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The Journal of Immunology, 2004, 172: 6345-6353.
Copyright © 2004 by The American Association of Immunologists

Intracellular Bacterial Infection-Induced IFN-{gamma} Is Critically but Not Solely Dependent on Toll-Like Receptor 4-Myeloid Differentiation Factor 88-IFN-{alpha}{beta}-STAT1 Signaling1

Antonio Gigliotti Rothfuchs, Christian Trumstedt, Hans Wigzell and Martin E. Rottenberg2

Microbiology and Tumorbiology Center, Karolinska Institute, Stockholm, Sweden


   Abstract
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 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Infection of murine bone marrow-derived macrophages (BMM{phi}) with Chlamydia pneumoniae induces IFN-{alpha}{beta}-dependent IFN-{gamma} secretion that leads to control of the intracellular bacterial growth. Enhanced growth of C. pneumoniae in Toll-like receptor (TLR) 4–/– and myeloid differentiation factor (MyD) 88–/– (but not TLR2–/–, TLR6–/–, or TLR9–/–) BMM{phi} is shown in this study. Reduced accumulation of IFN-{alpha} and IFN-{gamma} mRNA was also observed in TLR4–/–- and MyD88–/–-infected cells. IL-1R and IL-18R signaling did not account for differences between MyD88–/– and wild-type BMM{phi}. Surprisingly, infection-induced NF-{kappa}B activation as well as TNF-{alpha}, IL-1, or IL-6 mRNA expression were all normal in TLR4–/– and MyD88–/– cells. Phosphorylation of the transcription factor STAT1 during bacterial infection is IFN-{alpha}{beta} dependent, and necessary for increased IFN-{gamma} mRNA accumulation and chlamydial growth control. Signaling through common cytokine receptor {gamma}-chain and RNA-dependent protein kinase both mediated IFN-{alpha}{beta}-dependent enhancement of IFN-{gamma} mRNA levels. Accumulation of IFN-{gamma} mRNA and control of C. pneumoniae growth required NF-{kappa}B activation. Such NF-{kappa}B activation was independent of IFN-{alpha}{beta}, STAT1, and RNA-dependent protein kinase. In summary, C. pneumoniae-induced IFN-{gamma} expression in BMM{phi} is controlled by a TLR4-MyD88-IFN-{alpha}{beta}-STAT1-dependent pathway, as well as by a TLR4-independent pathway leading to NF-{kappa}B activation.


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Mammalian Toll-like receptors (TLRs)3 constitute a family of closely related transmembrane, primary signal-transducing proteins that respond to an array of microbial products (1). They recognize different pathogen-associated molecular patterns such as LPS, flagellin, unmethylated CpG motifs in DNA, dsRNA, mycobacterial lipoarabinomannan, yeast zymosan, and bacterial lipoproteins (1). Upon pathogen-associated molecular pattern recognition, the intracellular domains of all known TLRs interact with the adaptor molecule myeloid differentiation factor (MyD) 88 and initiate a common signaling cascade that leads to nuclear translocation of the transcription factors NF-{kappa}B and AP-1. This signaling cascade can also be activated upon IL-1 or IL-18R engagement (2). TLR can induce IFN-{alpha}{beta} in the presence of MyD88 (3, 4, 5) or in its absence (6, 7) by using the adaptor named Toll-IL-1R domain-containing adaptor molecule/TIR domain-containing adaptor inducing IFN-{beta} (8, 9, 10).

IFN-{alpha}{beta} are key immunoregulatory cytokines produced directly after cell exposure to many pathogens. IFN-{alpha}{beta} interfere with virus replication through induction of, e.g., double-stranded RNA-dependent protein kinase (PKR) and 2'-5'-oligoadenylate synthetase (2'5'OAS). Besides the antiviral effect exerted by inhibition of eukaryotic protein synthesis, PKR plays a catalytic or structural role to activate I-{kappa}B kinase or directly phosphorylate I-{kappa}B. Stress-activated protein kinases p38 and c-Jun kinase are also regulated by PKR in a pathway that leads to production of proinflammatory cytokines (11).

