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The Journal of Immunology, 2007, 179, 1166 -1177
Copyright © 2007 by The American Association of Immunologists, Inc.
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Type I IFN Induction in Response to Listeria monocytogenes in Human Macrophages: Evidence for a Differential Activation of IFN Regulatory Factor 3 (IRF3)1

Thornik Reimer2, Matthias Schweizer and Thomas W. Jungi

Institute of Veterinary Virology, University of Bern, Bern, Switzerland


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Listeria monocytogenes is a prototypic bacterium for studying innate and adaptive cellular immunity as well as host defense. Using human monocyte-derived macrophages, we report that an infection with a wild-type strain, but not a listeriolysin O-deficient strain, of the Gram-positive bacterium L. monocytogenes induces expression of IFN-beta and a bioactive type I IFN response. Investigating the activation of signaling pathways in human macrophages after infection revealed that a wild-type strain and a hemolysin-deficient strain of L. monocytogenes activated the NF-{kappa}B pathway and induced a comparable TNF response. p38 MAPK and activating transcription factor 2 were phosphorylated following infection with either strain, and IFN-beta gene expression induced by wild-type L. monocytogenes was reduced when p38 was inhibited. However, neither IFN regulatory factor (IRF) 3 translocation to the nucleus nor posttranslational modifications and dimerizations were observed after L. monocytogenes infection. In contrast, vesicular stomatitis virus and LPS triggered IRF3 activation and signaling. When IRF3 was knocked down using small interfering RNA, a L. monocytogenes-induced IFN-beta response remained unaffected whereas a vesicular stomatitis virus-triggered response was reduced. Evidence against the possibility that IRF7 acts in place of IRF3 is provided. Thus, we show that wild-type L. monocytogenes induced an IFN-beta response in human macrophages and propose that this response involves p38 MAPK and activating transcription factor 2. Using various stimuli, we show that IRF3 is differentially activated during type I IFN responses in human macrophages.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Listeria monocytogenes is a Gram-positive, facultative intracellular bacterium invading a variety of cells from different species (1). Upon phagocytosis by macrophages, L. monocytogenes escapes phagosomal compartments through lysis of the phagosomal membrane. This is achieved by a cholesterol-dependent cytolysin, listeriolysin O (LLO)3 (2). The requirements for lysing the phagosomal membrane have been shown to vary depending on the cell type and the activation state of the cell (3). In macrophages, L. monocytogenes depends on LLO to lyse the phagosomal membrane and escape into the cytosol. Thus, strains deficient in the hemolysin (hly) gene ({Delta}hly) are unable to escape into the cytosol and are restricted to phagosomal compartments (1). Although L. monocytogenes has been widely used as a pathogen for intracellular infections in murine in vivo and in vitro models, only a few studies have been conducted in human cells.

In 1967, Lukas and Hruskova described how an infection with L. monocytogenes induces a "virus inhibitor" in the blood of chickens (4). This initial observation was confirmed decades later and one of the putative "virus inhibitors" was identified as IFN-beta (5, 6, 7, 8, 9). The cytosolic pattern recognition receptors mediating the induction of this IFN-beta response have to date remained elusive, although a putative trigger has been identified as cytosolic DNA (10). As cells derived from mice deficient in TLR-2, TLR-4, TLR-9, Nod2, or the adaptor proteins MyD88, TRIF, TRAM, or Rip-2 still mount an IFN-beta response after infection with L. monocytogenes, it has been concluded that type I IFN induction occurs independently of these proteins (11, 12). However, a role was attributed to IFN regulatory factor (IRF) 3 and TANK-binding kinase (TBK) 1, as murine cells deficient in either of these components failed to induce IFN-beta gene expression in response to L. monocytogenes (10, 11, 12). Whereas the contribution of type I IFNs to the control of viral infections is essential, their contribution to the control of an infection with L. monocytogenes is rather detrimental (13, 14).

Induction of a type I IFN response is tightly regulated and occurs only after several transcription factors have bound to their DNA binding sites (15). The model showing how IFN-beta gene expression is initiated has been studied in great detail (16, 17, 18, 19). The ifnb promoter contains four positive regulatory domains (PRD) with binding sites for transcription factors such as NF-{kappa}B (binding PRD II), IRF3, and IRF7 (binding PRD III-I) as well as activating transcription factor (ATF) 2 and c-Jun (binding PRD IV) (15, 20). A critical role in the induction of IFN-beta gene expression has been attributed to IRF3 and IRF7, as murine cells deficient in either IRF3, IRF7, or the upstream kinase TBK-1 are unable to express IFNs or IFN-inducible genes (21, 22, 23, 24). IRF3 is a constitutively expressed transcription factor and localizes predominantly to the cytoplasm in its unphosphorylated state (25). Upon appropriate triggering of a cell, the upstream kinases TBK-1 or I{kappa}B kinase {epsilon} phosphorylate IRF3 (26), thereby inducing a conformational change leading to its dimerization (27, 28). The transcriptional activity of IRF3 depends on interactions with the coactivator complex CBP/p300 (29, 30, 31, 32). ATF-2 and c-Jun have been shown to bind the ifnb promoter in a heterodimeric complex (20). ATF-2 and c-Jun can be phosphorylated by either p38 MAPK or JNK (33). Although the crystal structures of ATF-2 and c-Jun bound to PRD IV of the ifnb promoter are available (20), much less is known about their contribution to the induction of a type I IFN response.

The mechanism of type I IFN induction described has been established using murine knockout models, and data regarding potential mechanisms leading to IFN-beta gene expression in human macrophages are rare. However, given the potentially fatal outcome of an infection with L. monocytogenes in humans, a further understanding of the biology of IFN induction in human macrophages in the context of an infection with L. monocytogenes is desirable. Thus, the use of cell culture models using primary innate immune cells of human origin might prove helpful to gain an understanding into disease pathogenesis.

Using monocyte-derived macrophages (MDM), we found that cytosol-reaching L. monocytogenes, but not a phagosomally restricted isogenic strain ({Delta}hly), induces the expression of IFN-beta mRNA, protein, and a bioactive type I IFN response. Moreover, infection with both strains of L. monocytogenes induced an activation of NF-{kappa}B signaling. Phosphorylation of p38 MAPK and the downstream transcription factor ATF-2 was detected after infection with either strain of L. monocytogenes. When p38 MAPK was inhibited, IFN-beta mRNA expression was suppressed, revealing a contribution of the p38 pathway to type I IFN induction. When investigating the activation of IRF3, we found that neither strain of L. monocytogenes induces detectable activation of IRF3, whereas vesicular stomatitis virus (VSV) and LPS triggered this response. Furthermore, IRF3 gene knockdown did not affect a L. monocytogenes-induced IFN-beta response but reduced a VSV-induced response 100-fold.

We thus provide evidence that a robust type I IFN response in human macrophages can be triggered in the absence of a detectable activation of IRF3 after infection with L. monocytogenes. Furthermore, we suggest a differential involvement of the transcription factor IRF3 in the induction of a type I IFN response in human macrophages depending on the stimulus applied.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Bacterial and viral strains

L. monocytogenes strain 10403s (wild-type strain) and its isogenic mutant L. monocytogenes {Delta}hly (strain DP-L2161) were provided by Dr. W. Goebel (University of Wuerzburg, Wuerzburg, Germany). For frozen aliquots, 10-ml broth cultures were grown overnight, 10-fold diluted, and grown to logarithmic phase. Cultures were washed in PBS and frozen as aliquots in PBS containing 20% glycerol (Merck). The VSV strain Indiana was provided by Dr. Hengartner (University of Zurich, Zurich, Switzerland) and grown on BHK-1 cells in DMEM (Invitrogen Life Technologies) supplemented with 7% FCS (Biochrom). Frozen aliquots were kept at –80°C. Infection of MDM was done at a multiplicity of infection (MOI) of 3. Sendai virus was grown and maintained as previously described (34).

