Muramyl-dipeptide-induced mitochondrial proton leak in macrophages is associated with upregulation of uncoupling protein and the production of reactive oxygen and reactive nitrogen species Takla G El-Khoury, Georges M Bahr and Karim S Echtay Faculty of Medicine and Medical Sciences and Faculty of Sciences, University of Balamand, Tripoli, Lebanon Keywords mitochondria; muramylpeptides; nitric oxide; respiratory control ratio; superoxide anion; UCP2 Correspondence K S Echtay, Faculty of Medicine and Medical Sciences, University of Balamand, PO Box 100, Tripoli, Lebanon Fax: +961 930279 Tel: +961 714125 E-mail: karim.echtay@balamand.edu.lb (Received May 2011, revised 13 June 2011, accepted 28 June 2011) doi:10.1111/j.1742-4658.2011.08226.x The synthetic immunomodulator muramyl dipeptide (MDP) has been shown to induce, in vivo, mitochondrial proton leak In the present work, we extended these findings to the cellular level and confirmed the effects of MDP in vitro on murine macrophages The macrophage system was then used to analyse the mechanism of the MDP-induced mitochondrial proton leak Our results demonstrate that the cellular levels of superoxide anion and nitric oxide were significantly elevated in response to MDP Moreover, isolated mitochondria from cells treated with MDP presented a significant decrease in respiratory control ratio, an effect that was absent following treatment with a non-toxic analogue such as murabutide Stimulation of cells with MDP, but not with murabutide, rapidly upregulates the expression of the mitochondrial protein uncoupling protein (UCP2), and pretreatment with vitamin E attenuates upregulation of UCP2 These findings suggest that the MDP-induced reactive species upregulate UCP2 expression in order to counteract the effects of MDP on mitochondrial respiratory efficiency Introduction Uncoupling proteins (UCPs) are members of the anion carrier family molecules present in the inner mitochondrial membrane Mammals express five UCP homologues, UCP1–UCP5 UCP2 and UCP3 have 59% and 57% identity, respectively, with UCP1, and 73% identity with each other [1], whereas UCP4 and UCP5 (also referred to as brain mitochondrial carrier protein 1, BMCP1) have much lower sequence identity with UCP1 [2,3] UCP1 is the best characterized of these proteins, mediating non-shivering thermogenesis in brown adipose tissue by catalysing proton leak activated by long-chain fatty acids and inhibited by purine nucleotides [4] UCP2 is widely expressed in many tissues with high levels detected in the spleen, thymus, pancreatic b-cells, heart, lung, white and brown adipose tissue, stomach, testis and macrophages, whereas low levels have been reported in the brain, kidney, liver and muscle [5] UCP3 is expressed predominantly in skeletal muscles and brown adipose tissues [6,7], at hundred-fold lower concentration than UCP1 in brown adipose tissue [8] UCP4 and UCP5 are only present in the brain [2,3] Due to their homology to UCP1 and their distribution in several mammalian tissues, it has been initially postulated that these proteins can regulate mitochondrial oxidative phosphorylation through uncoupling activity However, the physiological function of UCPs other than UCP1 has remained controversial Suggested functions include mild uncoupling, adaptive Abbreviations FCCP, fluorocarbonyl cyanide phenylhydrazone; LPS, lipopolysaccharide; MB, murabutide; MDP, muramyl dipeptide; PI, propidium iodide; RCR, respiratory control ratio; ROS, reactive oxygen species; RNS, reactive nitrogen species; UCP, uncoupling protein 3054 FEBS Journal 278 (2011) 3054–3064 ª 2011 The Authors Journal compilation ª 2011 FEBS T G El-Khoury et al thermogenesis, protection against obesity, regulation of the ATP ⁄ ADP ratio, export of fatty acids, and mediation of insulin secretion (reviewed in [9]) The hypothesis that has good experimental support is the function of UCP2 to attenuate mitochondrial production of free radicals and to protect against oxidative damage [10,11] This is mainly based on the activation of mitochondrial proton conductance mediated through UCPs by reactive oxygen species (ROS) or by-products of lipid peroxidation [12,13], resulting in a negative feedback loop that decreases ROS production by lowering both the proton-motive force and local oxygen consumption UCP2 was shown to play a regulatory role in macrophage-mediated immune and ⁄ or inflammatory responses [14,15] Infected peritoneal macrophages of UCP2) ⁄ ) mice are resistant to infection by the intracellular parasite Toxoplasma gondii through a mechanism proposed to involve higher production of intracellular ROS [14] On the other hand, studies in cells overexpressing UCP2 have reinforced the belief that UCP2 plays a role in limiting intracellular ROS production, as has been shown in the murine macrophage cell line Raw-264 [16] Moreover, cardiomyocytes transfected with a UCP2-expressing adenovirus were able to regulate ROS production and protect against doxorubicin-mediated cardiotoxicity [17] Therefore, by acting as a modulator of ROS production, particularly in monocytes ⁄ macrophages, UCP2 may impact the outcome of an innate response However, whether UCP2 functions to attenuate ROS production by simply catalysing mild uncoupling remains to be tested Muramyl peptides are a family of immunomodulators with diverse biological effects Their immunological activities include adjuvanticity, enhancement of non-specific resistance to viral and bacterial