Phagocytosis of bacteria is enhanced in macrophages undergoing nutrient deprivation

Phagocytosis of bacteria is enhanced in macrophagesundergoing nutrient deprivationWim Martinet1,*, Dorien M. Schrijvers1,*, Jean-Pierre Timmermans2, Arnold G. Herman1 andGuido R. Y. De Meyer1

1 Division of Pharmacology, University of Antwerp, Belgium

2 Laboratory of Cell Biology and Histology, University of Antwerp, Belgium

Nutrient deprivation triggers a variety of signaling

events that enable energy conservation by cells. Among

the different nutrient-sensing pathways, it is worth not-

ing: (a) AMP-activated protein kinase, a metabolic

stress sensor that is stimulated by elevated AMP ⁄ATP

ratios; (b) Per-Arnt-Sim kinase, which acts as a molec-

ular sensor of oxygen, redox status, ATP and other

indicators of the cellular metabolism; and (c) the

hexosamine biosynthetic pathway that produces uri-

dine 5¢-diphospho-N-acetylglucosamine as a substrate

for O-N-acetylglucosamine transferase [1]. One energy

conservation strategy that has attracted much attention

is enhanced autophagocytosis, also known as macro-

autophagy or simply autophagy [2]. This process is a

catabolic pathway involving the engulfment and degra-

dation of a cell’s own components through the lyso-

somal machinery [3,4]. Autophagic signaling in

response to nutrients is mainly relayed through the

serine-threonine kinase mammalian target of rapa-

mycin (mTOR). Indeed, mTOR is activated by nutri-

ent-rich conditions, especially high levels of amino

acids and insulin. Blocking mTOR function using

rapamycin or its analogs mimics nutrient deprivation

and triggers autophagy [1,4].

Keywords

autophagy; heterophagy; p38 MAP kinase;

scavenger receptor A; starvation

Correspondence

W. Martinet, Division of Pharmacology,

University of Antwerp, Universiteitsplein 1,

B-2610 Antwerp, Wilrijk, Belgium

Fax: +32 3 820 25 67

Tel: +32 3 820 26 79

E-mail: [email protected]

*These authors contributed equally to this

work

(Received 28 November 2008, revised 28

January 2009, accepted 5 February 2009)

doi:10.1111/j.1742-4658.2009.06951.x

Phagocytosis represents a mechanism used by macrophages to remove

pathogens and cellular debris. Recent evidence suggests that phagocytosis

is stimulated under specific conditions of stress, such as extracellular pres-

sure and hypoxia. In the present study, we show that amino acid or glucose

deprivation caused an increase in the phagocytosis of heat-inactivated

Escherichia coli and Staphylococcus aureus by macrophages, but not the

uptake of platelets, apoptotic cells or beads. Increased phagocytosis of bac-

teria could be blocked by phagocytosis inhibitors and was found to be

dependent on p38 mitogen-activated protein kinase activity and scavenger

receptor A. Although nutrient deprivation is a strong stimulus of auto-

phagy, autophagosome formation was not critical for the uptake of bacte-

ria because phagocytic clearance was not inhibited after down-regulation of

the autophagy essential gene Atg7. Moreover, enhanced uptake of bacteria

should not be considered as a general stress response because phagocytosis

of bacteria was not stimulated after exposure of macrophages to the

genotoxic agent camptothecin, heat (40 �C) or thapsigargin-induced endo-

plasmic reticulum stress. Overall, the results obtained in the present

study indicate that nutrient deprivation can stimulate macrophages to fight

bacterial infections.

Abbreviations

AC, apoptotic cell; CHOP, C ⁄ EBP homologous protein; EBSS, Earle’s balanced salt solution; eGFP, enhanced green fluorescent protein; ER,

endoplasmic reticulum; LPS, lipopolysaccharide; MAP, mitogen-activated protein; MARCO, macrophage receptor with collagenous structure;

mTOR, mammalian target of rapamycin; NDRG1, N-myc downstream-regulated gene 1; PI, propidium iodide; PI3-kinase, phosphoinositide

3-kinase; PLT, platelet; RORa, RAR-related orphan receptor alpha gene; siRNA, small interfering RNA; SR-A, scavenger receptor A; TLR,

Toll-like receptor.

FEBS Journal 276 (2009) 2227–2240 ª 2009 The Authors Journal compilation ª 2009 FEBS 2227

Beside autophagocytosis, cells have the ability to

internalize and degrade extracellular particles via het-

erophagocytosis (hereafter referred to as phagocytosis)

[5,6]. Lower organisms use phagocytosis for the acqui-

sition of nutrients, whereas phagocytosis in metazoa

primarily occurs in specialized cells, such as macro-

phages and neutrophils, where it has evolved into an

extraordinarily complex process. Phagocytosis by mac-

rophages is critical for the uptake and removal of

infectious agents and senescent or dying cells and is

stimulated under specific conditions of stress, such as

extracellular pressure [7] and hypoxia [8]. Several lines

of evidence indicate that many nutrients regulate phag-

ocytic activity in macrophages. For example, a choles-

terol-rich diet inhibits macrophage phagocytosis by

down-regulating plasma membrane fluidity and inter-

ference with receptor movement [9]. By contrast, ascor-

bate, as well as some trace elements, such as zinc, lead

and cadmium, stimulate the phagocytic capacity of

macrophages [10–12]. Furthermore, a diminished avail-

ability of iron may impair the ability of phagocytic

cells to kill ingested bacteria and fungi by down-regu-

lating myeloperoxidase activity [13].

Recent evidence was provided showing that auto-

phagy is essential for the phagocytosis of apoptotic

corpses during embryonic development because the

autophagic process contributes to the generation of

engulfment signals in apoptotic cells (ACs) by main-

taining cellular ATP production [14]. Moreover, engag-

ing the autophagic pathway via Toll-like receptor

(TLR) signaling also enhances phagosome maturation

and the destruction of engulfed material [15]. It should

also be noted that members of the phosphoinositide

3-kinase (PI3-kinase) family participate in autophago-

some formation [16,17] and in phagocytosis through

the delivery of membranes into extending pseudopodia

[18]. These findings clearly indicate that the autophagic

pathway is tightly linked to phagocytosis. In the pres-

ent study, we examined whether nutrient deprivation,

one of the main triggers of autophagy, stimulates the

phagocytosis capacity of macrophages.