Binding of IFN-{alpha}{beta} to their cellular receptor results in phosphorylation of STATs by receptor-associated Janus kinases, resulting in formation of homodimeric (STAT1 · STAT1), heterodimeric (STAT1 · STAT2), or heterotrimeric (STAT1 · STAT2 · IFN regulatory factor-9) protein complexes. These multimeric complexes translocate to the nucleus, where they bind to distinct DNA elements, leading to expression of IFN-inducible genes (12). The key macrophage-activating cytokine, IFN-{gamma}, mediates resistance against intracellular bacteria and protozoa via activation of antimicrobial effector molecules (13). Although involved in different responses, both IFN-{alpha}{beta}R and IFN-{gamma}R signaling share in common STAT1. In turn, both IFNs have partially overlapping biological effects.

The obligate intracellular Gram-negative bacterium Chlamydia pneumoniae is a common cause of acute respiratory disease (14) and has been associated with development of atherosclerosis (15). Chlamydia are internalized by macrophages, but by avoiding phagolysosomal fusion are able to replicate intracellularly. IFN-{gamma} is central in resistance to this pathogen both in vivo and in vitro (reviewed in Ref. 16). Several studies show that, besides NK and T cells, myeloid cells such as macrophages, dendritic cells (DCs), and neutrophils can also express IFN-{gamma} (17). In accordance, we have shown that mouse bone marrow-derived macrophages (BMM{phi}) infected in vitro with C. pneumoniae express IFN-{gamma} that in turn protects these cells against chlamydial growth. This IFN-{gamma} production was IL-12 independent, but required IFN-{alpha}{beta} (18).

DCs, smooth muscle cells, macrophages, endothelial cells, and PBMCs are all activated by chlamydial infection or acellular chlamydial components in a TLR2- or TLR4-dependent way (19, 20, 21, 22). However, further details of such activation, in particular with regards to IFN-{gamma} {epsilon} expression, are unknown.

IFN-{alpha}{beta}-dependent induction of IFN-{gamma} in splenocytes and T cells has been suggested to be STAT4 dependent (23, 24). However, IFN-{alpha}{beta} might also activate IFN-{gamma} in an IL-15-mediated way: Expression of IL-15 depends on presence of a functional IFN-{alpha}{beta}R (25, 26), and IFN-{gamma} secretion by splenic DCs and macrophages was markedly increased after treatment with IL-15 (27). In this study, we have explored how C. pneumoniae recognition and the ensuing signaling pathways lead to protective IFN-{gamma} expression in BMM{phi}.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

Mutant mouse strains without IFN-{alpha}{beta}R (28), IFN-{gamma}R (29), STAT1 (30, 31), MyD88 (32), TLR2 (33), TLR4 (34), TLR6 (35), TLR9 (36), IL-1-converting enzyme (ICE) (37), common cytokine receptor chain ({gamma}cR) (38), or recombination-activating gene (RAG)-1 (39) were generated by homologous recombination in embryonic stem cells. RAG-1–/–/{gamma}cR–/– mice were purchased from Taconic Farms (Germantown, NY). Animals were bred and kept under specific pathogen-free conditions. Mice of the C57BL/6 background were used as controls for IFN-{gamma}R–/–, IFN-{beta}–/–, STAT1–/–, MyD88–/–, TLR2–/–, TLR4–/–, TLR6–/–, TLR9–/–, and ICE–/– mice, and 129Sv/Ev mice as controls for IFN-{alpha}{beta}R–/– mice.

Generation of mouse BMM{phi}

Mouse BMM{phi} were obtained from 6- to 10-wk-old mice, as described (18). Mice were euthanized, and the femur and tibia of the hind legs were dissected. Bone marrow cavities were flushed with 5 ml of cold, sterile PBS. The bone marrow cells were washed and resuspended in DMEM (Sigma-Aldrich, St. Louis, MO) 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-Aldrich), 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 (1.2 x 107 cells/well, 2 x 106 cells/ml), and incubated for 7 days at 37°C, 5% CO2. Before use, BMM{phi} cultures were washed vigorously to remove nonadherent cells. Cells from several wells were also harvested and counted by trypan blue exclusion. Typically, bone marrow cells from one mouse yielded 2–3 x 106 BMM{phi} after 7 days in culture. We have previously shown by immunofluorescence staining that these BMM{phi} are F4/80+, CD14+, and Mac-3+ (18).

