FEBS Letters 587 (2013) 2241–2246

journal homepage: www.FEBSLetters.org

High glucose modulates IL-6 mediated immune homeostasis through

impeding neutrophil extracellular trap formation
Manjunath B. Joshi a, Apurva Lad a, Alevoor S. Bharath Prasad a, Aswath Balakrishnan a,
Lingadakai Ramachandra b, Kapaettu Satyamoorthy a,⇑
a
Department of Biotechnology, Manipal Life Sciences Center, Manipal University, Manipal, India
b
Department of Surgery, Kasturba Medical College, Manipal, India

a r t i c l e i n f o a b s t r a c t

Article history: Neutrophils serve as an active constituent of innate immunity and are endowed with distinct ability
Received 17 January 2013 for producing neutrophil extracellular traps (NETs) to eliminate pathogens. Earlier studies have
Revised 18 May 2013 demonstrated a dysfunction of the innate immune system in diabetic subjects leading to increased
Accepted 27 May 2013
susceptibility to infections; however, the influence of hyperglycemic conditions on NETs is
Available online 2 June 2013
unknown. In the present study we demonstrate that (a) NETs are influenced by glucose homeostasis,
(b) IL-6 is a potent inducer of energy dependent NET formation and (c) hyperglycemia mimics a state
Edited by Laszlo Nagy of constitutively active pro-inflammatory condition in neutrophils leading to reduced response to
external stimuli making diabetic subjects susceptible to infections.
Keywords: Ó 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
Neutrophil extracellular trap
Innate immunity
Diabetes
Interleukin-6
Hyperglycemia

1. Introduction suggest hyperglycemia induced abnormalities in neutrophil che-

motaxis, phagocytosis and bactericidal properties as causative fac-
Extrinsic and intrinsic infection is one of the problems associ- tors for increased susceptibility and severity of infections in T2D
ated with type 2 diabetes (T2D). Elevated risk for common commu- [3]. Lowering of blood glucose levels using anti-diabetic drugs
nity acquired infections due to poor glycemic control in T2D has been shown to improve and re-sensitize neutrophil activity [4].
patients is well documented. Several factors such as hyperglycemia Apart from canonically known phagocytosis mechanism, neu-
and hyperlipidemia are attributed to predispose diabetic subjects trophils are endowed with a unique ability of producing extracel-
to urinary and respiratory tract infections, periodontal diseases lular traps referred as neutrophil extracellular traps (NETs) to kill
and soft tissue infections [1]. Multiple studies have indicated spe- pathogens by expelling DNA coated with bactericidal proteins
cific defects in innate and adaptive immune function in diabetic and histones [5]. NETosis is stimulated by diverse bacteria and
patients. Innate immune system acts as the first line of defense their products, fungi, protozoans, cytokines, phorbol esters and
against microbial, chemical, physical and psychological injury by by activated platelets. Besides being beneficial for antimicrobial
facilitating damage repair, avoidance of insults by pathogens and processes, NETs are also associated with autoimmune diseases
restoration of homeostasis [2]. Neutrophils play an important role such as lupus erythematous and small vessel vasculitis, where
in eliminating pathogens as an active constituent of innate im- either excess NETs are produced or impairment in its clearance is
mune system. Clinical studies and data from experimental models noted [6,7]. A recent study indicates NETs as a causative factor
for tumor associated thrombosis [8]. Stimulus dependent role of
several key molecules for NETosis has been proposed. Chronic
Abbreviations: 2-DG, 2-deoxy-D-glucose; CGD, Chronic Granulomatous disease;
Granulomatous disease (CGD) patients with mutations in genes
IL-6, interleukin-6; LPS, lipopolysaccharide; NETs, neutrophil extracellular traps;
G
L-NAME, N -nitro-L-arginine methyl ester; NCM, neutrophil culture medium;
coding for nicotinamide adenine dinucleotide phosphate (NADPH)
NADPH, nicotinamide adenine dinucleotide phosphate; NOS, nitric oxide synthase; oxidase subunits and pharmacological inhibitors of NADPH oxidase
PMA, phorbol myristate acetate; TNF-a, Tumor necrosis factor-alpha; T2D, type 2 inhibit formation of NETs, suggesting that the process of NETosis is
diabetes NADPH oxidase dependent [9]. Interestingly Staphylococcus aureus
⇑ Corresponding author. Address: Manipal Life Sciences Center, Planetarium
Complex, Manipal University, Manipal 576104, India. Fax: +91 820 2571919.
induces rapid and oxidant independent NETs without neutrophil
E-mail address: [email protected] (K. Satyamoorthy). lysis [10]. NG-Nitro-L-arginine methyl ester (L-NAME), inhibitor of

