Intracellular bacteria recognition contributes to maximal interleukin by JOSE LUIS CONSTANTINO

Clinical and Experimental Immunology

O R I G I N A L ART I CLE

doi:10.1111/j.1365-2249.2011.04318.x

Intracellular bacteria recognition contributes to maximal interleukin (IL)-12 production by IL-10-deﬁcient macrophages cei_4318 137..144

H. Naruse, T. Hisamatsu, Y. Yamauchi, J. E. Chang, K. Matsuoka, M. T. Kitazume, K. Arai, S. Ando, T. Kanai, N. Kamada and T. Hibi Division of Gastroenterology and Hepatology, Department of Internal Medicine, Keio University School of Medicine, Tokyo, Japan

Accepted for publication 3 December 2010 Correspondence: T. Hibi, Department of Internal Medicine, School of Medicine, Keio University, 35 Shinano machi, Shinjuku-ku, Tokyo 160-8582, Japan. E-mail: thibi@sc.itc.keio.ac.jp

Summary Interleukin (IL)-12 is a key factor that induces T helper cell type 1-mediated immunity and inflammatory diseases. In some colitis models, such as IL-10 knock-out (KO) mice, IL-12 triggers intestinal inflammation. An abundant amount of IL-12 is produced by intestinal macrophages in response to stimulation by commensal bacteria in IL-10 KO mice. Intact bacteria are more potent inducers of macrophage IL-12 production than cell surface components in this model. This suggested that cell surface receptor signalling and intracellular pathogen recognition mechanisms are important for the induction of IL-12. We addressed the importance of intracellular recognition mechanisms and demonstrated that signal transducers and activator of transcription 1 (STAT1) signalling activated bacterial phagocytosis and was involved in the induction of abnormal IL-12 production. In IL-10 KO mouse bone marrow-derived (BM) macrophages, Escherichia coli stimulation induced increased IL-12p70 production compared to lipopolysaccharide combined with interferon (IFN)-g treatment. Significant repression of IL-12 production was achieved by inhibition of phagocytosis with cytochalasin D, and inhibition of de novo protein synthesis with cycloheximide. Induction of IFN regulatory factors-1 and -8, downstream molecules of STAT1 and the key transcription factors for IK-12 transcription, following E. coli stimulation, were mediated by phagocytosis. Interestingly, STAT1 was activated after stimulation with E. coli in IL-10 KO BM macrophages, although IFN-g could not be detected. These data suggest that molecules other than IFN-g are involved in hyper-production mechanisms of IL-12 induced by E. coli stimulation. In conclusion, enteric bacteria stimulate excessive IL-12p70 production in IL-10 KO BM macrophages via phagocytosis-dependent signalling. Keywords: bacteria, IL-10, IL-12, macrophage, phagocytosis

Introduction Prompt and thorough responses of the body’s immune system to foreign antigens are of vital importance in its defence against foreign intrusion. However, improper or uncontrolled responses to such antigens result ultimately in excessive immune activity and disruption of normal homeostasis. Because the intestinal mucosa is always exposed to numerous commensal bacteria, it is believed that the gut may possess regulatory mechanisms that prevent excessive inflammatory responses against commensal bacteria. When this homeostatic regulation is disrupted, the constant presence of food and commensal bacteria antigens may cause excessive immune activation.

Macrophages, the major population of tissue-resident mononuclear phagocytes, play key roles in bacterial recognition and elimination, as well as in the polarization of innate and adaptive immunity. Besides these classical anti-bacterial immune roles, it has become evident recently that several macrophage subsets also play an important role in immunological homeostasis maintenance. For example, they are involved in inflammation dampening via the production of anti-inflammatory cytokines such as interleukin (IL)-10 and transforming growth factor (TGF)-b [1]. This is particularly important for the contribution of intestinal macrophages to gut mucosal homeostasis. It has been reported previously that intestinal macrophages do not express innate response receptors [2,3], and although these cells retain their

