Báo cáo khoa học: Phagocytosis of bacteria is enhanced in macrophages undergoing nutrient deprivation ppt

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 Ear

undergoing nutrient deprivation

Wim Martinet1,*, Dorien M Schrijvers1,*, Jean-Pierre Timmermans2, Arnold G Herman1 and Guido 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 macro-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: wim.martinet@ua.ac.be

*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 (40C) 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.

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 avail-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 nutripres-ent 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

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-Agene 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 l M ) 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).

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 NH 4 Cl (10 m M ) 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 NH 4 Cl) *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 (40C) or thapsigargin-induced endoplasmic reticulum (ER) stress for 6 h Despite up-regulation of heat shock protein 70 and the ER stress marker C⁄ EBP homologous 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 l M ) and 3-methyladenine (3-MA; 10 m M ) 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 l M ) 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).

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 n M 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 l M ) 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 l M ) 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 siRsiR-NA 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⁄ or

phagocytosis 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