infections, potentiation of anti-tumour activity of macrophages, manipulation of cytokine release and restoration of haematopoiesis [18–20] The parent molecule of this family is muramyl dipeptide (MDP), which has been reported as the minimal adjuvant-active structure of bacterial peptidoglycan [21] However, MDP administration into different hosts was associated with serious toxicity Therefore, attempts have been made to generate analogues with desirable properties and reduced toxicities One of these derivatives is murabutide (MB), a hydrophilic derivative of MDP that has eventually reached a clinical stage of development [20,22] It has been tested in vivo comparing its pharmacological, inflammatory and toxic effects with those of the parent molecule MDP The results reported establish the safety of MB, the absence of undesirable effects on the UCP2 modulates MDP-induced mitochondrial inefficiency central nervous system, and the lack of induction of inflammatory responses [22] Despite a long-standing interest in the field of muramyl peptides, the impact of these molecules at the mitochondrial level has not yet been examined Recently the effect of these derivatives on mitochondrial bioenergetics has been studied [23] MDP induced in vivo a significant decrease in respiratory control ratio (RCR) in isolated mouse liver and spleen mitochondria versus non-toxic analogues such as MB The decrease in RCR in mitochondria of MDP-treated mice is attributed to an increase in mitochondrial proton leak (i.e mitochondrial uncoupling) In the present study we use the immunomodulators to reveal the mechanism of action of toxic MDPs on mitochondrial respiration by correlating the uncoupling effect induced by these molecules with the level and function of UCP2 and free radical production in macrophages We find that MDP induces reactive oxygen and nitrogen species production and upregulates UCP2 protein level, whereas MB does not We further show that the activity of UCP2 is consistent with the level of free radicals Results In vitro effect of muramyl peptides and lipopolysaccharide on respiratory mitochondrial activity of murine peritoneal macrophages Measurement of oxygen consumption represents a potent technique to characterize the respiratory function in mitochondria isolated from tissues or cultured cells and to thoroughly localize the sites of impairment of oxidative phosphorylation In this study, the activities of the respiratory chain complexes are examined as the oxygen consumption rates after addition of various substrates and inhibitors The mitochondrial respiratory function is conventionally separated into different states State is the oxygen consumption rate of substrate (succinate) oxidation State is defined as the phosphorylation state and is dependent on the oxygen consumption in the presence of ADP, thus reflecting the mitochondrial respiration coupled to ATP production State 4, the non-phosphorylation state, is a measure of oxygen consumption in the presence of oligomycin (ATP synthase inhibitor) This state represents the mitochondrial basal proton leak activity State ⁄ state 4, termed the RCR, is used as an indicator to evaluate mitochondrial efficiency since it reflects the coupling between oxidative phosphorylation and the mitochondrial electron transport chain activity FEBS Journal 278 (2011) 3054–3064 ª 2011 The Authors Journal compilation ª 2011 FEBS 3055 UCP2 modulates MDP-induced mitochondrial inefficiency T G El-Khoury et al 60 A RCR inhibition (%) 50 * 40 * 30 20 10 Time (h) 1.6 B 1.2 Respiration rate (nmol O·min–1·mg–1) Figure 1A shows the time-dependent inhibition of succinate-linked RCR in mitochondria extracted from MDP-treated (100 lgỈmL)1) macrophages A maximum decrease in RCR (about 42% compared with untreated cells) was noted after h of treatment and the value returned to its basal level after h Figure 1B shows that the decrease in RCR in mitochondria of MDPtreated macrophages was attributed to an increase in state respiration No significant changes were observed in state 2, state and fluorocarbonyl cyanide phenylhydrazone (FCCP) rates between untreated and MDP-treated cells The conditions at which MDP exerted its maximum effects on mitochondria were applied to examine the impact of the other derivatives Figure 1C and Table summarize the effect of MB (non-toxic muramyl peptide) and lipopolysaccharide (LPS) on the mitochondrial bioenergetics of macrophage-treated cells The results demonstrate clearly the inability of MB and LPS to induce any impairment in mitochondrial function after h of treatment RCR and states 2, 3, and FCCP rates of MB- and LPStreated cells were the same as those of unstimulated cells These results demonstrate clearly the ability of only toxic muramyl peptides (such as MDP) to impair mitochondrial function whereas non-toxic muramyl peptides (such as MB) and LPS have no effect on mitochondrial respirations of peritoneal macrophages after h of treatment 0.8 * 0.4 Effect of MDP on cell viability Fig Effects of muramyl peptides and LPS on respiration rates and RCR in murine peritoneal macrophage mitochondria in vitro (A) Oxygen consumption was measured in the presence of 100 lgỈmL)1 of MDP after 1, 2, and h of incubation The decrease in RCR is presented as a percentage of inhibition (B) Mitochondrial respiratory states were measured in mitochondria after h of treatment with MDP (100 lgỈmL)1) Data are normalized to state rates of unstimulated mitochondria (black