H. Naruse et al.

phagocytic and bactericidal functions, they do not produce proinflammatory cytokines in response to several inflammatory stimuli, including microbial components [4,5]. Another recent study has revealed that murine intestinal macrophages express several anti-inflammatory molecules, such as IL-10. Moreover, such intestinal macrophages suppress intestinal dendritic cell (DC)-derived helper T cell (Th) 1 and Th17 immunity, in both a regulatory T cell (Treg)-dependent and an independent manner [6]. Intestinal macrophages show a similar phenotype to in vitro macrophage colony-stimulating factor (M-CSF) differentiated macrophages, which have phagocytic ability and produce the anti-inflammatory cytokine IL-10 [4]. Consistent with this observation, M-CSF is expressed at the lamina propria in both mouse and human intestine, and the number of intestinal macrophages in op/op mice, which lack M-CSF signalling, was decreased significantly [7,8]. Furthermore, in mice lacking intestinal macrophages, gut homeostasis is disrupted [9]. Taken together, these findings suggest that, by producing IL-10, intestinal macrophages prevent excessive immune responses to commensal organisms. IL-10 knockout (KO) mice develop spontaneous Th1-dominant chronic colitis and are used widely as an animal model for human inflammatory bowel disease (IBD) [10]. As enteric flora are required for the development of colitis in IL-10 KO mice, it is thought that proper recognition and responses to commensal bacteria are impaired in IL-10 KO mice, and that this dysfunction plays a key role in the development of intestinal inflammation akin to mouse and human IBD pathogenesis [11]. Previously, we demonstrated that IL-10 KO intestinal macrophages produce abnormally high levels of proinflammatory cytokines, such as IL-12, in response to bacteria [4]. In those experiments, we found that stimulation with whole bacteria, but not pathogen-associated molecular patterns (PAMPs), can induce the production of large amounts of IL-12. This suggested that not only cell surface receptor signalling, such as Toll-like receptors (TLRs), but also intracellular pathogen recognition mechanisms are important for the production of IL-12 by IL-10 KO macrophages. Here, we show that phagocytosis and de novo protein synthesis are necessary stages leading to the activation of the signal transducers and activator of transcription (STAT)1-IFN regulatory factor (IRF)1/8 signalling pathway, which results subsequently in induction of maximal IL-12 production by IL-10 KO macrophages.

Materials and methods Reagents Recombinant mouse M-CSF and IFN-g were purchased from R&D Systems (Minneapolis, MN, USA). Gel filtrated grade lipopolysaccharide (LPS; Escherichia coli O111:B4), cytochalasin D (CyD) and cycloheximide (CHX) were purchased from Sigma-Aldrich (St Louis, MO, USA). 138

Bacteria heat-killed antigen A Gram-negative non-pathogenic strain of E. coli (ATCC25922) was cultured with Luria–Bertani (LB) medium. The bacteria were harvested and washed twice with ice-cold phosphate-buffered saline (PBS). Bacterial suspensions were heated at 80°C for 30 min, then washed and resuspended in PBS, and stored at -80°C. The complete killing was confirmed with 24 h incubation at 37°C on plates.

Mice Specific pathogen-free wild-type (WT) C57BL/6J mice were purchased from Charles River Japan, Inc. (Tokyo, Japan). These WT and IL-10 KO (C57BL/6J background) were housed at the animal centre of Keio University. All experiments using mice were approved by and performed according to the guidelines of the animal committee of Keio University.

Preparation of bone marrow (BM)-derived macrophages WT and IL-10 KO mice aged 7–12 weeks were used to isolate BM cells from the femora. The BM-mononuclear cells were separated by gradient centrifugation, and purified CD11b+ cells were obtained using a magnetic cell separation system (MACS; Miltenyi Biotech, Bergisch Gladbach, Germany) with anti-mouse CD11b microbeads. To generate BMmacrophages, CD11b+ cells (5 ¥ 105 cells/ml) were cultured with M-CSF (20 ng/ml) for 7 days. BM-macrophages cells were plated at a density of 1 ¥ 105 cells per 6·4-mm-diameter culture dish and cultured overnight.