Cell culture and infection

Human MDM were isolated from buffy coats of healthy blood donors (Blood Bank Service, Bern, Switzerland) following a modified Ficoll separation procedure. PBMCs were sorted using magnetic beads against CD14 (Miltenyi Biotech). CD14+ cells were allowed to differentiate into monocyte-derived macrophages in Teflon bags for 6 days in RPMI 1640 with Glutamax (Invitrogen Life Technologies) supplemented with 1% vitamins (Biochrom), nonessential amino acids (Biochrom), 1 mM sodium pyruvate (Biochrom), 50 µM mercaptoethanol (Merck), 100 µg/ml streptomycin (Biochrom), 100 U/ml penicillin (Biochrom), and 15% heat-inactivated human AB serum (Blood Bank Service).

MDBK cells (catalog no. CCL-22) were purchased from American Type Culture Collection and maintained as previously described (34).

Infection of MDM was performed in either 24-well plates (Techno Plastics Products) at a cell density of 1 x 105 cells per well or in 6-well plates (BD Biosciences) at a cell density of 1 x 106 cells per well. L. monocytogenes was added to adherent cells at an MOI of 10 in RPMI 1640 supplemented with 10% fresh human serum for opsonization of the bacteria. To determine infection titers, aliquots were plated on agar plates at appropriate dilutions and incubated overnight at 37° to assess the CFU. Phagocytosis of L. monocytogenes was allowed to occur for 1 h. Subsequently, nonphagocytosed bacteria were removed by washing with PBS. Incubation of infected MDM was done in RPMI 1640 supplemented with 2% heat-inactivated human serum and 5 µg/ml gentamycin (Sigma-Aldrich) to suppress extracellular growth of Listeria at 37°C in 5% CO2. p38 MAPK or JNK were inhibited using SB202190 (10 µM) and SP600125 (40 µM) (Sigma-Aldrich). Inhibitors were added during the infection and the incubation period. Leptomycin B (LMB) was purchased from Sigma-Aldrich and used at a concentration of 10 ng/ml. LPS (Sigma-Aldrich) was derived from Escherichia coli O55:B5 and used at a concentration of 100 ng/ml. Brefeldin (GolgiPlug) was purchased from BD Biosciences and used at a concentration of 1 µg/ml.

Immunofluorescence staining

For immunofluorescence staining, cells were grown on glass coverslips and stained following a standard indirect immunofluorescence protocol. Cells were fixed in 100% methanol for NF-{kappa}B p65 and IRF3 stainings or 4% paraformaldehyde for stainings of actin and L. monocytogenes. Fc receptors were blocked using human Ig (Globuman Berna) at a concentration of 10 mg/ml for 15 min. Primary Abs were diluted in PBS supplemented with 10% FCS and incubated for 1 h at 37°C. At the end of each incubation period, slides were washed with PBS containing 0.1% Tween 20. Secondary Abs were incubated for 1 h at room temperature in the dark. The following Abs were used: NF-{kappa}B p65: polyclonal rabbit Ab (Santa Cruz Biotechnology) diluted 1/50; IRF3: polyclonal rabbit Ab (Abcam) at 1/500; L. monocytogenes polyclonal rabbit Ab (Difco Laboratories) at 1/1,000; actin: phalloidin Texas Red (Invitrogen Life Technologies) at 1/40. Secondary Abs were purchased from Molecular Probes (Invitrogen Life Technologies) and used as follows: goat anti-rabbit Texas Red at 1/1,500 for NF-{kappa}B p65; and either Alexa-Fluor 488-labeled goat anti-rabbit or Alexa-Fluor 594-labeled goat anti-rabbit, both diluted 1/1,000 for polyclonal IRF3 and L. monocytogenes. DNA was visualized using 4',6'-diamidino-2-phenylindole (DAPI; Invitrogen Life Technologies). Stained cell cultures were mounted on microscope slides using a mounting medium (DakoCytomation, Glostrup). Images were recorded on a Nikon E800i epifluorescence microscope. Acquired images were processed using Openlab software (Improvision). For the determination of mean fluorescence intensities, DAPI-stained nuclei were set as regions of interest and the fluorescence of Texas Red-stained p65 was quantified in those regions. The data of three different blood donors were pooled and means and SD values were calculated. At least 100 cells per donor, time point, and infection were measured.

SDS-PAGE, native PAGE, and Western blotting

For SDS-PAGE and subsequent Western blotting, 1 x 106 adherent MDM were lysed using the M-PER mammalian protein extraction reagent (Pierce) supplemented with a set of protease inhibitors (Roche) and phosphatase inhibitor cocktails 1 and 2 (Sigma-Aldrich) according to the manufacturers’ instruction. Nuclear and cytoplasmic extracts were prepared using NE-PER nuclear and cytoplasmic extraction reagent (Pierce) according to the manufacturer’s instruction. Protein contents of cell lysates were determined by a Bradford assay using bovine IgG (Nordic Biotech) as a standard and Coomassie Plus (Pierce Biotechnology) as a protein stain. OD values were determined in a microplate reader (Molecular Devices) at a wavelength of 590 nm. Fifty micrograms of whole cell protein extracts, 30 µg of cytoplasmic protein extracts, or 10 µg of nuclear protein extracts were denatured in Laemmli buffer and separated on a SDS gel. Proteins were transferred onto a nitrocellulose membrane (Amersham Biosciences). Membranes were washed in distilled water, blocked for 1 h in PBS, 5% nonfat dry milk powder, and 0.1% Tween 20. Native-PAGE experiments were performed as described by others (35). Briefly, 7 µg of nondenatured whole cell protein extracts were loaded on a pre-electrophoresed 8% polyacrylamide gel and separated at 25 mA at 4°C. Transfer and Western blotting was done as described above. The following Abs were used: rabbit polyclonal Ab diluted 1/1,000 (Cell Signaling Technology) for phospho-Ser536 of NF-{kappa}B p65; rabbit polyclonal Ab diluted 1/200 (Santa Cruz Biotechnology) for pan-specific NF-{kappa}B p65, I{kappa}B-{alpha}, and IRF7; polyclonal rabbit Ab diluted 1/1,000 (Abcam) for pan-specific IRF3; mouse mAb MMHB-3 diluted 1/250 (PBL Biomedical Laboratories) for IFN-beta; mouse mAb 9H2 diluted 1/1,000 (Cell Signaling Technology) for Stat-1; polyclonal rabbit Ab diluted 1/1,000 (Cell Signaling Technology) for Tyr701-phosphorylated Stat-1; for phospho-ATF-2: mouse mAb diluted 1/200 (Santa Cruz Biotechnology); ATF-2: rabbit polyclonal Ab diluted 1:200 (Santa Cruz Biotechnology); rabbit polyclonal Ab diluted 1/1,000 (Cell Signaling Technology) for phospho-p38; mouse mAb diluted 1/2,000 (clone L53F8; Cell Signaling Technology) for p38; rabbit polyclonal Abs diluted 1/1,000 (Cell Signaling Technology) for phospho-JNK and JNK; a mAb provided by Dr. O. Haller (University of Freiburg, Freiburg, Germany) and diluted 1/750 for myxovirus resistance (Mx) protein; monoclonal mouse anti-actin Ab diluted 1/10,000 (Sigma-Aldrich) for actin; rabbit polyclonal Ab diluted 1/200 (Santa Cruz Biotechnology) for nucleolin; and mouse mAb diluted 1/5,000 (Abcam) for GAPDH. Rabbit Abs were detected using a monoclonal peroxidase-conjugated mouse anti-rabbit Ab (Sigma-Aldrich) at a dilution of 1:4,000. Mouse Abs were detected with a peroxidase-conjugated donkey anti-mouse Ab (Jackson ImmunoResearch Laboratories) diluted 1/7,000. ECL (Amersham Bioscience) was used as a substrate and signals were detected by a charge-coupled device camera (Fujifilm LAS3000, Raytest).