bars) (C) RCRs of mitochondria isolated from cells treated for h with MDP (100 lgỈmL)1), murabutide (MB, 100 lgỈmL)1) or LPS (1 lgỈmL)1) Data are normalized to the values of unstimulated cells (black bar, taken as 1) Data are means ± SEM of three independent experiments each performed in triplicate *P < 0.05 3056 State State State FCCP 1.2 C 0.8 RCR The viability of peritoneal macrophages under conditions of maximum impairment of mitochondrial activity of MDP-treated cells was examined The proportions of viable (Annexin V-FITCneg ⁄ propidium iodide (PIneg)), early apoptotic (Annexin V-FITCpos ⁄ PIneg) and late apoptotic ⁄ necrotic (Annexin V-FITCpos ⁄ PIpos) cells were identified (Fig 2A–C) The mean * 0.6 0.4 0.2 l ro nt Co DP M B M S LP percentage of viable cells in unstimulated and in MDP-treated cells was 69.05% and 65.45% respectively (P > 0.05) Moreover, no significant difference FEBS Journal 278 (2011) 3054–3064 ª 2011 The Authors Journal compilation ª 2011 FEBS T G El-Khoury et al UCP2 modulates MDP-induced mitochondrial inefficiency Table Effects of MB and LPS on respiration rates in murine peritoneal macrophage mitochondria in vitro Mitochondria were isolated from murine peritoneal macrophages after h of treatment with MB (100 lgỈmL)1) or LPS (1 lgỈmL)1) Data are presented as the percentage of unstimulated cells Data are means ± standard error of the mean of three independent experiments each performed in triplicate Percentage unstimulated cells State State FCCP rate B 100 101 PI 102 103 A 104 MB (100 lgỈmL)1) 100 ± 112 ± 14.2 107 ± 10.8 95.68 ± 4.6 LPS (1 lgỈmL)1) 100 ± 1.5 102 ± 12.3 98 ± 7.6 98.27 ± 8.2 100 101 102 103 Annex-FITC 104 100 101 102 103 Annex-FITC In order to investigate the mechanism of action of MDP on the mitochondrial bioenergetics system and since mitochondria are an important source of ROS production and especially of superoxide anion, we investigated the effect of MDP (100 lgỈmL)1) on total cellular superoxide anion production by murine peritoneal macrophages As shown in Fig 3, total superoxide production was unchanged after 30 but was significantly elevated at 60 and 120 (P < 0.05) in MDP-treated cells Interestingly, the OÀ level decreased after h of stimulation, returning almost to the resting level after h On the other hand, stimulation with MB failed to induce superoxide production (Fig 3), even after h of treatment, whereas stimulation with LPS only induced significant enhancement of superoxide production after a period of h of stimulation (data not shown) The effect of muranyl peptides on the total NO (nitrite and nitrate) production of murine peritoneal macrophages was determined by Griess assay The NO concentration of the culture supernatant was significantly increased after stimulation with 104 * * 80 60 Fold increase of superoxide % peritoneal macrophages C 40 20 – n – /An PI + n – /An PI + n – /An PI * 2 Fold increase of total NO State Time course effect of MDP on ROS and reactive nitrogen species production by murine peritoneal macrophages + n + /An PI Fig The percentage of viable, dead and apoptotic cells in treated and untreated cells is shown in (C) Data (A,B) represent one of three separate experiments with similar results The percentage of decrease in cell viability (C) is the mean ± SEM of three independent experiments was noted between stimulated and MDP-treated samples in the percentage of apoptotic or necrotic cells (Fig 2C) 0 Time (h) Fig Effect of MDP and MB on OÀ and NOÀ =NOÀ production 2 by murine peritoneal macrophages Macrophages (106 well)1) were stimulated with 100 lg of MDP (closed symbols) or MB (open symbols) per millilitre for various time intervals, and OÀ and NOÀ =NOÀ 2 were measured as described in Experimental procedures Results for OÀ (circle) and total NO (square) production were expressed as fold increase of unstimulated cells Data are means ± SEM of five independent experiments each performed in duplicate *P < 0.05 FEBS Journal 278 (2011) 3054–3064 ª 2011 The Authors Journal compilation ª 2011 FEBS 3057 UCP2 modulates MDP-induced mitochondrial inefficiency T G El-Khoury et al Free radical generation contributes to UCP2 upregulation To determine if MDP-induced UCP2 upregulation correlated with free radical generation, cells stimulated with MDP were pretreated with an antioxidant (vitamin E) Figure 5A shows that both OÀ and total NO significantly decreased in MDP-treated cells Figure 5B clearly demonstrates that vitamin E significantly reduced the MDP-induced UCP2 upregulation, thus showing that free radicals contribute to UCP2 upregulation Evidence for the involvement of UCP2 in the mitochondrial impairment caused by MDP The results obtained suggested a role of UCP2 in macrophage activation by MDP The question raised at this stage is whether UCP2 is responsible for the increase in mitochondrial proton permeability (state 4) induced in macrophages after stimulation with MDP Purine nucleotides (such as GDP) are recognized inhibitors of UCP1 [4] Also for UCP2 a purine nucleotide binding domain has been predicted from the translated 3058 US M D M P B LS P GAPDH MDP MB LPS 6h US 2h B UCP2 * US 1h Stimulation of peritoneal macrophages by MDP increased cellular ROS and reactive nitrogen species (RNS) production The increased production of reactive species was apparent after h of stimulation Since UCP2 is described as a regulator of ROS production, the expression of UCP2 in macrophages stimulated or not with immunomodulators was then