Analysis of phagocytosis Luminol-bound microbeads (Kamakura Techno-Science, Kamakura, Japan) at a concentration of 2 mg/200 ml were added to recultured macrophages, from which luminescence was generated by reactive oxygen within phagosomes. The luminescence of each dish was quantified in a luminometer (Synergy 4; Biotek, Winooski, VT, USA) by integrating the signal for 5 s every minute at a sensitivity level of 200. We also examined the phagocytic ability of macrophages using whole bacteria antigens. After a 30-min stimulation with E. coli [K-12 strain, multiplicity of infection (MOI) = 1] labelled with Alexa Fluor 488 (Invitrogen, Carlsbad, CA, USA) with or without CyD (1 mM), BM-macrophages were washed with PBS, harvested using ethylenediamine tetraacetic acid (EDTA) and washed with MACS buffer. Cells were incubated with 0·5 mg/ml CD11b monoclonal antibody (eBiosciences, San Diego, CA, USA) for 20 min at 4°C. After staining, cells were washed with MACS buffer, stained with propidium iodide and analysed using a fluorescence activated

Intracellular bacteria recognition for IL-12 production in macrophage

cell sorter (FACS)Calibur (BD Pharmingen, San Diego, CA, USA). CellQuest software was used for data analysis.

Activation of BM-macrophages by PAMPs and heat-killed bacteria Macrophages were washed to remove the secreted or supplemented cytokines in the supernatant. Macrophages were then stimulated with 100 ng/ml LPS and 100 ng/ml of IFN-g, or heat-killed bacteria (MOI = 100). Culture supernatants were collected and passed through a 0·22-mm pore size filter and then stored at -80°C for later analysis of cytokines.

Signaling, Beverly, MA, USA) was used. Blots were visualized using an enhanced chemiluminescence kit (GE Healthcare, Buckinghamshire, UK). The antibodies used were antiphospho-STAT1 tyrosine (Tyr) 701, anti-phospho-STAT1 serine (Ser) 727 and anti-STAT1 (Cell Signaling).

Statistical analysis Statistical differences between two groups were tested using Student’s t-test using GraphPad PRISM5 (GraphPad Inc., San Diego, CA, USA).

Cytokine measurement

Results

A mouse inflammatory cytometric beads array (CBA) kit (BD Pharmingen) was used for measurement of cytokines in the culture supernatants. Samples were analysed using a FACSCalibur (BD Pharmingen) according to the manufacturer’s specifications.

IL-10 KO BM-macrophages produce IL-12p70 in response to whole bacteria (E. coli)

(a) Luminescence (cps)

After 8 h stimulation by bacterial antigen, total RNA was isolated from macrophages using the RNeasy Mini kit (Qiagen Inc., Valencia, CA, USA) and cDNA was synthesized with Omniscript reverse transcriptase (Qiagen). For qRT– PCR, TaqMan Universal PCR Master Mix and TaqMan Gene Expression Assays for murine IL-12p35, p40, IRF1, IRF8, IFN-g and b-actin (purchased from Applied Biosystems, Foster City, CA, USA) were used. The PCRs were carried out in a thermocycler DNA engine, OPTICON2 (MJ Research, Waltham, MA, USA). Cycling conditions for PCR amplification were 50°C for 2 min, 95°C for 10 min and then 40 cycles of 95°C for 15 s followed by 60°C for 1 min.

Luminescence (cps)

Analysis of cytokine transcription by quantitative reverse transcription–polymerase chain reaction (qRT–PCR)

We confirmed that the phagocytic ability of WT and IL-10 KO macrophages were not significantly different (Fig. 1a). As we reported previously, IL-10 KO BM-macrophages differentiated by M-CSF produced a large amount of IL-12p70 in response to whole bacteria stimulation compared with those in WT mice (Fig. 1b) [4]. These results led us to investigate whether innate immune receptors on the cell surface, such as TLR-4 for LPS, or intracellular pathogen recognition mechanisms are involved.