Results

Nutrient deprivation leads to an increase in the

phagocytosis of heat-inactivated bacteria by

macrophages in vitro

Mouse J774A.1 macrophages were incubated in amino

acid-free Earle’s balanced salt solution (EBSS) or con-

trol medium supplemented with 10% fetal bovine

serum for 6 or 24 h, followed by 1 h of incubation

with fluorescently-labeled platelets (PLT), U937 ACs,

carboxylated beads (0.1 or 1 lm in diameter) or heat-

inactivated Escherichia coli bacteria. Flow cytometric

analysis demonstrated that the uptake of PLTs, U937

AC or beads was not changed in EBSS-starved macro-

phages versus control cells (Fig. 1A). By contrast, the

uptake of E. coli bacteria was clearly increased after 6

and 24 h (Fig. 1A). To examine the potential role of

TLR signaling in the enhanced clearance of bacteria,

phagocytosis of beads was measured in the presence of

10 lgÆmL)1 lipopolysaccharide (LPS), which is a TLR

ligand and important surface constituent of E. coli.

Despite activation of macrophages by LPS, as deter-

mined by nitrite measurements in the culture medium

(12 ± 2 lm nitrite versus < 0.1 lm after 24 h of incu-

bation with or without LPS, respectively), phagocytosis

of beads was not stimulated. Increased phagocytosis of

E. coli in macrophages undergoing EBSS-induced

nutrient deprivation was confirmed by confocal

microscopy (Fig. 1B,C). Optical slides in the three

perpendicular axes showed that most bacteria were

surrounded by macrophage cytoplasm in all dimen-

sions (Fig. 1B). Moreover, enhanced uptake of bacteria

was blocked by the phagocytosis inhibitor cytochala-

sin D (Fig. 1D). We may therefore assume that the

flow cytometry data truly reflect phagocytosis and not

merely adherence of the bacteria to the macrophage

surface. Apart from amino acid deprivation, glucose

deprivation significantly increased the internalization

of heat-inactivated E. coli, whereas incubation in

serum-free medium had no effect (Fig. 2B). Similar

findings were obtained after phagocytosis of the

Gram-positive bacterium Staphylococcus aureus

(Fig. 2B). Titration experiments demonstrated that glu-

cose in the medium must be below 1 mgÆdL)1 to trig-

ger enhanced bacterial uptake (not shown).

Nutrient deprivation was also found to cause an

increase in the extent of phagocytosis by peritoneal

macrophages in vitro, as reflected by an increase in

mean fluorescence of macrophages after phagocytosis

of propidium iodide (PI)-labeled E. coli in amino

acid deprivation versus control conditions (mean fluo-

rescence: 116 ± 14 versus 48 ± 5, respectively;

P < 0.001; unpaired Student’s t-test, n = 5).

Enhanced phagocytosis of bacteria by starved

J774A.1 macrophages is scavenger receptor A

(SR-A) dependent

Both SR-A and macrophage receptor with collagenous

structure (MARCO) mediate the binding of unopson-

ized bacteria in vitro and in vivo, and are suggested to

play a pivotal role in bacterial clearance [19]. Although

MARCO is the dominant receptor for unopsonized

Phagocytosis of bacteria in starved macrophages W. Martinet et al.

2228 FEBS Journal 276 (2009) 2227–2240 ª 2009 The Authors Journal compilation ª 2009 FEBS

bacteria on human alveolar macrophages [20], western

blot analysis of J774A.1 lysate before and after starva-

tion showed that these macrophages did not express

MARCO (Fig. 2A). SR-A was overexpressed after

amino acid or glucose deprivation, but also after

serum withdrawal. Co-treatment of macrophages with

anti-SR-A serum inhibited uptake of E. coli and

S. aureus (Fig. 2B). Moreover, down-regulation of

SR-A gene expression with gene-specific small interfer-

ing RNA (siRNA) gave similar results (Fig. 2C), indi-

cating that SR-A is essential for enhanced bacterial

clearance. TLR4, also known as the LPS receptor, and

the accessory molecule CD14 are involved in the

phagocytosis of Gram-negative bacteria [21,22], but a

potential role in the phagocytosis of E. coli after

induction of autophagy is unlikely because TLR4 is

down-regulated in starved J774A.1 cells (Fig. 2A).

Moreover, phagocytosis of heat-inactivated E. coli was

unaltered in nutrient-deprived peritoneal macrophages

from TLR4 knockout mice (Fig. 2D).

Starvation-induced autophagy is not involved in

enhanced phagocytosis of bacteria by J774A.1

macrophages

Because nutrient deprivation is a powerful inducer of

autophagy, we next aimed to assess whether autophagy

is induced in starved J774A.1 cells and whether this

process affects the phagocytosis of bacteria. Transmis-

sion electron microcopy revealed that both amino acid

Fig. 1. Phagocytosis of heat-inactivated

E. coli is enhanced in EBSS-treated J774A.1

macrophages. (A) Cells were incubated in

serum-containing RPMI 1640 medium (con-

trol) or treated with EBSS for 6 or 24 h.

Subsequently, PLTs, U937 ACs, beads

(1 lm) or E. coli bacteria (labeled with PI or

CellTracker Red) were added to the culture

medium. After 1 h of phagocytosis, the

mean fluorescence of macrophages was

measured by flow cytometry. *P < 0.05,

***P < 0.001 versus control (one-way

ANOVA, followed by Dunnett’s or Dunnett’s

T3 post-hoc tests, n = 8–16). (B) Confocal

microscopy of CellTracker Green-stained

J774A.1 macrophages after phagocytosis of

PI-labeled E. coli for 1 h. Macrophages

were incubated in serum-containing RPMI

medium (control) or EBSS for 6 h prior to

phagocytosis of E. coli. Bacteria were

surrounded by macrophage cytoplasm in the

three perpendicular optical sections. (C)

Quantification of E. coli bacteria that

adhered or were engulfed by J774A.1

macrophages after incubation in serum-

containing RPMI medium (control) or EBSS

for 6 h followed by 1 h of phagocytosis.