DNA binding activity

An EMSA for the detection of NF-{kappa}B DNA binding activity was performed as described previously (36) with the following modifications. 3'-Biotinylated oligonucleotides were obtained from Microsynth. The {kappa}B binding sequence used was 5'-AGTTGAGGGGACTTTCCCAGGC-3'. For supershift experiments, 0.4 µg of a rabbit polyclonal Ab against p65 (Santa Cruz Biotechnology) was added 20 min after the addition of the probe and incubated on ice for 1 h. Biotinylated DNA was detected using a chemiluminescent nucleic acid detection module (Pierce) according to the manufacturer’s instruction. Signals were captured on a charge-coupled device camera (Fujifilm LAS3000, Raytest). IRF3 DNA binding was detected using a transcription factor assay kit from Active Motif according to the manufacturers instruction. OD values were determined at 650 nm using an automated ELISA plate reader (Molecular Devices). Data are expressed as "fold induction" after normalization to uninfected cells at each time point tested.

ELISA

TNF was measured using an Ab capture ELISA kit purchased from Biosource International and used according to the manufacturer’s instruction. OD values were determined at 650 nm using an automated ELISA plate reader (Molecular Devices). TNF was quantified using an international standard (National Institute for Biological Standards and Control, Potters Bar, U.K.).

Real-time RT-PCR

To collect RNA, 1 x 106 adherent MDM were infected or mock infected in 6-well plates. Cell cultures were lysed using an Applied Biosystems nucleic acid purification lysis solution. Isolation of RNA was performed on an ABI Prism 6100 nucleic acid prep station using total RNA purification trays (Applied Biosystems) according to the manufacturer’s instruction. Concentrations of isolated RNA were determined at 260 nm in a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies). Samples were subsequently frozen at –80°C until use. Quantitative RT-PCR was performed in a Mx4000 multiplex quantitative RT-PCR system (Stratagene). Primers and probes were designed using the Primer Design software package (Applied Biosystems) and purchased from Microsynth. Sequences were as follows. IFN-beta: 5'-ACAAAGAAGCAGCAATTTTCAGTGTCAGAAGCT (probe), 5'-GAGCTACAACTTGCTTGGATTCC-3' (forward), and 5'-CAAGCCTCCCATTCAATTGC (reverse); IRF7: 5'-CGCA GCGTGAGGGTGTGTCTTCC-3' (probe), 5'-TGTGCCGAGTGCA CCTAGAG-3' (forward), and 5'-GAAGCACTCGATGTCGTCATAG AG-3' (reverse); IRF3: 5'-ACCCTCACGACCCACATAAAATCTACGAGTTTG-3' (probe), 5'-AGCAGAGGACCGGAGCAA-3' (forward), and 5'-AGAGGTGTCTGGCTGGGAAA (reverse); Mx1: 5'-AAGGTTGTGG ACGTGGTGCGGAA-3' (probe), 5'-GATCTGGTGGACAAAGGAACTGA-3' (forward), and 5'-TTGACAATCATGTAACCCTTCTTCA-3' (reverse); GAPDH: 5'-CCCCCATGTTCGTCATGGGTGTG-3' (probe), 5'-CTCTGCCCCCTCTGCTGAT-3' (forward), 5'-TGATGATCTTGAGGCTGTTGTCA-3' (reverse). 18S rRNA was quantified using a predesigned assay kit (catalog no. 4310893E; Applied Biosystems). Data are presented as expression levels (1/2{Delta}Ct) to either GAPDH mRNA or to 18S rRNA as indicated in the respective figures.

Small interfering RNA (siRNA) and transfections

siRNA was ordered from Dharmacon. IRF3 (GenBank accession no. NM_001571), IRF7 (GenBank accession no. NM_004030), and GAPDH (GenBank accession no. NM_002046) were targeted using a predesigned ON-TARGET plus SMARTpool siRNA (catalog nos. L-006875-00-0020, L-011810-00-0010, and D-001140-01-05, respectively; Dharmacon). Nontargeting siRNA was used as a control in an identical manner as that of relevant siRNA. Transfections were done using a human macrophage Nucleofector kit from Amaxa according to the manufacturer’s instruction. siRNA (1.4 µg) was used to transfect 1 x 106 macrophages 72 h before infection with L. monocytogenes or VSV for the IRF3 siRNA and 48 h before infection for the IRF7 siRNA. Knockdown efficiency was monitored using quantitative RT-PCR and Western blotting. For the IRF3 and IRF7 proteins the greatest knockdown efficiency was observed at 72 and 48 h posttransfection respectively (data not shown).

Pathogenicity reduction assay

Pathogenicity reduction assays for detecting type I IFN-like antiviral activity were done as described (34) with the following modifications. MDBK cells (1 x 106) were used in a 96-well plate. MDBK cells were primed with standards or culture supernatants for 18–24 h. VSV was used as a challenge virus at an MOI of 0.1. A cytopathic effect was assessed 24 h after VSV challenge. Samples containing L. monocytogenes were incubated with 50 µg/ml gentamicin to kill L. monocytogenes and exclude the possible interference by live bacteria with the detection of type I IFN-like antiviral activity.

Statistical analysis

Statistically significant differences were detected using either Kruskal-Wallis test (Figs. 1A and 6A) or Friedman’s test (Fig. 3B), both followed by Dunn’s multiple comparison test. Significances are indicated as follows: *, p < 0.05; **, p < 0.01; ***, p < 0.001.


