Edital FAPERJ N.º 15/2014 PROGRAMA COOPERAÇÃO BILATERAL FAPERJ / ASSOCIAÇÃO COLUMBIA GLOBAL CENTER/BRASIL 2014

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1 Edital FAPERJ N.º 15/214 PROGRAMA COOPERAÇÃO BILATERAL FAPERJ / ASSOCIAÇÃO COLUMBIA GLOBAL CENTER/BRASIL 214 Título do Projeto: Biomarcadores associados à Depressão e TB ativa e TB latente Title: The pivotal role of Tryptophan-kynurenine metabolism in Mycobacterium tuberculosis-infected macrophages and its potential involvement in depressive disorder Leonardo Ribeiro Batista Silva Federal University of Rio de Janeiro / Oswaldo Cruz Foundation - FIOCRUZ July 216

2 Depression Serious public health problem with high prevalence worldwide; 2 million Americans per year; Depressed people less medical care to investigate respiratory symptoms and also have lower adherence to chemotherapy. High levels of IFN-γ, TNF-α and IL-6 Increased hypothalamic-pituitaryadrenal activity and cortisol levels can lead to immunological changes.

3 Tuberculosis and Depression Individuals with depression tend to seek less medical care to investigate respiratory symptoms and also have lower adherence to drug treatment (Pachi et al, 213; Prince, 27; Di Matteo, Lepper & Croghan, 2). Recently, a systematic review of 31 studies conducted in 11 countries, it was observed that depression can occur in high proportion (48.9%) of TB patients (Sweetland, Oquendo, Wickramaratne et al, in press).

4 Some questions... Is the chronical inflammation caused by tuberculosis infection promoting depressive behavior? What is the immunological mechanism involved in depression development? How can we treated TB patients with depression?

5 In vitro evaluation of kynurenine metabolism in Mycobacterium tuberculosisinfected macrophages and its potential involvement on depressive disorder the development

6 Introduction Inflammatory immune responses from chronic infection might affect the central nervous system and increase the risk of depressive disorder; Tryptophan catabolism mediated by indoleamine (IDO) activation are involved in tuberculosis chronic infection; Tryptophan catabolism product, Kynurenine, is linked with major depressive disorders divert available tryptophan away from serotonin production towards further catabolism; Cytokine-induced activation of IDO in peripheral macrophages leads to the production of plasma Kynurenine. However, the temporal association between tuberculosis chronic infection and incident depressive disorder has not been evaluated.

7 Kynurenine pathway and depression

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9 Latent TB Active TB IDO Kyn/Try IFN-γ IL-6 TNF-α type I IFN IDO Kyn/Try Major depressive disorder

10 Preliminary in vitro results

11 I D O e x p r e s s ã o r e l a t i v a A F M I D e x p r e s s ã o r * H 3 7 R v H 3 7 R v + M IN O Macrófagos primários humanos N I H 3 7 R v * H 3 7 R v + M I N O K y n u e x p r e s s ã o r e l a t i v a 1 1 * H 3 7 R v H 3 7 R v + M IN O

12 Tryptophan pathway KYNU

13 Kynurenine/Tryptophan ratio activity Macrofagos primários 24 horas 2. ** 1.5 * H37Rv H37Rv+ MINO M. tuberculosis MOI 5.1 = uninfected H37Rv = 5.1 bacteria cell ratio Rif = Rifampicin HPLC chromatography assay

14 Cellular Viability MTT Cellular Viability MTT Resultados Ensaio de citotoxicidade da droga Minociclina na linhagem monocítica THP THP-1 24hs 15 THP-1 48hs Não estimulado MINO 1µg/mL MINO 2µg/mL MINO 4µg/mL MINO 8µg/mL Não estimulado MINO 1µg/mL MINO 2µg/mL MINO 4µg/mL MINO 8µg/mL

15 IDO/RPL13 (fold change) Kynurenine/Tryptophan ratio activity B C G M. b o v i s 2 C F U 1 Evaluation of M. bovis BCG capacity on Kynurenine pathway modulacion T H P h s THP1 24 hs BCG 1:1 THP1 24 hs 2. * BCG 24h * * N O - H A R M A N E * M I N O C I C L I N E

16 IL1 pg/ml IFN pg/ml IL1 pg/ml IL6 pg/ml TNF- pg/ml Análise da produção de citocians em macrófagos THP1 infectados com M. bovis BCG após 24h *** *** *** BCG 1 BCG 1 + MINO BCG 1 + NOH 5 * BCG 1 BCG 1 + MINO BCG 1 + NOH * ** BCG 1 BCG 1 + MINO BCG 1 + NOH BCG 1 BCG 1 + MINO BCG 1 + NOH 8 ** * BCG 1 BCG 1 + MINO BCG 1 + NOH

17 CCL3 pg/ml CCL4 pg/ml IL1 pg/ml ILR pg/ml Cytokine production in M. tuberculosis-infected macrophages treated with minocycline. 8 6 * 15 1 *** *** *** 6 4 * BCG 1 BCG 1 + MINO BCG 1 + NOH BCG 1 BCG 1 + MINO BCG 1 + NOH BCG 1 BCG 1 + MINO BCG 1 + NOH BCG 1 BCG 1 + MINO BCG 1 + NOH

18 Kynurenine/Tryptophan ratio activity TNF pg/ml Resultados Minociclina não altera a produção de TNF-α em macrófagos primários infectados com M. bovis BCG. Macrófagos primários 24 horas (2 indivíduos) Macrofagos primários 24 horas BCG BCG + Min 2 g/ml BCG + Min 4 g/ml Controle BCG BCG+MIN BCG+RIF NOHARMANE BCG 1.1 MOI N= 3

19 M. tuberculosis IFNR Cortisol BLOOD NERVOUS SYSTEM TNF-α IDO Tryptophan TDO 5-Hydroxytryptophan Kynurenine Kynurenine Kynurenine IL6 KYNU 3-Hydroxykynurenine Kynurenic acid Serotonin Peripheral macrophages Quinolinic acid NMDA receptor Neurodegeneration Oxidative stress Excitotoxicity Glial cells

20 Acknowledgement : Dr. Afranio Kritski (supervisor) Dr. Milton Ozório (FIOCRUZ) Priscilla Helen (Graduate student) Dr. Elisangela (UFRJ) Leprosy Laboratory - IOC - FIOCRUZ LMM Laboratory - UFRJ Financial support: FAPERJ