cellulose (n = 10). After 16 weeks, learning and memory was assessed by Morris Water Maze (MWM) and microbiota composition was analyzed by 16S rRNA sequencing. Despite identical treatment, we observed differences in learning and memory abilities between cohorts, along with distinct microbiotas. In C1, pectin-fed mice spent a higher percentage of time in the target quadrant at the 24-h probe trial of the MWM versus cellulose-fed mice; in C2, no effect of pectin was observed. In both cohorts, UniFrac distance revealed significant differences in gut microbial communities between cellulose-fed and pectin-fed mice. UniFrac analysis also revealed significantly different bacterial communities between cohorts. Further analysis demonstrated that the microbial genera Oscillospira, Bilophila, and Peptostreptococcoceae were more abundant in C1 versus C2, and positively associated with distance from the platform during the 24-h probe test. These data support previous findings that differences in the gut microbiota may play a role in host response to a dietary intervention and could partly explain irreproducibility in psychological and behavioral experiments. Further research is needed to determine if a causal relationship exists.

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.

Long-term supplementation with EGCG and beta-alanine decreases mortality but does not affect cognitive or muscle function in aged mice.

We have previously shown that 6weeks of a diet containing epigallocatechin gallate (EGCG) and beta-alanine (B-ALA) was not effective in improving either cognitive or muscle function in aged (18month) mice (Gibbons et al. Behav Brain Res 2014). However, diet reduced oxidative stress in the brain, and previous studies using longer-term interventions have documented beneficial effects in cognitive, but not muscle, function. Therefore, we investigated the effect of 6months of feeding on measures of cognitive and muscle function in mice. Mice (12months, N=15/group) were fed AIN-93M containing 0.15% EGCG and 0.34% B-ALA or standard AIN-93M for 6months, then underwent a battery of tests for cognitive and muscle function at 18months. Interestingly, a higher percentage of mice receiving EGCG and B-ALA (E+B, 80%) survived to study end compared to control (Ctrl, 40%) mice (p=0.02). E+B did not affect arm preference in the Y-maze test (p=0.74, novel arm) and did not alter performance in an active avoidance test (p=0.16, avoidances per 50 trials). E+B increased rotarod performance (p=0.03), did not affect grip strength (p=0.91), and decreased time to exhaustion in a treadmill fatigue test (p=0.02) compared to Ctrl. In conclusion, E+B reduced mortality, had no effect on cognitive function and variable effects on muscle function.

Acute fasting inhibits central caspase-1 activity reducing anxiety-like behavior and increasing novel object and object location recognition.

BACKGROUND: Inflammation within the central nervous system (CNS) is frequently comorbid with anxiety. Importantly, the pro-inflammatory cytokine most commonly associated with anxiety is IL-1ß. The bioavailability and activity of IL-1ß are regulated by caspase-1-dependent proteolysis vis-a-vis the inflammasome. Thus, interventions regulating the activation or activity of caspase-1 should reduce anxiety especially in states that foster IL-1ß maturation. METHODS: Male C57BL/6j, C57BL/6j mice treated with the capase-1 inhibitor biotin-YVAD-cmk, caspase-1 knockout (KO) mice and IL-1R1 KO mice were fasted for 24h or allowed ad libitum access to food. Immediately after fasting, caspase-1 activity was measured in brain region homogenates while activated caspase-1 was localized in the brain by immunohistochemistry. Mouse anxiety-like behavior and cognition were tested using the elevated zero maze and novel object/object location tasks, respectively. RESULTS: A 24h fast in mice reduced the activity of caspase-1 in whole brain and in the prefrontal cortex, amygdala, hippocampus, and hypothalamus by 35%, 25%, 40%, 40%, and 40% respectively. A 24h fast also reduced anxiety-like behavior by 40% and increased novel object and object location recognition by 21% and 31%, respectively. IL-1ß protein, however, was not reduced in the brain by fasting. ICV administration of YVAD decreased caspase-1 activity in the prefrontal cortex and amygdala by 55%, respectively leading to a 64% reduction in anxiety like behavior. Importantly, when caspase-1 KO or IL1-R1 KO mice are fasted, no fasting-dependent reduction in anxiety-like behavior was observed. CONCLUSIONS: Results indicate that fasting decrease anxiety-like behavior and improves memory by a mechanism tied to reducing caspase-1 activity throughout the brain.

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.

Non-tumor cell IDO1 predominantly contributes to enzyme activity and response to CTLA-4/PD-L1 inhibition in mouse glioblastoma.