investigated Results shown in Fig 4A clearly demonstrate that stimulation of macrophages with MDP (100 lgỈmL)1) for h results in significant increase in UCP2 expression (3.6-fold, P < 0.05) On the other hand, analysis of the kinetics of induction of UCP2 protein in MDP-treated macrophages revealed a significant increase starting h after stimulation (2.2-fold, P < 0.05), a peak level after h (3.6-fold, P < 0.05) and a return to baseline level after h of treatment (Fig 4B) Relative expression of UCP2/GAPDH Macrophage activation by MDP leads to overexpression of UCP2 A UCP2 Relative expression of UCP2/GAPDH 100 lgỈmL)1 MDP for h (Fig 3) (unstimulated cells 2.48 nmol NO ⁄ 106 cells ± 0.29; MDP treated cells 16.99 nmol NO ⁄ 106 cells ± 0.31; P < 0.05) However, stimulation with MB (100 lgỈmL)1) failed to generate NO (Fig 3), whereas stimulation with LPS only induced a high and significant level of NO after 48 h of treatment (data not shown) * GAPDH * 1h 2h 6h Fig Immunodetection of UCP2 in murine peritoneal macrophages Total cell lysates were prepared from unstimulated (US) and MDP (100 lgỈmL)1), MB (100 lgỈmL)1) or LPS (1 lgỈmL)1) treated macrophages, and 50 lg of total cell lysate proteins were loaded onto an SDS ⁄ 12% PAGE gel (A) (B) Time course effect of MDP on UCP2 expression in macrophages Western blot analysis was performed as described under Experimental procedures Inserts in (A) and (B) show western immunoblot analysis Data are relative to the value for unstimulated cells (black bars, taken as 1) Each result shown is the mean ± SEM of three independent experiments *P < 0.05 GAPDH, glyceraldehydes-3-phosphate dehydrogenase mRNA sequence [4], and any effect of GDP on respiration (proton permeability) has broadly been equated with the involvement of the relevant UCP (here UCP2) in the process Therefore, the effect of GDP on mitochondrial respiration in macrophages was analysed Figure 6A shows that GDP added to mitochondria extracted from the cells treated with MDP for h induced a significant decrease in state (14.94%) Consequently, the RCR value increased significantly by 15.15% in GDP-treated mitochondria (Fig 6B) These results clearly suggest that the mitochondrial inefficiency caused by MDP (100 lgỈmL)1) after h of incubation in peritoneal macrophages occurs partially through UCP2 FEBS Journal 278 (2011) 3054–3064 ª 2011 The Authors Journal compilation ª 2011 FEBS T G El-Khoury et al UCP2 modulates MDP-induced mitochondrial inefficiency 10 ** ** 120 80 40 US * UCP2 GAPDH MDP + Vit E M + V DP it E Vit E DP Vi tE MDP M US B 100 ** 80 ** RCR (%) Relative expression of UCP2/GAPDH ** 0 B * 160 State (%) * * A 200 Fold increase of total NO Fold increase of superoxide A ** 60 * 40 20 US MDP Vit E MDP + Vit E Fig Effect of vitamin E on UCP2 expression Macrophages (106 well)1) were pretreated with vitamin E (100 lM) for 10 and then stimulated with MDP (100 lg) for h, and OÀ and NOÀ =NOÀ were measured as described in Experimental proce2 dures Results for OÀ (open bars) and total NO (black bars) produc2 tion were expressed as fold increase of unstimulated cells Data are means ± range of two independent experiments each performed in duplicate (B) UCP2 western blot analysis Conditions are as described in the legend to Fig *P < 0.05 versus unstimulated; **P < 0.05 versus MDP stimulation US DP M DP M DP +G Fig Effect of GDP on respiration rates of mitochondria extracted from murine peritoneal macrophages Cells were treated for h with 100 lgỈmL)1 of MDP and oxygen consumption of extracted mitochondria was analysed in the presence or absence of mM of GDP Respiration states (A) and RCR (B) of treated cells are presented as a percentage of unstimulated samples Data are means ± SEM of three independent experiments each performed in duplicate *P < 0.05 versus control **P < 0.05 versus MDP treated Discussion The results obtained in this study demonstrate the ability of toxic MDP to potently induce impairment in mitochondrial bioenergetics in murine peritoneal macrophages The effect of MDP was observed in vitro at a concentration of 100 lgỈmL)1 and after an incubation period of 1–2 h In contrast, the nontoxic muramyl dipeptide derivative MB was not able to provoke any defect in macrophage mitochondria since the RCR and the respiration rate values obtained after h of treatment and at 100 lgỈmL)1 concentration were identical to those of the unstimulated cells This view is consistent with a previous report showing that MDP, but not a safe analogue such as MB, is capable of inducing mitochondrial proton leak in the spleen and liver of injected mice Moreover, it is of importance to note that the maximum in vivo effect of MDP and some of its derivatives on mitochondrial respiration was observed h after administration, a time peak which has been reported for several of the toxicological effects of MDP in vivo [24] The results obtained in this study and in the previous report [23] shed light on mitochondria as a new target affected by MDP and FEBS Journal 278 (2011) 3054–3064 ª 2011 The Authors Journal compilation ª 2011 FEBS 3059 UCP2 modulates MDP-induced mitochondrial inefficiency T G El-Khoury et al reveal a new approach by which muramyl peptides could exert their toxic effect Furthermore, LPS, which constitutes a chemically different immunomodulator from muramyl dipeptides but exerts a high toxic effect in vivo, does not show any significant effect on mitochondrial respiration rates within the time period studied