Muramyl-dipeptide-induced mitochondrial proton leak in
macrophages is associated with upregulation of
uncoupling protein 2 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 6 930279
Tel: +961 3 714125
E-mail:
(Received 5 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 expres-

late 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
thermogenesis, protection against obesity, regulation of
the ATP ⁄ ADP ratio, export of fatty acids, and media-
tion 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 oxi-
dative damage [10,11]. This is mainly based on the
activation of mitochondrial proton conductance medi-
ated through UCPs by reactive oxygen species (ROS)
or by-products of lipid peroxidation [12,13], resulting
in a negative feedback loop that decreases ROS pro-
duction 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 perito-
neal macrophages of UCP2
) ⁄ )
mice are resistant to
infection by the intracellular parasite Toxoplasma gon-
dii through a mechanism proposed to involve higher
production of intracellular ROS [14]. On the other
hand, studies in cells overexpressing UCP2 have rein-

molecule MDP. The results reported establish the
safety of MB, the absence of undesirable effects on the
central nervous system, and the lack of induction of
inflammatory responses [22].
Despite a long-standing interest in the field of mura-
myl peptides, the impact of these molecules at the
mitochondrial level has not yet been examined.
Recently the effect of these derivatives on mitochon-
drial bioenergetics has been studied [23]. MDP induced
in vivo a significant decrease in respiratory control
ratio (RCR) in isolated mouse liver and spleen mito-
chondria versus non-toxic analogues such as MB. The
decrease in RCR in mitochondria of MDP-treated
mice is attributed to an increase in mitochondrial pro-
ton 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 nitro-
gen 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

returned to its basal level after 4 h. Figure 1B shows
that the decrease in RCR in mitochondria of MDP-
treated macrophages was attributed to an increase in
state 4 respiration. No significant changes were
observed in state 2, state 3 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 1 summarize the effect of MB
(non-toxic muramyl peptide) and lipopolysaccharide
(LPS) on the mitochondrial bioenergetics of macro-
phage-treated cells. The results demonstrate clearly the
inability of MB and LPS to induce any impairment in
mitochondrial function after 2 h of treatment. RCR
and states 2, 3, 4 and FCCP rates of MB- and LPS-
treated 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 mito-
chondrial respirations of peritoneal macrophages after
2 h of treatment.
Effect of MDP on cell viability
The viability of peritoneal macrophages under condi-
tions of maximum impairment o f mitochondrial activity
of MDP-treated cells was examined. The proportions
of viable (Annexin V-FITC
neg
⁄ propidium iodide

)1
), murabutide (MB, 100 lgÆmL
)1
) or LPS (1 lgÆmL
)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.
*
0
0.2
0.4
0.6
0.8
1
1.2
RCR
MB
LPS
Control
MDP
C
A
1
2
4
6
*
*

(Fig. 2C).
Time course effect of MDP on ROS and reactive
nitrogen species production by murine peritoneal
macrophages
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 lgÆmL
)1
) on total
cellular superoxide anion production by murine perito-
neal macrophages. As shown in Fig. 3, total superox-
ide production was unchanged after 30 min but was
significantly elevated at 60 and 120 min (P < 0.05) in
MDP-treated cells. Interestingly, the O
À
2
level
decreased after 2 h of stimulation, returning almost to
the resting level after 4 h. On the other hand, stimula-
tion with MB failed to induce superoxide production
(Fig. 3), even after 6 h of treatment, whereas stimula-
tion with LPS only induced significant enhancement of
superoxide production after a period of 6 h of stimula-
tion (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

PI
–
/Ann
+
PI
–
/Ann
+
PI
+
/Ann
+
C
10
0
10
2
10
3
10
4
10
1
10
0
10
2
10
3
10

2
2
3
3
4
4
4
5
8
5
Fold increase of superoxide
Fold increase of total NO
Time (h)
*
*
*
Fig. 3. Effect of MDP and MB on O
À
2
and NO
À
2
=NO
À
3
production
by murine peritoneal macrophages. Macrophages (10
6
well
)1

