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Originally published online as doi:10.1189/jlb.0706456 on March 14, 2007

Published online before print March 14, 2007
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(Journal of Leukocyte Biology. 2007;81:1591-1598.)
© 2007 by Society for Leukocyte Biology

Role of IRAK4 and IRF3 in the control of intracellular infection with Chlamydia pneumoniae

Christian Trumstedt*, Emma Eriksson*, Anna M. Lundberg{dagger}, Tang-bin Yang*,{ddagger}, Zhong-qun Yan{dagger}, Hans Wigzell* and Martin E. Rottenberg*,1

* Microbiology and Tumor Biology Center and
{dagger} Center for Molecular Medicine, Karolinska Institutet, Stockholm, Sweden; and
{ddagger} Laboratory of Space Cellular and Molecular, Institute of Space and Medico-Engineering, Beijing, China

1 Correspondence: Microbiology and Tumor Biology Center, Karolinska Institute, Nobels väg 16, 171 77 Stockholm, Sweden. E-mail: martin.rottenberg{at}ki.se


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ABSTRACT
 
TLR signal transduction involves a MyD88-mediated pathway, which leads to recruitment of the IL-1 receptor (IL-1R)-associated kinase 4 (IRAK4) and Toll/IL-1R translation initiation region domain-containing adaptor-inducing IFN-ß-mediated pathway, resulting in the activation of IFN regulatory factor (IRF)3. Both pathways can lead to expression of IFN-ß. TLR-dependent and -independent signals converge in the TNF receptor-associated factor 6 (TRAF6) adaptor, which mediates the activation of NF-{kappa}B. Infection of murine bone marrow-derived macrophages (BMM) with Chlamydia pneumoniae induces IFN-{alpha}/ß- and NF-{kappa}B-dependent expression of IFN-{gamma}, which in turn, will control bacterial growth. The role of IRAK4 and IRF3 in the regulation of IFN-{alpha}/ß expression and NF-{kappa}B activation was studied in C. pneumoniae-infected BMM. We found that levels of IFN-{alpha}, IFN-ß, and IFN-{gamma} mRNA were reduced in infected IRAK4–/– BMM compared with wild-type (WT) controls. BMM also showed an IRAK4-dependent growth control of C. pneumoniae. No increased IRF3 activation was detected in C. pneumoniae-infected BMM. Similar numbers of intracellular bacteria, IFN-{alpha}, and IFN-{gamma} mRNA titers were observed in C. pneumoniae-infected IRF3–/– BMM. On the contrary, IFN-ß–/– BMM showed lower IFN-{alpha} and IFN-{gamma} mRNA levels and higher bacterial titers compared with WT controls. C. pneumoniae infection-induced activation of NF-{kappa}B and expression of proinflammatory cytokines were shown to be TRAF6-dependent but did not require IRAK4 or IRF3. Thus, our data indicate that IRAK4, but not IRF3, controls C. pneumoniae-induced IFN-{alpha} and IFN-{gamma} secretion and bacterial growth. IRAK4 and IRF3 are redundant for infection-induced NF-{kappa}B activation, which is regulated by TRAF6.

Key Words: macrophage • IFN-{alpha} • IFN-ß • IFN-{gamma} • TLR • NF-{kappa}B • TRAF6


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INTRODUCTION
 
TLRs play a critical role in innate immune responses in mammals [1 ]. TLR signaling can lead to activation of NF-{kappa}B and of various MAPKs. These signals contribute critically to the induction of a variety of inflammatory cytokines, including TNF-{alpha}, IL-1, IL-6, and IL-12, and the expression of costimulatory molecules such as CD80 and CD86 on the cell surface [2 ]. Upon specific ligand-binding to TLR, the receptor associates with intracellular adaptor proteins, including MyD88 [3 ], which recruits IL-1 receptor (IL-1R)-associated kinases (IRAKs) to the receptor complex [4 5 6 ]. In particular, IRAK4 is required for inflammatory cytokine production and optimal expression of costimulatory molecules in response to TLR signaling [7 , 8 ]. Once phosphorylated, IRAK4 dissociates from the receptor complex and then associates with the TNF receptor-associated factor 6 (TRAF6), a member of the TRAF family. TRAF proteins are ubiquitin E3 ligases, which have a pivotal role in signaling pathways by many cell-surface receptors, including TLR, leading to the activation of NF-{kappa}B and induction of proinflammatory cytokines [9 ].

