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This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/imm.12168 This article is protected by copyright. All rights reserved.

Received Date : 27-Feb-2013

Revised Date : 15-Aug-2013

Accepted Date : 30-Aug-2013

Article type : Original Article

Autophagy activation by interferon-γ via the p38 MAPK signalling pathway is involved

in macrophage bactericidal activity

Short title: Macrophage bactericidal activity involves IFN-γ-mediated autophagy via p38

MAPK

Takeshi Matsuzawa,1* Eri Fujiwara,2 and Yui Washi2

1Division of Veterinary Science, Graduate School of Life and Environmental Sciences,

Osaka Prefecture University, Izumisano, Osaka, Japan

2School of Veterinary Science, College of Life, Environment, and Advanced Sciences, Osaka

Prefecture University, Izumisano, Osaka, Japan

1-58 Rinku-Ourai Kita, Izumisano, Osaka 598-8531, Japan

*Correspondence should be addressed to:

Takeshi Matsuzawa

Division of Veterinary Science, Graduate School of Life and Environmental Sciences, Osaka

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Prefecture University, 1-58 Rinku-Ourai Kita, Izumisano, Osaka 598-8531, Japan

Phone: +81-72-463-5709

Fax: +81-72-463-5711

E-mail: t.matsuzawa@icloud.com

Keywords: interferon-γ; macrophage; autophagy; cell-autonomous innate immunity

Summary

Macrophages are involved in many essential immune functions. Their role in

cell-autonomous innate immunity is reinforced by interferon-γ (IFN-γ), which is mainly

secreted by proliferating type 1 T helper cells and natural killer cells. Previously, we showed

that IFN-γ activates autophagy via p38 mitogen-activated protein kinase (p38 MAPK), but

the biological importance of this signalling pathway has not been clear. Here, we found that

macrophage bactericidal activity increased by 4 h after IFN-γ stimulation. Inducible nitric

oxide synthase (NOS2) is a major downstream effector of the Janus kinase (JAK)-signal

transducer and activator of transcription1 (STAT1) signalling pathway that contributes to

macrophage bactericidal activity via nitric oxide (NO) generation. However, no NO

generation was observed after 4 h of IFN-γ stimulation, and macrophage bactericidal activity

at early stages after IFN-γ stimulation was not affected by the NOS inhibitors,

NG-methyl-L-arginine acetate salt (L-NMMA) and diphenyleneiodonium chloride (DPI).

These results suggest that an NOS2-independent signalling pathway is involved in

IFN-γ-mediated bactericidal activity. We also found that this macrophage activity was

attenuated by the addition of the p38 MAPK inhibitors, PD 169316, SB 202190, and SB

203580, or by the expression of shRNA against p38α or the essential factors for autophagy,

Atg5 and Atg7. Collectively, our results suggest that the IFN-γ-mediated autophagy via p38

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MAPK, without the involvement of NOS2, also contributes to the ability of macrophages to

kill intracellular bacteria. These observations provide direct evidence that p38

MAPK-mediated autophagy can support IFN-γ-mediated cell-autonomous innate immunity.

Introduction

Macrophage participates in many essential functions such as tissue homeostasis and clearance

of infection.1, 2 The type II interferon (IFN), IFN-γ, a cytokine that is mainly secreted by

activated Th1 T lymphocytes and NK cells, is a powerful macrophage activator.3, 4 IFN-γ

induces tyrosine phosphorylation of the signal transducer and activator of transcription1

(STAT1) protein via Janus kinase1(JAK1) and JAK2.5-7 Subsequently, STAT1 dimers bind to

IFN-stimulated response elements and induce the transcription of many IFN-stimulated

genes (ISGs).5, 6 In addition to the JAK-STAT1 pathway, expression of another gene is

mediated by IFN-γ via STAT1-independent pathways and modulates various signalling

cascades, including those involving myeloid differentiating factor 88 (MyD88), p38

mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K), and protein

kinase C (PKC).7-14 The major immune factors in the signalling pathway downstream of

