Preclinical And Clinical Assessment Of Tryptophan Catabolites (Trycats) As A Measure Of Neuroinflammation And Utility In Compound Screening.

s | 79 University College of Medicine, Seoul, South Korea 3Department of Physiology, Seoul National University College of Medicine, Seoul, South Korea 4Department of Brain & Cognitive Science, Seoul National University Graduate School, Seoul, South Korea 5Ischemic/Hypoxic Disease Institute, Seoul National University College of Medicine, Seoul, South Korea (* equally contribution) Abstract Microglia are commonly known as the resident immune cells and have emerged as key players for neuro-immune system. However, the underlying mechanisms of neuro-glia interaction are poorly understood. Here, we investigated how microglia affect neural functions and behavioral responses. To make concrete, we used microglia specific STAT3 knock-out (KO) mice via Cre-loxP recombination system. Primarily, we found that immobility time in forced swim test and tail suspension test were reduced not only in microglial STAT3 KO group but also in the groups exposed to acutely and chronically induced restraint stress. From cytokine profiling assays, we identified that level of M-CSF was increased in microglial STAT3 KO mice. To decipher the mechanisms, we employed in vitro co-culture system between BV2 cells and HT22 cells. Surprisingly, level of M-CSF mRNA was higher in HT22 cells cultured with STAT3 knocked-down BV2 cells. Likewise, secreted form of M-CSF was increased in the same co-cultured media. Moreover, M-CSF treatment on HT22 cells significantly induced phosphorylation of ERK1/2, Akt, GSK3β, which were known as signal mediators for synaptic activities and especially antidepressant pathways. Interestingly, we discovered the increased level of BDNF in HT22 cells and co-culture systems after M-CSF treatment. The same results were confirmed in microglial STAT3 KO mice. Concerned with synaptic functions, mEPSC frequency of medial prefrontal cortex was increased in the microglial STAT3 KO mice and M-CSF treatment group. Our findings open the possibility that microglial STAT3 modulates neuronal cytokine expression and synaptic activity, and consequently regulates depression and stressrelated behaviors.Microglia are commonly known as the resident immune cells and have emerged as key players for neuro-immune system. However, the underlying mechanisms of neuro-glia interaction are poorly understood. Here, we investigated how microglia affect neural functions and behavioral responses. To make concrete, we used microglia specific STAT3 knock-out (KO) mice via Cre-loxP recombination system. Primarily, we found that immobility time in forced swim test and tail suspension test were reduced not only in microglial STAT3 KO group but also in the groups exposed to acutely and chronically induced restraint stress. From cytokine profiling assays, we identified that level of M-CSF was increased in microglial STAT3 KO mice. To decipher the mechanisms, we employed in vitro co-culture system between BV2 cells and HT22 cells. Surprisingly, level of M-CSF mRNA was higher in HT22 cells cultured with STAT3 knocked-down BV2 cells. Likewise, secreted form of M-CSF was increased in the same co-cultured media. Moreover, M-CSF treatment on HT22 cells significantly induced phosphorylation of ERK1/2, Akt, GSK3β, which were known as signal mediators for synaptic activities and especially antidepressant pathways. Interestingly, we discovered the increased level of BDNF in HT22 cells and co-culture systems after M-CSF treatment. The same results were confirmed in microglial STAT3 KO mice. Concerned with synaptic functions, mEPSC frequency of medial prefrontal cortex was increased in the microglial STAT3 KO mice and M-CSF treatment group. Our findings open the possibility that microglial STAT3 modulates neuronal cytokine expression and synaptic activity, and consequently regulates depression and stressrelated behaviors. PS216 Preclinical and clinical assessment of tryptophan catabolites (TRYCATS) as a measure of neuroinflammation and utility in compound screening. Kelly Allers, S Alastalo, Tom Bretschneider, Bastian Hengerer, J Hoke, Oliver Kleiner, O Kontakanen, Andre