***P < 0.001 versus adherent (two-way

ANOVA, n = 25). (D) J774A.1 cells were

incubated in serum-containing RPMI

medium (control) or EBSS in the presence

or absence of cytochalasin D (2 lM) for 6 h

prior to phagocytosis of fluorescently-labeled

E. coli for 1 h. ***P < 0.001 versus without

cytochalasin D, §§§P < 0.001 versus control

(two-way ANOVA, n = 9–15).

W. Martinet et al. Phagocytosis of bacteria in starved macrophages


Fig. 2. Phagocytosis of heat-inactivated E. coli and S. aureus is enhanced in J774A.1 cells after nutrient starvation and is SR-A-dependent.

(A) J774A.1 cells were incubated in RPMI 1640 medium supplemented with 10% fetal bovine serum (control), serum-free RPMI 1640 med-

ium (without serum), EBSS [amino acid deprivation; without serum and amino acids (AA)] or glucose-free DMEM [without serum and

glucose (glu)] for 6 h, followed by western blot analysis of macrophage receptors that are potentially involved in phagocytosis of heat-inacti-

vated bacteria, such as MARCO, SR-A, TLR4 and CD14. In vitro translated MARCO cDNA served as a positive control for MARCO expres-

sion. b-actin was used as a loading control. (B) Cells were incubated in RPMI 1640 medium supplemented with 10% fetal bovine serum

(control), serum-free RPMI 1640 medium (serum deprivation), EBSS (serum and amino acid deprivation) or glucose-free DMEM (serum and

glucose deprivation) for 6 h prior to phagocytosis of fluorescently-labeled E. coli or S. aureus for 1 h. Incubations were performed in the

presence or absence of SR-A antibodies or nonspecific immunoglobulins (negative control antibodies). ++P < 0.01, +++P < 0.001 versus

control (one-way ANOVA, followed by Dunnett’s test, n = 5–10); *P < 0.05, ***P < 0.001 versus without antibody and negative control anti-

body (one-way ANOVA, followed by Bonferroni test, n = 5–10). (C) J774A.1 cells were transfected with SR-A-specific siRNA or siControl

nontargeting siRNA. Three days after transfection, siRNA-treated cells were incubated in EBSS (serum and amini acid deprivation) for 6 h

prior to phagocytosis of fluorescently-labeled E. coli for 1 h. **P < 0.01 versus control (unpaired Student’s t-test, n = 4). (D) Peritoneal mac-

rophages from wild-type or TLR4 knockout (KO) mice were incubated in RPMI 1640 medium supplemented with 10% fetal bovine serum

(control) or EBSS (serum and amino acid deprivation) for 6 h prior to phagocytosis of fluorescently-labeled E. coli for 1 h. ***P < 0.001 ver-

sus control (two-way ANOVA, n = 7–9).



and glucose deprivation stimulated the formation of

cytoplasmic vacuoles containing partially degraded

cellular debris (Fig. 3A,B), a hallmark of autophagy.

To evaluate whether these vacuoles represent autopha-

gic vesicles, we assayed the intracellular formation of

autophagosomes in RAW264.7 macrophages stably

transfected with GFP-LC3 (Fig. 3C). Under nutrient-

rich conditions, GFP-LC3 was found diffusely in the

cytoplasm, with few punctuate dots. Amino acid depri-

vation in EBSS induced the formation of autophago-

somes that appeared as GFP-LC3 positive dots in the

cytoplasm (Fig. 3C). Macrophages that underwent

glucose starvation did not show significant changes in

GFP-LC3 (Fig. 3C) compared to controls, despite the

presence of large vacuoles in the cytosol early (6 h)

after treatment (Fig. 3A). Instead, glucose-starved cells

developed morphological features of necrotic death at

later time points (24 h), such as organelle swelling and

disruption of the plasma membrane (not shown), most

likely as a result of ATP depletion and the lack of a

supply of energy. Apart from GFP-LC3, we analyzed

endogenous LC3 via western blotting. Because LC3 is

poorly expressed in J774A.1 macrophages, the protein

could be detected only in a reproducible way after

transfection of LC3-encoding plasmid DNA. LC3 from

transfected control cells, grown in serum-containing

medium, was present mainly as the membrane-bound,

autophagy-specific form LC3-II (Fig. 3D). Under

EBSS conditions (i.e. without serum and amino acids),

LC3-II markedly increased in the presence of the lyso-

somal enzyme inhibitor NH4Cl, whereas it decreased

in the absence of this inhibitor (Fig. 3D). These find-

ings indicate that EBSS stimulates autophagosome

formation, but that LC3-II is rapidly degraded by

lysosomal hydrolases after fusion of autophagosomes

with lysosomes. Neither amino acid-deprived cells, nor

glucose-deprived J774A.1 cells underwent apoptosis

because they did not demonstrate cleavage of caspase-

3, chromatin condensation, nuclear fragmentation or

DNA fragmentation (not shown). Untreated macro-

Fig. 3. Autophagy is induced in macrop-

hages after amino acid deprivation, but not

after glucose and ⁄ or serum deprivation. (A,

B) Cells were incubated in RPMI 1640 med-

ium supplemented with 10% fetal bovine

serum (control), serum-free RPMI 1640

medium (without serum), EBSS [amino acid

deprivation; without serum and amino acids

(AA)] or glucose-free DMEM [without serum

and glucose (glu)] for 6 h followed by trans-

mission electron microscopy (A) and manual

counting of vacuolated cells in transmission

electron microscopy sections (B). Scale

bar = 2 lm. **P < 0.01 versus control

(one-way ANOVA, followed by Dunnett’s

test, n = 5). (C) RAW264.7 macrophages

expressing GFP-LC3 underwent nutrient

deprivation for 2 h. Amino acid deprivation

induced the formation of autophagosomes

(arrowheads) in the cytoplasm. Scale

bar = 20 lm. (D) Western blot analysis of

LC3 in J774A.1 cells 24 h after LC3 trans-

fection. Cells underwent nutrient deprivation

in the presence or absence of the lysosomal

enzyme inhibitor NH4Cl (10 mM) for 2 h.