IFN-γ are major histocompatibility complex (MHC) class I and II; inflammatory and

pyrogenic cytokines; chemokines; and antimicrobial proteins, such as inducible nitric oxide

synthase (NOS2; also known as iNOS), phagocyte oxidase, and immune guanosine

triphosphatases (GTPases).5-7, 15-18

Notably, IFN-γ is critical for cell-autonomous innate immunity against

intracellular bacteria, such as Listeria, Mycobacteria, and Salmonella.17, 19-21 The

antimicrobial enzyme NOS2 is largely considered responsible for the bactericidal activity,

via nitric oxide (NO) generation, of IFN-γ-activated macrophages against intracellular

bacteria.22, 23 However, NOS2-knockout (NOS2-/-) mice that had been infected with virulent

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Mycobacterium tuberculosis have been found to survive significantly longer and exhibit

some control of lung M. tuberculosis growth when compared with mice lacking IFN-γ or

IFN-γ receptor.24 This observation suggested that IFN-γ-dependent, NOS2-independent

immunity against intracellular bacteria exists. Recently, it has been shown that, in addition to

NOS2, IFN-γ-inducible immune GTPases, including p47 immunity-related GTPases (p47

IRGs) and p65 guanylate-binding proteins (p65 GBPs), regulate autophagy and contribute to

the disposal of intracellular pathogens.17, 18, 20, 24-28

Autophagy has emerged as a major immune defence pathway and this cascade can

be provoked by host-derived cytokines, IFN-γ, or pattern-recognition receptors, including

Toll-like receptors (TLRs) and nucleotide-binding oligomerization domain (NOD)-like

receptors (NLRs).25, 26, 29-35 It has been shown that IFN-γ controls autophagy via several

types of IFN-γ-inducible immune GTPases belonging to the IRG family and the GBP

family.18, 25-28, 36, 37 More recently, we have shown that, in addition to the IFN-inducible

GTPase pathway, the p38 MAPK pathway contributes to autophagy activation in the

IFN-γ-stimulating cells.38 IFN-γ is able to activate autophagy through at least 2 different

pathways, the conventional STAT1- and Irgm1-dependent pathway and an alternative p38

MAPK-dependent, STAT1-independent pathway. However, the biological role of

IFN-γ-induced autophagy via p38 MAPK remains unclear.

In this study, we demonstrated that macrophage bactericidal activity increased at 4

h after IFN-γ stimulation in a STAT1- and NOS2-independent manner. Furthermore, this

macrophage bactericidal activity that occurred early after IFN-γ stimulation was attenuated

by inhibition of p38 MAPK or autophagic function. These results suggest that the autophagy

mediated by p38 MAPK, without the influence of NOS2, also contributes to the ability of

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macrophages to kill intracellular bacteria. To our knowledge, this study is the first to

document that p38 MAPK-mediated autophagy can activate IFN-γ-mediated

cell-autonomous innate immunity.

Materials and Methods

Reagents

Recombinant mouse IFN-γ was purchased from R&D Systems (Minneapolis, MN, USA)

and used at a concentration of 200 U/ml. NG-methyl-L-arginine acetate salt (L-NMMA) and

diphenyleneiodonium chloride (DPI) were obtained from Sigma (St Louis, MO, USA) and

used at a concentration of 500 µM or 10 µM. PD 169316 and SB 202190 were obtained

from Cayman (Ann Arbor, MI, USA) and used at a concentration of 10 µM. SB 203580 was

bought from Calbiochem (Darmstadt, Germany) and used at a concentration of 5 µM.

Mammalian cell culture

RAW 264.7 cells were obtained from the American Type Culture Collection

(Manassas, VA, USA) and maintained in RPMI-1640 medium containing 10% fetal bovine

serum (FBS), 10 mM HEPES, and 1 mM sodium pyruvate. The primary bone-marrow

derived macrophages (BMMs) were generated from C57BL/6 mice, as reported previously.38