It has been demonstrated previously that LPS requires a period of 16 h to induce a significant impact on rat mitochondrial respiration in vivo [25] Therefore, the mechanism of action of LPS is completely different from MDP in inducing mitochondrial proton leak MDP decreases mitochondrial RCR by increasing state respiration (non-phosphorylation state), without affecting state (succinate-link respiration) or state (phosphorylation state) This increase in the basal proton leak activity of mitochondria (i.e state 4) from MDP-treated cells could be the result of activation or an induction of expression of a mitochondrial membrane protein such as UCP adenine nucleotide translocase or others which can induce a proton leak and thus increase the inefficiency of oxidative phosphorylation In this regard, the effect on state is similar to an uncoupling effect UCP2 acts as a mild uncoupler, controlling both ATP synthesis and the production of ROS (reviewed in [9]) Several lines of evidence emphasize a role for UCP2 in immunity First, UCP2 is expressed in immune cells such as phagocytes and lymphocytes [15] Second, Ucp2) ⁄ ) mice are more resistant to a Toxoplasma gondii or Listeria monocytogenes infection than Ucp2+ ⁄ + mice [14,15] Third, the development of unstable atherosclerotic plaques is greater in the Ucp2) ⁄ ) mouse model of atherosclerosis [26] Fourth, transgenic mice overexpressing UCP2 show a reduced inflammatory response following LPS treatment [27] Moreover, macrophages from ob ⁄ ob mice were reported to express lower UCP2 and higher ROS levels than lean mice [28] These findings agree with the hypothesis [29] that an increase in the mitochondrial membrane potential would slow the transport of electrons through the respiratory chain, increasing the time of interaction between these electrons and molecular oxygen and facilitating the formation of ROS Activation of innate immune cells by MDP is known to be crucial for stimulating host antimicrobial defence reactions [30] ROS are rapidly produced from macrophages after stimulation with MDP and are involved in cellular signalling Also, nitric oxide (NO) production after stimulation plays a pivotal role in numerous and diverse biological functions, in particular as a principal mediator of the microbicidal and tumoricidal actions of macrophages [31] Furthermore, OÀ and NO combine to form the potent oxidant peroxynitrite 3060 (ONOO)) which mediates bactericidal activity [32] Thus, both ROS and NO are important mediators of cellular immune response It is well established that mitochondria are the main source of ROS Moreover, mitochondrial ROS production is particularly sensitive to membrane potential and to mild uncoupling [33] However, the role of mild uncoupling in the regulation of the response to MDP has not been elucidated Thus, we aimed in the present study (a) to demonstrate the involvement of mitochondria in MDP-induced ROS signalling and (b) to identify the mitochondrial protein UCP2 as a physiological brake on this phenomenon As anticipated, both ROS and RNS were markedly higher in MDP-treated macrophages than in unstimulated cells and the overexpression of UCP2 protein correlated with the production of both reactive species However, cells stimulated with MB did not present any modification in the level of detectable ROS or UCP2 expression This finding indicated that UCP2 is a constitutive modulator of reactive species production, suggesting a role for UCP2 in the regulation of intracellular redox state and macrophage-mediated immunity As stated earlier, mitochondria are the major source of ROS production and the primary ROS generated is superoxide anion as a consequence of monoelectronic reduction of O2 Moreover, the main sites of OÀ genera2 tion at the level of the mitochondrial electron transport chain are complexes I and III [34] The ROS generated in mitochondria are removed by local superoxide dismutases and peroxidases and by reaction with low molecular weight reductants and sulfhydryl-containing protein reductants The mechanisms for removal of mitochondrial ROS are thus well described (reviewed in [9]) Additionally, regulated expression of UCP2 would provide a mechanism for adjusting mitochondrial ROS production in cell types such as macrophages by lowering membrane potential and thereby limit ROS production Taken together, our data support a model of UCP2 regulation consisting of a late phase response to MDP At this stage, to h after MDP stimulation, oxidative stress has been induced and there is a need to counteract the toxic effects of inflammation and overstimulation of immune cells Upregulation of UCP2 expression may be seen as a response to reduce the production of ROS in immune cells in a negative feedback regulatory cycle Finally, these data suggest the interesting possibility that UCP2 may serve as an antioxidant, guarding against an excess of oxygen free radicals Further studies on signal transduction cascades that participate in the positive ⁄ negative regulation of UCP2 expression would contribute to designing possible drugs that control bacterial infections FEBS Journal 278 (2011) 3054–3064 ª 2011 The Authors Journal compilation ª 2011 FEBS T G El-Khoury et al Experimental procedures Animals Experiments were done on Balb ⁄ C mice weighing 30–40 g Animals were housed under standard conditions (12 h light ⁄ dark cycle, 22 ± °C) All experiments were approved by the Institutional Animal Care and Use Committee of the University