Figure 1
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  		FIGURE 1. Induction of IFN-beta expression and a bioactive type I IFN response after infection with L. monocytogenes. A, Induction of IFN-beta mRNA expression in response to the wild-type strain (WT-Lm) or the hemolysin deficient ({Delta}hly) strain of L. monocytogenes in human MDM. Sendai virus served as a positive control for the induction of IFN-beta gene expression. Data from 3–6 blood donors were pooled and are expressed as means and SD. Data are normalized to the expression of GAPDH mRNA and expressed as relative expression levels (1/2{Delta}Ct). *, p < 0.05; **, p < 0.01; ***, p < 0.001. uninf; Uninfected. B, Western blotting to detect IFN-beta protein. MDM were infected for 1 h with the wild-type strain of L. monocytogenes (WT-Lm) or the Sendai virus as a positive control for IFN-beta induction, and whole cell extracts were prepared at 12 h p.i. Where indicated, cells were incubated in the presence of 1 µg/ml brefeldin to block cytokine release. A representative blood donor of 5 is shown. uninf, Uninfected. C, Tyrosine 701 (Y701) phosphorylation of Stat-1 (P-Stat-1) and induction of the IFN-inducible protein Mx1 (Mx). MDM were infected with the wild-type strain (WT-Lm) or the {Delta}hly strain of L. monocytogenes. At 8 h p.i., whole cell protein extracts were analyzed by SDS-PAGE and subsequent Western blotting. Whole Stat-1 and actin served as loading control. One blood donor of two is shown. D, Detection of type I IFN-like antiviral activity. Culture supernatant of MDM infected with the wild-type strain (WT-Lm), the {Delta}hly strain of L. monocytogenes, or Sendai virus were probed for type I IFN-like antiviral activity on MDBK cells in a pathogenicity reduction assay using VSV as a challenge virus as stated in Materials and Methods. The result of a representative experiment of four is shown.

 

Figure 6
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  		FIGURE 6. IRF7 is only expressed as a type I IFN inducible protein in human macrophages. A, Quantitative RT-PCR analysis to detect the expression of IRF7 mRNA in macrophages infected with the hly-deficient strain ({Delta}hly) or the wild-type strain of L. monocytogenes (WT-Lm) for the time points indicated. Data are expressed as relative expression levels to GAPDH mRNA. Bars indicate the means and the error bars indicate SEM. Data are pooled from 3–6 blood donors tested. *, <0.05; **, <0.01. uninf, Uninfected. B, Representative Western blot analysis of whole cell extracts to show the protein expression of IRF7 in uninfected macrophages (uninf.) and macrophages infected with the above mentioned stimuli. Actin served as a loading control. One representative blood donor of three is shown. WT-Lm, wild-type L. monocytogenes strain.

 

Figure 3
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  		FIGURE 3. Contribution of p38 MAPK in the induction of IFN-beta gene expression in response to L. monocytogenes. A, Human MDM were infected with the wild-type strain of L. monocytogenes (WT-Lm) or the hly-deficient strain ({Delta}hly). At the indicated time points, whole cell protein extracts were prepared and separated by SDS-PAGE. Western blotting was performed to detect the phosphorylation state of p38 MAPK (P-p38 and p38) and ATF-2 (P-ATF-2 and ATF-2). One representative blood donor of seven is shown. B, MDM were infected with the wild-type strain of L. monocytogenes in the presence or absence of a p38 MAPK inhibitor (SB202190) or a JNK inhibitor (SP600125). SB202190 and SP600125 were used at 10 and 40 µM, respectively. DMSO served as solvent control. At 4 h p.i. cells were lysed, RNA was isolated, and quantitative RT-PCR was performed. The expression of IFN-beta was normalized to the expression of GAPDH. Subsequently, the expression of IFN-beta detected in DMSO treated cells was set as 100% to exclude donor variability. Data from six blood donors were pooled. Each symbol represents one blood donor, and the lines indicate the means of each group. *, p < 0.05; **, p < 0.01.

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Wild-type but not phagosomally restricted {Delta}hly L. monocytogenes induce a bioactive type I IFN response in human macrophages

It has been reported that L. monocytogenes induces IFN-beta gene expression in murine cells upon reaching the cytoplasm of a host cell (8, 9). To determine whether this also occurs in human macrophages, we infected human macrophages for 4, 6, and 8 h with the cytosol-reaching wild-type strain of L. monocytogenes or a phagosomally restricted but isogenic {Delta}hly mutant or Sendai virus as a positive control (Fig. 1A). The cytosol-reaching wild-type strain of L. monocytogenes induced IFN-beta gene expression ~100-fold within 4 h, whereas the phagosomally restricted {Delta}hly strain only weakly induced IFN-beta expression at the mRNA level. The priming of macrophages with IFN-{gamma} did not affect a L. monocytogenes-triggered IFN-beta mRNA expression (data not shown). In comparison, the stimulation of macrophages with LPS also induced a 100-fold increase in IFN-beta mRNA (data not shown). This induction is similar to a L. monocytogenes triggered response. Sendai virus infection of macrophages yielded higher amounts of IFN-beta mRNA independently of whether cells were unprimed or had been primed with IFN-{gamma} (Fig. 1A and data not shown). The mRNA induction was sustained and lasted at least until 12 h postinfection (p.i.) (data not shown).

To determine whether IFN-beta protein is detectable, macrophages were infected with either the cytosol-reaching wild-type strain of L. monocytogenes or Sendai virus in the presence or absence of 1 µg/ml brefeldin to block cytokine release (Fig. 1B). After 12 h of infection, the amount of IFN-beta protein detected in response to Sendai virus exceeded that produced by L. monocytogenes by far. Sendai virus-induced IFN-beta protein expression by macrophages was even detected in the absence of brefeldin.

The bioactivity of the type I IFN response was determined by several means. We assessed the phosphorylation state of Stat-1, the induction of the IFN-inducible protein Mx1, and type I IFN-like antiviral activity in the cell culture supernatants of L. monocytogenes- or Sendai virus-infected cells (Fig. 1, C and D). Only wild-type L. monocytogenes, but not the phagosomally restricted {Delta}hly strain, induced the phosphorylation of Stat-1 and the expression of Mx1 (Fig. 1C). Because Sendai virus interferes with Jak-Stat signaling, the expression of Mx is not induced in Sendai-infected macrophages (data not shown). Culture supernatants of wild-type L. monocytogenes- and Sendai virus-infected macrophages reduced a VSV-induced cytopathic effect, pointing to the presence of bioactive type I IFN (Fig. 1D). The culture supernatant of mock-treated cells and of cells infected with the {Delta}hly strain of L. monocytogenes did not have cytoprotective effects on MDBK cells (Fig. 1D). Thus, the infection of human macrophages with wild-type L. monocytogenes but not with the {Delta}hly strain induces IFN-beta mRNA and protein expression and a bioactive type I IFN response.

The NF-{kappa}B pathway is activated in response to L. monocytogenes

NF-{kappa}B is a transcription factor to which most TLR- and nucleotide-binding oligomerization domain-mediated signaling pathways converge (37), and it plays a major role in the initiation of an innate immune response. We asked whether cytosolic or phagosomally restricted L. monocytogenes might differ in their ability to activate the NF-{kappa}B pathway.