The lentiviral vectors used for expressing short hairpin (sh)RNA against IFN-γR1, STAT1,

and Atg7 have been described previously.38 The plasmids for expressing shRNA as a

non-target control and for expressing shRNA against Atg5 or p38α were constructed using

pLKD.neo38 and the Addgene pLKO.1 protocol (http://www.addgene.org). The RNAi

sequences were as follows: for the non-target shRNA control,

5′-CAACAAGATGAAGAGCACCAA-3′; for Atg5, 5′-

GCAGAACCATACTATTTGCTT-3′; for p38α, 5′-CCTCTTGTTGAAAGATTCCTT-3′. The

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ViraPower Lentiviral Expression system (Invitrogen, Carlsbad, CA, USA) was used to

cotransfect the viral vector into 293FT (Invitrogen) to produce lentiviruses. The resulting

viral supernatant was used for the transfection of RAW 264.7 cells or BMMs, and then stable

knockdown (KD) cells were selected with G418 (BD Clontech, Palo Alto, CA, USA).

Bacterial culture

Listeria monocytogenes EGD (serovar 1/2a) was a generous gift from Dr. Masao

Mitsuyama (Kyoto University Graduate School of Medicine, Kyoto, Japan). It was grown

overnight in brain heart infusion (BHI) broth (BD Biosciences, Sparks, MD, USA) at 37ºC

and shaken. L. monocytogenes cells were washed with RPMI medium once and used in an

infection assay. Salmonella enterica serovar Typhimurium (RIMD1985009) was provided by

the Research Institute for Microbial Diseases, Osaka University (Osaka, Japan), and was

grown overnight in Luria-Bertani (LB) broth (Sigma).

Measurement of bacterial growth in the macrophages

RAW 264.7 cells or BMMs were infected with L. monocytogenes for 1 h at a

multiplicity of infection (MOI) of 5 or with S. Typhimurium for 10 min at an MOI of 10, and

then the cells were washed 3 times with PBS. Following this, the cell culture medium was

changed to new RPMI-1640 containing 50 µg/ml of gentamicin (Sigma) to exclude the

bacteria, which were not taken up by the macrophages. After 0 h or 4 h of Listeria infection,

or after 1 or 4 h of Salmonella infection, the macrophages were lysed with 0.1% Triton

X-100, and living bacteria were quantified by the colony-forming unit (CFU) method. The

growth rate of bacteria in the cells over a 4-h period was calculated. For each time point,

counts were obtained from 3 independent experiments.

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Measurement of NO production

Levels of NO were measured as the accumulation of nitrite in the cell culture

medium. The nitrite level was determined spectrophotometrically with Griess reagent

(Sigma). RAW264.7 cells or BMMs were treated with or without 200 U/ml IFN-γ for 4 or 24

h. Briefly, 250 µl of cell culture supernatant was mixed with an equal volume of Griess

reagent. Following incubation for 10 min, the absorbance at 550 nm was measured, and

values were quantified against a standard curve of sodium nitrite.

Western blotting

The macrophages were lysed in cell lysis buffer (50 mM Tris [pH 7.5], 1% Triton X-100,

and 150 mM NaCl) plus protease inhibitor mixture (Roche, Mannheim, Germany), and

centrifuged at 13,000 rpm for 20 min. The supernatants were used as cell lysates, and were

subjected to SDS-PAGE before transferring to polyvinylidene fluoride membranes. Western

blotting was carried out with the following antibodies (Abs): anti-IFN-γR polyclonal Ab

(pAb) and anti-Atg7 pAb (GeneTex, Irvine, CA, USA), anti-STAT1 pAb (GenScript,

Piscataway, NJ, USA), anti-GAPDH monoclonal Ab (mAb) (6C5; Santa Cruz Biotechnology,

Santa Cruz, CA, USA), anti-phospho-p38, p38, Atg5 pAbs (Cell Signaling Technology,

Danvers, MA, USA), ant-LC3 mAb (2G6; Nano Tools, Hamburg, Germany), Peroxidase

(HRP)-conjugated anti-mouse IgG Ab and HRP-conjugated anti-rabbitIgG Ab (Jackson

ImmunoResearch Laboratories, West Grove, PA, USA). The expression of GAPDH was

used as the internal control. The intensity of bands was quantified using ImageJ software.

Statistical analysis

Statistical analyses for differences between group means were analysed by Student’s t test.