0014-5793/$36.00 Ó 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.febslet.2013.05.053
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2242 M.B. Joshi et al. / FEBS Letters 587 (2013) 2241–2246

nitric oxide synthase (NOS) and Rac2 null mouse inhibited NETs nologies) to detect extracellular DNA as described previously [13].
formation, suggesting participation of reactive nitrogen species in Experiments were performed in 96-well culture plates. NETs were
NETosis [11]. quantified after direct stimulation of freshly isolated neutrophils
An inter-relationship between metabolic and inflammatory (1  105 cells/well) with phorbol 12-myristate 13-acetate (phorbol
pathways in T2D has been well studied. Considering deregulation myristate acetate (PMA); 25 nM), Lipopolysaccharide (LPS 2 lg/
of metabolic and immune response pathways during pathological ml), Tumor necrosis factor (Tumor necrosis factor-alpha (TNF-a);
state of diabetes and NETosis as a potential mechanism for killing 20 ng/ml) or interleukin-6 (IL-6) (all from Sigma Chemicals, St.
bacteria, we therefore, in the present study, investigated whether Louis, MO, USA). At the end of indicated incubation periods, SYTOX
hyperglycemic conditions modulate formation of neutrophil NETs and green was added (5 lM) and after 15 min, fluorescence was mea-
attempted to identify the underlying immunoregulatory mechanisms. sured at 504 nm (excitation) and 530 nm (emission) respectively.
NETs formation is expressed as the fold-increase in fluorescence
2. Materials and methods relative to unstimulated neutrophils. Details of experimental con-
ditions for NETs activity during high glucose conditions are pro-
2.1. Neutrophil isolation vided under the respective figure legends. To visualize NETs, live-
cell cultures were imaged with an Olympus IX-51 inverted fluores-
Blood samples (5 ml each) were obtained from healthy donors cence microscope using EM-C2 Rolera camera (Q imaging, Surrey,
and diabetic subjects at the Kasturba Hospital, Manipal and used Canada) and photomicrographs were analyzed by Image-Pro Plus
for neutrophil isolation. Neutrophils were isolated using well software V 7.0.
established Ficoll–Dextran methods as described previously
[12,13]. Purified neutrophils were resuspended in neutrophil cul- 2.3. Subject selection
ture medium (NCM) which comprised of RPMI 1640 medium sup-
plemented with 2% heat inactivated human serum, 2 mM L- The study involved randomly selected elderly male subjects
glutamine, 100 U/ml penicillin and 100 lg/ml streptomycin among whom 17 were healthy and 18 presented with T2D. The pro-
(Himedia, Mumbai, India). 2-Deoxy-D-glucose (2-DG) was pur- tocol was approved by Kasturba Hospital Ethical Committee, Mani-
chased from Sigma–Aldrich. Cell viability was 95–97% as deter- pal, India and informed written consent was obtained from all
mined by trypan blue dye exclusion method. subjects. Fasting and postprandial serum glucose levels of above
126 mg/dl (7.0 mmol/l) and 200 mg/dl (11.1 mmol/l) respectively
2.2. Neutrophil activation and analysis of NETs were defined as diabetic. Mean value of glycosylated hemoglobin
levels of diabetic subjects and normal individuals in our study was
NETs were quantified using the membrane-impermeable DNA 10.64 ± 2.47% and 5.75 ± 1.12% respectively. The study excluded
binding dye SYTOX green (Molecular Probes, Invitrogen Life Tech- subjects suffering from systemic diseases (i.e., immunologic and