The LC3-II bands from three independent

experiments were quantified and are shown

as a percent of control (serum-containing

medium without NH4Cl). *P < 0.05,

**P < 0.01 versus control (one-way

ANOVA, followed by Dunnett’s test, n = 3).



phages or cells incubated in medium without serum

showed a normal cell morphology and did not undergo

autophagy, apoptosis or necrotic death (Fig. 3A–D).

Treatment of J774A.1 cells with the PI3-kinase

inhibitors LY294.002 or 3-methyladenine blocked star-

vation-induced phagocytosis of E. coli (Fig. 4A). To

further test whether autophagosome formation is

essential for the enhanced phagocytosis of bacteria,

gene-silencing experiments were performed with siR-

NA specific for the essential autophagy gene Atg7.

Down-regulation of Atg7 gene expression with Atg7-

specific siRNA was demonstrated at the mRNA level

(91 ± 1% silencing after 24 h) and protein level

(Fig. 4B), but not with siControl nontargeting siRNA.

Vacuolization could not be stimulated in Atg7 siRNA

transfected cells after EBSS treatment (Fig. 4B) and

thus appear to be autophagy deficient. Atg7 silencing

did not affect phagocytosis of E. coli (Fig. 4C), indi-

cating that autophagosome formation is not critical

for this process.

Enhanced phagocytosis of bacteria by starved

J774A.1 macrophages is p38 mitogen-activated

protein (MAP) kinase dependent

Amino acid or glucose deprivation, but not serum star-

vation, substantially increased the phosphorylation of

p38 MAP kinase (Fig. 5A). Inhibition of p38 MAP

kinase during amino acid deprivation with the

p38-specific inhibitor SB202190 blocked the enhanced

clearance of heat-inactivated E. coli and S. aureus

(Fig. 5B). Because p38 MAP kinase is potently and

preferentially activated by a variety of environmental

stresses [23], it is tempting to speculate that enhanced

uptake of bacteria is a general stress response. To test

this possibility, J774A.1 cells were exposed to heat

(40 �C) or thapsigargin-induced endoplasmic reticulum

(ER) stress for 6 h. Despite up-regulation of heat

shock protein 70 and the ER stress marker C ⁄EBPhomologous protein (CHOP), respectively (Fig. 6A),

Fig. 4. Autophagy is not essential for enhanced phagocytosis of

heat-inactivated E. coli by amino acid-deprived J774A.1 macro-

phages. (A) J774A.1 cells were incubated in serum-containing

RPMI medium (control) or EBSS in the presence or absence of the

autophagy inhibitors LY294.002 (LY; 50 lM) and 3-methyladenine

(3-MA; 10 mM) for 6 h prior to phagocytosis of fluorescently-

labeled E. coli for 1 h. ***P < 0.001 versus control; §§P < 0.01,§§§P < 0.001 versus EBSS-treated cells (one-way ANOVA,

followed by Dunnett’s test, n = 12). (B) J774A.1 cells were trans-

fected with Atg7-specific siRNA or siControl nontargeting siRNA.

Silencing of Atg7 expression was evaluated after 0–3 days by

western blotting. To evaluate the inhibition of autophagy, the

number of vacuolated cells was counted via transmission electron

microscopy after 6 h of EBSS treatment. ***P < 0.001 versus

control (unpaired Student’s t-test, n = 5). (C) siRNA transfected

cells were incubated in EBSS for 6 h prior to phagocytosis of

fluorescently-labeled E. coli for 1 h. Differences between siControl

and Atg7 siRNA-treated cells were not statistically significant

(two-way ANOVA, n = 5).



phagocytosis of E. coli was not affected (Fig. 6B). How-

ever, p38 was not hyperphosphorylated under these

conditions (Fig. 6C) and, thus, we sought other stress

situations that activated p38. Treatment of cells with

the DNA damaging agent camptothecin caused strong

phosphorylation of p38 (Fig. 6D), although without

stimulating the uptake of heat-inactivated E. coli

(Fig. 6E). We therefore concluded that enhanced

phagocytosis of bacteria is not a general stress response

and that p38 activation is required, but not sufficient, to

increase the rate of bacterial clearance.

To examine potentially important downstream path-

ways linked to EBSS-induced starvation and p38

MAP kinase activation, a full genome microarray rep-

resenting over 41 000 mouse genes or transcripts was

probed with cDNA isolated from J774A.1 cells that

were treated with EBSS, EBSS supplemented with the

p38 inhibitor SB202190 or control medium supple-

mented with 10% fetal bovine serum. The microarray

data are available via the National Center for Bio-

technology Information Gene Expression Omnibus

(http://www.ncbi.nlm.nih.gov/geo/; accession number

GSE14293). Among the genes that were differentially

expressed, only N-myc downstream-regulated gene 1

(NDRG1) and RAR-related orphan receptor alpha

(RORa), were up-regulated upon EBSS-induced star-

vation. Up-regulation of NDRG1 and RORa was

inhibited in the presence of SB202190. Microarray

data were confirmed by real-time RT-PCR (Fig. 7A)

and western blotting (Fig. 7B), with the exception of

RORa, which could not be detected at the protein

level (Fig. 7B). To examine whether NDRG1 and

RORa are involved in the phagocytosis of bacteria,

both genes were silenced via siRNA. Down-regulation

of NDRG1 and RORa gene expression with gene-

specific siRNA was demonstrated at the mRNA level

(90 ± 1% and 84 ± 1% silencing of NDRG1 and

RORa, respectively, after 24 h) and protein level

(detectable only for NDRG1; Fig. 7C), but not with

siControl nontargeting siRNA. Silencing of either

gene did not affect the phagocytosis of E. coli

(Fig. 7C), indicating that they do not play a critical

role in this process.