Generation of fibroblasts from mouse lung

Primary fibroblast cultures were generated, as described (40). The pulmonary vasculature was perfused, and lungs were aseptically removed, excised into small pieces, and subjected to collagenase digestion at 37°C for 15 min under agitation. The resulting cell suspension was collected. Enzymatic digestion and collection of cell suspensions were repeated twice. Cell suspensions were then pooled, passed through a 100-µm cell strainer, washed, and plated in tissue culture flasks with IMDM supplemented with 2 mM L-glutamine, 5% FCS, 10 mM HEPES, 100 µg/ml streptomycin, and 100 U/ml penicillin at 37°C, 5% CO2. Fresh medium was added every 7 days until fibroblast cultures attained confluence, ~7–14 days later. Lung primary fibroblasts were then washed vigorously, trypsinated, and replated onto six-well plates (2.5 x 105 cells/well, 0.42 x 105 cells/ml).

Infection and infectivity assay

Mycoplasma-free C. pneumoniae isolate Kajaani 6 (41) 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{phi} were infected with C. pneumoniae by centrifugation for 1 h, 500 x g at 35°C. A multiplicity of infection of 3 was used for both BMM{phi} and fibroblast experiments. At different time points after infection, cells were washed with PBS and then lysed in SPG buffer. Assessment of IFU in SPG lysates was done in HEp-2 cells. Aliquots of SPG lysates diluted 10- 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/ml streptomycin (DMEM/Strep) on round 13-mm2 glass coverslides in 24-well plates. Inoculated cells were centrifuged for 1 h, 500 x g at 35°C. Thereafter, supernatant was removed and DMEM/Strep containing 0.5 µg/ml cycloheximide was added. Cells were incubated at 35°C for 72 h, 5% CO2, thereafter washed gently with PBS, and fixed in methanol. Glass coverslides were then stained for 0.5 h at room temperature with a FITC-conjugated Chlamydia genus-specific mAb (1/5 dilution; Pathfinder Chlamydia Confirmation System; Bio-Rad, Hercules, CA). Coverslides were mounted with fluorescent mounting medium (DAKO, Carpinteria, CA), and IFU of C. pneumoniae were quantified by fluorescence microscopy. The infectivity was expressed as IFU of C. pneumoniae per well.

Competitive RT-PCR assay

Cultures of C. pneumoniae-infected BMM{phi} were disrupted in RNAzol B (Nordic Biosite, Täby, Sweden), and total RNA was isolated according to the instructions of the manufacturer and reversed transcribed into cDNA, as described (42). Specific primer pairs for IFN-{alpha}, IFN-{gamma}, inducible NO synthase (iNOS), PKR, 2'5'OAS, IL-1{alpha}, IL-6, IL-15, TNF-{alpha}, and {beta}-actin were used to amplify cDNA. Amplified cDNAs were visualized in ethidium bromide-stained 2% agarose gels or quantified by competitive PCR assays. Briefly, competitor fragments with a different length, but using the same primers as the target cDNA, were constructed using composite primers and an exogenous DNA fragment (43). Three- to 4-fold serial dilutions of the competitor were amplified in the presence of a constant amount of cDNA. Reactions were conducted for 23–41 cycles in a thermal cycler (PerkinElmer, Cetus, CT) using an annealing step of 65°C for 2'5'OAS; 62°C for IL-15, 60°C for IFN-{alpha}, IFN-{gamma}, iNOS, IL-1{alpha}, IL-6, and {beta}-actin; and 54°C for PKR and TNF-{alpha}. All primer sequences are given in Table I.


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  		Table I. PCR primers used in this study