of Balamand and complied with the principles of laboratory animal care Chemicals and reagents Muramyl peptides (MDP and MB) used in this work were kindly provided by ISTAC-SA (Lille, France) and were synthesized as described previously [35] LPS, derived from Escherichia coli (0127:B8), was purchased from Sigma (Steinheim, Germany) Macrophage harvesting and cultivation Macrophages were obtained from mice peritoneum following the method described in [36] BALB ⁄ c mice were intraperitoneally injected with 3% thioglycollate (Difco, Lawrence, KS, USA) broth Four days later, the animals were killed by neck dislocation, and the peritoneal exudates were collected and centrifuged at 400 g The cell sediment was resuspended in Dulbecco’s modified Eagle’s medium (DMEM) phenol red free, supplemented with 10% fetal bovine serum Cells were seeded in 75 cm2 flasks to a final concentration of · 105 cellsỈcm)2 Non-adherent cells were washed with NaCl ⁄ Pi Analysis of murine peritoneal macrophages After h of adherence, cells were washed twice with cold NaCl ⁄ Pi to remove non-adherent cells; then they were detached by trypsinization, rewashed twice with cold NaCl ⁄ Pi and finally resuspended at a final concentration of 106 cellsỈ100 lL)1 in cold NaCl ⁄ Pi Cells were labelled with PE-Cy7-conjugated rat anti-mouse CD11b monoclonal antibody or its isotype control PE-Cy7-conjugated rat IgG2b, j monoclonal immunoglobulin for 30 at room temperature (25 °C) Cells were washed once with NaCl ⁄ Pi, resuspended in 500 lL cell fix solution (containing formaldehyde and 1% sodium azide) and subjected to flow cytometry analysis Data from the experiments were analysed using cellquest software The collected events per sample were 10 000 Isolation of mitochondria Mitochondria from murine peritoneal macrophages were prepared as described previously [12], with all steps carried out at °C Cells were homogenized using a glass Dounce homogenizer in isolation medium consisting of 250 mm UCP2 modulates MDP-induced mitochondrial inefficiency sucrose, mm Tris ⁄ HCl (pH 7.4) and mm EGTA The homogenate was centrifuged at 1047 g for The supernatant was centrifuged at 11 360 g for 11 Mitochondrial pellets were resuspended in the isolation medium and protein concentration was determined by the Biuret method [37] All results are expressed per milligram mitochondrial protein Measurement of oxygen consumption Measurements of oxygen consumption were performed using an oxygen electrode (Clark electrode; Rank Brothers Ltd, Cambridge, UK) Oxygen consumption rates were calculated assuming that the concentration of oxygen in the air-saturated incubation medium was 406 nmolỈmL)1 [12] Mitochondria (3 mgỈmL)1) isolated from culture cells were incubated in standard assay medium (500 lL) containing 120 mm KCl, mm KH2PO4, mm HEPES, mm EGTA supplemented with 0.3% defatted BSA and lm rotenone (pH 7.2, 37 °C) Respiration was initiated with mm succinate as substrate State respiration was measured in the presence of 200 lm ADP and state respiration by adding lgỈmL)1 oligomycin Electrode linearity was checked by following the uncoupled respiration rate in the presence of mm FCCP from 100% to 0% air saturation RCRs were calculated as state divided by state respiration rates Assay for superoxide anion generation Superoxide anion release was determined by superoxide dismutase inhibitable reduction of ferricytochrome c Briefly, macrophages (1 · 106 well)1) were covered with 450 lL of Kreeb’s ringer phosphate buffer (123 mmolỈL)1 NaCl, 1.23 mmolỈL)1 MgCl2, 4.9 mmolỈL)1 KCl and 16.7 mmolỈL)1 Na phosphate buffer, pH 7.4), containing mmolỈL)1 glucose, 0.5 mmolỈL)1 CaCl2 and mmolỈL)1 NaN3 and supplemented with 80 lmolỈL)1 cytochrome c (Sigma) After 10 incubation at 37 °C (5% CO2), cells were treated with MDP (100 lgỈmL)1), MB (100 lgỈmL)1) or LPS (1 lgỈmL)1) A 350 lL aliquot from each well was aspirated at different time intervals and diluted : with cold buffer The reduced cytochrome c was measured by analysing the difference in absorbency at 550–468 nm using a micromolar extinction coefficient of 0.0245 [38] All assays were performed in duplicate Controls containing 30 lgỈmL)1 superoxide dismutase (Sigma) were also made in order to provide correction for the OÀ independent reduction of cytochrome c The results were expressed as nanomoles of superoxide anion per million cells ) ) Measurement of NO2 and NO3 as readout for NO production NO production was evaluated by spectrophotometric determination of its stable decomposition products nitrate and nitrite using Griess’s reaction [39] Nitrate was detected FEBS Journal 278 (2011) 3054–3064 ª 2011 The Authors Journal compilation ª 2011 FEBS 3061 UCP2 modulates MDP-induced mitochondrial inefficiency T G El-Khoury et al after reduction to nitrite using a commercially available preparation of nitrate reductase from Aspergillus (Sigma) Macrophages were seeded in 24-well plates to a final concentration of · 106 cellsỈmL)1 in DMEM phenol red free The supernatants were collected after the appropriate incubation period with MDP (100 lgỈmL)1) or MB (100 lgỈmL)1) or LPS (1 lgỈmL)1) and stored at )20 °C until analysis A mixture at : of 0.1% naphthylenediamine dihydrochloride and 1% sulfanilamide in 5% H3PO4 was added and incubated at room temperature for 10 The absorbance was measured at 540 nm in a microplate automated multiscan reader (Thermo, Runcorn, UK) The results were expressed as nanomoles of NO per million cells Western