Glioblastoma (GBM) is the most common malignant brain tumor in adults with a median survival of 14.6months. A contributing factor to GBM aggressiveness is the intratumoral expression of the potently immunosuppressive enzyme, indoleamine 2,3 dioxygenase 1 (IDO1). The enzymatic activity of IDO1 is associated with the conversion of tryptophan into downstream kynurenine (Kyn), which has previously been hypothesized to contribute toward the suppression of tumor immunity. Utilizing the syngeneic, immunocompetent, intracranial GL261 cell GBM model, we previously demonstrated that tumor cell, but not non-tumor cell IDO1, suppresses T cell-mediated brain tumor regression in mice. Paradoxically, we also showed that the survival advantage mediated by immune checkpoint blockade is abrogated by non-tumor cell IDO1 deficiency. Here, we have built on our past observations and confirm the maladaptive role of tumor cell IDO1 in a novel mouse GBM model. We also demonstrate that, non-tumor cells, rather than mouse GBM cells, are the dominant contributor to IDO1-mediated enzyme activity. Finally, we show the novel associations between maximally-effective immune-checkpoint blockade-mediated survival, non-tumor cell IDO1 and intra-GBM Kyn levels. These data suggest for the first time that, GBM cell-mediated immunosuppression is IDO1 enzyme independent, while the survival benefits of immune checkpoint blockade require non-tumor cell IDO1 enzyme activity. Given that current clinical inhibitors vary in their mechanism of action, in terms of targeting IDO1 enzyme activity versus enzyme-independent effects, this work suggests that choosing an appropriate IDO1 pharmacologic will maximize the effectiveness of future immune checkpoint blockade approaches.

Dose-dependent decrease in mortality with no cognitive or muscle function improvements due to dietary EGCG supplementation in aged mice.

We have previously shown that a diet containing epigallocatechin gallate (EGCG) and beta-alanine is not effective in improving either cognitive or muscle function in aged (18 month) mice (Gibbons et al., Behav. Brain Res., 2014, 272:131-140; Pence et al., Appl. Physiol. Nutr. Metab., 2016, 41(2): 181-190). However, this diet reduced oxidative stress in the brain, and previous studies using longer term interventions and other doses have documented beneficial effects in cognitive and muscle function, especially with EGCG. Here we hypothesized that a different dose of EGCG or longer feeding period would be more efficacious in improving cognition. Aged (21-25 mo) Balb/cByJ male mice underwent 63 days of feeding with EGCG at 0, 0.091, or 3.67 mg/g AIN-93M diet and were then subjected to a battery of cognitive and muscle function tests. EGCG feeding at either of the 2 doses did not alter preference for novel versus familiar arm in the Y-maze test (p = 0.29) and did not affect learning in the active avoidance test (p = 0.76). Similarly, EGCG did not affect preference for novel versus familiar mice in a social discrimination test (p = 0.17). Likewise, there was no effect of EGCG on muscle function by grip strength (p = 0.16), rotarod (p = 0.18), or treadmill test to exhaustion (p = 0.25). EGCG reduced mortality in a dose-dependent fashion (p = 0.05, log-rank test for trend), with 91% of high EGCG, 72% of low EGCG, and 55% of control mice surviving to the end of the study. In conclusion, EGCG improves survival in aged mice but does not affect cognitive or muscle function.

Glial and tissue-specific regulation of Kynurenine Pathway dioxygenases by acute stress of mice.

Stressors activate the hypothalamic-pituitary-adrenal (HPA) axis and immune system eliciting changes in cognitive function, mood and anxiety. An important link between stress and altered behavior is stimulation of the Kynurenine Pathway which generates neuroactive and immunomodulatory kynurenines. Tryptophan entry into this pathway is controlled by rate-limiting indoleamine/tryptophan 2,3-dioxygenases (DOs: Ido1, Ido2, Tdo2). Although implicated as mediating changes in behavior, detecting stress-induced DO expression has proven inconsistent. Thus, C57BL/6J mice were used to characterize DO expression in brain-regions, astrocytes and microglia to characterize restraint-stress-induced DO expression. Stress increased kynurenine in brain and plasma, demonstrating increased DO activity. Of three Ido1 transcripts, only Ido1-v1 expression was increased by stress and within astrocytes, not microglia, indicating transcript- and glial-specificity. Stress increased Ido1-v1 only in frontal cortex and hypothalamus, indicating brain-region specificity. Of eight Ido2 transcripts, Ido2-v3 expression was increased by stress, again only within astrocytes. Likewise, stress increased Tdo2-FL expression in astrocytes, not microglia. Interestingly, Ido2 and Tdo2 transcripts were not correspondingly induced in Ido1-knockout (Ido1KO) mice, suggesting that Ido1 is necessary for the central DO response to acute stress. Unlike acute inflammatory models resulting in DO induction within microglia, only astrocyte DO expression was increased by acute restraint-stress, defining their unique role during stress-dependent activation of the Kynurenine Pathway.