MDM were infected with a cytosol-reaching wild-type strain of L. monocytogenes or a phagosomally restricted, hemolysin-deficient ({Delta}hly) strain. Cells were fixed at 30 min p.i. and stained for NF-{kappa}B p65 and with DAPI to assess a nuclear translocation (NTL) of the NF-{kappa}B subunit p65 (Fig. 2A). NF-{kappa}B p65 translocated to the nucleus irrespective of the strain used. Similar results were obtained when macrophages were primed with IFN-{gamma} (data not shown). To quantify the NTL of p65, photomicrographs of p65 and DAPI were analyzed as described in Materials and Methods (Fig. 2B). We found an increase in the p65 mean fluorescence intensity in the nucleus at 30 min p.i. when compared with uninfected cells. Quantification of nuclear p65 fluorescence did not reveal differences in cells infected with bacteria of either strain. Next, nuclear protein extracts were prepared at 30 min p.i. with either strain of L. monocytogenes and probed with a phospho-specific Ab against Ser536 of p65 and a pan-specific Ab against NF-{kappa}B p65 (Fig. 2C). Nucleolin served as a loading control for nuclear protein extracts and GAPDH was used to exclude contamination by the cytoplasmic fraction. Immunoblot experiments confirmed the results of a nuclear translocation of p65 detected in microscopic studies. To establish a kinetic of the activation of NF-{kappa}B signaling in more detail, whole cell extracts were prepared between 15 min and 4 h p.i. with the {Delta}hly strain or the cytosol-reaching wild-type strain of L. monocytogenes and probed for the degradation of I{kappa}B-{alpha} and the phosphorylation of NF-{kappa}B p65 at Ser536 (Fig. 2D). We found that I{kappa}B-{alpha} was degraded within 15 min p.i. At 2 h and 4 h p.i., I{kappa}B-{alpha} was expressed at considerably higher levels when compared with mock-treated cells, pointing to transcriptional activity and the termination of NF-{kappa}B signaling. EMSA was performed to detect DNA binding activity of NF-{kappa}B to a {kappa}B consensus sequence at 30 min after infection with either strain of L. monocytogenes (Fig. 2E). We found a similar band shift in response to both strains of L. monocytogenes, pointing to comparable DNA binding activities and similar compositions of the complexes detected. A 200-fold molar excess of the unlabeled competitor showed specificity of the binding reaction. Furthermore, supershift experiments with an Ab against p65 revealed that the NF-{kappa}B subunit p65 was involved in the formation of the complexes detected. Thus, the NF-{kappa}B pathway becomes activated regardless of the subcellular localization of L. monocytogenes.


Figure 2
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  		FIGURE 2. Activation of NF-{kappa}B signaling and release of TNF in response to L. monocytogenes. A, MDM were infected with the wild-type (WT) or the hly deficient ({Delta}hly) strain of L. monocytogenes. NF-{kappa}B p65 was stained following an indirect immunofluorescence protocol as stated in Materials and Methods. One representative blood donor of three is shown. B, Mean fluorescence intensity (MFI) of nuclear NF-{kappa}B p65 was quantified in macrophages of three different blood donors at 30 min, 2 h, and 4 h p.i. as stated in Materials and Methods. Data are presented as the means of three blood donors. Error bars indicate SD. C, Nuclear extracts of MDM infected for 30 min with either the wild-type strain (WT-Lm) or the {Delta}hly strain of L. monocytogenes. Western blot analysis for the phosphorylation at Ser536 (S536) and nuclear translocation of NF-{kappa}B p65 were performed. Nucleolin served as a loading control for nuclear extracts, and GAPDH was used to exclude contamination by the cytoplasmic fraction. A single blood donor is shown. uninf, Uninfected. D, Whole cell extracts were prepared at the time points indicated and probed to detect the degradation of I{kappa}B-{alpha} as well as the phosphorylation (P-) state of NF-{kappa}B p65 by Western blotting. GAPDH served as a loading control. One representative blood donor of three is shown. uninf, Uninfected; WT-Lm, wild-type L. monocytogenes; {Delta}hly, hly-deficient strain. E, EMSA to analyze the DNA binding activity to a {kappa}B consensus sequence. Nuclear extracts were prepared at 30 min p.i. with either strain of L. monocytogenes. Where indicated, a 200-fold molar excess of the unlabeled probe was added to show the specificity of the binding reaction. Supershifts were performed with an Ab against the p65 subunit of NF-{kappa}B. The arrow indicates the position of supershifted complexes. One representative blood donor of three is shown. uninf, Uninfected; WT-Lm, wild-type L. monocytogenes; {Delta}hly, hly-deficient strain. F, Culture supernatants of macrophages infected with either the wild-type strain (WT) or the {Delta}hly strain of L. monocytogenes were collected at 4 h p.i., and TNF release was quantified in an ELISA. Bars indicate means and error bars indicate SD.

 
TNF is a cytokine whose synthesis critically depends on NF-{kappa}B activation (38). We collected the supernatants of infected cell cultures at 4 h p.i. and measured the release of TNF in a sandwich ELISA (Fig. 2F). Both strains up-regulated TNF release ~100-fold when compared with mock-treated cells. We found no difference in the release of TNF when macrophages were infected with the wild-type strain or the {Delta}hly strain of L. monocytogenes.

Activation of p38 MAPK by L. monocytogenes

We next investigated whether p38 MAPK and JNK might contribute to the induction of IFN-beta gene expression. Protein extracts of macrophages infected with either the wild-type strain or the {Delta}hly strain of L. monocytogenes were probed for phosphorylated p38 MAPK and for phosphorylated ATF-2 (Fig. 3A). Both {Delta}hly and wild-type L. monocytogenes induced the phosphorylation of ATF-2 and p38 MAPK (Fig. 3A). Similar results were obtained when macrophages were primed with IFN-{gamma} (data not shown).

ATF-2 and c-Jun influence the transcription of multiple target genes. To assess the contribution of its upstream kinases p38 and JNK to the initiation of IFN-beta gene expression, p38 MAPK and JNK were inhibited with the small molecule inhibitors SB202190 and SP600125, respectively, and IFN-beta mRNA induction was assessed at 4 h p.i. (Fig. 3B). The inhibition of p38 MAPK but not JNK significantly reduced IFN-beta gene expression in cells infected with the wild-type strain of L. monocytogenes.

IRF3 is differentially activated in human monocyte-derived macrophages upon induction of type I IFN responses

IRF3 is a constitutively expressed transcription factor and thought to be mandatory for the induction of IFN-beta expression (15, 21, 23). IRF3 has to be phosphorylated at multiple serine and threonine residues to undergo a conformational change leading to dimerization and subsequent NTL (21, 26, 39). To detect posttranslational modifications of IRF3, either unprimed or IFN-{gamma} primed (data not shown), macrophages were infected with L. monocytogenes or VSV. VSV served as a positive control for activation of the IRF3 pathway, as this virus has been shown to be an activator of IRF3 signaling (40). SDS-PAGE and subsequent Western blotting was performed on whole cell protein extracts prepared at 1, 2, 4, and 8 h p.i. (Fig. 4A). Slower migrating bands of IRF3 that point to posttranslational modifications were only detected in response to VSV (Fig. 4A). Priming with IFN-{gamma} did not have any detectable effect on posttranslational modifications of IRF3 leading to slower migrating bands (data not shown). To rely on a more functional assay, we performed native-PAGE analysis to detect the dimerization of IRF3 in response to either strain of L. monocytogenes or VSV (Fig. 4B). At 4 h and 8 h p.i., dimerized forms of IRF3 were clearly detectable in response to an infection with VSV. The stimulation of macrophages with LPS also induced detectable IRF3 dimers (data not shown). Infection of macrophages with either strain of L. monocytogenes did not, however, lead to the formation of detectable IRF3 dimers (Fig. 4B).