All metrics are given as mean ± standard deviation values.

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Results

Macrophage bactericidal activity increased at 4 h after IFN-γ stimulation in an

IFN-γR1-dependent, but STAT1-independent manner

Previously, we have demonstrated that autophagy, which plays key roles in the

host defence pathway, is activated by IFN-γ stimulation for 4 h.38 In this study, we analysed

the function of macrophages that had been stimulated with IFN-γ for 4 h, to elucidate the

biological significance of IFN-γ-activated autophagy via p38 MAPK. Macrophage

bactericidal activity is potentiated by IFN-γ stimulation and this effect is predominantly

guided by NO, which is produced from arginine by IFN-inducible NOS2.22, 39 In mouse

macrophage-like RAW 264.7 cells, the expression of NOS2 mRNA was observed at 5 h after

IFN-γ (100 U/ml) stimulation, and NO production remained low until 10 h after IFN-γ

treatment.40, 41 Moreover, our result also showed that NO was barely produced at 4 h after

IFN-γ stimulation (Fig. 1a). However, an elevation in bactericidal activity against both

intracytosolic (L. monocytogenes) and intravacuolar (S. Typhimurium) pathogens was

observed in the macrophages that had been treated with IFN-γ for 4 h (Fig. 1b and c). This

kind of IFN-γ-mediated bactericidal activity was inhibited in IFN-γ receptor 1 KD cells, but

not in STAT1-KD cells (Fig. 2). These results suggested that bactericidal activity is

reinforced at an early stage of IFN-γ stimulation in a STAT1- and NO-independent manner.

Unlike NO production, autophagy activation has previously been observed in RAW 264.7

cells and BMMs at 4 h after IFN-γ stimulation38, we therefore expected that autophagy may

contribute to bactericidal activity at an early stage of IFN-γ stimulation.

NOS2 is not involved in macrophage bactericidal activity in the early stages after IFN-γ

stimulation

Next, we used the NOS inhibitors, L-NMMA and DPI, to rule out the possibility of

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participation of NOS2 and NO in macrophage bactericidal activity immediately after IFN-γ

stimulation. The bactericidal activity at 4 h after IFN-γ stimulation was not affected by either

NOS inhibitor (Fig. 3a and b). The effects of these NOS inhibitors were confirmed by NO

production in the macrophages that had been treated with IFN-γ for 24 h, because no NO

production was observed immediately after IFN-γ stimulation (Figs. 1a and 3c). L-NMMA

and DPI were effective at these concentrations (500 µM for L-NMMA and 10 µM for DPI).

These results suggested that macrophage bactericidal activity in the early stages following

IFN-γ stimulation is independent of NOS2 activity.

The p38 MAPK signalling pathway is employed for IFN-γ-mediated pathogen

clearance

IFN-γ activates various signalling cascades, including MyD88, p38 MAPK, PI3K, and

protein kinase C.8, 9, 12-14 In previous studies, we have demonstrated that 4 h after IFN-γ

stimulation, autophagy may be activated in macrophages.38 Therefore, we next investigated

whether p38 MAPK-mediated autophagy contributes to bactericidal activity (Figs. 4 and 5).

We found that IFN-γ-mediated autophagy activation and bactericidal activity was inhibited

in the presence of p38 MAPK inhibitors (PD 169316, SB 202190, and SB 203580) (Fig. 4)

or in the p38α KD cells (Fig. 5), suggesting that macrophage activation at an early stage

following IFN-γ stimulation depends on p38 MAPK.

Autophagy plays an important role in pathogen elimination by IFN-γ-stimulated

macrophages

Next, to confirm whether p38 MAPK-mediated autophagy is involved in the function of

IFN-γ-activated macrophages, autophagy-deficient Atg5 or Atg7 KD macrophage cells were

used (Fig. 6). The antibacterial effect observed at 4 h after IFN-γ treatment was blocked in

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autophagy-deficient cells, suggesting that macrophage bactericidal activity in the early stages

following IFN-γ stimulation is mediated by autophagy. Our findings collectively suggested

that, in IFN-γ-activated macrophages, bactericidal activity is quickly strengthened by p38

MAPK-mediated autophagy, rather than by NO. This action may be advantageous in

macrophages before full activation.