Liesener, Verena Nold, A Nurmi, Markus Otto, S Pleiner, J Puolivali, Carlos Schoenfeldt, Birgit Stierstorfer BI Pharma GmbH & Co. KG, Germany Abstract Numerous studies measuring pro-inflammatory cytokines in blood demonstrate that a subpopulation of psychiatric patients exhibit inflammation. In post-mortem and imaging studies, neuroinflammation was indicated by the presence of activated microglia [1,2]. Since tryptophan catabolism increases in activated microglia, tryptophan catabolites (TRYCATs) are possibly novel biomarkers of neuroinflammation-induced psychiatric symptoms. Specific objectives: A) Assess TRYCATs in animal models of neuroinflammation to determine utility as an endpoint for therapeutic intervention. B) Assess TRYCAT levels from psychiatric patients and controls as a potential clinical biomarker for patient stratification. Methods Used: LPS model: LPS was injected i.c.v. to mice, and brains were collected 24 hours later. Chronic Social Defeat model: Mice were subjected to chronic social defeat in a standard paradigm. Brains were collected on the final day of testing. Clinical samples were obtained from the Universitätsklinikum Ulm, Germany. TRYCATs in mouse brain tissue homogenates/ plasma and human CSF/plasma were analysed using liquid chromatography-tandem mass spectrometry. Results: LPS model: A robust increase in all TRYCATS from brain homogenates was observed in all LPS treated animals. Chronic social defeat model: No change in TRYCATs was observed in mice subjected to chronic stress. However, a significant proportion of microglia became activated following chronic social defeat. Inhibition of the IDO1 enzyme, which catalyses the first step in the TRYCAT pathway, reversed TRYCAT induction in the LPS model, as well as behavioral alterations in chronic social defeat. Results from the clinical samples indicate that patients exhibit an altered TRYCAT profile, and that CSF and plasma TRYCATs appear to be independently regulated. Conclusion: TRYCAT levels provide a useful tool for preclinical models of neuroinflammation and treatment. A first study of TRYCATs in a patient population suggests potential use as a clinical biomarker.Numerous studies measuring pro-inflammatory cytokines in blood demonstrate that a subpopulation of psychiatric patients exhibit inflammation. In post-mortem and imaging studies, neuroinflammation was indicated by the presence of activated microglia [1,2]. Since tryptophan catabolism increases in activated microglia, tryptophan catabolites (TRYCATs) are possibly novel biomarkers of neuroinflammation-induced psychiatric symptoms. Specific objectives: A) Assess TRYCATs in animal models of neuroinflammation to determine utility as an endpoint for therapeutic intervention. B) Assess TRYCAT levels from psychiatric patients and controls as a potential clinical biomarker for patient stratification. Methods Used: LPS model: LPS was injected i.c.v. to mice, and brains were collected 24 hours later. Chronic Social Defeat model: Mice were subjected to chronic social defeat in a standard paradigm. Brains were collected on the final day of testing. Clinical samples were obtained from the Universitätsklinikum Ulm, Germany. TRYCATs in mouse brain tissue homogenates/ plasma and human CSF/plasma were analysed using liquid chromatography-tandem mass spectrometry. Results: LPS model: A robust increase in all TRYCATS from brain homogenates was observed in all LPS treated animals. Chronic social defeat model: No change in TRYCATs was observed in mice subjected to chronic stress. However, a significant proportion of microglia became activated following chronic social defeat. Inhibition of the IDO1 enzyme, which catalyses the first step in the TRYCAT pathway, reversed TRYCAT induction in the LPS model, as well as behavioral alterations in chronic social defeat. Results from the clinical samples indicate that patients exhibit an altered TRYCAT profile, and that CSF and plasma TRYCATs appear to be independently regulated. Conclusion: TRYCAT levels provide a useful tool for preclinical models of neuroinflammation and treatment. A first study of TRYCATs in a patient population suggests potential use as a clinical biomarker.