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.

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.

Effects of exercise and dietary epigallocatechin gallate and ß-alanine on skeletal muscle in aged mice.

Aging leads to sarcopenia and loss of physical function. We examined whether voluntary wheel running, when combined with dietary supplementation with (-)-epigallocatechin-3-gallate (EGCG) and ß-alanine (ß-ALA), could improve muscle function and alter gene expression in the gastrocnemius of aged mice. Seventeen-month-old BALB/cByJ mice were given access to a running wheel or remained sedentary for 41 days while receiving either AIN-93M (standard feed) or AIN-93M containing 1.5 mg·kg(-1) EGCG and 3.43 mg·kg(-1) ß-ALA. Mice underwent tests over 11 days from day 29 to day 39 of the study period, including muscle function testing (grip strength, treadmill exhaustive fatigue, rotarod). Following a rest day, mice were euthanized and gastrocnemii were collected for analysis of gene expression by quantitative PCR. Voluntary wheel running (VWR) improved rotarod and treadmill exhaustive fatigue performance and maintained grip strength in aged mice, while dietary intervention had no effect. VWR increased gastrocnemius expression of several genes, including those encoding interleukin-6 (Il6, p = 0.001), superoxide dismutase 1 (Sod1, p = 0.046), peroxisome proliferator-activated receptor gamma coactivator 1-α (Ppargc1a, p = 0.013), forkhead box protein O3 (Foxo3, p = 0.005), and brain-derived neurotrophic factor (Bdnf, p = 0.008), while reducing gastrocnemius levels of the lipid peroxidation marker 4-hydroxynonenal (p = 0.019). Dietary intervention alone increased gastrocnemius expression of Ppargc1a (p = 0.033) and genes encoding NAD-dependent protein deacetylase sirtuin-1 (Sirt1, p = 0.039), insulin-like growth factor I (Igf1, p = 0.003), and macrophage marker CD11b (Itgam, p = 0.016). Exercise and a diet containing ß-ALA and EGCG differentially regulated gene expression in the gastrocnemius of aged mice, while VWR but not dietary intervention improved muscle function. We found no synergistic effects between dietary intervention and VWR.

The kynurenine to tryptophan ratio as a prognostic tool for glioblastoma patients enrolling in immunotherapy.

We hypothesized that peripheral tryptophan (Trp) and/or kynurenine (Kyn) levels would provide prognostic value for physicians planning to enroll glioblastoma multiforme (GBM) patients in immunotherapy. GBM is the most common form of malignant glioma in adults. Despite aggressive surgical resection, irradiation and chemotherapy, patients with GBM have a median survival of only 14.6 months after diagnosis. This poor outcome has led to the search for more effective treatments, including immunotherapy. However, the identification of parameters that proactively stratify GBM patients who have the potential for therapeutic benefit has been challenging. Given recent observations demonstrating high indoleamine 2,3 dioxygenase 1 (IDO1) expression in GBM, the immunosuppressive impact of IDO1-mediated Trp catabolism, as well as active transport of Trp and the IDO1-downstream Trp catabolite, Kyn, across the blood brain barrier, we hypothesized that peripheral blood analysis of this pathway would provide diagnostic utility. When comparing individuals without tumors to GBM patients prior to surgical resection, or at the 48 hour (48 h) and ⩾10 week (10 w+) postoperative time points, Trp levels were significantly decreased (p<0.0002). Similarly, Kyn levels were decreased in the pre- and 48 h postoperative GBM patients (p<0.0001), while there was no difference between individuals without tumors and 10 w+ GBM patients. Interestingly, those 10 w+ patients with a high Kyn/Trp ratio (⩾9.5) had a mean overall survival (OS) of 23.6 ± a standard error of 6.8 months, compared to an OS of 38.7 ± 4.9 months for patients with lower Kyn/Trp values. Since the 10 w+ blood draw and analyses occurred prior to patient enrollment in the heat shock protein peptide complex-96 clinical trial, these novel data suggest that the late Kyn/Trp index may be a relevant clinical benchmark, providing prognostic value for GBM patients who are enrolled in immunotherapeutic regimens.