In addition to inflammatory cytokines, a number of TLR will induce IFN-{alpha}/ß [10 11 12 ]. Such activation occurs in a MyD88-dependent and -independent manner [13 , 14 ]. Although most TLRs signal via the MyD88 adaptor, TLR3 signals exclusively through a MyD88-independent pathway in which Toll/IL-1R translation initiation region domain-containing adaptor-regulating IFN-ß (TRIF) is the adaptor. TLR4 uses TRIF as an alternative pathway to MyD88. TRIF signaling will lead to phosphorylation of the transcription factor IFN regulatory factor (IRF)3, which regulates IFN-ß induction [15 , 16 ].

TLR7, TLR8, and TLR9 can also activate expression of the Type I IFN genes; however, different to TLR3 and TLR4, these receptors use MyD88 for inducing the expression of IFN [17 ].

The obligate, intracellular Gram-negative bacterium Chlamydia pneumoniae is a common cause of high and low respiratory tract diseases and has been reported to be associated with development of atherosclerosis [18 , 19 ]. Chlamydia are internalized by macrophages but by avoiding phagolysosomal fusion, are able to replicate intracellularly. IFN-{gamma} is central in resistance to this pathogen in vivo and in vitro [20 21 22 23 24 ]. Macrophages infected with C. pneumoniae express IFN-{gamma}, which in turn, protects these cells against chlamydial growth. Such bacterial infection-induced IFN-{gamma} secretion is IL-12-independent but instead, requires IFN-{alpha}/ß [22 ]. It is interesting that this IFN-{alpha}/ß-dependent pathway of IFN-{gamma} expression is macrophage-specific and does not take place in T cells or dendritic cells during infection with C. pneumoniae [25 ].

MyD88-dependent signaling has been implicated in chlamydial recognition, growth control, and infection-induced disease [26 27 28 29 ]. We and others [28 , 30 ] have shown that the Chlamydia infection-induced Type I IFN secretion by bone marrow-derived macrophages (BMM) is dependent on MyD88. Moreover, optimal expression of IFN-{gamma} in infected BMM requires activation of NF-{kappa}B, which is at least in part IFN-{alpha}ß- and MyD88-independent [30 ]. Still, the detailed mechanisms behind the chlamydial-induced expression of IFN-{alpha} are unknown. We here describe that IFN-{alpha} and IFN-{gamma} expression and defense against C. pneumoniae depend on IRAK4 but not on IRF3 signaling, whereas activation of NF-{kappa}B requires the presence of TRAF6.


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MATERIALS AND METHODS
 
Mice
Mutant mouse strains with genomic deficiency in IRAK4 [31 ], IRF3 [32 ], IFN-ß [33 ], and TRAF6 [34 ] were generated by homologous recombination in embryonic stem cells. Animals were bred and kept under specific pathogen-free conditions. Mice of the C57Bl/6 background were used as controls for IRF3–/– and IFN-ß–/– mice, and 129Sv/Ev/B6 F2 mice were used as controls for IRAK4–/– mice.

Generation of mouse BMM
Mouse BMM were obtained from 6- to 10-week-old mice as described [35 ]. Mice were killed, and the femur and tibia of the hind legs were dissected. Bone marrow cavities were flushed with 5 ml cold, sterile PBS. The bone marrow cells were washed and resuspended in DMEM containing glucose and supplemented with 2 mM L-glutamine, 10% FCS, 10 mM Hepes, 100 µg/ml streptomycin, 100 U/ml penicillin (all from Sigma Chemical Co., St Louis, MO, USA), and 20–30% L929 cell-conditioned medium (as a source of M-CSF). Bone marrow cells were passed through a 100-µm cell strainer, plated in six-well plates (1.2x107 cells per well), and incubated for 7 days at 37°C, 5% CO2. Before use, BMM cultures were washed vigorously to remove nonadherent cells, and cells from several wells were also harvested and counted by trypan blue exclusion. Typically, bone marrow cells yielded 2–3 x 106 BMM per well after 7 days in culture. We have shown previously by immunofluorescence staining that these BMM are F4/80+, CD14+, and Mac-3+ [35 ].