 
SDS-PAGE and Western blotting

C. pneumoniae-infected BMM{phi} were lysed in 150 mM NaCl, 20 mM Tris-HCl, 2 mM EDTA, 1% Triton X-100, 10% glycerol, and 2 mM PMSF. The protein content in cell lysates was measured by Lowry assay (Bio-Rad). Sample buffer (Bio-Rad) containing 2-ME was added to samples that were then boiled for 5 min. A total of 10 µg of samples was 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 polyclonal rabbit anti-I-{kappa}B-{alpha}, monoclonal mouse anti-phosphorylated (Ser32/36) I-{kappa}B-{alpha}, polyclonal rabbit anti-phosphorylated (Tyr701) STAT1, polyclonal rabbit anti-STAT1 (all 1/1000 dilution; Cell Signaling, Beverly, MA), and polyclonal rabbit anti-actin (1/500 dilution; Sigma-Aldrich). Membranes were then washed and incubated with HRP-conjugated polyclonal rabbit anti-mouse Igs or HRP-conjugated polyclonal goat anti-rabbit Igs (both 1/2000 dilution; DAKO), developed using ECL-Plus (Amersham Pharmacia Biotech, Piscataway, NJ), and photographed using a Fuji intelligent darkbox II digital camera.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
TLR4 and MyD88 are necessary for enhanced IFN-{alpha} and IFN-{gamma} mRNA levels and control growth of C. pneumoniae in BMM{phi}

To investigate which TLR are essential for growth control of C. pneumoniae, TLR2–/–, TLR4–/–, TLR6–/–, or TLR9–/– BMM{phi} were infected with C. pneumoniae, respectively. Only TLR4–/– BMM{phi} showed enhanced bacterial levels in comparison with wild-type (WT) controls (Fig. 1A). On the contrary, TLR2–/–, TLR6–/–, and TLR9–/– BMM{phi} showed paradoxically lower C. pneumoniae levels than WT BMM{phi} (data not shown), an observation that remains to be investigated. Enhanced chlamydial growth in TLR4–/– BMM{phi} correlated with lack of enhanced IFN-{alpha} and IFN-{gamma} mRNA accumulation (Fig. 1, B and C). Signaling through TLR4 occurs at least in part through MyD88. In line with this, MyD88–/– BMM{phi} were more susceptible to C. pneumoniae (Fig. 2A) compared with WT BMM{phi}, and showed no increase in IFN-{alpha} and IFN-{gamma} mRNA levels (Fig. 2, B and C). To confirm that the observations made in MyD88–/– BMM{phi} indeed were linked to a defect in TLR signaling, ICE–/– BMM{phi} were infected with C. pneumoniae. ICE is required for proteolytic activation of IL-1 and IL-18, which signal through MyD88 (44). ICE–/– BMM{phi} showed nondiminished IFN-{gamma} mRNA and C. pneumoniae levels compared with WT cells (Fig. 2D, and data not shown). Thus, reduced IFN-{alpha} and IFN-{gamma} expression in infected MyD88–/– BMM{phi} is due to a defect in TLR signaling.



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  		FIGURE 1. TLR4 is necessary for enhanced IFN-{alpha} and IFN-{gamma} mRNA levels and controls growth of C. pneumoniae in BMM{phi}. A, WT and TLR4–/– BMM{phi} were infected with C. pneumoniae and lysed in SPG buffer at the indicated time points after infection. C. pneumoniae IFU in BMM{phi} lysates were quantified by HEp-2 infectivity assay. A representative from three independent experiments is shown. B and C, Total RNA was extracted from WT and TLR4–/– BMM{phi} at the indicated time points after infection with C. pneumoniae. Accumulation of IFN-{alpha} (B) and IFN-{gamma} (C) mRNA was measured by competitive PCR. Similar results were obtained in two separate experiments.

 


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  		FIGURE 2. MyD88 is necessary for enhanced IFN-{alpha} and IFN-{gamma} mRNA levels and controls growth of C. pneumoniae in BMM{phi}. A, WT and MyD88–/– BMM{phi} were infected with C. pneumoniae and lysed in SPG buffer at the indicated time points after infection. C. pneumoniae IFU in BMM{phi} lysates were quantified by HEp-2 infectivity assay. For each time point shown, bacterial levels were found to be at least 6 times higher in MyD88–/– BMM{phi} compared with WT BMM{phi}. A representative from three independent experiments is shown. B and C, Total RNA was extracted from WT and MyD88–/– BMM{phi} at the indicated time points after infection with C. pneumoniae. The accumulation of IFN-{alpha} (B) and IFN-{gamma} (C) mRNA was measured by competitive PCR. Comparable results were obtained in two separate experiments. D, Total RNA was extracted from WT, MyD88–/–, and ICE–/– BMM{phi} at 6 h after infection with C. pneumoniae. The accumulation of IFN-{gamma} mRNA was measured by competitive PCR. A representative from two independent experiments is shown.