Fig. 1. High glucose inhibits NETs formation: (A) Freshly isolated neutrophils were cultured under normal (5.5 mM) and high (30 mM) glucose conditions for 24 h followed by
stimulation with LPS (2 lg/ml), PMA (25 nM) and TNF-a (20 ng/ml) for 3 h. DNA lattices were stained with SYTOX green (5 lM) for 15 min in dark. NETs were quantified
using DNA binding dye SYTOX green by flourimetery. Significant decrease in NETs formation when cultured in hyperglycemic conditions compared to normal glucose are
indicated (⁄⁄⁄P < 0.001, ⁄⁄P < 0.01). (B) Fluorescence microscopy of Sytox green stained NETs induced by LPS and PMA in neutrophils cultured under normal glucose (top panel)
and high glucose (lower panel) conditions (scale bar 100 lm). (C) Kinetic analysis of neutrophil NETs formation under different glucose conditions upon treatment with LPS.
Significant accumulation of NETs formation at 3 and 6 h in normal glucose and at 6 h in high glucose compared to 0 h is indicated (⁄⁄⁄P < 0.001). (D) Effects of hyperosmolar
condition on NETs. Neutrophils were cultured in normal glucose (5.5 mM), high glucose (30 mM) and mannitol (30 mM) for 24 h. LPS induced NETs formation in normal
glucose and mannitol are represented (⁄⁄⁄P < 0.001). Data is expressed as percentage of maximal SYTOX green fluorescence ± S.E.M. against glucose concentrations in
neutrophils cultured from 8 individuals. Results of at least four independent experiments are represented.
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M.B. Joshi et al. / FEBS Letters 587 (2013) 2241–2246 2243

auto-immune disorders, mononuclear blood cell dysfunctions, Neutrophils isolated from control (n = 4) and diabetic subjects
infectious diseases, a history of cardiovascular disease, and other (n = 4) were activated with or without LPS (2 lg/ml) and PMA
chronic systemic diseases) or treated with immunosuppressive (25 nM) for 3 h. Supernatant were collected after centrifuging
medications. plates at 500g for 10 min and NETs associated elastase was re-
leased after treating with DNase (8 U/ml) (Sigma Chemicals, St.
2.4. Bacterial survival assay Louis, MO, USA) for 20 min. Total neutrophil elastase was quanti-
fied from resting neutrophils lysed with 0.02% triton X-100 in
Assay was performed as described in earlier studies [9]. 1 M NaCl. Elastase activity in cell lysate, supernatant and medium
Freshly isolated neutrophils from control (n = 4) and diabetic treated with DNase were measured, upon incubation with elastin
subjects (n = 4) were activated with or without LPS for 3 h. substrate conjugated with BODIPYÒ FL dye for 30 min, at 505/
Neutrophils were infected with Escherichia coli DH5a at the 515 nm using EnzCheck Elastase Assay Kit (Life Technologies,
multiplicity of infection 0.1 in 1.0 ml RPMI for 1 h. To ensure India).
the role of NETs, bacteria infected neutrophils were treated in
presence or absence of DNase (8 U/ml) along with LPS. Cellular 2.6. IL-6 ELISA
content was scrapped, mixed and subsequently, 100 ll was
spread onto nutrient agar plates to assess bacterial growth. Neutrophils isolated from normal and diabetic subjects were
After overnight incubation at 37 °C, colony forming units were seeded at a density of 1  105 cells per well in 96-well plates in
estimated. the presence or absence of LPS (2 lg/ml). Neutrophil conditioned
medium was collected after 3 and 16 h, centrifuged and the accu-
2.5. Elastase assay mulated IL-6 was measured using sandwich ELISA method accord-
ing to the manufacturer’s instruction (Biolegend’s ELISATM Set
NETs associated elastase in control and diabetic subjects were Deluxe, USA). Absorbance was read at 450 nm in an ELISA reader
measured as described previously [9] with minor modifications. (TECAN, Austria).