Generation of murine embryonic fibroblasts (MEFs)
TRAF6+/– heterozygous mice were crossed, and MEFs were isolated from embryos by trypsin EDTA treatment and cultured as described previously [36 ]. MEF DNA was extracted and screened for homozygosity of the disrupted TRAF6 gene by PCR analysis. MEFs were genotyped by PCR, using the following primer sequences: sense TRAF6, 5' ACG GAA GCA AGC CTC TGT TCA TAC CG 3', antisense TRAF6, 5' CTG CAG TGA AAG ATG ACA GCG TGA GT 3'; neomycin, 5' CCA AGT GCC CAG CGG GGC TGC TAA AG 3'.

All of the experiments were carried out and repeated with different lines of MEF. The MEFs used in this study were not passaged for more than five times.

Infection and infectivity assay
Mycoplasma-free C. pneumoniae isolate Kajaani 6 [37 ] was propagated in HEp-2 cells. Bacteria were stored in small aliquots in sucrose-phosphate-glutamate (SPG) solution at –70°C until further use. The infectivity as measured by inclusion forming units (IFU) of bacterial preparation was determined in HEp-2 cells as described below.

Cultures of BMM or MEF were infected with C. pneumoniae by centrifugation for 1 h, 500 g, at 35°C. A multiplicity of infection of 1 was used. At different time-points after infection, cells were washed with PBS and then lysed in SPG buffer. Assessment of IFU in cell lysates was done in HEp-2 cells. Aliquots of cell lysates diluted ten- to 200-fold were used in duplicate to infect overnight cultures of confluent HEp-2 cells. The latter were grown in DMEM containing glucose and supplemented with 2 mM L-glutamine, 5% FCS, 10 mM Hepes, and 25 µg per ml streptomycin (DMEM/Strep) on round, 13 mm2 glass coverslides in 24-well plates. Inoculated cells were centrifuged for 1 h, 500 g, at 35°C. Thereafter, supernatant was removed, and DMEM/Strep containing 0.5 µg per ml cycloheximide (Sigma Chemical Co.) was added. Cells were incubated at 35°C for 72 h 5% CO2 and thereafter, washed gently with PBS and fixed in methanol. Glass coverslides were then stained for 30 min at room temperature with a FITC-conjugated Chlamydia genus-specific mAb (1/5 dilution, Pathfinder Chlamydia confirmation system, Bio-Rad, Hercules, CA, USA). Coverslides were mounted with fluorescent-mounting medium (Dako, Glostrup, Denmark), and IFU of C. pneumoniae were quantified by fluorescence microscopy. The infectivity was expressed as IFU of C. pneumoniae per well.

Real-time PCR
Cytokine and hypoxanthine-guanine phosphoribosyltransferase (HPRT) transcripts in BMM at different time-points after C. pneumoniae infection were quantified by real-time PCR. Total RNA was to cDNA as described [30 ]. The real-time PCR was performed in duplicate, 25 µl reactions containing Platinum® SYBR® Green quantitative PCR Supermix-UDG (Invitrogen, Carlsbad, CA, USA), 150 nM forward and reverse primers, and 0.5 µl cDNA on an ABI Prism® 7500 sequence detection system (Applied Biosystems, Foster City, CA, USA). The following primer sequences were used: sense IFN-{gamma}, 5' GCT TTG CAG CTC TTC CTC AT 3', antisense IFN-{gamma}, 5' CAC ATC TAT GCC ACT TGA GTT AAA ATA GT 3'; sense IFN-ß, 5' CTG GAG CAG CTG AAT GGA AAG 3', antisense IFN-ß, 5' TCC GTC ATC TCC ATA GGG ATCT 3'; sense IFN-{alpha}, 5' TCT GAT GCA GCA GGT GGG 3', antisense IFN-{alpha}, 5' AGG GCT CTC CAG AYT TCT GCT CTG 3'; sense IL-1ß, 5' TGG TGT GTG ACG TTC CCA TT 3', antisense IL-1ß, 5' CAG CAC GAG GCT TTT TTG TTG 3'; sense IL-6, 5' ACA AGT CGG AGG CTT AAT TAC ACA T 3', antisense IL-6, 5' TTG CCA TTG CAC AAC TCT TTT C 3'; sense IRF7, 5' TGG AGC CAT GGG TAT GCA A 3', antisense IRF7, 5' CTA GAC AAG CAC AAG CCG AGA CT 3'; sense HPRT, 5' CCC AGC GTC GTG ATT AGC 3', antisense HPRT, 5' GGA ATA AAC ACT TTT TCC AAA TCC 3'.