 
Phosphorylation of I-{kappa}B-{alpha} is required before nuclear translocation of NF-{kappa}B, serving as marker of NF-{kappa}B activation (45, 46). Phosphorylation of I-{kappa}B-{alpha} increased in C. pneumoniae-infected WT BMM{phi} (Fig. 3, A and B). Surprisingly, similar levels of phosphorylated I-{kappa}B-{alpha} were detected in infected TLR4–/–, MyD88–/–, and WT BMM{phi} (Fig. 3, A and B). Levels of IL-1{alpha}, IL-6, and TNF-{alpha} mRNA in infected TLR4–/–, MyD88–/–, and WT BMM{phi} were also similar (Fig. 3, C and D).



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  		FIGURE 3. NF-{kappa}B activation in C. pneumoniae-infected BMM{phi} does not require TLR4 and MyD88 signaling. A and B, WT (A and B), TLR4–/– (A), and MyD88–/– (B) BMM{phi} were infected with C. pneumoniae. Protein extracts were prepared at the indicated time points after infection, separated by SDS-PAGE, electroblotted onto a nitrocellulosemembranes, and immunoblotted with Abs that specifically recognize actin and phosphorylated I-{kappa}B-{alpha}. Abs were detected with HRP-conjugated anti-IgG, followed by ECL detection. C and D, Total RNA was extracted from WT (C and D), TLR4–/– (C), and MyD88–/– (D) BMM{phi} at the indicated time points after infection with C. pneumoniae. The accumulation of IL-1{alpha}, IL-6, and TNF-{alpha} mRNA was visualized by RT-PCR. A representative from two independent experiments is shown.

 
STAT1 is necessary for enhanced IFN-{gamma} mRNA levels and controls growth of C. pneumoniae in BMM{phi}

Biological effects of IFN-{alpha}{beta} and IFN-{gamma} are in part mediated via STAT1 (30). Phosphorylation of STAT1 is noted in C. pneumoniae-infected WT BMM{phi}, but not in uninfected controls (Fig. 4). The level of phosphorylated STAT1 was relatively diminished in infected IFN-{gamma}R–/– and undetectable in IFN-{alpha}{beta}R–/– BMM{phi} (Fig. 4). Growth of C. pneumoniae was higher (Fig. 5A) and IFN-{gamma} mRNA accumulation lower (Fig. 5C) in STAT1–/– BMM{phi} compared with WT controls. PKR, 2'5'OAS, and iNOS mRNA levels as well as NO in culture supernatants were all reduced in STAT1–/– BMM{phi}, while mRNA levels of IL-1{alpha}, IL-6, and TNF-{alpha} were not or only slightly affected as compared with WT BMM{phi} (Fig. 5D, and data not shown). Primary cultures of lung fibroblasts generated from STAT1–/– mice also displayed enhanced C. pneumoniae growth compared with WT controls (Fig. 5B).



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  		FIGURE 4. IFN-{alpha}{beta}R and IFN-{gamma}R signaling completely account for STAT1 phosphorylation in C. pneumoniae-infected BMM{phi}. WT, IFN-{alpha}{beta}R–/–, and IFN-{gamma}R–/– BMM{phi} were infected with C. pneumoniae. Protein extracts were prepared at the indicated time points after infection and separated by SDS-PAGE, electroblotted onto nitrocellulose membranes, and immunoblotted with Abs that specifically recognize actin, total STAT1, and phosphorylated STAT1. Abs were detected with HRP-conjugated anti-IgG, followed by ECL detection. A representative from three independent experiments is shown.

 


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  		FIGURE 5. STAT1 is necessary for enhanced IFN-{gamma} mRNA levels and controls growth of C. pneumoniae in BMM{phi}. A and B, WT and STAT1–/– BMM{phi} (A) and lung fibroblasts (B) were infected with C. pneumoniae and lysed in SPG buffer at the indicated time points after infection. C. pneumoniae IFU in the BMM{phi} lysates were quantified by HEp-2 infectivity assay. For each time point shown, bacterial levels were found to be at least 5 times higher in STAT1–/– BMM{phi} and 8 times higher in STAT1–/– fibroblasts compared with WT controls, respectively. A representative from three and two independent experiments for BMM{phi} and fibroblasts, respectively, is shown. C and D, Total RNA was extracted from WT and STAT1–/– BMM{phi} at the indicated time points after infection with C. pneumoniae. The accumulation of IFN-{gamma} (C) mRNA was measured by competitive PCR, while that of iNOS, PKR, 2'5'OAS, IL-1{alpha}, IL-6, and TNF-{alpha} was visualized by RT-PCR (D).