Discussion

Autophagy has emerged as a major immune defence pathway; it also contributes to

inflammation by facilitating an IFN-γ response and signal transduction.34, 42 We have

previously shown that the p38 MAPK signalling cascade also activates autophagy in

IFN-γ-stimulated macrophages, but the effect of p38 MAPK-mediated autophagy on

macrophage function was unknown.38 In this study, we found that macrophage bactericidal

activity increased at 4 h after IFN-γ stimulation in a STAT1- and NOS2-independent, p38

MAPK- and autophagy-dependent manner. Furthermore this macrophage action was

effective against both intracytosolic and intravacuolar pathogens. These findings suggest that

p38 MAPK-mediated autophagy can help stimulate IFN-γ-mediated cell-autonomous

immunity, with implications for understanding how IFN-γ-induced autophagy is mobilized

within macrophages for inflammation and host defence.

Both type I and type II IFNs upregulate gene transcription via the JAK-STAT, p38

MAPK, and PI3K signalling pathways, but slight differences exist in the regulation enacted

by each type of IFN.7 Studies using a pharmacological inhibitor of p38 MAPK,

overexpression of kinase-inactive p38α mutant, or mouse embryonic fibroblasts from p38α

knockout mice, showed that p38 MAPK is required for gene transcription via the

IFN-stimulated response element (ISRE) and IFN-γ-activated site (GAS) in response to type

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I IFNs, such as IFN-α and IFN-β.43, 44 However, p38 MAPK plays no role in type II IFN or

IFN-γ-dependent gene transcription.44 In our previous study, phosphorylation of p38 MAPK

by IFN-γ stimulation was observed after STAT1 knockdown in RAW 264.7 cells and in

BMMs from STAT1-knockout mice.38 Collectively, these data demonstrate that STAT1- and

p38 MAPK-dependent pathways operate separately from each other in IFN-γ signalling.

NOS2 is responsible for bactericidal activity in IFN-γ-stimulated macrophages that

combat intracellular bacteria via NO generation.22, 23 In addition to NOS2, Irgm1-a member

of the IFN-γ-inducible p47 IRG family-activates autophagy, and this contributes to the

defence mechanism of macrophages acting against M. tuberculosis.25, 26 IFN-γ-induced

macrophage bactericidal activity via NOS2 and Irgm1 depend on STAT1, because this

molecule mediates the expression of NOS2 and Irgm1.24, 45

In this study, we showed that macrophage bactericidal activity increased at 4 h

following IFN-γ stimulation. This phenomenon was not attenuated in STAT1-KD cells,

suggesting that the macrophage bactericidal activity occurring in the early stages following

IFN-γ stimulation is not due to NOS2 or Irgm1 (Fig. 2). In fact, the NOS inhibitors

L-NMMA and DPI failed to affect this kind of bactericidal activity (Fig. 3). The synergistic

effects that are initiated via NOS2-, Irgm1-, and p38 MAPK-dependent signalling cascades

are likely to contribute to IFN-γ-mediated host defence.

Acknowledgments

We thank Masao Mitsuyama (Kyoto University Graduate School of Medicine, Kyoto, Japan)

for the generous gift of a L. monocytogenes strain EGD (serovar 1/2a).

This work was supported by JSPS KAKENHI grant numbers 22659088 and 24790422.

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Figure Legends

Fig. 1. Macrophage bactericidal activity increased on IFN-γ stimulation for 4 h. RAW 264.7

cells or the primary bone-marrow derived macrophages (BMMs) were treated with 200 U/ml

IFN-γ for 4 or 24 h. The nitrite level in the cell culture medium was determined by the Griess

method (a). The cells were then infected with Listeria monocytogenes (b) or Salmonella

Typhimurium (c), and bacterial growth in the macrophages was calculated on the basis of

CFU. The values shown are the mean ± the standard deviation values obtained from 3

independent experiments. * p < 0.01; ** p < 0.02.