Serial-fold dilutions of a cDNA sample were amplified to control amplification efficiency for each primer pair. Thereafter, the comparative threshold cycle (Ct) values for all cDNA samples were obtained. HPRT was used as a control gene to calculate the {Delta}Ct values for individual samples. The relative amount of cytokine/HPRT transcripts was calculated using the 2–({Delta}{Delta}Ct) method as described [38 ]. These values were then used to calculate the relative expression of cytokine mRNA in uninfected and infected BMM.

SDS-PAGE and Western blotting
C. pneumoniae-infected BMM were lysed in 150 mM NaCl, 20 mM Tris-HCl, 2 mM EDTA, 1% Triton, 10% glycerol, and 2 mM PMSF. The protein content in cell lysates was measured by Lowry assay (Bio-Rad). Sample buffer (Bio-Rad) containing ß-ME was added to samples, which were then boiled for 5 min. Samples (10 µg) were separated at 100 V, 250 mA, on 10% separating/5% stacking SDS-polyacrylamide gels. Samples were then transferred onto nitrocellulose membranes (Bio-Rad) by electroblotting at 100 V, 250 mA, for 1 h. Immunostaining was performed using monoclonal mouse antiphosphorylated (Ser 32/36) I{kappa}B-{alpha} (anti-pI{kappa}B), rabbit anti-IRF3, rabbit antiphosphorylated IRF3 (anti-pIRF3; all from Cell Signaling, Danvers, MA, USA), and polyclonal rabbit antiactin (1/500 dilution, Sigma Chemical Co.). Membranes were then washed and incubated with HRP-conjugated polyclonal rabbit antimouse Igs or HRP-conjugated polyclonal goat antirabbit Igs (both 1/2000 dilution, Dako), developed using ECL-Plus (Amersham Pharmacia Biotech, Little Chalfont, UK), and photographed using a Fuji Intelligent Darkbox II digital camera.

EMSA
BMM or MEFs were infected with C. pneumoniae as described above. At different time-points after infection, cells were collected and lysed for 10 min in hypotonic lysis buffer [10 mM Hepes, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, Nonidet P-40, 1 mM DTT, and protein inhibitor cocktail (Roche Diagnostics GmbH, Mannheim, Germany)]. Cytosolic proteins were removed by centrifugation (13,000 gfor 1 min), supernatants were aspirated, and the pellet was washed once with hypotonic lysis buffer without supplements. The nucleic fraction was resuspended in hypertonic lysis buffer (20 mM Hepes, pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, protein inhibitor cocktail, 1 mM DTT) and incubated at 4°C for 1 h. Cellular debris were removed (13,000 g for 15 min), and the supernatant containing the nuclear proteins was collected. Nuclear extracts (1–3 µg) were incubated with a P32 double-stranded oligonucleotide probe (Promega, Madison, WI, USA) as described [39 ]. The resulting protein-DNA complexes were separated on a native polyacrylamide gel and autoradiographed. Unlabeled (cold) excess probe was added to binding reactions using nuclear extracts from LPS-stimulated BMM to confirm probe specificity.

ELISA
The amount of IFN-{gamma} (R&D Systems, Minneapolis, MN, USA), IL-12 (BD PharMingen, Belgium), and IL-6 (Nordic Biosite, Stockholm, Sweden) in the culture supernatants of C. pneumoniae-infected BMMs was measured by ELISA according to the instructions of the manufacturers.


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RESULTS
 
IRAK4 is required for control of C. pneumoniae and expression of IFN genes
In a first set of experiments, we studied the role of IRAK4 in expression of cytokines in C. pneumoniae-infected BMM. The levels of IFN-ß, IFN-{alpha}, and IFN-{gamma} mRNA were all increased during C. pneumoniae infection of wild-type (WT) BMM (Fig. 1A 1B 1C ). IRAK4-deficient BMM showed, however, reduced induction of IFN-ß, IFN-{alpha}, and IFN-{gamma} mRNA compared with WT controls in response to the infection (Fig. 1A 1B 1C) . Accordingly, protein levels of IFN-{gamma} also increased in supernatants from WT but not from IRAK4–/– BMM after 3 and 6 h of infection with C. pneumoniae (Fig. 1D) . In contrast, similar levels of IL-12 were measured in supernatants from IRAK4–/– and WT BMM during infection with C. pneumoniae (Fig. 1E) . IRAK4–/– BMM also showed higher intracellular titers of C. pneumoniae than WT controls (Fig. 1F) .