 
Increased IFN-{gamma} mRNA levels in C. pneumoniae-infected BMM{phi} are {gamma}cR dependent

IL-15 signals through the {gamma}cR and can trigger release of IFN-{gamma} from stimulated macrophages and DCs (25, 27). Enhanced IL-15 mRNA accumulation was observed in C. pneumoniae-infected WT and IFN-{gamma}R–/– BMM{phi}, but not in IFN-{alpha}{beta}R–/– or STAT1–/– BMM{phi} (Fig. 6A). RAG-1–/–/{gamma}cR–/– BMM{phi} showed higher C. pneumoniae numbers, whereas IFN-{gamma} was reduced in comparison with infected RAG-1–/– BMM{phi} (Fig. 6, B and D). RAG-1–/–/{gamma}cR–/– and RAG-1–/– BMM{phi} showed similar levels of IFN-{alpha}, IL-1{alpha}, IL-6, or TNF-{alpha} mRNA (Fig. 6, C and E).



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  		FIGURE 6. Increased IL-15 mRNA accumulation in C. pneumoniae-infected BMM{phi} is dependent on signaling by IFN-{alpha}{beta}R and STAT1. A, Total RNA was extracted from WT, IFN-{alpha}{beta}R–/–, IFN-{gamma}R–/–, and STAT1–/– BMM{phi} at the indicated time points after infection with C. pneumoniae. The accumulation of IL-15 was visualized by RT-PCR. For the sake of clarity, only the C57BL/6 WT control is represented, although similar results were obtained with 129Sv/Ev WT BMM{phi} (data not shown). A representative from two independent experiments is shown. B–E, Increased IFN-{gamma} mRNA accumulation in C. pneumoniae-infected BMM{phi} is {gamma}cR dependent. RAG-1–/– and RAG-1–/–/{gamma}cR–/– BMM{phi} were infected with C. pneumoniae and lysed in SPG buffer at the indicated time points after infection. C. pneumoniae IFU in BMM{phi} lysates were quantified by HEp-2 infectivity assay. For each time point shown, bacterial levels were found to be at least 5 times higher in RAG-1–/–/{gamma}cR–/– BMM{phi} compared with RAG-1–/– BMM{phi} (B). Total RNA was extracted from RAG-1–/– and RAG-1–/–/{gamma}cR–/– BMM{phi} at the indicated time points after infection with C. pneumoniae (C–E). The accumulation of IFN-{alpha} (C) and IFN-{gamma} (D) mRNA was measured by a competitive PCR, while IL-1{alpha}, IL-6, and TNF-{alpha} were visualized by RT-PCR (E). A representative from three independent experiments is shown.

 
PKR mediates IFN-{alpha}{beta}-dependent expression of IFN-{gamma} mRNA

Enhanced PKR mRNA accumulation observed in C. pneumoniae-infected BMM{phi} requires IFN-{alpha}{beta}R and STAT1 (Fig. 5D, and data not shown). To investigate whether PKR was involved in IFN-{alpha}{beta}-dependent IFN-{gamma} mRNA accumulation, 2-aminopurine (2-AP), a specific pharmacological inhibitor of PKR (47), was used. Treatment of WT, but not IFN-{alpha}{beta}R–/– BMM{phi} with 2-AP increased C. pneumoniae growth (Fig. 7A), suggesting that PKR acts downstream of IFN-{alpha}{beta}R signaling. In line with this, C. pneumoniae-induced IFN-{gamma}, but not IFN-{alpha} mRNA accumulation was dramatically reduced in 2-AP-treated WT BMM{phi} (Fig. 7, B and C). These results suggest a positive role for PKR in IFN-{gamma} expression. PKR can activate NF-{kappa}B through phosphorylation of I-{kappa}B-{alpha} or the I{kappa}B kinase complex components (11). However, treatment of BMM{phi} with 2-AP did not affect I{kappa}B-{alpha} phosphorylation (Fig. 7D). Also, IFN-{alpha}{beta}R–/– BMM{phi} showed similar levels of phosphorylated I-{kappa}B-{alpha} as WT controls (Fig. 7D).