Fig. 2. Neutrophils from diabetic subjects make inadequate and inactive NETs. (A) Neutrophils isolated from non-diabetic (n = 17) and diabetic subjects (n = 18) were
stimulated with LPS for 3 h and stained with SYTOX green. NETs were quantified by measuring fluorescence of SYTOX green. Scatter graph represents ± S.E.M. of arbitrary
units measured in quadruplicate for each subject. Significant changes in NETs formation within and between control and diabetic subsets are indicated (⁄⁄⁄P < 0.001,
⁄⁄
P < 0.01). (B) Representative fluorescence photomicrographs of NETs stained with SYTOX green of control donor (top panel) and diabetic donor (lower panel). (Scale bar
100 lm). (C) NETs associated elastase activity was measured in neutrophils isolated from control (n = 4) and diabetic (n = 4) subjects. Cells were either treated with LPS and
PMA or left untreated for 3 h, followed by addition of DNase (8 U/ml) to release elastase for 20 min. Percentage of elastase levels ± S.E.M. in neutrophils from control and
diabetic subjects is indicated. Statistically significant differences in NET bound elastase levels in neutrophils from control and diabetic individuals are represented
(⁄⁄⁄P < 0.001). (D) Bacterial survival was assessed upon infecting neutrophils from control (n = 4) and diabetic (n = 4) subjects. Naïve neutrophils were activated with LPS to
make NETs and infected with E. coli for 1 h in presence or absence of DNase, subsequently cellular content was spread on nutrient agar plates. Bacterial survival was
determined by enumerating colony forming units. Representative data of three independent experiments are expressed as percentage bacterial survival ± S.E.M. Significant
decrease (⁄⁄⁄P < 0.001) in CFU is observed when bacteria infected with LPS treated compared to untreated and DNase treated neutrophils. Increase in survival rate in LPS
treated diabetic neutrophils as compared to normal subjects are also indicated (⁄⁄⁄P < 0.001).
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2244 M.B. Joshi et al. / FEBS Letters 587 (2013) 2241–2246

2.7. Statistics creased release of myeloperoxidase and elastase from azurophilic

granules. This suggests that increased circulating glucose under
Each experiment was performed in triplicates and on three sep- inflammatory conditions abrogates neutrophil degranulation
arate occasions. Statistical analyses were performed using Graph- [16,17]. Hence, to examine any modulation in NETs associated pro-
Pad Prism 5.0. A One-way Repeated Measure Analysis of Variance teins in diabetic subjects, we measured elastase activity. Pre-stim-
followed by post hoc Bonferroni’s multiple comparison tests was ulated neutrophils from non-diabetic individuals exhibited 2.5-
used to calculate the statistical significance differences between fold increase in NETs associated elastase activity (DNase treated)
experimental groups. P values of less than 0.05 were considered relative to DNase untreated neutrophils (Fig. 2C). Interestingly,
as significant. NETs bound elastase activity was reduced in diabetes subjects
when compared to non-diabetic individuals, indicating a dysfunc-
3. Results and discussion tion of one of the important protein component of NETs during
diabetes.
3.1. High glucose condition impairs and delays neutrophil NETs We next examined the biological function of NETs by perform-
formation ing bacterial survival assay upon infecting neutrophils from control
and diabetic subjects with E. coli DH5a (Fig. 2D). We observed sig-
To assess the impact of high glucose conditions on neutrophil nificant increase in survival of bacteria when infected with neutro-
NETs formation, we cultured neutrophils from normal subjects in phils from diabetic subjects in unstimulated condition, suggesting
the presence or absence of high glucose followed by stimulation a constitutively decreased neutrophil function during diabetes.
either with LPS, PMA or TNF-a. Three hours after induction, neutro- Bacterial growth decreased from 20% to 3% in LPS stimulated neu-
phils initiated expelling DNA lattices to form NETs in normal con- trophils from control subjects; such effects were not observed in
ditions. Both microscopy and fluorimetry analysis suggested neutrophils from diabetic subjects. LPS stimulated neutrophils
drastic impairment in NETs formation under high glucose condi- from control subjects upon treatment with DNase showed a robust
tions. Extracellular DNA lattices formed in hyperglycemic condi- increase in bacterial survival rate. We also observed increased bac-
tions were short-lived and unstable leading to rapid teria killing in DNase treated neutrophil NETs from diabetic sub-
disintegration (Fig. 1A and B). Subsequently, we carried out a time jects albeit lesser than under similar conditions from neutrophils
course experiment to determine whether NETs production was de- of normal individuals (Fig. 2D). This clearly suggests existence of
layed in hyperglycemic conditions. After 6 h of stimulation with alternative mechanisms in diabetic environment for the regulation
LPS, we observed a gradual formation and accumulation of NETs of anti-microbial effect. Regardless, our study does not rule out a
in high glucose conditions, albeit the density of NETs was not as role for other prominent anti-bacterial mechanisms such as phago-
high as under normal culture conditions (Fig. 1C). Mannitol did cytosis. As reported in earlier studies [9] DNase per se does not
not show any effect on LPS induced NETs, suggesting inhibition interfere with bacterial survival (data not shown). Taken together
of NETs in high glucose condition was not due to hyperosmolarity our data suggest neutrophils NETs in diabetic environment show
conditions (Fig. 1D). To validate our findings more closely to clini- reduced antimicrobial property.
cal conditions, we investigated the neutrophil activation and NETs
formation in diabetic patients. Upon stimulation with LPS for 3 h, 3.2. Pro-inflammatory conditions during hyperglycemia favor
neutrophils from diabetic subjects responded weakly to LPS and constitutive NETs formation leading to weak response to stimuli
lesser NETs were formed (Fig. 2A and B); whereas, neutrophils
from normal individuals showed robust release of NETs. In some We observed neutrophils forming NETs without external stim-
patients we found short and imperfect NETs in basal conditions ulation in a significant number (50%) of diabetic patients
suggesting constitutive activation of neutrophils in diabetic sub- (Fig. 3A). These NETs produced under basal conditions were smal-
jects. Granular proteins such as elastase and myeloperoxidase are ler and disintegrated rapidly. To address this pre-activated state of
important components of NETs. Degranulation followed by trans- neutrophils, we evaluated the differential response of neutrophils
location of elastase and myeloperoxidase to the nucleus, and sub- exposed to various concentrations of glucose on NETs formation.
sequent chromatin decondensation is a prerequisite for NETs There was a strong NETs formation in neutrophils cultured under
formation. Both elastase knock out mouse and myeloperoxidase normal glucose (5.5 mM) conditions but these cells displayed var-
deficient patients have been reported to produce reduced amount ied response to LPS under higher glucose conditions (Fig. 3A).
of NETs [14,15]. Hyperglycemic conditions have been shown to Interestingly at higher concentration (20 mM) of glucose, neutro-
attenuate LPS induced neutrophil degranulation resulting in a de- phils produced NETs in the absence of LPS. At very high concentra-