Figure 1
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  		Figure 1. IRAK4 controls IFN-{alpha}, IFN-ß, and IFN-{gamma} mRNA accumulation in C. pneumoniae-infected BMM. Total RNA was extracted from WT and IRAK4–/– BMM at the indicated time-points after infection with C. pneumoniae. The accumulation of IFN-ß (A), IFN-{alpha} (B), and IFN-{gamma} (C) mRNA was quantified by real-time PCR. The levels of IFN-{gamma} and IL-12 in the supernatants from WT and IRAK4–/– BMM at the indicated time-points after infection with C. pneumoniae were measured by ELISA (D and E). Triplicate wells of IRAK4–/– and WT BMM were infected with C. pneumoniae and lysed with SPG buffer at the indicated time-points after infection. The number of C. pneumoniae IFU/well ± SEM was quantified by infecting Hep-2 cells with 100 µl BMM lysate. A representative from two independent experiments is shown (F). *, Differences with WT BMM are significant (P<0.05 Student’s t test).

IFN-ß but not IRF3 is required for increased IFN-{alpha} and IFN-{gamma} mRNA accumulation in chlamydial-infected macrophages
The role of IRF3 in the outcome of control of C. pneumoniae infection by BMM was then studied. Phosphorylation of IRF3 was used as a measurement of activation, as it is required for its nuclear translocation [40 ]. We found similar levels of pIRF3 in IRAK4–/– and WT BMM before or after infection with C. pneumoniae (Fig. 2A ). In accordance, IFN-{alpha} and IFN-{gamma} mRNA levels in WT and IRF3–/– BMM were similar (Fig. 2B and 2C) . Moreover, comparable numbers of C. pneumoniae were recorded in IRF3–/– and WT BMM (Fig. 2D) .


Figure 2
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  		Figure 2. IRF3 is not required for IFN-{alpha} and IFN-{gamma} mRNA accumulation in chlamydial-infected BMM or for the control of bacterial growth. Protein extracts were prepared at the indicated time-points after infection of WT and IRAK4–/– BMM, separated by SDS-PAGE, electroblotted onto nitrocellulose membranes, and immunoblotted with antibodies, which specifically recognize actin, total IRF3, and pIRF3. Antibody binding was detected with HRP-conjugated anti-IgG, followed by ECL detection (A). Total RNA was extracted from WT and IRF3–/– BMM at the indicated times after infection with C. pneumoniae. The accumulation of IFN-{alpha} (B) and IFN-{gamma} (C) mRNA was measured by real-time PCR. A representative from two independent experiments is shown. WT and IRF3–/– BMM were infected with C. pneumoniae and lysed in SPG buffer at the indicated time-points after infection. C. pneumoniae IFU in BMM lysates were quantified by HEp-2 infectivity assay (D).

We then asked whether the presence of IFN-ß was required for efficient growth control of C. pneumoniae in BMM. Expression of IFN-{alpha} and IFN-{gamma} mRNA was reduced markedly in IFN-ß–/– BMM (Fig. 3A and 3B ). IRF7 has been shown to be a main regulator of IFN-{alpha} production. IRF7 mRNA titers in infected BMM were reduced as compared with WT controls (Fig. 3C) . Higher titers of C. pneumoniae were detected in IFN-ß–/– BMM (Fig. 3D) .


Figure 3
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  		Figure 3. IFN-ß is required for IFN-ß, IFN-{alpha}, and IFN-{gamma} mRNA accumulation and for control of C. pneumoniae growth in BMM. Total RNA was extracted from WT and IFN-ß–/– BMM at the indicated times after infection with C. pneumoniae. The accumulation of IFN-{alpha} (A), IFN-{gamma} (B), and IRF7 (C) mRNA was measured by real-time PCR. A representative from two independent experiments is shown. WT and IFN-ß–/– BMM were infected with C. pneumoniae and lysed in SPG buffer at the indicated time-points after infection. C. pneumoniae IFU in BMM lysates were quantified by HEp-2 infectivity assay (D). *, Differences with WT BMM are significant (P<0.05 Student’s t test).