Fig. 5. Activation of p38 MAP kinase is required for enhanced

phagocytosis of bacteria by J774A.1 macrophages. (A) J774A.1

cells were incubated in RPMI 1640 medium supplemented with

10% fetal bovine serum (control), serum-free RPMI 1640 medium

(without serum), EBSS [amino acid deprivation; without serum and

amino acids (AA)] or glucose-free DMEM [without serum and glu-

cose (glu)] for 6 h, followed by western blot analysis of p38 and

phospho-p38 (Thr180 ⁄ Tyr182) in crude cell lysate. (B) J774A.1 cells

were incubated in serum-containing RPMI medium (control),

serum-free RPMI (serum deprivation), EBSS (serum and amino acid

deprivation) or glucose-free DMEM (serum and glucose deprivation)

for 6 h in the presence or absence of the p38 inhibitor SB202190

(10 lM) prior to phagocytosis of fluorescently-labeled E. coli or

S. aureus for 1 h. ***P < 0.001 versus without SB202190

(unpaired Student’s t-test, n = 5).



Discussion

In the present study, we have demonstrated that the

exposure of mouse J774A.1 cells or primary mouse

peritoneal macrophages to nutrient deprivation (i.e.

the withdrawal of amino acids or glucose in combina-

tion with serum withdrawal) leads to a striking

increase in bacterial phagocytosis. Furthermore, we

have determined that the starvation effect on phagocy-

tosis is relatively selective for bacteria because phago-

cytosis of other extracellular particles, including

platelets, ACs or carboxylated beads, was not stimu-

lated. Phagocytosis of beads was not stimulated in the

presence of LPS. We therefore consider that TLRs are

not involved in the enhanced clearance of bacteria

after nutrient deprivation. Furthermore, treatment of

starved macrophages with beads resembling the size of

bacteria (0.1 lm diameter instead of the regular 1 lm)

did not improve their uptake, suggesting that the selec-

tive engulfment of bacteria is not a consequence of

particle size. However, internalization of beads in mac-

rophages might occur via a phagocytosis-independent

mechanism (e.g. emperipolesis) because the uptake of

beads cannot be blocked with the phagocytosis inhibi-

tor cytochalasin D [24]. Therefore, the possibility that

the efficiency of phagocytosis depends on particle size

cannot be entirely ruled out. Of note, nutrient depriva-

tion is a strong stimulus of autophagy. Despite recent

evidence linking the phagocytosis and autophagy path-

ways [14,15], down-regulation of the autophagy essen-

tial gene Atg7 in J774A.1 macrophages did not reveal

a plausible connection between the enhanced phagocy-

tosis of bacteria and the induction of autophagy.

Nonetheless, starvation-induced phagocytosis of E. coli

was blocked with LY294.002 or 3-methyladenine,

which are two compounds that are widely used to inhi-

bit autophagy. However, they act as PI3-kinase inhibi-

tors [25] and, thus, may inhibit the uptake of bacteria

by preventing the formation of pseudopodia, which is

the primary role of class I PI3-kinase activity in

Fig. 6. Enhanced phagocytosis of bacteria by J774A.1 macrophages is not a general stress response. (A–C) J774A.1 cells were incubated in

serum-containing medium at 40 �C (heat) or in medium supplemented with 10 nM thapsigargin (to evoke ER stress) for 0–6 h followed by

western blot analysis of heat shock protein 70 (Hsp70), ER stress marker CHOP, p38 and phospho-p38 (Thr180 ⁄ Tyr182). In addition, phago-

cytosis of fluorescently-labeled E. coli was analyzed by flow cytometry using amino acid deprivation (EBSS) as a positive control. **P < 0.01

versus control (one-way ANOVA, followed by Dunnett’s test, n = 4). (D, E) J774A.1 cells were incubated in serum-containing medium sup-

plemented with the genotoxic agent camptothecin (CT; 10 lM) for 6 h followed by western blot analysis of p38 and phospho-p38

(Thr180 ⁄ Tyr182). Phagocytosis of fluorescently-labeled E. coli was analyzed by flow cytometry using amino acid deprivation (EBSS) as a posi-

tive control. **P < 0.01 versus control (one-way ANOVA, followed by Dunnett’s test, n = 5).



phagocytosis [18]. Moreover, even though the autopha-

gic machinery enhances the maturation of phagosomes

[15], it is not known whether it contributes to the

engulfment of bacteria. Our findings suggest that the

role of the autophagy machinery is limited to the

maturation of the phagosome and that it does not

participate in the initial internalization of bacteria.

Macrophages express a broad spectrum of receptors

that participate in bacterial recognition and internali-

zation [5,6,26]. These receptors either recognize serum

components (opsonins) that opsonize the bacteria (e.g.

integrins, Fc- or complement-receptors) or directly rec-

ognize molecular determinants on the bacterial surface.

Because the incubation of bacteria with starved macro-

phages took place in the absence of serum, phago-

cytosis was opsonin-independent. Molecules of the

scavenger receptor family have been implicated in the

bacterial phagocytosis of unopsonized bacteria by mac-

rophages. For example, SR-A can bind Gram-positive

[27] and Gram-negative bacteria [28], including the

species used in the present study (i.e. S. aureus and

E. thinsp;coli). Studies with SR-A knockout mice

revealed that SR-A plays an important role in host

defense against bacterial infection; it enhances sensitiv-

ities for S. aureus [29] and Listeria infection [30], as

well as for LPS-mediated septic shock [31]. The results

obtained in the present study indicate that SR-A was

up-regulated in J774A.1 macrophages after nutrient

starvation and that treatment of starved cells with an

SR-A-specific antibody blocked the enhanced phagocy-

tosis of E. coli and S. aureus after nutrient deprivation.

It should be noted, however, that serum withdrawal

also triggered SR-A expression without significant

stimulation of bacterial phagocytosis. This finding indi-

cates that SR-A is required, but not sufficient, for the

enhanced uptake of bacteria after nutrient deprivation.