WT 300 200 100 0 0

IL-10KO microbeads(–) microbeads(+)

300 200 100 0 0

30 (min)

(b)

Immunoblotting

IL-12p70

Macrophages (1 ¥ 105 cells/well) were washed with ice-cold PBS containing protease inhibitors, then lysed in 30 ml of lysis buffer (Pierce, Rockford, IL, USA) containing protease inhibitor cocktail (Sigma-Aldrich, Poole, UK), 1 mM phenylmethylsulphonylfluoride (PMSF) and 1 mM Na3VO4. After centrifugation, the supernatants were mixed with lithium dodecyl sulphate (LDS) sample buffer and reducing agent (Invitrogen) and electrophoresed on a Bis–Tris 4–12% gradient polyacrylamide gel (Invitrogen). Separated proteins were transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA) and immunoblotted for specific antibodies as per the manufacturer’s instructions. After blocking, primary antibodies were added at a 1:1000 dilution overnight at 4°C. Horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody (Cell

(pg/ml)

150

WT IL-10KO

100 50 0

Time (h) Fig. 1. Interleukin (IL)-10 knock-out (KO) bone marrow (BM)-macrophages produced IL-12p70 in response to whole bacteria (Escherichia coli). (a) After luminol-bound microbeads were added, the luminescence of each dish was quantified in a luminometer by integrating the signal. (b) BM-macrophages from wild-type (WT) and IL-10 KO were stimulated. The amount of IL-12p70 proteins was measured with a cytometric beads array (CBA) kit after stimulation for the indicated time with E. coli. Data shown are representative of three independent experiments.

H. Naruse et al. (a)

E. coli stimulation induces large amounts of IL-12p70 production compared with LPS and IFN-g combined treatments

IL-12p70

***

600

To resolve this issue, we have investigated the difference in IL-12 mRNA transcription, protein production and signalling activation following stimulation with LPS plus IFN-g or whole bacteria antigen stimulation. LPS, a cell wall component of Gram-negative bacteria such as E. coli, is a typical PAMP. LPS stimulation alone is incapable of inducing excessive IL-12p70 production independently in IL-10 KO BM-macrophages (data not shown), but when administered with IFN-g, IL-12p70 production is increased substantially. Whole bacteria stimulation induced large amounts of IL-12 (Fig. 2a). The mRNA transcription kinetics of p40 and p35 showed a higher maximum amplitude 9 h after stimulation and sustained higher transcription levels in the E. colistimulated group, while the LPS and IFN-g group only had a small, transient level of transcription at 3 h (Fig. 2b).

400 200 0 LPS + IFNγ EC

IL-12p35

0·008 0·006 0·004 0·002 0·000

Time (h)

Relative gene expression

(b) IL-12p40

0·0001

LPS + IFNγ EC

0·0001 0·0000 0·0000

Time (h)

Fig. 2. In interleukin (IL)-10 knock-out (KO) bone marrow (BM)-macrophages, Escherichia coli stimulation induced large amounts of IL-12p70 production, compared with the combined lipopolysaccharide (LPS) and interferon (IFN)-g treatment. BM-macrophages from IL-10 KO mice were stimulated. (a) The amount of IL-12p70 protein was measured with a cytometric beads array (CBA) kit after 12 h of stimulation with LPS and IFN-g or E. coli. Data indicate the expression as the mean ⫾ standard error of the mean from 12 independent experiments. (b) The transcripts of IL-12p35 and p40 mRNA were measured by quantitative reverse transcription–polymerase chain reaction (qRT–PCR) after stimulation with LPS and IFN-g or E. coli at the indicated times. Data shown are representative of three independent experiments. ***P < 0·001 compared with the control groups.

