DESCRIPTION (provided by applicant): Proinflammatory cytokines act in the brain to regulate important biobehavioral processes that are critical for physical and mental health. Patients receiving cytokine immunotherapy for treatment of viral infections or malignancies and those suffering from chronic inflammatory diseases are at an increased risk of developing depressive disorders. The neurobiological mechanisms and immune substrates underlying these immune-brain interactions remain unclear. Acute and chronic activation of the peripheral innate immune system in laboratory rodents precipitates depressive-like biobehaviors. New data from our laboratory have demonstrated that these biobehaviors are mediated by cytokine-induced activation of the tryptophan-degrading enzyme indoleamine 2, 3 dioxygenase (IDO), which is the first and rate-limiting enzyme of the kynurenine pathway. We demonstrated that pharmacologic inhibition or genetic deletion of IDO prevents depressive-like biobehaviors following peripheral immune activation. Our most recent data point to the generation of neuroactive kynurenine metabolites in the brain as the likely neurobiological mechanism for increased depressive-like biobehaviors. Clinically, reduced circulating tryptophan and increased kynurenine concentrations (the first catabolic product of IDO activity) are correlated with increased depression scores in patients undergoing cytokine immunotherapy. Our new data extend these clinical observations to show that depressive-like behavior in immune-stressed mice requires cytokine-induced IDO activity. In fact, direct administration of kynurenine induces depressive-like behavior in naive mice, but kynurenine is not neuroactive. Instead, kynurenine is compartmentally metabolized in the brain to generate free radical-producing or glutamatergically-active kynurenine metabolites. Microglia produce the free radical forming metabolites 3-hydroxykynurenine and 3-hydroxyanthranilic acid en route to generating the ionotropic glutamate receptor agonist, quinolinic acid. Conversely, endothelial cells and astrocytes produce kynurenic acid, which is a glutamate and 17-nicotinic acetylcholine receptor antagonist. Based on these data, we hypothesize that kynurenine metabolism in activated microglia drives development of inflammation-induced depressive-like biobehaviors. We will first determine if microglial activation is responsible for IDO-dependent depressive-like behavior following peripheral immune stress. Objective two will define the regulatory pathways that link peripheral immune activation to changes in brain kynurenine metabolism, and objective three will utilize ex vivo and in vitro techniques to specifically define how kynurenine metabolism is regulated at the cellular level. The last objective will use pharmacologic and genetic approaches to target kynurenine pathway enzymes that might alleviate cytokine-induced depressive-like biobehaviors. These preclinical experiments are needed to understand the neurobiological mechanisms by which proinflammatory cytokines cause psychopathology and to identify novel therapeutic targets for the treatment of inflammation-induced depression.

PUBLIC HEALTH RELEVANCE: Depression represents one of the most frequent, debilitating and costly comorbidities of chronic physical illness and infection. Unfortunately, the molecular and cellular mechanisms that mediate these neuro-immune interactions remain unclear. We have recently identified a metabolic pathway (the kynurenine pathway) that is upregulated in the brain in response to activation of the peripheral innate immune system. It appears that the generation of neuroactive kynurenine metabolites by brain support cells called microglia, following peripheral immune activation, is responsible for precipitating depressive behaviors in mice. Successfully unraveling these molecular networks will enable the development of novel therapeutic strategies to treat inflammation-induced depression. Narrative Depression represents one of the most frequent, debilitating and costly comorbidities of chronic physical illness and infection. Unfortunately, the molecular and cellular mechanisms that mediate these neuro-immune interactions remain unclear. We have recently identified a metabolic pathway (the kynurenine pathway) that is upregulated in the brain in response to activation of the peripheral innate immune system. It appears that the generation of neuroactive kynurenine metabolites by brain support cells called microglia, following peripheral immune activation, is responsible for precipitating depressive behaviors in mice. Successfully unraveling these molecular networks will enable the development of novel therapeutic strategies to treat inflammation-induced depression. __SpecificAimsTextDelimiter__ Specific Aims The broad goal of this research application is to test the hypothesis that kynurenine metabolism by activated microglia drives development of inflammation-induced depressive-like biobehaviors. If we are correct, the kynurenine pathway could well be the next pharmacologic target that could be translated form mouse to man in the form of new treatment approaches for clinical depression. Functional biobehavioral consequences of inflammation in the brain, rather than in the periphery, are the primary focus of this application. Over the past 15 years, our laboratory and others have demonstrated that cytokines produced by activated innate immune cells not only communicate immune status to the brain, but these cytokines are also produced within the CNS (5) where they act to regulate a variety of important brain functions. These centrally produced cytokines are responsible for the transient behavioral response to infection known as