Fig. 7. Expression of NDRG1 and RORa in J774A.1 macrophages

after nutrient deprivation. J774A.1 cells were incubated in

RPMI 1640 medium supplemented with 10% fetal bovine serum

(control), serum-free RPMI 1640 medium (without serum), EBSS

[amino acid deprivation; without serum and amino acids (AA)] or

glucose-free DMEM [without serum and glucose (glu)] for 6 h in

the presence or absence of the p38 inhibitor SB202190 (10 lM)

prior to real-time RT-PCR (A) or western blotting (B). *P < 0.05 ver-

sus without SB202190 (unpaired Student’s t-test, n = 4). (C)

J774A.1 cells were transfected with NDRG1- or RORa-specific siR-

NA or siControl nontargeting siRNA. Silencing of NDRG1 expres-

sion was evaluated after 0–3 days via western blotting. Three days

after transfection, siRNA-treated cells were incubated in EBSS for

6 h prior to phagocytosis of fluorescently-labeled E. coli for 1 h. Dif-

ferences between siControl and siRNA-treated cells were not sta-

tistically significant (two-way ANOVA, n = 5).



Analogous to SR-A, the scavenger receptor MARCO

can adhere to Gram-positive and Gram-negative bacte-

ria. MARCO is considered to be the major binding

receptor for unopsonized bacteria on human alveolar

macrophages [20], but was not detectable in J774A.1

macrophages, suggesting that its involvement in

enhanced bacterial clearance, as reported in the present

study, is unlikely. Similarly, the changes in TLR4 and

CD14 cannot fully explain the enhanced uptake of

bacteria by starved J774A.1 cells because these mole-

cules only bind Gram-negative bacteria [21,22]. More-

over, these receptors were not up-regulated after

nutrient deprivation. Possibly, other receptors on the

surface of J774A.1 cells are involved in binding and ⁄orphagocytosis of bacteria [19]. The macrophage

mannose receptor and lectin-like oxidized low-density

lipoprotein receptor 1, for example, support the adhe-

sion of Gram-positive and Gram-negative bacteria, but

these receptors are not professional phagocytic recep-

tors, and only bind receptors that require a partner to

trigger efficient phagocytosis [32,33].

Although macrophage receptors are key proteins

involved in the phagocytosis of bacteria [19], several

lines of evidence suggest that other proteins may be

equally important. For example, Anand et al. [8] dem-

onstrated that hypoxia triggers phagocytosis of bacte-

ria by macrophages in a p38 MAP kinase-dependent

manner. Analogous with hypoxia, nutrient deprivation

(both amino acids and glucose, but not serum) sub-

stantially increased the phosphorylation of p38 MAP

kinase. Inhibition of p38 MAP kinase activation by

the p38-specific inhibitor SB202190 attenuated bacte-

rial phagocytosis induced by nutrient deprivation. p38

MAP kinase regulates cell growth, cell differentiation,

cell activation and cell death, and responses to inflam-

mation and stress stimuli at the transcriptional and

translational levels [34]. Many downstream substrates

of p38 MAP kinase have been described, both in the

cytoplasm and in the nucleus, that mediate these

effects. Doyle et al. [35] reported that numerous TLR

ligands specifically enhance the phagocytosis of bacte-

ria through myeloid differentiation factor 88, interleu-

kin-1 receptor-associated kinase-4 and p38, and that

activation of this pathway is essential for the up-regu-

lation of several scavenger receptors, including

MARCO and SR-A, as well as lectin-like oxidized

low-density lipoprotein receptor 1 and interstitial cell

adhesion molecule-1. Hypoxia-induced phagocytosis of

bacteria by macrophages occurs under the control of

hypoxia-inducible factor-1a, whose expression is

reversed after p38 inhibition [8]. In the present study, a

microarray screening of the whole mouse genome did

not reveal up-regulation of macrophage receptors or

hypoxia-inducible factor-1a in starved J774A.1 cells,

but led to the identification of two hypoxia-regulated

genes (i.e. NDRG1 and RORa) that were strongly

up-regulated at the mRNA level after amino acid or

glucose deprivation, but not after serum withdrawal.

The expression of both genes was controlled by p38

because up-regulation of NDRG1 and RORa mRNA

was inhibited in the presence of SB202190. The overex-

pression of NDRG1 was confirmed via western blot-

ting; however, expressed protein from RORa was

undetectable in J774A.1 cells even after starvation.

Given that the uptake of bacteria is enhanced after

hypoxia in macrophages [8] and that NDRG1 is

up-regulated after hypoxia [36], it is tempting to specu-

late that NDRG1 overexpression is associated with the

induction of bacterial clearance. However, gene-silenc-

ing experiments showed that neither NDRG1 nor

RORa are essential for the phagocytosis of bacteria.

Most likely, other factors may be expressed or released

during starvation, which are undetectable via micro-

array technology, and serve to augment phagocytosis

independent of NDRG1 and RORa. Indeed, macro-

phages are specialized immune cells that, once acti-

vated, may release large amounts of different cytokines

and ⁄or reactive oxygen species. Secretion of interleu-

kin-10 is a well-known stimulus for phagocytosis in

human monocytes [37,38]. We previously demonstrated

that J774A.1 macrophages secrete large amounts of

the pro-inflammatory cytokines interleukin-6 and

tumor necrosis factor a upon amino acid deprivation

[39]; however, Anand et al. [8] demonstrated that treat-

ment of macrophages with interleukin-1, tumor necro-

sis factor a or reactive oxygen species (H2O2 or NO)

does not affect phagocytosis significantly.

In summary, exposure of macrophages to amino

acid or glucose deprivation selectively stimulates the

phagocytosis of heat-inactivated Gram-positive and

Gram-negative bacteria via an SR-A- and p38-depen-

dent mechanism. From a clinical perspective, these

results are promising with respect to the development

of nutritional strategies for the treatment of patients

who are infected with multi-resistant bacteria.