Role of IRAK4 and IRF3 in C. pneumoniae-induced NF-{kappa}B activation
Infection of WT BMM with C. pneumoniae resulted in increased titers of IL-1ß and IL-6 mRNA along with IL-6 protein in supernatants and activation of NF-{kappa}B signal transduction pathway characterized by increased levels of pI{kappa}B and nuclear translocation of NF-{kappa}B (Fig. 4A 4B 4C 4D 4E ). Similar levels of IL-6 protein (Fig. 4 C ) and IL-1ß and IL-6 mRNA (Fig. 4A and 4B) were measured in infected WT and IRAK4–/– BMM. Furthermore, similar levels of NF-{kappa}B activation were detected in C. pneumoniae-infected IRAK4–/– and WT BMM by Western blot (Fig. 4D) and EMSA (Fig. 4E) . Levels of IL-6 and IL-1ß mRNA and pI{kappa}B in infected IRF3–/–, IFN-ß–/–, and WT BMM were similar (Fig. 4F 4G 4H , and data not shown).


Figure 4
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  		Figure 4. IRAK4 or IRF3 is not required for expression of proinflammatory cytokines and activation of NF-{kappa}B after infection with C. pneumoniae. The accumulation of IL-1ß (A and F) and IL-6 (B and G) mRNA in total RNA extracted from WT, IRAK4–/– (A and B), and IRF3–/– (F and G) BMM at the indicated time-points after infection was quantified by real-time PCR. The levels of IL-6 in the supernatants from WT and IRAK4–/– BMM at the indicated time-points after infection with C. pneumoniae were measured by ELISA (C). Protein extracts were prepared at the indicated time-points after infection of WT and IRAK4–/– (D) and IRF3–/– (H) BMM, separated by SDS-PAGE, electroblotted onto nitrocellulose membranes, and immunoblotted with antibodies, which recognize actin and pI{kappa}B specifically. Antibody binding was detected with HRP-conjugated anti-IgG followed by ECL detection. Nuclear extracts were obtained at the indicated time-points after infection of WT or IRAK4–/– BMM with C. pneumoniae. The nuclear samples were examined for the level of NF-{kappa}B DNA-binding ability by EMSA (E).

Role of TRAF6 in C. pneumoniae-induced NF-{kappa}B activation
The role of TRAF6 in expression of proinflammatory genes during infection with C. pneumoniae was then studied. Different TLR-dependent and -independent pathways converge in TRAF6, which will trigger activation of NF-{kappa}B [9 ]. As TRAF6 is essential for perinatal and postnatal survival [34 ], TRAF6–/– and TRAF6+/+ embryonic fibroblasts (MEF) were generated. Similar to BMM, infection of WT MEF with C. pneumoniae induced high IL-1ß, IL-6, or TNF-{alpha} mRNA, IL-6 protein, and NF-{kappa}B activation levels (Fig. 5A 5B 5C 5D 5F and 5G ). In contrast, no increase of IL-1ß, IL-6, and TNF-{alpha} mRNA or IL-6 protein levels was detected in C. pneumoniae-infected TRAF6–/– MEFs (Fig. 5A 5B 5C 5D) . In addition, pI{kappa}B and nuclear translocation of NF-{kappa}B were strickingly diminished in infected TRAF6–/– MEFs (Fig. 5F and 5G) . C. pneumoniae-induced expression of IFN-{alpha}, IFN-ß, and IFN-{gamma} mRNA in MEF was generally low as compared with BMM (Fig. 5E and data not shown). However, the increased expression of IFN-ß mRNA was not affected by the absence of TRAF6 (Fig. 5E) .


Figure 5
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  		Figure 5. TRAF6 is required for expression of proinflammatory cytokines and activation of NF-{kappa}B after infection with C. pneumoniae. Total RNA was extracted from WT and TRAF6–/– MEFs at the indicated times after infection with C. pneumoniae. The accumulation of IL-1ß (A), IL-6 (B), TNF-{alpha} (C), and IFN-ß (E) mRNA was measured by real-time PCR. A representative from two independent experiments is shown. The levels of IL-6 in the supernatants from WT and IRAK4–/– BMM at the indicated time-points after infection with C. pneumoniae were measured by ELISA (D). Protein extracts were prepared at the indicated time-points after infection of WT and TRAF6–/– MEFs, and a Western blot against actin and pI{kappa}B was performed as described above (F). Nuclear extracts were obtained at the indicated time-points after infection of WT or TRAF6–/– MEF with C. pneumoniae. The nuclear samples were examined for the level of NF-{kappa}B DNA-binding ability by EMSA (G).