Fig. 3. Constitutively activated neutrophils exist in high glucose conditions. (A) Isolated neutrophils from normal individuals were cultured under indicated concentrations of
glucose for 24 h, followed by stimulation with LPS for 3 h and DNA lattices were stained with SYTOX green. Significant accumulation of NETs in LPS stimulated (relative to
untreated ⁄⁄⁄P < 0.001) and in cells cultured in the presence of 20 mM glucose (relative to untreated ⁄⁄P < 0.01) are indicated. Data are expressed as percentage of maximal
SYTOX green fluorescence ± S.E.M. against glucose concentrations. (B) Neutrophils from non-diabetic control (n = 8) and Diabetic subjects (n = 8) were cultured in presence or
absence of LPS for 0, 3 and 16 h. U, unstimulated; S, stimulated. Conditioned medium was measured for IL-6 levels by ELISA. Statistically significant levels of IL-6 (pg/105 cells)
expressed by diabetic neutrophils relative to normal individuals is indicated by (⁄⁄⁄P < 0.001).
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M.B. Joshi et al. / FEBS Letters 587 (2013) 2241–2246 2245

Fig. 4. High glucose negatively modulates IL-6 induced NETosis. (A) Neutrophils isolated from normal individual were cultured in presence or absence of LPS (2 lg/ml) for 3 h
and IL-6 (20, 40 and 60 ng/ml) for 16 h. NETs were quantified using SYTOX green fluorescence. Average data from 4 different individuals are presented. Significant values of
(⁄⁄P < 0.01) and (⁄⁄⁄P < 0.001) relative to untreated control are represented. (B) Neutrophils cultured under 5.5 and 30 mM glucose for 24 h were subsequently treated with IL-
6 (20 ng/ml) for 16 h. NETs were measured by SYTOX green fluorescence. Statistical significance for reduction in IL-6 induced NETs formation in high glucose relative to
normal glucose is indicated (⁄⁄P < 0.01). Data are expressed as percentage of maximal SYTOX green fluorescence ± S.E.M. against glucose concentrations.