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  		FIGURE 7. PKR mediates IFN-{alpha}{beta}-dependent expression of IFN-{gamma} mRNA. A, WT and IFN-{alpha}{beta}R–/– BMM{phi} were treated with the PKR inhibitor 2-AP and infected with C. pneumoniae. Three days after infection, BMM{phi} were lysed in SPG buffer, and C. pneumoniae IFU in BMM{phi} lysates were quantified by HEp-2 infectivity assay. B and C, Total RNA was extracted from 2-AP-treated WT BMM{phi} 6 h after infection with C. pneumoniae, and the accumulation of IFN-{alpha} (B) and IFN-{gamma} (C) mRNA was measured by competitive PCR. A representative from two independent experiments is shown. D, WT, IFN-{alpha}{beta}R–/–, and 2-AP-treated WT BMM{phi} were infected with C. pneumoniae. Protein extracts were prepared at the indicated time points after infection, separated by SDS-PAGE, electroblotted onto nitrocellulose membranes, and immunoblotted with Abs that specifically recognize actin and phosphorylated I-{kappa}B-{alpha}. Abs were detected with HRP-conjugated anti-IgG, followed by ECL detection. A representative from three independent experiments for IFN-{alpha}{beta}R–/– BMM{phi} and from two independent experiments for 2-AP-treated BMM{phi} is shown.

 
NF-{kappa}B activation is necessary for enhanced IFN-{gamma} mRNA levels and control of C. pneumoniae growth in BMM{phi}

We accordingly asked whether activation of NF-{kappa}B was linked to protection against C. pneumoniae infection of BMM{phi}. For this purpose, BAY 11-7082, a pharmacological inhibitor of I-{kappa}B-{alpha} phosphorylation (48), was used. BAY 11-7082 treatment inhibited infection-induced I-{kappa}B-{alpha} phosphorylation in WT BMM{phi} without affecting total I-{kappa}B-{alpha} levels (Fig. 8B). Treatment of C. pneumoniae-infected BMM{phi} with BAY 11-7082 also increased chlamydial growth (Fig. 8A) and reduced IFN-{gamma} (Fig. 9A) and iNOS (Fig. 9B), but had no impact on IFN-{alpha} mRNA accumulation (Fig. 9C). As expected, IL-1{alpha}, IL-6, and TNF-{alpha} levels were reduced following BAY 11-7082 treatment (Fig. 9C).



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  		FIGURE 8. NF-{kappa}B activation is necessary for control of C. pneumoniae growth in BMM{phi}. A, WT BMM{phi}, treated with 1 µM BAY 11-7082 (4-methylphenyl)sulfonyl-2-properinitrite; Calbiochem, La Jolla, CA), solubilized in DMSO or left untreated, were infected with C. pneumoniae. Cells were lysed in SPG buffer at the indicated time points after infection. C. pneumoniae IFU in BMM{phi} lysates were quantified by HEp-2 infectivity assay. For each time point shown, bacterial levels were found to be at least 5 times higher in BAY 11-7082-treated BMM{phi} compared with untreated BMM{phi}. A representative from two independent experiments is shown. B, WT BMM{phi} were treated with 1 µM BAY 11-7082 and infected with C. pneumoniae. Protein extracts were prepared at the indicated time points after infection, separated by SDS-PAGE, electroblotted onto nitrocellulose membranes, and immunoblotted with Abs that specifically recognize actin and phosphorylated I-{kappa}B-{alpha}. Abs were detected with HRP-conjugated anti-IgG, followed by ECL detection.