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DISCUSSION
 
We show here that IRAK4 is required for C. pneumoniae-induced BMM expression of IFN-{alpha}, IFN-ß, and IFN-{gamma} genes as well as for C. pneumoniaegrowth control. This is in agreement with previous data showing that infection of macrophages with Chlamydia induces IFN-{alpha} in a MyD88-dependent manner [28 , 30 ] and with experiments showing that MyD88–/– mice or macrophages showed increased susceptibility to chlamydial infection [29 , 30 ].

The infection of BMM with C. trachomatis has been shown to induce nuclear translocation of IRF3 [28 ]. Contrary to this result, we found no enhanced activation of IRF3 in C. pneumoniae-infected BMM. Moreover, IRF3 was neither required for expression of IFN-{alpha} and IFN-{gamma} mRNA during C. pneumoniae infection nor for control of C. pneumoniae infection in BMM. In agreement with our data obtained with C. pneumoniae-infected macrophages, IRF3–/– MEF showed normal IFN-mediated, antiviral response against vesicular stomatitis virus and HSV [41 42 43 ]. Of importance, induction of IFN-{alpha}/ß is, in the absence of IRF3, dependent on IRF7 expression and on the presence of IFN-ß in nontreated cells [41 ]. In accordance, presence of IFN-ß was found to be required for IRF7, IFN-{alpha}, and IFN-{gamma} mRNA expression and resistance against C. pneumoniae. We hypothesize that infection with C. pneumoniae will induce a MyD88/IRAK4-dependent activation of IRF7, resulting in IFN-{alpha} expression [44 , 45 ].

IRAK4–/– mice have previously been shown to be highly resistant to LPS-induced septic shock, surviving a dose of LPS, which causes 100% lethality in WT animals [31 ]. No residual, inflammatory cytokine responses to LPS or various TLR ligands were observed in IRAK4–/– cells [31 ]. Activation of NF-{kappa}B and stress kinases could be detected in LPS-stimulated IRAK4–/– BMM, but the kinetics was delayed [7 ]. However, the MyD88-IRAK4 signaling pathway was found to be redundant for NF-{kappa}B activation and expression of proinflammatory cytokines during C. pneumoniae infection, suggesting that signaling pathways mediated by other innate receptors such as NOD1 could also participate in NF-{kappa}B activation during C. pneumoniae infection [46 ]. In contrast to the redundancy observed for MyD88 [4 ], IRAK4, and IRF3, TRAF6 was found to be required for activation of NF-{kappa}B and expression of proinflammatory cytokine genes during infection with C. pneumoniae. Activation of TRAF6 could be mediated by TRIF, MyD88, and NOD1 signaling [47 48 49 ].

In summary, C. pneumoniae infection will induce an IRAK4-dependent IFN-ß, -{alpha}, and -{gamma} gene expression in macrophages. IFN-{alpha} and IFN-{gamma} expression requires IFN-ß. IRF3 and IRAK4 are redundant for C. pneumoniae-induced NF-{kappa}B activation, which is itself dependent on TRAF6 (Fig. 6 ).


Figure 6
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  		Figure 6. Molecular pathways controlling macrophage secretion of IFN-{gamma} after infection with C. pneumoniae, which induces a MyD88/IRAK4-dependent induction of IFN-ß and IFN-{alpha}. Presence of IFN-ß is required for bacterial-induced expression of IFN-{alpha}. Infection with C. pneumoniae activates NF-{kappa}B in a TRAF6-dependent manner. We hypothesize that during bacterial infection, MyD88-IRAK4, TRIF, and possibly other intracellular signaling pathways contribute to NF-{kappa}B activation. NF-{kappa}B and IFN-{alpha} play a role in the bacterial-induced expression of IFN-{gamma} [30 ].


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ACKNOWLEDGEMENTS
 
This work was supported by the European Community QLK2-CT-2002-00846 grant, the Karolinska Institute, The Swedish Health Insurance Company AFA, and The Swedish Research Council, Sweden. We thank Dr. Tak W. Mak for providing us TRAF6–/+ mice. We thank Berit Olsson for excellent technical assistance.

Received July 19, 2006; revised January 28, 2007; accepted February 4, 2007.


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