Figure 4
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  		FIGURE 4. Differential activation of IRF3 upon type I IFN induction. A, Human MDM were infected with the hly-deficient strain of L. monocytogenes ({Delta}hly), the wild-type strain of L. monocytogenes (WT-Lm), or VSV for 1, 2, 4, or 8 h. Whole cell extracts were separated by SDS-PAGE and blotted for IRF3 to detect mobility shifts in SDS-PAGE. One representative blood donor of six is shown. B, Native PAGE analysis to detect the dimerization of IRF3 in response to the above-mentioned stimuli. Whole cell extracts were prepared at 1, 2, 4, or 8 h, separated on a polyacrylamide gel, and Western blot analysis was performed with an Ab against IRF3. One representative blood donor of three is shown. uninf, Uninfected; WT-Lm, wild-type L. monocytogenes strain; {Delta}hly, hly-deficient L. monocytogenes. C, Nuclear and cytoplasmic protein extracts of MDM that had been infected with the wild-type strain (WT-Lm), the hly-deficient strain of L. monocytogenes ({Delta}hly), or VSV for 2, 4, and 8 h were separated using SDS-PAGE and blotted for IRF3. Nucleolin and GAPDH served as loading controls for the nuclear and the cytoplasmic fraction respectively. One representative blood donor of three is shown. uninf, Uninfected. D, Immunofluorescence staining to reveal the subcellular distribution of IRF3 after infection with the wild-type strain, the {Delta}hly strain of L. monocytogenes (one of seven blood donors shown), VSV (one of three blood donors shown), or LMB treatment (one of two blood donors shown). MDM were fixed in methanol at the indicated time points. Original magnification was x400 (x600 for VSV 4 h p.i.). Of note, IRF3 was stained with either a green- or red-labeled secondary antibody. E, DNA binding activity of IRF3 to an IRF-binding sequence. Nuclear protein extracts of MDM were assayed as described in Materials and Methods. Data obtained with two blood donors are expressed as induction compared with uninfected control cells. In all experiments, VSV served as a positive control for an activation of the IRF3 pathway. uninf, Uninfected; WT-Lm, wild-type L. monocytogenes strain; {Delta}hly, hly-deficient L. monocytogenes.

 
IRF3 is retained in the nucleus only after activation-induced phosphorylation, subsequent dimerization, and association with CBP/p300 (29, 31). Therefore, the subcellular localization of IRF3 in response to L. monocytogenes and VSV was determined (Fig. 4, C and D). Nuclear and cytoplasmic extracts were prepared and probed for IRF3 (Fig. 4C). Nucleolin served as a loading control for nuclear extracts, and GAPDH as a loading control for cytoplasmic extracts. IRF3 translocated to the nucleus only in response to an infection with VSV, and neither strain of L. monocytogenes induced a detectable nuclear translocation of IRF3 at 2, 4, and 8 h p.i (Fig. 4C).

The results obtained from Western blotting experiments were confirmed when we investigated the subcellular localization of IRF3 at a single cell level using fluorescence microscopy; when either unprimed or IFN-{gamma} primed macrophages infected with either strain of L. monocytogenes or with VSV were fixed at various time points between 30 min and 12 h p.i. and stained for the subcellular distribution of IRF3, we only detected a nuclear translocation of IRF3 in response to VSV and LPS (Fig. 4D and data not shown). This response was maintained until 12 h p.i., which was the latest time point investigated. We also observed a nuclear accumulation of IRF3 after treatment with the nuclear export inhibitor LMB (Fig. 4D), revealing the sensitivity of the method. Only a minority of cells responded to an infection with the wild-type strain of L. monocytogenes by inducing a nuclear translocation of IRF3. Corresponding stainings with DAPI revealed that the majority of the cells harbor bacterial DNA (Fig. 4D). Additionally, immunofluorescence staining for L. monocytogenes and actin confirmed that every cell was infected (data not shown). To rule out the possibility that L. monocytogenes actively interfered with an activation of IRF3, macrophages were preinfected with L. monocytogenes and challenged with VSV. Staining for the subcellular localization of IRF3 revealed that a VSV-mediated nuclear translocation was not affected by L. monocytogenes (data not shown). To investigate the DNA binding activity of IRF3, we performed a transcription factor binding assay. The principle of this assay is that oligonucleotides with an IRF binding sequence are coated to an ELISA plate. Protein extracts are added and activated IRF3 bound to DNA is detected with immunoperoxidase staining in accordance with a general ELISA protocol. Consistent with previous results, we detected DNA binding of IRF3 in response to an infection with VSV at 4 h p.i. but failed to detect DNA binding of IRF3 after infection with either strain of L. monocytogenes (Fig. 4E). The addition of competing oligonucleotides revealed the specificity of the binding reaction observed after infection with VSV (Fig. 4E).

IRF3 differentially contributes to an IFN-beta response in human macrophages

To assess the contribution of IRF3 to IFN-beta expression, we decided to target IRF3 for an siRNA-mediated knockdown (Fig. 5). IRF3 protein levels could be reduced to 33% of the amount of untreated macrophages (Fig. 5A). IFN-beta mRNA induction was assessed following infection with the cytosol-reaching wild-type strain of L. monocytogenes, the phagosomally restricted {Delta}hly strain, or VSV after 72 h of siRNA transfection (Fig. 5B). We found that an siRNA against IRF3 reduced a VSV-mediated IFN-beta mRNA induction 100-fold, whereas a L. monocytogenes-triggered IFN-beta mRNA response remained largely unaffected (Fig. 5B). However, we observed a statistically significant decrease triggered by an irrelevant control siRNA after infection with VSV. Because this nonspecific decrease was not observed after infection with L. monocytogenes (Fig. 5B) or stimulation with LPS (data not shown), we hypothesize that the inhibition detected might be an artifact. Furthermore, a statistically significant difference was still detected between control siRNA- and IRF3 siRNA-treated macrophages after VSV infection. Next, we assessed how far the expression of the IFN-inducible gene Mx1 is affected by a down-regulation of IRF3 (Fig. 5C). We found that following IRF3 knockdown, L. monocytogenes-induced Mx1 expression is not significantly altered. Because the expression of Mx1 in the human system solely depends on IFN-receptor signaling and not on IRF3 transcriptional activity (41), this points to comparable levels of bioactive type I IFN after IRF3 knockdown in response to an infection with L. monocytogenes. As the VSV M protein inhibits mRNA export (42), type I IFN-like bioactivity is absent from VSV-infected cells (Fig. 5C and data not shown). Therefore, the mRNA levels of Mx1 in response to VSV infection remain unchanged. We thus show that IRF3 differentially contributes to the induction of an IFN-beta response in human macrophages depending on the trigger applied.


Figure 5
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  		FIGURE 5. Stimulation-specific contribution of IRF3 to the expression of IFN-beta. A, IRF3 knockdown efficiency was assessed by using Western blotting of macrophages left untreated (untr) or transfected with a nontargeting control siRNA (siCTRL) or IRF3 siRNA (IRF3) 72 h after siRNA transfection. IRF3 protein expression was normalized to the expression of tubulin and set as 100% in untreated cells. The graph shows mean and SD of six blood donors, and a representative Western blot is shown. B, IFN-beta quantitative RT-PCR (QRT-PCR) of untreated macrophages (–), nontargeting control siRNA-transfected macrophages (siCTRL), or IRF3 siRNA-transfected macrophages (IRF3). At 72 h after siRNA transfection macrophages were infected with the hly-deficient strain of L. monocytogenes ({Delta}hly), a wild-type strain of L. monocytogenes (WT-Lm), or VSV. At 4 h p.i. the cells were lysed and RNA was isolated and assessed for the expression of IFN-beta mRNA. Data are normalized to the expression of 18S rRNA and expressed as relative mRNA expression levels (1/2{Delta}Ct). Mean and SEM of six blood donors are shown. uninf, Uninfected. C, Expression of the IFN-inducible gene Mx1. Untreated (–), control siRNA (siCTRL), or IRF3 siRNA (IRF3)-transfected macrophages were infected with the {Delta}hly strain or the wild-type strain of L. monocytogenes (WT-Lm) or VSV. At 4 h p.i. RNA was isolated and assessed for the expression of the IFN-inducible gene Mx1. Data are presented as mRNA expression levels normalized to 18S rRNA (1/2{Delta}Ct). uninf, Uninfected.