Phagocytosis is necessary for production of IL-12 after stimulation with E. coli in IL-10 KO mice BM-macrophages In the experiments using microbeads and labelled heatkilled E. coli, treatment with CyD inhibited phagocytotic activity in IL-10 KO BM-macrophages (Fig. 3a and b). Microbeads did not stimulate cytokine production or STAT1 phosphorylation (Fig. S1a and b). Having inhibited phagocytosis of E. coli with CyD, we then measured production of

140

Luminescence (cps)

(a)

(c)

IL-10KO 250 200 150 100 50 0

CyD (–) CyD (+)

(min)

IL-12p70 1500 *** 1000 500 0 CyD (–) CyD (+)

(b) 100 80 % of max

Fig. 3. Phagocytosis was necessary for production of interleukin (IL)-12 after stimulation with Escherichia coli in IL-10 knock-out (KO) bone marrow (BM)macrophages. IL-10 KO BM-macrophages were treated with or without cytochalasin D (CyD) for 1 h before stimulation. (a) After luminol-bound microbeads were added, the luminescence of each dish was quantified in a luminometer by integrating the signal. (b) BM-macrophages from IL-10 KO mice were used. After a 30-min stimulation with E. coli (K-12 strain, multiplicity of infection = 1) labelled with Alexa Fluor 488 with or without CyD (1 mM), cells were washed with phosphate-buffered saline (PBS) and fluorescence analysed in CD11b+ cells by flow cytometry. (c) The amount of IL-12p70 protein was measured with a cytometric beads array (CBA) kit after 12 h of stimulation with E. coli. Data are expressed as the mean ⫾ standard error of the mean from three independent experiments. ***P < 0·001 compared with the control groups.

IL-12p70 (pg/ml)

800

Sample name

(–) 40

E. coli E. coli + CyD

100

101

102

103

Mean, FL1-H 12·97 134·20 81·24

104

FITC-labelled E. coli

Intracellular bacteria recognition for IL-12 production in macrophage IL-12p40

IL-12p35 1·5

0·0

Phagocytosis and new protein synthesis is necessary for E. coli-stimulated STAT1 activation in IL-10 KO BM-macrophages

0·5

0·0 CHX (–) CHX (+)

CHX (–) CHX (+)

Fig. 4. Protein synthesis was necessary for Escherichia coli-stimulated interleukin (IL)-12p35/p40 transcriptional up-regulation in IL-10 knock-out (KO) bone marrow (BM)-macrophages. IL-10 KO BM-macrophages were pretreated with or without cycloheximide (CHX) for 1 h, followed by stimulation with E. coli. The IL-12p35 and p40 mRNA transcripts were measured by quantitative reverse transcription–polymerase chain reaction (qRT–PCR) after 3 h of stimulation with E. coli. Data indicate the fold expression compared with non-CHX-treated macrophages and are expressed as the mean ⫾ standard error of the mean from three independent experiments. ***P < 0·001, compared with the control groups.

inflammatory cytokines, such as IL-12, tumour necrosis factor (TNF)-a, monocyte chemotactic protein (MCP)-1 and IL-6. Expression of IL-12 was decreased strongly, although TNF-a, MCP-1 and IL-6 were not decreased significantly (Fig. 3c, Fig. S3).

De novo protein synthesis is necessary for E. coli-stimulated IL-12p70 transcriptional up-regulation in IL-10 KO BM-macrophages After inhibition of translation with CHX, E. coli stimulation showed complete ablation of IL-12p35 and p40 transcription, as assessed by qRT–PCR (Fig. 4). These results suggested that transcriptional up-regulation of IL-12p35 and p40 was induced by E. coli via the de novo synthesis of signalling molecules.

It is well known that IFN-g activates STAT1. Activation of STAT1 leads subsequently to synthesis and secretion of IL-12p35 and p40. STAT1 is activated by E. coli stimulation in IL-10 KO macrophages, not in WT (Fig. 6a). Therefore, the combination of LPS and IFN-g treatment resulted immediately in STAT1 activation in IL-10 KO macrophages, (a) 25 20 15 10 5 0

(b)

