Immunobiology 217 (2012) 808–815 Contents lists available at SciVerse ScienceDirect Immunobiology j o ur nal homep ag e: www.elsevier.com/locate/imbio Nitric oxide affects immune cells bioenergetics Long-term effects of nitric-oxide derivatives on leukaemic Jurkat cell metabolism Ines Hammami a , Marie Bertrand a , Jingkui Chen a , Vincenzo Bronte b , Gregory De Crescenzo a , Mario Jolicoeur a,∗ a Department of Chemical Engineering, Ecole Polytechnique de Montreal, Joseph-Armand Bombardier Building, 5155 Decelles Avenue, Montreal, Quebec, Canada H3T-2B2 b Department of Pathology, Immunology Section, Verona University, P.le L.A. Scuro, 10, 37134 Verona, Italy a r t i c l e i n f o Article history: Received 9 April 2011 Accepted 7 May 2012 Keywords: AT38 Bioenergetics Cyclosporine A Jurkat cells Metabolism Nitric oxide a b s t r a c t Major advances in dissecting mechanisms of NO-induced down-regulation of the anti-tumour specific T-cell function have been accomplished during the last decade. In this work, we studied the effects of a NO donor (AT38) on leukaemic Jurkat cell bioenergetics. Culturing Jurkat cells in the presence of AT38 triggered irreversible inhibition of cell respiration, led to the depletion of 50% of the intracellular ATP content and induced the arrest of cell proliferation and the loss of cell viability. Although a deterioration of the overall metabolic activity has been observed, glycolysis was stimulated, as revealed by the increase of glucose uptake and lactate accumulation rates as well as by the up-regulation of GLUT-1 and PFK- 1 mRNA levels. In the presence of NO, cell ATP was rapidly consumed by energy-requiring apoptosis mechanisms; under a glucose concentration of about 12.7 mM, cell death was switched from apoptosis into necrosis. Exposure of Jurkat cells to DMSO (1%, v/v), SA and AT55, the non-NO releasing moiety of AT38, failed to modulate neither cell proliferation nor bioenergetics. Thus, as for all NSAIDs, beneficial effects of AT38 on tumour regression are accompanied by the suppression of the immune system. We then showed that pre-treating Jurkat cells with low concentration of cyclosporine A, a blocker of the mitochondrial transition pore, attenuates AT38-induced inhibition of cell proliferation and suppresses cell death. Finally, we have studied and compared the effects of nitrite and nitrate on Jurkat cells to those of NO and we are providing evidence that nitrate, which is considered as a biologically inert anion, has a concentration and time-dependent immunosuppressive potential. © 2012 Elsevier GmbH. All rights reserved. Introduction The metabolic, energetic and biosynthetic demands of immune cells increase dramatically after their activation by antigens and mitogens. Mounting a functional immune response requires rapid and extensive cell growth, proliferation, activation and produc- tion of effector proteins (Maciver et al. 2008). Immunoregulatory functions are known to be ATP-dependant and sensitive to dis- turbances in intracellular nutrient levels (Buttgereit et al. 2000). Co-stimulation of T-cells by T-cell receptor and CD28 was shown to lead in PI3K/Akt-dependent up-regulation of glucose transporter 1 Abbreviations: CsA, cyclosporine A; DMSO, dimethyl sulfoxide; DO, dissolved oxygen; GLUT, glucose transporter; IL-2, interleukin-2; iNOS, inducible nitric oxide synthase; MPTP, mitochondrial permeability transition pore; NO, nitric oxide; NSAID, non-steroidal anti-inflammatory drug; OUR, oxygen uptake rate; PFK, phos- phofructokinase; SA, salicylic acid. ∗ Corresponding author at: PO 6079 Station Centre-Ville, Montreal, Quebec, Canada H3C 3A7. Tel.: +1 514 340 4711x4525; fax: +1 514 340 4159. E-mail address: mario.jolicoeur@polymtl.ca (M. Jolicoeur). (GLUT-1) gene expression, glucose uptake and oxygen metabolism (Frauwirth et al. 2002). In aerobic organisms, energy is provided by glycolysis or respi- ration via oxidative phosphorylation, which has the higher energy production efficiency. However, the tumoural microenvironment is characterized by low concentrations of oxygen and glucose, as well as high levels of lactate and reductive and oxidative species (Raghunand et al. 2003). Specifically, a myeloid-derived suppres- sor cell population ensures high output of NO via the enzymatic activity of inducible NO synthase (iNOS) (Serafini et al. 2006). NO diffuses freely and reacts rapidly with various biomolecules and intracellular compounds (e.g. O 2 - , H 2 O 2 ) within both NO- generating cells and also target cells to produce reactive nitrogen oxide species (Mocellin et al. 2007). The primary decomposition product of NO in aerobic aqueous solution is nitrite (NO 2 - ), which can be further oxidized to generate nitrate (NO 3 - ) in the presence of additional oxidizing species such as oxyhemoproteins (Ignarro et al. 1993). NO is known to be responsible for various physiological functions and is implicated in multiple pathologies (Mocellin et al. 2007), but special attention is accorded to its role in cancer pathogenesis and tumour-related immunosuppression 0171-2985/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.imbio.2012.05.005