 
IRF7 protein is only expressed in a type I IFN-inducible manner in human macrophages

A recent study suggested that the transcription factor IRF7 functions as a "master regulator" of type I IFN-mediated responses in a murine knockout model (22). As we did not detect an activation of IRF3 signaling despite a bioactive type I IFN response, we hypothesized that IRF7 might act in place of IRF3, especially as both transcription factors can bind to the same PRD of the ifnb promoter. We found that IRF7 mRNA is expressed in uninfected macrophages (Fig. 6A). When cells were infected with the wild-type strain of L. monocytogenes, we observed a ~10-fold increase in mRNA expression within 4 h p.i. Stimuli that failed to induce a bioactive type I IFN response, such as {Delta}hly L. monocytogenes, or interfered with type I IFN receptor signaling, such as VSV or the Sendai virus (data not shown), did not significantly enhance the expression of IRF7 mRNA. We next analyzed whole cell protein extracts for the expression of IRF7 protein (Fig. 6B). When macrophages were left uninfected, the expression of IRF7 protein was not detected. Only stimuli that induced a bioactive type I IFN response lead to the detection of IRF7 protein. Thus, IRF7 protein is expressed and detected in a type I IFN-inducible manner.

IRF7 does not contribute to IFN-beta mRNA expression

To rule out an involvement of IRF7 in the induction phase of a type I IFN response, we performed siRNA experiments to knock down IRF7. Because the IRF7 protein is only detected in the course of a type I IFN response but not in resting macrophages (Fig. 6B), we treated siRNA-transfected macrophages with IFN-beta and assessed whether an siRNA against IRF7 suppressed expression of IRF7 protein (Fig. 7A). We found a marked reduction of IRF7 protein after transfection of an IRF7 siRNA when compared with GAPDH siRNA or a nontargeting control siRNA. However, the nontargeting siRNA induced a nonspecific reduction of IRF7 protein. Expression of Mx, another IFN-inducible protein, was also assessed to prove that type I IFN signaling remains functional after siRNA transfection. Mx expression was not affected by any siRNA applied (Fig. 7A). To assess the impact of IRF7 on the induction of IFN-beta gene expression, macrophages were transfected with an siRNA against IRF7 or a control siRNA and the cells were infected with either strain of L. monocytogenes (Fig. 7B). After infection of macrophages with L. monocytogenes, expression of IFN-beta mRNA was not significantly affected by the IRF7 siRNA when compared with the control siRNA. Additionally, supernatants were collected and the release of TNF at 4 h after infection with L. monocytogenes was measured (Fig. 7C). We found that TNF release was not significantly affected when the expression of IRF7 was suppressed by siRNA, further revealing the functionality of the transfected cells. Thus, these data do not support the hypothesis that IRF7 might function in a redundant manner to that of IRF3 in the IFN-beta induction pathway in human macrophages.


Figure 7
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  		FIGURE 7. IRF7 is not involved in the initial induction of IFN-beta mRNA expression. A, Macrophages were mock treated or transfected with a nontargeting control siRNA (siCTRL), GAPDH siRNA (GAPDH), or IRF7 siRNA (IRF7) and left untreated or primed with recombinant human IFN-beta for 24 h. Protein extracts were prepared and probed for expression of IRF7 and Mx. Actin served as a loading control. Western blot of one blood donor of two is shown. B and C, Macrophages were left untreated (untr.) or transfected with a control siRNA (siCTRL) or an IRF7 siRNA (IRF7), and at 48 h after transfection macrophages were infected with the {Delta}hly strain ({Delta}hly) or the wild-type strain (WT-Lm) of L. monocytogenes. At 4 h after infection, RNA was harvested to assess IFN-beta mRNA expression in a quantitative RT-PCR (QRT-PCR) (B) and the supernatant was collected to assess TNF release in an ELISA (C). Quantitative RT-PCR data are presented as mRNA expression levels normalized to 18S rRNA (1/2{Delta}Ct). uninf, Uninfected.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
It has been reported that an infection with wild-type L. monocytogenes induces a type I IFN response in murine in vivo and in vitro models (5, 6, 7, 8, 9, 12) that depends on IRF3 and TBK-1 (11). However, type I IFN induction triggered by L. monocytogenes has never been studied in the human system. In this study we show that human monocyte-derived macrophages also responded to an infection with cytosol-reaching wild-type L. monocytogenes, but not to a phagosomally restricted isogenic strain, by inducing IFN-beta expression and a bioactive type I IFN response (Fig. 1). Yet, this response occurred without detectable activation of the IRF3 pathway (Fig. 4). Despite a robust and sustained induction of IFN-beta mRNA starting at 4 h p.i. and lasting at least for 12 h, we did not detect a gel mobility shift of IRF3 in SDS-PAGE, the dimerization of IRF3 in native PAGE, nuclear translocation of IRF3 in immunoblots or fluorescence microscopic studies, or DNA binding activity of IRF3 (Fig. 4). However, virtually every cell was infected as revealed by immunofluorescence staining of L. monocytogenes (data not shown), as well as activation of the NF-{kappa}B pathway (Fig. 2). Because VSV is a potent activator of IRF3 signaling (40), we used this virus as a positive control to detect IRF3 activation. Indeed, in response to VSV, we detected mobility shifts, dimers, nuclear translocations, and DNA binding activity of IRF3 (Fig. 4). LPS, which has been shown to activate IRF3 via TLR-4/TRIF (43), also induced an activation of IRF3 signaling as assessed by nuclear translocation and dimer formation (data not shown). With IRF3 activated after LPS challenge, the observed induction of IFN-beta mRNA reached ~100-fold (data not shown), an extent similar to that after an infection with L. monocytogenes, which did not induce IRF3 dimers or nuclear translocation. We also observed a nuclear accumulation of IRF3 following treatment of macrophages with LMB, as has been previously reported (44). When using LMB on L. monocytogenes-infected macrophages, an accelerated nuclear accumulation of IRF3 could not be detected (data not shown). Consistently, when IRF3 was knocked down using siRNA a L. monocytogenes-triggered IFN-beta mRNA response was not affected, whereas a VSV-triggered mRNA response was reduced ~100-fold (Fig. 5). Similarly, the expression of Mx1 mRNA was not affected by IRF3 knockdown in L. monocytogenes-infected macrophages (Fig. 5C). Thus, at no level investigated could an activation of IRF3 in the IFN-beta mRNA response triggered by L. monocytogenes be observed. In contrast, infection with VSV or stimulation with LPS did induce the activation of IRF3 signaling. Similar results were obtained in the human monocytoid cell line THP-1. Thus, the wild-type strain of L. monocytogenes induced IFN-beta mRNA expression, whereas only a minority of cells exhibited a nuclear translocation of IRF3 (data not shown). Given that L. monocytogenes is adapted to an intracellular life style, we speculated whether L. monocytogenes might actively interfere with an activation of IRF3. Thus, if L. monocytogenes interfered with an IRF3 activation, a reduction in the VSV-mediated IRF3 nuclear translocation would be observed. However, VSV still induced a robust IRF3 nuclear translocation in L. monocytogenes-preinfected cells (data not shown). Thus, we propose that IRF3 is differentially involved in the induction of a type I IFN response in human macrophages, depending on the stimulus applied. Whether this contribution of IRF3 signaling to the induction of IFN-beta gene expression is due to a cell type difference or even a species difference remains to be investigated.