–

Several IRFs participated in the synthesis of IL-12p70. The activation of IRF1 by IFN-g or TLR ligands was shown to be required for the activation of the p35 gene, whereas it did not affect p40 gene expression [12,13]. IRF8, another factor inducible by IFN-g, was shown to be involved in the expression of both the p35 and p40 genes in conditions in which IFN-g was used as a priming agent before TLR stimulation [14]. Therefore, we hypothesized that IFN-g-like protein expression is induced after E. coli recognition by the macrophages, and these proteins activate macrophages in an autocrine manner. Both IRF1 and IRF8 induction were

8 6 4 2 0

–

IRF8 (3 h)

IRF1 (3 h) 1·5

1·5 ***

** 1·0

0·5

0·0

1·0

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0·0 CyD (–)

Phagocytosis is necessary for IRF1 and IRF8 induction after stimulation with E. coli in IL-10 KO BM-macrophages

IRF8 (3 h)

IRF1 (3 h)

Fold induction

0·5

1·0

Fold induction

1·0

n=3 ***

Fold induction

n=3 ***

Fold induction

1·5

up-regulated following E. coli stimulation (Fig. 5a). Induction of IRF1 and IRF8 was dependent on phagocytosis, as their expression was suppressed by CyD treatment (Fig. 5b).

CyD (+)

CyD (–)

CyD (+)

Fig. 5. Phagocytosis was necessary for interferon (IFN) regulatory factor (IRF)1 and IRF8 expression after stimulation with Escherichia coli in interleukin (IL)-10 knock-out (KO) bone marrow (BM)-macrophages. (a) The IRF1 and IRF8 mRNA transcripts were measured by quantitative reverse transcription–polymerase chain reaction (qRT–PCR) after 3- or 6-h stimulation with E. coli. Data indicate the fold expression compared with non-stimulated macrophages and are expressed as the mean ⫾ standard error of the mean (s.e.m.) from nine independent experiments at 3 h. (b) IL-10 KO BM-macrophages were pretreated with or without cytochalasin D (CyD) for 1 h, followed by stimulation with E. coli. The amounts of IRF1 and IRF8 mRNA transcripts were measured after 3 h of stimulation. Data indicate the fold expression compared with non-treated macrophages and are expressed as the mean ⫾ s.e.m. from seven independent experiments at 3 h. *P < 0·05; **P < 0·01; ***P < 0·001 compared with the control groups.

H. Naruse et al. (a)

Fig. 6. Phagocytosis and protein synthesis were necessary for Escherichia coli-stimulated signal transducers and activator of transcription (STAT)1 activation in interleukin (IL)-10 knock-out (KO) bone marrow (BM)-macrophages. (a) Macrophages from wild-type (WT) and IL-10 KO mice were stimulated with E. coli at the indicated time-points. Western blot analysis was used to determine phospho-STAT1 and total STAT1 expression levels. (b) IL-10 KO BM-macrophages were pretreated with or without cytochalasin D (CyD) for 1 h or cycloheximide (CHX) for 1 h, followed by stimulation with E. coli for 3 h. Western blot analysis was used to determine phospho-STAT1 and total STAT1 expression levels. Data shown are representative of three independent experiments.

EC stimulation WT IL-10KO 0 15 30 60 180 0 15 30 60 180 (min) Y701

STAT1

S727 whole

(b)

CyD (–) –

CHX (–)

CyD (+) –

–

STAT1

S727 whole

Discussion IL-12 is a key cytokine that drives naive T cells to differentiate into Th1 cells. In Crohn’s disease, it has also been reported that the excessive production of IL-12 by macrophages and dendritic cells in response to commensal bacteria promotes the Th1 response [17]. Furthermore, IFN-g can