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Fig. 2. Macrophage bactericidal activity increased on 4 h of IFN-γ stimulation in an

IFN-γR-dependent, STAT1-independent manner. (a and b) RAW 264.7 cells, BMMs, or

stable knockdown cells were treated with 200 U/ml IFN-γ for 4 h. The cells were then

infected with L. monocytogenes (a) or S. Typhimurium (b), and bacterial growth in the

macrophages was calculated on the basis of CFU. The values shown are the mean ± the

standard deviation values obtained from 3 independent experiments. * p < 0.01; ** p < 0.1;

NS, not significant. (c) The cells were lysed in cell lysis buffer containing protease inhibitors,

and the cell lysates were then subjected to SDS-PAGE analysis and western blotting.

Fig. 3. NOS2 activity is not required for macrophage activation in the early stages after

IFN-γ stimulation. RAW 264.7 cells or BMMs were treated with 200 U/ml IFN-γ in the

presence or absence of 500 µM of L-NMMA or 10 µM of DPI for 4 h (a and b) or 24 h (c).

(a and b) The cells were then infected with L. monocytogenes (a) or S. Typhimurium (b), and

bacterial growth in the cells was calculated on the basis of CFU. (c) The nitrite level in the

cell culture medium was determined by the Griess method. The values shown are the mean ±

the standard deviation values obtained from 3 independent experiments. * p < 0.01; ** p <

0.02; *** p < 0.05; **** p < 0.1; NS, not significant.

Fig. 4. Macrophage activation in the early stages after IFN-γ stimulation is reduced by the

p38 MAPK inhibitors. RAW 264.7 cells or BMMs were treated with 200 U/ml IFN-γ in the

absence or presence of p38 MAPK inhibitors (10 µM of PD 169316, 10 µM of SB202190,

or 5 µM of SB 203580) for 4 h. (a and b) The cells were then infected with L.

monocytogenes (a) or S. Typhimurium (b), and bacterial growth in the cells was calculated on

the basis of CFU. The values shown are the mean ± the standard deviation values obtained

from 3 independent experiments. * p < 0.01; ** p < 0.05; *** p < 0.1. (c) The cells were

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lysed in cell lysis buffer containing protease inhibitors, and the cell lysates were then

subjected to SDS-PAGE analysis and western blotting. Densitometric LC3-II/GAPDH ratios

are shown underneath the blot.

Fig. 5. Macrophage activation in the early stages after IFN-γ stimulation was reduced in

p38α knockdown cells. (a and b) RAW 264.7 cells, BMMs, or stable knockdown cells were

treated with 200 U/ml IFN-γ for 4 h. The cells were then infected with L. monocytogenes (a)

or S. Typhimurium (b), and bacterial growth in the macrophages was calculated on the basis

of CFU. The values shown are the mean ± the standard deviation values obtained from 3

independent experiments. * p < 0.01; ** p < 0.02; *** p < 0.05. (c) The cells were lysed in

cell lysis buffer containing protease inhibitors, and the cell lysates were then subjected to

SDS-PAGE analysis and western blotting. Densitometric LC3-II/GAPDH ratios are shown

underneath the blot.

Fig. 6. Autophagy plays a critical role in macrophage activation in the early stages after

IFN-γ stimulation. RAW 264.7 cells, BMMs, and stable knockdown cells were treated with

200 U/ml IFN-γ for 4 h. (a and b) The cells were infected with L. monocytogenes (a) or S.

Typhimurium (b). The bacterial growth in the cells was calculated on the basis of CFU. The

values shown are the mean ± the standard deviation values obtained from 3 independent

experiments. * p < 0.01; ** p < 0.05; *** p < 0.1; NS, not significant. (c) The cells were

lysed in cell lysis buffer containing protease inhibitors, and the cell lysates were then

subjected to SDS-PAGE analysis and western blotting. Densitometric LC3-II/GAPDH ratios

are shown underneath the blot.

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This article is protected by copyright. All rights reserved.

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This article is protected by copyright. All rights reserved.

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This article is protected by copyright. All rights reserved.

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This article is protected by copyright. All rights reserved.