sickness behavior (6-8). When neuroinflammation inappropriately persists, both psychopathology and neuropathology can develop (7). Dysregulated activation of the innate immune response can precipitate development of depressive symptoms or depressive-like behaviors that are temporally and mechanistically distinct from the transient sickness behavior response and are mediated by cytokine-dependent activation of the tryptophan degrading enzyme indoleamine 2, 3-dioxygenase (IDO) (3, 9, 10). In humans, costly and debilitating chronic inflammatory states, such as cardiovascular disease, obesity and autoimmune disorders, are associated with increased prevalence of clinical depression (1, 11, 12). Direct evidence for the relationship between inflammation and depression can be found in cancer patients receiving interferon-¿ therapy for the treatment of kidney cancer, metastatic melanoma and hepatitis C (9, 13-15). These patients develop depressive symptoms that include psychomotor retardation, anhedonia, sadness, inability to feel and even suicidal thoughts. In each of these clinical situations, development of depressive symptoms is associated with IDO activation, as measured by reduced tryptophan and increased kynurenine in plasma (12, 16, 17). We have now significantly extended these correlative findings by demonstrating for the first time that IDO is a necessary mediator of depressive-like behaviors that occur in response to both acute (lipopolysaccharide, LPS) (3) and chronic (Mycobacterium bovis, BCG) (2) peripheral immune activation in mice. In these experiments, pharmacological inhibition as well as genetic deletion of IDO prevented depressive-like behaviors without altering sickness behavior. We initially hypothesized that depressive-like biobehaviors were caused by depletion of the serotonin precursor tryptophan. However, we have now collected evidence showing that depressive-like behavior is not related to brain serotonin levels. Instead, we have discovered that direct administration of kynurenine induces depressive-like behavior (3). These findings indicate that proinflammatory cytokines lead to activation of IDO, which degrades tryptophan along the kynurenine pathway to generate kynurenine. Since kynurenine itself is not neuroactive (18, 19), here we will test the likely possibility that catabolic metabolites of kynurenine are directly responsible for causing inflammation-induced depressive-like behaviors. In accord with this hypothesis, we have generated exciting new preliminary data that implicate kynurenine metabolites produced by activated microglia leads to the development of depressive-like behaviors in response to peripheral immune activation. The rate limiting enzyme of the kynurenine pathway, IDO, is upregulated by proinflammatory signals in various brain cells, including endothelial cells, astrocytes and microglia (20, 21). Although each of these cell types increase kynurenine production in response to proinflammatory signals, such as LPS, interferon (IFN)-¿ and tumor necrosis factor (TNF)-¿ (21, 22), kynurenine, per se, is not neuroactive (18, 19). Rather, kynurenine is further metabolized in the brain by enzymes downstream of IDO that are also regulated by proinflammatory cytokines and are expressed in a cell-type specific pattern (18). Microglia produce the free radical forming metabolite, 3-hydroxykynurenine, and the ionotropic glutamate receptor agonist, quinolinic acid. Conversely, astrocytes and brain endothelial cells do not degrade kynurenine to quinolinic acid. Instead, these cells produce the glutamate and ¿7-nicotinic acetylcholine receptor antagonist, kynurenic acid (18). Peripheral inflammation activates brain microglia (23, 24), and suppression of microglial activation using the tetracycline derivative, minocycline, prevents upregulation of brain IDO, the subsequent increase in brain kynurenine levels and the ensuing development of depressive-like behaviors in response to peripheral immune stress (3, 25). Experiments in this application will directly test the hypothesis that production of these neuroactive kynurenine metabolites by activated microglia is responsible for depressive-like behaviors precipitated by peripheral immune activation. Successful completion of the project will yield several significant and innovative benefits: (a) Provide the first successful demonstration that activated microglia play a causal role in the psychopathogy of inflammation- induced depressive symptoms via the generation of neuroactive metabolites; (b) Define the molecular regulation of brain kynurenine metabolism following peripheral immune activation; (c) Create two vital and entirely novel murine genetic models that will enable unprecedented studies of kynurenine metabolism in the brain; and (d) Identify novel therapeutic strategies that target downstream kyneurenine-catabolizing enzymes that could be used for preventing or treating depressive disorders that are initially driven by systemic inflammation. The specific questions that will be answered by the proposed experiments are highlighted below: Objective 1: Is kynurenine metabolism in the brain sufficient and necessary to induce inflammation-associated depressive-like biobehaviors? Objective 2: Are inflammation-induced depressive-like behaviors associated with a shift in balance of brain kynurenine metabolism toward the production of microglial-derived neurotoxic metabolites? Objective 3: What is the role of microglia/astrocytes in the generation of neuroactive kynurenine metabolites in response to proinflammatory signals? Objective 4: Does in vivo inhibition of microglial kynurenine metabolism alleviate development of depressive- like behaviors in response to peripheral immune stimulation?