CHX (+) –

Y701

whereas E. coli treatment did not result in STAT1 activation until after 180 min (Fig. S6a). This difference in kinetics suggests different mechanisms of signal transduction. As shown previously, IFN-g can induce STAT1 activation directly and rapidly. Therefore, we examined the possibility of IFN-g up-regulation in response to E. coli stimulation by IL-10 KO BM-macrophages. However, inconsistent with previously reported findings [15,16], IFN-g was not detected following E. coli treatment at both the mRNA and protein levels in IL-10 KO BM-macrophages (Fig. S4). We also examined the effect of CyD-treatment on STAT1 phosphorylation at Tyr701 and Ser727 in E. coli-stimulated IL-10 KO BM-macrophages. As expected, STAT1 was not activated in samples in which phagocytosis was inhibited (Fig. 6b). Thus, these data suggest that phagocytic activity is necessary to mount a significant IL-12 response to bacterial stimulation in IL-10 KO BM-macrophages. Furthermore, the loss of E. coli-induced IL-12p35/p40 in the presence of CHX corresponded to a lack of STAT1 phosphorylation at Tyr701 and Ser727 (Fig. 6b). Taken together, these results suggest that phagocytosis and de novo protein synthesis are required for bacteria-induced STAT1 activation in IL-10 KO BM-macrophages. Conversely, STAT1 activation from LPS + IFN-g did not decrease with either CHX or CyD (Fig. S5).

142

STAT1

S727 whole

promote the up-regulation of IL-12 production [18–20]. This locally Th1-skewed environment promotes the chronic intestinal inflammation seen in Crohn’s disease. WT intestinal macrophages produce large amounts of IL-10 and suppress proinflammatory cytokine production, especially IL-12. Conversely, IL-10 KO mice are used as a model of Th1-dominant colitis because their intestinal macrophages produce abnormally high levels of proinflammatory cytokines in response to commensal bacteria. In previous studies the induction of IL-12 in monocytic cell lineages, such as monocytes, dendritic cells and cell lines such as THP-1, has been studied mainly under the stimulation of PAMPs (for example, LPS and peptidoglycan). However, regarding the physiological function of phagocytic cells such as macrophages, intracellular recognition mechanisms following bacterial phagocytosis should be investigated. Indeed, recent studies have identified several molecules that are related to intracellular bacterial recognition as susceptibility genes of IBD [21,22]. In the present study, we have demonstrated that intracellular bacterial recognition is important for IL-12 production by IL-10 KO BM-macrophages. Phagocytosis of bacteria followed by intracellular recognition activates STAT1–IRF signalling inducing production of higher amounts of IL-12p70. Our results demonstrate that stimulation with whole bacteria could induce relatively higher levels of IL-12 compared with the combined LPS and IFN-g stimulation, which is known to be a typical IL-12 inducer in the monocytic cell lineage [12,14,23]. The production of IFN-g by innate immune cells, such as natural killer cells, provides a strong feedback loop to sustain the production of high levels of IL-12p70 by activated macrophages [24]. IFN-g phosphorylates STAT1 and induces downstream signalling and