Fig. 5. 2-DG restores NETs formation. (A) Freshly isolated neutrophils were cultured under normal (NG) and high glucose (HG) in presence or absence of 2-DG (500 lM) for
24 h and subsequently stimulated with LPS for 3 h and IL-6 for 16 h. NETs were quantified after staining with SYTOX green dye and represented as percentage of maximal
SYTOX green fluorescence ± S.E.M. against glucose concentration. Significant differences in NETs formation are indicated (⁄⁄P < 0.01, ⁄⁄⁄P < 0.001). (B) Neutrophils isolated
from control subjects (NCS) and diabetic subjects (NDS) (n = 4) were treated with or without LPS in presence of 2-DG (200 lM) for 3 h and NETs were stained with SYTOX
green. Significant accumulation of NETs restored upon 2-DG treatment is represented (⁄⁄⁄P < 0.001).

tion of glucose (30 mM), DNA lattices disintegrated and did not re- cose-dependent priming of neutrophils produces superoxides con-
spond to LPS, suggesting neutrophils are constitutively active in stitutively and may contribute towards pre-activation of
hyperglycemic conditions with reduced response to further stim- neutrophils. However, peritoneal neutrophils from alloxan induced
uli. Diabetes is associated with low grade, sub-clinical and chronic diabetic rats showed impaired and delayed NADPH oxidase activity
inflammation characterized by abnormal cytokine production, in response to Candida albicans infection [17], suggesting role of
upregulated acute phase reactants and pre-activated inflammatory multiple mechanisms for neutrophil activation in T2D conditions.
signaling networks [18]. Monocytes and macrophages are hyperac-
tivated during T2D by constitutively producing TLR4 thereby influ- 3.3. IL-6 induces NETosis and is abrogated by high glucose condition
encing a pro-inflammatory ‘feed-forward loop’. To support the data
for pre-activation of neutrophils under our conditions, we mea- Diabetes is associated with a chronic inflammatory condition
sured IL-6 levels from cultured neutrophils isolated from normal and is reported to produce elevated levels of TNF-a, IL-8, IL-6
and diabetic subjects treated with or without LPS at various time and C-reactive proteins [18]. IL-6 is one of the main pro-inflamma-
points (Fig. 3B). Neutrophils isolated from T2D patients produced tory cytokines produced during diabetes. At the same time, neutro-
higher basal levels of IL-6 and did not respond to LPS treatment phils are known to secrete IL-6 upon autocrine and paracrine
as compared to cells from normal individuals. A similar observa- stimulation. Therefore, we investigated whether IL-6 secreted by
tion was made for NETs formation in T2D subjects where in hyper- neutrophils has any influence due to autocrine effect on NETosis.
glycemic condition, pre-activated neutrophils showed stimulant For the first time, we report that indeed IL-6 is a potent inducer
independent NETs formation and failed to respond to treatment of NETs. IL-6 induced NETs after 16 h of stimulation and was com-
with LPS. Taken together, our study shows that neutrophils in parable to that of LPS (Fig. 4A). Interestingly IL-6 failed to produce
T2D produce excess levels of IL-6 and may be responsible for NETs in neutrophils when cultured under high glucose (30 mM)
inducing a constitutively active state. Omori et al. (2008) demon- conditions (Fig. 4B).
strated that high glucose induces ERK1/2 mediated premature
translocation of p47phox subunit of NADPH oxidase to cell mem- 3.4. Glycolysis inhibitor 2-deoxyglucose restores NETs formation
brane in diabetic neutrophils leading to constitutive generation
of superoxides [19]. Clinical and experimental data strongly sug- Since glucotoxicity is known to show causal influence on neu-
gests that NADPH oxidase derived oxygen free radicals are the trophil dysfunction in diabetes, we investigated whether inhibition
key components required for NETosis. This suggests that high glu- of glycolysis has any role in the formation of NETs. Addition of 2-
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2246 M.B. Joshi et al. / FEBS Letters 587 (2013) 2241–2246

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ipal University, Manipal, India. Authors thank Ms. Vandana Singh, treatment of endothelial cells induces autophagy by reactive oxygen species-
Ms. Deepika, Mr. Ganesh and Mr. Vishwanath for technical mediated activation of the AMP-activated protein kinase. PLoS ONE 6, e17234.
assistance.