Experimental procedures

Cell culture

Mouse J774A.1 macrophages and U937 monocytic cells

were grown in RPMI 1640 medium (Invitrogen, Carlsbad,

CA, USA) supplemented with antibiotics and 10% fetal

bovine serum. RAW264.7 macrophages stably expressing

GFP-LC3 [15] (a gift from M. Sanjuan, St Jude Children’s

Research Institute, Memphis, TN, USA) were grown in



serum-containing DMEM medium. Peritoneal macrophages

were isolated 5 days after injection of 2 mL of Brewer’s

thioglycolate medium (Sigma, St Louis, MO, USA) into the

peritoneal cavity of fasting C57Bl ⁄ 6 mice. To generate

ACs, U937 cells were incubated with 50 lm etoposide for

4 h. Macrophages, U937 ACs and human blood platelet

concentrates (kindly provided by the blood transfusion cen-

ter of the University Hospital of Antwerp, Belgium) were

fluorescently labeled with 10 lm CellTracker Green (Molec-

ular Probes, Eugene, OR, USA) for 30 min at 37 �C. Forphagocytosis experiments, macrophages were washed in

NaCl ⁄Pi and incubated in RPMI 1640 medium with or

without serum, DMEM without glucose or EBSS in the

presence or absence of the autophagy ⁄phagocytosis inhibi-

tors LY294.002 (50 lm; Sigma) or 3-methyladenine

(10 mm; Sigma), the phagocytosis inhibitor cytochalasin D

(2 lm; Sigma) or the p38 MAP kinase inhibitor SB202190

(10 lm; Sigma). These inhibitors did not affect macrophage

viability within the studied time frame. In some phagocyto-

sis experiments, the medium was supplemented with

10 lgÆmL)1 LPS from E. coli O111:B4 (Sigma) to assess the

potential role of TLR signaling. Activation of macrophages

by LPS was assessed by measuring nitrite, a stable NO

metabolite and indicator of NO synthase activity, using the

Griess reaction [40]. In other experiments, SR-A was

blocked prior to phagocytosis of bacteria by addition of rat

anti-SR-A (2F8; AbD Serotec, Oxford, UK) to the culture

medium (10 lgÆmL)1). Immunoglobulin G from rat serum

(Sigma) was used as a negative control.

Bacteria

E. coli and S. aureus bacteria were grown in LB medium at

37 �C. Bacteria were harvested by centrifugation and

washed three times with NaCl ⁄Pi to remove LB. Subse-

quently, bacteria were heat inactivated (1 h at 70 �C) and

fluorescently labeled with 10 lgÆmL)1 PI or 10 lm Cell-

Tracker Red (Molecular Probes) for 1 h on a shaker at

room temperature. Finally, bacteria were washed two times

in NaCl ⁄Pi and diluted to a concentration of 108 bacte-

riaÆmL)1.

Phagocytosis assay

Macrophages (106) were washed with NaCl ⁄Pi and incu-

bated for 1 h with PLTs (2 · 108), U937 ACs (3 · 106),

bacteria (5 · 107) or carboxylate-modified fluospheres

(5 · 107, 0.1 or 1.0 lm; Molecular Probes) at 37 �C. To

remove adherent and unphagocytosed particles, macrophag-

es were washed with cold NaCl ⁄Pi and briefly treated with

a solution of 0.05% trypsin and 0.02% EDTA. Finally,

macrophages were scraped from the plate and 20 000 cells

per sample were analyzed on a FACSort cytometer (BD

Biosciences, Franklin Lakes, NJ, USA). The acquisition

threshold was set to include cells but to exclude debris

and remaining unbound particles from further analysis.

Data were analyzed using cell quest pro Software (BD

Biosciences).

Plasmid construction, transfection and in vitro

translation

The enhanced green fluorescent protein (eGFP) coding

sequence was excised from plasmid pEGFP-N3 (BD Bio-

sciences Clontech, Mountain View, CA, USA) by digestion

with EcoRI and NotI. Vector ends were blunted with

Klenow polymerase and self ligated to produce plasmid pN3.

Full-length cDNA encoding mouse LC3 protein was ampli-

fied by PCR from C2C12 cells using Pfu DNA polymerase

(Stratagene, La Jolla, CA, USA) and primers 5¢-CAAGA

TCTCGCGCGATGCCCTCMGACCGG-3¢ and 5¢-CAAA

GCTTTCAGAAGCCGAAGGTTTCYTGGGAG-3¢. The

resultant PCR product was BglII ⁄HindIII digested and

cloned in the similarly opened pN3 to produce pMuLC3.

J774A.1 macrophages were transiently transfected using

Fugene HD transfection reagent (Roche, Mannheim,

Germany) at a ratio of 5 lL of reagent to 2 lg of plasmid

DNA.

In vitro translation of mouse MARCO was performed

using XbaI linearized pcDNA3MARCO plasmid (a gift

from T. Pikkarainen, Karolinska Institute, Stockholm, Swe-

den) and the Rabbit Reticulocyte Lysate System (Promega,

Madison, WI, USA). To obtain a positive control for

mouse RORa in western blot experiments, mouse RORacDNA was amplified by PCR using the oligonucleotides

5¢-TCAAAGCTTGCGATGAAAGCTCAAATTGAAATTA

TTCC-3¢ and 5¢-TCAGGATCCTTACCCATCGATTTG

CATGGCTGGCTC-3¢. The PCR product was HindIII ⁄BamHI digested and cloned in the similarly opened plasmid

pUC19 to yield pUC19RORa. Plasmid pGEM4ZGFP-A64

(gift from V. Van Tendeloo, University Hospital of

Antwerp, Edegem, Belgium) containing the eGFP coding

sequence downstream of a T7 promoter was BamHI

digested to remove eGFP and self ligated to produce

pGEM4Z-A64. RORa was then excised from pUC19RORaand cloned into the HindIII ⁄BamHI sites of pGEM4Z-A64

to yield pGEM4Z-RORa. In vitro translation of RORa was

finally performed using nonlinearized pGEM4Z-RORa and

the TNT Quick Coupled Transcription ⁄ translation System

(Promega). All constructs were confirmed by sequencing.

siRNA-mediated gene silencing

J774A.1 macrophages were transfected with 50 nm

ON-TARGETplus SMARTpool siRNA specific to mouse

Atg7, SR-A, NDRG1 or RORa (Dharmacon, Lafayette,

CO, USA) using HiPerfect transfection reagent (Qiagen,

Valencia, CA, USA) according to the manufacturer’s

instructions. siControl RISC-free siRNA (Dharmacon) was

used as a negative control.