Intracellular bacteria recognition for IL-12 production in macrophage

synthesis of proteins such as IRF1, IRF8, major histocompatibility complex class II and TLR-4. Thus, STAT1 signalling is known as a proinflammatory signal. Once activated, STAT1 binds to IFN-stimulated response elements and IFN-gactivated sequence domains in the promoter of genes such as the IRF family. IRF1 binds to the promoter region of the IL-12p35 gene, while IRF8 binds to the IL-12p40 promoter. Upon priming with IFN-g, IRF8 enhances expression of both the p35 and p40 genes [12,14]. Interestingly, LPS and IFN-g stimulation could only induce IL-12p40 and p35 temporarily, whereas E. coli stimulated the sustained induction of p40 and p35. Furthermore, the combined LPS and IFN-g stimulation did not fully enhance IL-12p35 transcription followed by lesser production of IL-12p70 (Fig. S2). These results suggest that transcriptional regulation of the IL-12 genes is quite different between E. coli stimulation and the combined LPS and IFN-g stimulation. E. coli is constructed with various components, including LPS, single-stranded DNA and flagellin. Because these factors bind to specific receptors and stimulate various signalling pathways, it is possible that they act synergistically to increase IL-12p70 production. Importantly, the inhibition of phagocytosis by CyD reduced E. coli stimulation-induced IL-12 production. As macrophages phagocytose bacteria and digest them in lysosomes, our results suggest that intracellular signalling pathways, in addition to surface receptor signalling, are important to IL-12 induction following bacterial stimulation. Furthermore, our results from experiments using the translation inhibitor CHX indicate that de novo protein synthesis following phagocytosis is required for E. coli stimulation-induced IL-12 production. As we expected, both IRF1 and IRF8 were up-regulated at the transcriptional and protein levels by bacterial stimulation. Importantly, up-regulation of both transcription factors was also dependent on phagocytosis. The process of phagocytosis and de novo protein synthesis may contribute to STAT1 activation. We hypothesized that macrophages produce IFN-g by phagocytosing E. coli, and that IFN-g activates macrophages through an autocrine loop [16]. While IFN-g could not be detected at the mRNA or protein levels, we cannot deny the possibility that undetectable levels of IFN-g play a functional role. Therefore, it is possible that IFN-g-like factors may play a role in STAT1 activation. In IL-10 KO BM-macrophages, combined LPS and IFN-g rapidly induced both STAT1 and STAT3 phosphorylation. In contrast, E. coli stimulation did not induce STAT3 phosphorylation and only induced STAT1 phosphorylation at the late phase (Figs S6 and S7). Thus, this imbalance of STAT1 and STAT3 activation may contribute to bacteria-induced IL-12 hyper-production by phagocytic cells. In conclusion, we show here that phagocytosis, intracellular recognition of bacteria and new protein synthesis contribute to the maximum production of IL-12 by IL-10 KO BM-macrophages. We believe that the identification of intracellular signalling pathways activated in response to bacterial

engulfment by innate immune cells is important in the context of the pathophysiology of chronic intestinal inflammation in IBD.

Acknowledgements The authors thank Drs T. Takayama, R Saito, T. Kobayashi, M. Kurokouchi, N. Tsuzuki and T. C. Lee for helpful discussions and critical comments. The authors thank Dr Y. Wada for manuscript preparation. This study was supported in part by grants-in-aid from the Japanese Ministry of Education, Culture and Science, the Japanese Ministry of Health, Labor and Welfare, Keio University and Keio Medical Foundation, Tokyo, Japan.

Disclosure The authors declare no financial or commercial conflict of interest.

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Supporting information Additional Supporting Information may be found in the online version of this article: Fig. S1. Microbeads did not stimulate cytokine production and signal transducers and activator of transcription (STAT)1 phosphorylation. Fig. S2. Comparison of cytokine production by interleukin (IL)-10 knock-out (KO) bone marrow (BM)macrophages following stimulation with lipopolysaccharide (LPS) plus interferon (IFN)-g or heat-killed Escherichia coli. Fig. S3. The amount of tumour necrosis factor (TNF)-a, monocyte chemotactic protein (MCP)-1 and interleukin (IL)-6 protein was measured with a cytometric beads array (CBA) kit after a 12-h stimulation with Escherichia coli. Data are expressed as the mean ⫾ standard error of the mean from three independent experiments. Fig. S4. Interleukin (IL)-10 knock-out (KO) bone marrow (BM)-macrophages were stimulated with a heat-killed Gram-negative strain of Escherichia coli for the indicated time-points. Fig. S5. Interleukin (IL)-10 knock-out (KO) bone marrow (BM)-macrophages were pretreated with or without cycloheximide (CHX) or cytochalasin D (CyD) for 1 h, followed by stimulation with lipopolysaccharide (LPS) and interferon (IFN)-g for 1 h. Fig. S6. Interleukin (IL)-10 knock-out (KO) bone marrow (BM)-macrophages were stimulated with lipopolysaccharide (LPS) and interferon (IFN)-g or with Escherichia coli for the indicated time-points. Western blot analysis was used to determine phospho-signal transducers and activator of transcription (STAT)1, total STAT1, phospho-STAT3 and total STAT3 expression levels. Fig. S7. Interleukin (IL)-10 knock-out (KO) bone marrow (BM)-macrophages were stimulated with the indicated dilution of IL-10 and Western blot analysis was performed to determine phosphorylation of signal transducers and activator of transcription (STAT)1. Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.