blot analysis About 50 lg of total cell lysate proteins were resolved by SDS ⁄ PAGE and then transferred to poly(vinylidene difluoride) membranes (GE Healthcare, Chalfont St Giles, UK) that were probed with either an anti-UCP2 antibody or a mouse anti-glyceraldehyde-3-phosphate dehydrogenase antibody used as a loading control The immunoblots were developed by enhanced chemiluminescence (GE Healthcare), and the band intensity was recorded using high performance chemiluminescence films (GE Healthcare) at room temperature The films were scanned using the Gel Documentation System (Biorad) and quantification of the proteins was achieved using quantity one software (Biorad, Marnesla-Coquette, France) Viability test AnnexinV-FITC Apoptosis Detection Kit II was used to determine the percentage of viable, apoptotic and dead cells after treatment, or not, with MDP (100 lgỈmL)1) for h Cells were washed twice with cold NaCl ⁄ Pi, detached by trypsinization (1· trypsin), rewashed twice with cold NaCl ⁄ Pi and finally resuspended at a final concentration of 106 cellsỈmL)1 in 1· binding buffer (10· binding buffer contains 0.1 m HEPES ⁄ NaOH (pH 7.4), 1.4 m NaCl, 25 mm CaCl2) The solution (100 lL, · 105 cells) was transferred to a mL culture tube containing lL of FITC Annexin V and lL propidium iodide The cells were gently mixed and incubated for 15 at room temperature (25 °C) in the dark Finally, 400 lL of 1· binding buffer was added to each tube The suspension was analysed by flow cytometry within h using a FACSCalibur (Becton Dickinson, Erembodegem, Belgium) equipped with a 488-nm argon laser and a 635-nm red diode laser Data from the experiments were analysed using cellquest software The collected events per sample were 10 000 Statistical analysis All results are shown as the mean of data from at least three independent experiments The statistical significance 3062 of the differences was calculated using Student’s t-test and values of P < 0.05 were accepted as statistically significant Data were analysed using the spss 11.0 software Acknowledgements We would like to thank Samer Bazzi and Michel Zakhem for technical assistance This work is supported by grants from the University of Balamand Research Council References Krauss S, Zhang CY & Lowell BB (2005) The mitochondrial uncoupling protein homologues Nat Rev Mol Cell Biol 6, 248–261 Sanchis D, Fleury C, Chomiki N, Goubern M, Huang Q, Neverova M, Gregoire F, Easlick J, Raimbault S, Levi-Meyrueis C et al (1998) BMCP1, a novel mitochondrial carrier with high expression in the central nervous system of humans and rodents, and respiration uncoupling activity in recombinant yeast J Biol Chem 273, 34611–34615 Mao W, Yu XX, Zhong A, Li W, Brush J, Sherwood SW, Adams SH & Pan G (1999) UCP4, a novel brainspecific mitochondrial protein that reduces membrane potential in mammalian cells FEBS Lett 443, 326–330 Klingenberg M & Echtay KS (2001) Uncoupling proteins: the issues from a biochemist point of view Biochem Biophys Acta 1504, 128–143 Pecqueur C, Alves-Guerra MC, Gelly C, Levi-Meyrueis C, Couplan E, Collins S, Ricquier D, Bouillaud F & Miroux B (2001) Uncoupling protein 2, in vivo distribution, induction upon oxidative stress, and evidence for translational regulation J Biol Chem 276, 8705–8712 Boss O, Samec S, Paoloni Giacobino A, Rossier C, Dulloo A, Seydoux J, Muzzin P & Giacobino JP (1997) Uncoupling protein-3: a new member of the mitochondrial carrier family with tissue-specific expression FEBS Lett 408, 39–42 Vidal-Puig A, Solanes G, Grujic D, Flier JS & Lowell BB (1997) UCP3: an uncoupling protein homologue expressed preferentially and abundantly in skeletal muscle and brown adipose tissue Biochem Biophys Res Commun 235, 79–82 Harper JA, Stuart JA, Jekabsons MB, Roussel D, Brindle KM, Dickinson K, Jones RB & Brand MD (2002) Artifactual uncoupling by uncoupling protein in yeast mitochondria at the concentrations found in mouse and rat skeletal-muscle mitochondria Biochem J 361, 49–56 Echtay KS (2007) Mitochondrial uncoupling proteins – what is their physiological role? Free Radic Biol Med 43, 1351–1371 FEBS Journal 278 (2011) 3054–3064 ª 2011 The Authors Journal compilation ª 2011 FEBS T G El-Khoury et al 10 Negre-Salvayre A, Hirtz C, Carrera G, Cazenave R, Troly M, Salvayre R, Penicaud L & Casteilla L (1997) A role for uncoupling protein-2 as a regulator of mitochondrial hydrogen peroxide generation FASEB J 11, 809–815 11 Brand MD, Buckingham AJ, Esteves TC, Green K, Lambert AJ, Miwa S, Murphy MP, Pakay JL, Talbot DA & Echtay KS (2004) Mitochondrial superoxide and aging: uncoupling-protein activity and superoxide production Biochem Soc Sym 71, 203–213 12 Echtay KS, Roussel D, St-Pierre J, Jekabsons MB, Cadenas S, Stuart JA, Harper JA, Roebuck SJ, Morrison A, Pickering S et al (2002) Superoxide activates mitochondrial uncoupling proteins Nature (London) 415, 96–99 13 Echtay KS, Esteves TC, Pakay JL, Jekabsons MB, Lambert AJ, Portero-Otin M, Pamplona R, Vidal-Puig AJ, Wang S, Roebuck SJ et al (2003) A signalling role for 4-hydroxy-2-nonenal in regulation of mitochondrial uncoupling EMBO J 22, 4103–4110 14 Arsenijevic D, Onuma H, Pecqueur C, Raimbault S, Manning BS, Miroux B, Couplan E, Alves-Guerra MC, Goubern M, Surwit R et al (2000) Disruption of the uncoupling protein-2 gene in mice reveals a role in immunity and reactive oxygen species production Nat Genet 26, 435–439 15 Rousset S, Emre Y, Join-Lambert O, Hurtaud C, Ricquier D & Cassard-Doulcier AM (2006) The uncoupling protein modulates the cytokine balance in innate immunity Cytokine 35, 135–142 16 Kizaki T, Suzuki K, Hitomi Y, Taniguchi N, Saitoh D, Watanabe K, Onoe K, Day KN, Good AR & Ohno H (2002) Uncoupling protein plays an important role in nitric oxide production of lipopolysaccharide-stimulated macrophages Proc Natl Acad Sci USA 99, 9392–9397 17 Teshima Y, Akao M, Jones SP & Marban E (2003) Uncoupling protein-2 overexpression inhibits mitochondrial death pathway in cardiomyocytes Circ Res 93, 192–200 18 Lederer E (1988) Natural and synthetic immunomodulators derived from the mycobacterial cell wall In Advances in Immunomodulation (Bizzini B & Bonmassar E eds), pp 9–36 Pythagora Press, Rome 19 Chedid L, Audibert F, Lefrancier P, Choay JP & Lederer E (1976) Modulation of the immune response by a synthetic adjuvant and analogs Proc Natl Acad Sci USA 73, 2472–2475 20 Bahr GM, Darcissac E, Bevec D, Dukor P & Chedid L (1995) Immunopharmacological activities and clinical development of muramyl peptides with particular emphasis on murabutide Int J Immunopharmacol 17, 117–131 21 Ellouz F, Adam A, Ciorbaru R & Lederer E (1974) Minimal structural requirements for adjuvant activity of UCP2 modulates MDP-induced mitochondrial inefficiency 22 23 24 25 26 27 28 29 30 31 32 33 34 35 bacterial peptidoglycan derivatives Biochem Biophys Res Commun 59, 1317–1325 Chedid LA, Parant MA, Audibert FM, Riveau GJ, Parant FJ, Lederer E, Choay JP & Lefrancier PL (1982) Biological activity of a new synthetic muramyl peptide adjuvant devoid of pyrogenicity Infect Immun 35, 417–424 El-Jamal N, Bahr GM & Echtay KS (2009) Effect of muramyl peptides on mitochondrial respiration Clin Exp Immunol 155, 72–78 Colditz IG & Cybulsky MI (1987) Some characteristics of inflammation induced by muramyl dipeptide, endotoxin and concanavalin A Inammation 11, 111 ă Kozlov AV, Staniek K, Haindl S, Piskernik C, Ohlinger W, Gille L, Nohl H, Bahrami S & Redl H (2006) Different effects of endotoxic shock on the respiratory function of liver and heart mitochondria in rats Am J Physiol Gastrointest Liver Physiol 290, 543–549 Blanc J, Alves-Guerra MC, Esposito B, Rousset S, Gourdy P, Ricquier D, Tedgui A, Miroux B & Mallat Z (2003) Protective role of uncoupling protein in atherosclerosis Circulation 107, 388–390 Horvath TL, Diano S, Miyamoto S, Barry S, Gatti S, Alberati D, Livak F, Lombardi A, Morreno M, Goglia F et al (2003) Uncoupling proteins-2 and influence obesity and inflammation in transgenic mice Int J Obes Relat Metab Disord 27, 433–442 Lee FY, Li Y, Yang EK, Yang SQ, Lin HZ, Trush MA, Dannenberg AJ & Diehl AM (1999) Phenotypic abnormalities in macrophages from leptin-deficient, obese mice Am J Physiol 276, C386–C394 Korshunov SS, Skulachev VP & Starkov AA (1997) High protonic potential actuates a mechanism of production of reactive oxygen species in mitochondria FEBS Lett 416, 15–18 Ulevitch RJ & Tobias PS (1999) Recognition of gramnegative bacteria and endotoxin by the innate immune system Curr Opin Immunol 11, 19–22 MacMicking J, Xie Q & Nathan C (1997) Nitric oxide and macrophage function Annu Rev Immunol 15, 323–350 Beckman JS, Beckman TW, Chen J, Marshall PA & Freeman BA (1990) Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide Proc Natl Acad Sci USA 87, 1620–1624 Miwa S, St-Pierre J, Partridge L & Brand MD (2003) Superoxide and hydrogen peroxide production by Drosophila mitochondria Free Radic Biol Med 35, 938–948 St-Pierre J, Buckingham JA, Roebuck SJ & Brand MD (2002) Topology of superoxide production from different sites in the mitochondrial electron transport chain J Biol Chem 277, 44784–44790 Lefrancier P, Choay JP, Derrien M & Lederman I (1977) Synthesis of N-acetyl-muramyl-L-alanyl-D-iso- FEBS Journal 278 (2011) 3054–3064 ª 2011 The Authors Journal compilation ª 2011 FEBS 3063 UCP2 modulates MDP-induced mitochondrial inefficiency T G El-Khoury et al glutamine, an adjuvant of the immune response, and of some n-acetyl-muramyl-peptide analogs Int J Pept Protein Res 9, 249–257 36 Robertson TA, Maley MLA, Grounds MD & Papadimitriou JM (1993) The role of macrophages in skeletal muscle regeneration with particular reference to chemotaxis Exp Cell Res 207, 321–331 37 Gornall AG, Bardawill CJ & David MM (1994) Determination of serum proteins by means of the biuret reaction J Biol Chem 177, 751–766 3064 38 Bellavite P, Dri P, Della Bianca V & Serra MC (1983) The measurement of superoxide anion production by granulocytes in whole blood A clinical test for the evaluation of phagocyte function and serum opsonic capacity Eur J Clin Invest 13, 363–368 39 Green LC, Wagner DA, Glogowski J, Skipper PL, Wishnok JS & Tannenbaum SR (1982) Analysis of nitrate, nitrite, and 15Nnitrate in biological fluids Anal Biochem 126, 131–138 FEBS Journal 278 (2011) 3054–3064 ª 2011 The Authors Journal compilation ª 2011 FEBS ... correlating the uncoupling effect induced by these molecules with the level and function of UCP2 and free radical production in macrophages We find that MDP induces reactive oxygen and nitrogen species. .. effects on the UCP2 modulates MDP-induced mitochondrial inefficiency central nervous system, and the lack of induction of in? ??ammatory responses [22 ] Despite a long-standing interest in the field of muramyl... (3.6-fold, P < 0.05) On the other hand, analysis of the kinetics of induction of UCP2 protein in MDP-treated macrophages revealed a significant increase starting h after stimulation (2. 2-fold, P < 0.05),