Microarray analysis

Total RNA was prepared from cultured cells using the

Absolutely RNA Miniprep Kit (Stratagene). All RNA

samples were treated with RNase-free DNase I. RNA

quality was verified on an Agilent 2100 Bioanalyser using

the RNA 6000 Nano LabChip kit (Agilent Technologies,

Palo Alto, CA, USA). Samples were then analyzed by the

Microarray Facility of the Flanders Interuniversity Insti-

tute for Biotechnology (VIB, Leuven, Belgium) using the

Whole Mouse Genome Oligo Microarray Kit (Agilent

Technologies) representing over 41 000 mouse genes and

transcripts.

Real-time quantitative RT-PCR

cDNA was prepared from cultured cells using the Fastlane

Cell cDNA kit (Qiagen, Venlo, The Netherlands). TaqMan

gene expression assays for Atg7 (assay Id:

Mm01183587_m1), SR-A (assay Id: Mm01197876_m1),

NDRG1 (assay Id: Mm00440447_m1) and RORa (assay Id:

Mm00443103_m1) (Applied Biosystems, Foster City, CA,

USA) were then performed in duplicate on an ABIPrism

7300 sequence detector system (Applied Biosystems) in

25 lL reaction volumes containing 1· Universal PCR

Master Mix (Applied Biosystems). The parameters for

PCR amplification were 95 �C for 10 min followed by 40

cycles of 95 �C for 15 s and 60 �C for 1 min. The relative

expression of mRNA species was calculated using the

comparative threshold cycle method. All data were con-

trolled for quantity of cDNA input by performing measure-

ments on the endogenous reference gene b-actin (assay Id:

Mm00607939_s1; Applied Biosystems).

Immunoblot assays

Cultured cells were lysed in an appropriate volume of

Laemmli sample buffer (Bio-Rad, Richmond, CA, USA).

Cell lysates were then heat-denatured for 4 min in boiling

water and loaded onto SDS ⁄PAGE. After electrophoresis,

proteins were transferred to an Immobilon-P Transfer

Membrane (Millipore, Bedford, MA, USA) according to

standard procedures. Membranes were blocked in NaCl ⁄Tris containing 0.05% Tween-20 (NaCl ⁄Tris-T) and 5%

nonfat dry milk (Bio-Rad) for 1 h. After blocking, mem-

branes were probed overnight at 4 �C with primary anti-

bodies in antibody dilution buffer (NaCl ⁄Tris-Tcontaining 1% nonfat dry milk), followed by 1 h of incu-

bation with secondary antibody at room temperature.

Antibody detection was accomplished with SuperSignal

West Pico or SuperSignal West Femto Maximum Sensi-

tivity Substrate (Pierce, Rockford, IL, USA) using a

Lumi-Imager (Roche).

The primary antibodies used were: goat anti-MARCO

and rat anti-CD14 (R&D Systems, Abingdon, UK); rat

anti-SR-A (clone 2F8) (AbD Serotec); mouse anti-caspase-3

(clone 19) and anti-HSP70 (clone 7) (BD Transduction

Laboratories, Lexington, KY, USA); rabbit anti-Atg7, anti-

p38 and anti-phospho-p38 (Thr180 ⁄Tyr182) (Cell SignalingTechnology, Beverly, MA, USA); rabbit anti-LC3 (Novus

Biologicals, Littleton, CO, USA); chicken anti-NDRG1

(Abcam, Cambridge, UK); rabbit anti-RORa (clone H-65)

and mouse anti-CHOP (clone B-3) (Santa Cruz Biotechnol-

ogy, Santa Cruz, CA, USA); mouse anti-b-actin (clone

AC-15) (Sigma); and anti-Toll-like receptor 4 (eBioscience,

San Diego, CA, USA). Peroxidase-conjugated secondary

antibodies were purchased from DakoCytomation (Glost-

rup, Denmark).

Confocal microscopy

Macrophages were grown on coverslips, fluorescently

labeled with 10 lm CellTracker Green or 1 lm LysoTracker

(Molecular Probes) and incubated with PI-labeled bacteria

for 1 h. Finally, coverslips were formalin fixed and

mounted on microscope slides using Slowfade Gold

Reagent (Molecular Probes). Dual-channel images were

taken with a LSM510 confocal microscope (Carl Zeiss,

Jena, Germany), using an Argon laser (488 nm line) and a

HeNe laser (543 nm line) with a bandpass emission filter

(BP500-530) and a longpass emission filter (LP560), respec-

tively. Regions of interest containing individual macro-

phages were selected and Z-stacks were further analyzed

using the ortho and gallery displays of the LSM510 imag-

ing software. After confocal imaging, adherent and

engulfed bacteria were quantified via manual counting of

PI-labeled particles.

Transmission electron microscopy

J774A.1 macrophages were fixed in 0.1 m sodium cacody-

late-buffered (pH 7.4), 2.5% glutaraldehyde solution for

2 h and then rinsed (3 · 10 min) in 0.1 m sodium cacody-

late-buffered (pH 7.4), 7.5% saccharose and postfixed in

1% OsO4 solution for 1 h. After dehydration in an ethanol

gradient (70% ethanol for 20 min; 96% ethanol for 20 min;

100% ethanol for 2 · 20 min), samples were embedded in

Durcupan ACM. Ultrathin sections were stained with

uranyl acetate and lead citrate. Sections were examined in

a Philips CM 10 microscope (Philips, Eindhoven, The

Netherlands) at 80 kV.

Statistical analysis

Results are expressed as the mean ± SEM. All analyses

were performed using spss software, version 14.0 (SPSS

Inc., Chicago, IL, USA). The statistical tests used in the

present study are noted in the appropriate figure legends.

P < 0.05 was considered statistically significant.



Acknowledgements

Research was supported by the Fund for Scientific

Research (FWO)-flanders (Belgium) (projects No.

G.0308.04, G.0113.06 and G.0112.08), the University

of Antwerp (NOI-BOF and TOP-BOF) and the

Bekales Foundation. The authors thank Jan Van

Daele, Lieve Svensson, Francis Terloo and Dominique

De Rijck for their excellent technical assistance.

Wim Martinet and Dorien M Schrijvers are post-

doctoral fellows of the FWO-Flanders.

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