)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).
Macrophage activation by MDP leads to
overexpression of UCP2
Stimulation of peritoneal macrophages by MDP
increased cellular ROS and reactive nitrogen species
(RNS) production. The increased production of reac-
tive species was apparent after 2 h of stimulation.
Since UCP2 is described as a regulator of ROS pro-
duction, the expression of UCP2 in macrophages stim-
ulated or not with immunomodulators was then
investigated. Results shown in Fig. 4A clearly demon-
strate that stimulation of macrophages with MDP
(100 lgÆmL
)1
) for 2 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 signif-
icant increase starting 1 h after stimulation (2.2-fold,
P < 0.05), a peak level after 2 h (3.6-fold, P < 0.05)
and a return to baseline level after 6 h of treatment
(Fig. 4B).
Free radical generation contributes to UCP2
upregulation
To determine if MDP-induced UCP2 upregulation cor-
related with free radical generation, cells stimulated

These results clearly suggest that the mitochondrial
inefficiency caused by MDP (100 lgÆmL
)1
) after 2 h of
incubation in peritoneal macrophages occurs partially
through UCP2.
1
3
2
4
0
5
Relative expression of
UCP2/GAPDH
*
A
US MDP MB LPS
*
*
1 h
1
3
2 h
2
4
6 h
0
5
Relative expression of
UCP2/GAPDH

UCP2 modulates MDP-induced mitochondrial inefficiency T. G. El-Khoury et al.
3058 FEBS Journal 278 (2011) 3054–3064 ª 2011 The Authors Journal compilation ª 2011 FEBS
Discussion
The results obtained in this study demonstrate the abil-
ity of toxic MDP to potently induce impairment in
mitochondrial bioenergetics in murine peritoneal mac-
rophages. The effect of MDP was observed in vitro at
a concentration of 100 lgÆmL
)1
and after an incuba-
tion period of 1–2 h. In contrast, the nontoxic mura-
myl dipeptide derivative MB was not able to provoke
any defect in macrophage mitochondria since the RCR
and the respiration rate values obtained after 2 h of
treatment and at 100 lgÆmL
)1
concentration were iden-
tical 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 2 h after administration, a time peak
which has been reported for several of the toxicologi-
cal 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
1

US MDP MDP + Vit EVit E
*
*
US
MDP
Vit E
MDP
+ Vit E
B
*
*
Fig. 5. Effect of vitamin E on UCP2 expression. Macrophages
(10
6
well
)1
) were pretreated with vitamin E (100 lM) for 10 min
and then stimulated with MDP (100 lg) for 2 h, and O
À
2
and
NO
À
2
=NO
À
3
were measured as described in Experimental proce-
dures. Results for O
À

**
Fig. 6. Effect of GDP on respiration rates of mitochondria extracted
from murine peritoneal macrophages. Cells were treated for 2 h
with 100 lgÆmL
)1
of MDP and oxygen consumption of extracted
mitochondria was analysed in the presence or absence of 1 m
M 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.
T. G. El-Khoury et al. UCP2 modulates MDP-induced mitochondrial inefficiency
FEBS Journal 278 (2011) 3054–3064 ª 2011 The Authors Journal compilation ª 2011 FEBS 3059
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 mito-
chondrial respiration rates within the time period stud-
ied. 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
4 respiration (non-phosphorylation state), without
affecting state 2 (succinate-link respiration) or state 3
(phosphorylation state). This increase in the basal pro-

membrane potential would slow the transport of elec-
trons 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 macro-
phages after stimulation with MDP and are involved
in cellular signalling. Also, nitric oxide (NO) produc-
tion 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, O
À
2
and
NO combine to form the potent oxidant peroxynitrite
(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