The hypothesis that IRF7 might act in place of IRF3 to induce a type I IFN response is intriguing, given that it has been shown that IRF7 can function as a "master regulator" of type I IFN-mediated responses (22). Furthermore, a study by Lu et al. revealed a role of IRF7 during the monocyte-to-macrophage transition and suggested that human primary macrophages also express IRF7 protein (45). Despite the detection of IRF7 mRNA in unstimulated macrophages of several blood donors (Fig. 6A), we only observed IRF7 protein as a consequence of a type I IFN response (Fig. 6B and data not shown). Thus, it seems unlikely to us that IRF7 functions in the place of IRF3, as IRF7 protein was detected only at later time points following type I IFN bioactivity. In addition, IRF1 has also been implicated in the induction of a type I IFN response (46). Using nuclear translocation assays we assessed an activation of IRF1 in macrophages following an infection with L. monocytogenes. However, we could not detect a nuclear translocation of IRF1 following infection with L. monocytogenes, whereas IFN-{gamma} priming induced a robust IRF1 nuclear translocation (data not shown).

NF-{kappa}B is a transcription factor that binds to PRD II within the ifnb promoter. We assessed the activation of the NF-{kappa}B pathway in our system and detected a robust nuclear translocation of the NF-{kappa}B subunit p65 (Fig. 2). Immunoblotting for Ser536-phosphorylated p65 in the nuclear fraction revealed a phospho-specific signal and an increase in the amount of nuclear p65 already observed by fluorescence microscopy. The degradation of I{kappa}B-{alpha} and its increased expression at later time points also point to the activation of the NF-{kappa}B pathway (Fig. 2D). DNA binding activity to a {kappa}B consensus sequence revealed comparable DNA binding activity in response to either strain of L. monocytogenes (Fig. 2E). However, NF-{kappa}B signaling occurred in a similar manner in response to both strains of L. monocytogenes. Given that only wild-type L. monocytogenes induce an IFN-beta response, NF-{kappa}B cannot be the transcription factor discriminating between cytosolic L. monocytogenes or {Delta}hly infection with regard to IFN-beta expression. Nevertheless, NF-{kappa}B might contribute to gene expression through binding to its PRD within the ifnb promoter. Consistent with an activation of NF-{kappa}B, we found that TNF release was up-regulated ~100-fold with either strain of L. monocytogenes (Fig. 2F). Because TNF response is critically dependent on NF-{kappa}B (38), we hypothesize that the transcriptional activity of NF-{kappa}B is similar after infection with either strain of L. monocytogenes. Although several studies suggested the capability of LLO to induce an activation of the NF-{kappa}B pathway and the release of TNF (47, 48, 49), we did not detect an enhancing effect on the activation of NF-{kappa}B signaling or TNF release when human macrophages were infected with the wild-type strain of L. monocytogenes as compared with an infection with the {Delta}hly strain. Given the variety of L. monocytogenes-specific virulence factors that have been shown to possess the ability to activate the NF-{kappa}B pathway and the general proinflammatory properties of bacterial cell wall components (50, 51, 52, 53, 54), it is not surprising that the cytosol-reaching wild-type strain as well as the phagosomally restricted {Delta}hly strain induce comparable NF-{kappa}B-dependent responses.

Another set of transcription factors that bind to PRD IV within the ifnb promoter are ATF-2 and c-Jun (15, 20), which can be phosphorylated and activated by p38 MAPK or JNK. In response to an infection with L. monocytogenes, we detected a phospho-specific signal of p38 and ATF-2 (Fig. 3). Accordingly, when inhibiting p38 MAPK by using the small molecule inhibitor SB202190, we were able to suppress a L. monocytogenes-induced IFN-beta response (Fig. 3B), providing evidence that the p38 pathway contributes to the induction of IFN-beta gene expression. Remarkably, both the {Delta}hly strain as well as the wild-type strain of L. monocytogenes induced the phosphorylation of p38 in our hands. This contrasts with a study performed by O’Riordan et al. (8) that observed the phosphorylation of p38 only after infection with cytosol-reaching bacteria. We currently do not know how far this is due to a species difference. As we never observed actin tail formations in macrophages infected with the {Delta}hly strain (data not shown), infections are likely to be contained to the phagosomal compartments as described by O’Riordan (8). Differences regarding the contribution of p38 to IFN-beta expression may be downstream of p38, e.g., substrate selectivity or kinase activity, because the inhibition of p38 reduced levels of IFN-beta in our system but both strains induced comparable phosphorylations of p38. Nevertheless, it is not surprising to observe p38 phosphorylation in response to a bacterial infection. Yates et al. (55) observed a TLR-2-dependent phosphorylation of p38 in murine macrophages after infection with Staphylococcus aureus.

To our knowledge, this is the first study showing that human macrophages induce a type I IFN response after infection with cytosol-reaching L. monocytogenes, but not with phagosomally restricted L. monocytogenes. Thus, the activation of p38 MAPK and NF-{kappa}B are not sufficient for IFN-beta gene expression after infection with L. monocytogenes, as both pathways were equally activated by cytosolic as well as phagosomally restricted bacteria. In addition, we show that cytoplasmic L. monocytogenes, in contrast to other stimuli, induce IFN-beta expression in the absence of a detectable activation of the IRF3 pathway in human monocyte-derived macrophages. This reveals a differential involvement of the transcription factor IRF3 in the induction of an IFN-beta response in human monocyte-derived macrophages.


   Acknowledgments
 
We thank Mary O’Riordan, Werner Goebel, Hans Hengartner, and John Hiscott for sharing reagents, Giuseppe Bertoni and Daniel Kuemin of our institute for critical review of the manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Grants 3200-665036 and 3200B0-105642 of the Swiss National Science Foundation (to T.W.J.). Back

2 Address correspondence and reprint requests to Dr. Thornik Reimer, Institute of Veterinary Virology, University of Bern, Laenggassstrasse 122, Bern, Switzerland. E-mail address: reimer{at}ivv.unibe.ch Back

3 Abbreviations used in this paper: LLO, listeriolysin O; ATF, activating transcription factor; DAPI, 4',6'-diamidino-2-phenylindole; IRF, interferon regulatory factor; hly, hemolysin; LMB, leptomycin B; MDM, monocyte-derived macrophages; MOI, multiplicity of infection; Mx1, myxovirus resistance 1 (gene/protein); NTL, nuclear translocation; p.i., postinfection; PRD, positive regulatory domain; siRNA, small interfering RNA; TBK, TANK-binding kinase; VSV, vesicular stomatitis virus. Back

Received for publication February 6, 2007. Accepted for publication May 1, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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