duction 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, 1 to 2 h after MDP stimulation, oxida-
tive stress has been induced and there is a need to
counteract the toxic effects of inflammation and over-
stimulation 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 feed-
back regulatory cycle. Finally, these data suggest the
interesting possibility that UCP2 may serve as an anti-
oxidant, guarding against an excess of oxygen free rad-
icals. Further studies on signal transduction cascades
that participate in the positive ⁄ negative regulation of
UCP2 expression would contribute to designing possi-
ble drugs that control bacterial infections.
UCP2 modulates MDP-induced mitochondrial inefficiency T. G. El-Khoury et al.
3060 FEBS Journal 278 (2011) 3054–3064 ª 2011 The Authors Journal compilation ª 2011 FEBS
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 ± 2 °C). All experiments were
approved by the Institutional Animal Care and Use Com-
mittee 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

10
6
cellsÆ100 lL
)1
in cold NaCl ⁄ P
i
. Cells were labelled with
PE-Cy7-conjugated rat anti-mouse CD11b monoclonal anti-
body or its isotype control PE-Cy7-conjugated rat IgG
2
b, j
monoclonal immunoglobulin for 30 min at room tempera-
ture (25 °C). Cells were washed once with NaCl ⁄ P
i
, resus-
pended in 500 lL cell fix solution (containing formaldehyde
and 1% sodium azide) and subjected to flow cytometry anal-
ysis. Data from the experiments were analysed using cell-
quest software. The collected events per sample were 10 000.
Isolation of mitochondria
Mitochondria from murine peritoneal macrophages were pre-
pared as described previously [12], with all steps carried out
at 4 °C. Cells were homogenized using a glass Dounce
homogenizer in isolation medium consisting of 250 mm
sucrose, 5 mm Tris ⁄ HCl (pH 7.4) and 2 mm EGTA. The
homogenate was centrifuged at 1047 g for 3 min. The super-
natant was centrifuged at 11 360 g for 11 min. 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.

Briefly, macrophages (1 · 10
6
well
)1
) were covered with
450 lL of Kreeb’s ringer phosphate buffer (123 mmolÆL
)1
NaCl, 1.23 mmolÆL
)1
MgCl
2
, 4.9 mmolÆL
)1
KCl and
16.7 mmolÆL
)1
Na phosphate buffer, pH 7.4), containing
5 mmolÆL
)1
glucose, 0.5 mmolÆL
)1
CaCl
2
and 2 mmolÆL
)1
NaN
3
and supplemented with 80 lmolÆL
)1
cytochrome c

)
as readout for NO
production
NO production was evaluated by spectrophotometric deter-
mination of its stable decomposition products nitrate and
nitrite using Griess’s reaction [39]. Nitrate was detected
T. G. El-Khoury et al. UCP2 modulates MDP-induced mitochondrial inefficiency
FEBS Journal 278 (2011) 3054–3064 ª 2011 The Authors Journal compilation ª 2011 FEBS 3061
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 con-
centration of 1 · 10
6
cellsÆmL
)1
in DMEM phenol red free.
The supernatants were collected after the appropriate
incubation period with MDP (100 lgÆmL
)1
)orMB
(100 lgÆmL
)1
) or LPS (1 lgÆmL
)1
) and stored at )20 °C
until analysis. A mixture at 1 : 1 of 0.1% naphthylenedi-
amine dihydrochloride and 1% sulfanilamide in 5% H
3
PO
4

6
cellsÆmL
)1
in 1· binding buffer (10· binding buffer contains
0.1 m HEPES ⁄ NaOH (pH 7.4), 1.4 m NaCl, 25 mm CaCl
2
).
The solution (100 lL, 1 · 10
5
cells) was transferred to a 5 mL
culture tube containing 5 lL of FITC Annexin V and 5 lL
propidium iodide. The cells were gently mixed and incubated
for 15 min at room temperature (25 °C) in the dark. Finally,
400 lLof1· binding buffer was added to each tube. The sus-
pension was analysed by flow cytometry within 1 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 cell-
quest 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
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 Zak-
hem for technical assistance. This work is supported
by grants from the University of Balamand Research
Council.

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