 


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  		FIGURE 9. NF-{kappa}B activation is necessary for increased IFN-{gamma} mRNA levels in C. pneumoniae-infected BMM{phi}. A–C, Total RNA was obtained from WT BMM{phi} treated with different concentrations of BAY 11-7082, 6 h after infection with C. pneumoniae. The accumulation of IFN-{gamma} (A) and iNOS (B) mRNA was measured by competitive PCR, while that of IFN-{alpha}, IL-1{alpha}, IL-6, and TNF-{alpha} was visualized by RT-PCR (C). Similar results were obtained in two separate experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We show in this study that macrophage infection with C. pneumoniae induces IFN-{gamma} mRNA accumulation and bacterial growth control in a TLR4-MyD88-IFN-{alpha}{beta}-STAT1-dependent manner (Fig. 10). PKR and {gamma}cR signaling participate downstream of IFN-{alpha}{beta}R in C. pneumoniae infection-induced IFN-{gamma} expression and bacterial growth control. However, accumulation of IFN-{gamma} mRNA does not solely depend on signals via TLR4-IFN-{alpha}{beta} pathway. NF-{kappa}B activation was observed in C. pneumoniae-infected macrophages and DCs (19, 21). We now show that NF-{kappa}B activation after C. pneumoniae infection is required for IFN-{gamma} expression and chlamydial growth control, but can take place in a TLR4- and IFN-{alpha}{beta}R-independent way. C. pneumoniae-induced proinflammatory cytokine transcripts are neither reduced in MyD88–/– nor TLR4–/– BMM{phi} (Fig. 10).



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  		FIGURE 10. Molecular pathways controlling macrophage secretion of IFN-{gamma} after infection with C. pneumoniae. BMM{phi} infection with C. pneumoniae activates a TLR4-MyD88-dependent IFN-{alpha} expression. IFN-{alpha}{beta}-dependent, STAT1-mediated signaling is necessary for expression of IFN-{gamma}. Activation of NF-{kappa}B, which can occur in a TLR4-MyD88- and IFN-{alpha}{beta}-independent way, is also critical for IFN-{gamma} induction.

 
In MyD88–/– macrophages, nuclear translocation of NF-{kappa}B and phosphorylation of the mitogen-activated protein kinases (MAPK) in response to LPS are somewhat delayed (32, 49). In contrast, TLR4–/– macrophages show no NF-{kappa}B and MAPK activation after exposure to LPS (34). Likewise, no activation of NF-{kappa}B or c-Jun kinase was observed in MyD88–/– macrophages in response to other TLR ligands such as peptidoglycan, lipoprotein, CpG DNA, or the imidazoquinolines (reviewed in Ref. 1).

Activation of NF-{kappa}B can, however, occur in a TLR4- and MyD88-independent manner in C. pneumoniae-infected BMM{phi}. Thus, a TLR-independent signaling pathway probably also participates in bacterial induced NF-{kappa}B activation. It is also unlikely that other TLR mediate MyD88-independent proinflammatory cytokine expression (e.g., via Toll-IL-1R domain-containing adaptor molecule/TIR domain-containing adaptor inducing IFN-{beta}) in our experimental model, because expression of proinflammatory cytokines in response to different specific TLR ligands is all reduced in MyD88–/– cells (32, 49). However, TLR2 has been shown to be involved in TNF-{alpha} and IL-6 secretion in Chlamydia trachomatis-infected macrophages (50) and C. pneumoniae-infected DCs (19).

Activation of NF-{kappa}B is needed for IFN-{gamma} expression, and thereby for growth control of C. pneumoniae. In line with this, NF-{kappa}B-binding elements have been identified in the IFN-{gamma} promoter important for enhancement of gene transcription (51, 52). However, an IL-18-independent role for NF-{kappa}B in IFN-{gamma} gene expression has not been previously reported.

Together, our results thus suggest that Chlamydia can induce both TLR-dependent and -independent pathways that cooperate for IFN-{gamma} expression. TLR-analogous detection systems for microorganisms inside cells have been described (53). Nucleotide-binding oligomerization domain proteins in the cytoplasm are candidate among the molecules for such detection systems inside cells and recognize products from both Gram-positive and -negative bacteria (53, 54, 55). We plan to study the role for nucleotide-binding oligomerization domain proteins in IFN-{gamma} secretion and chlamydial growth control.

In other system, listerial infection and LPS stimulation were shown to activate IFN regulatory factor 3 inducing the synthesis of IFN-{beta} and IFN-{alpha}4 (7, 56). IFN regulatory factor 3 and IFN-{alpha}{beta} can be induced after activation of certain TLR in both MyD88-dependent and -independent ways (3, 4, 6, 10). However, MyD88-independent signaling was not sufficient for enhanced IFN-{alpha} mRNA accumulation in C. pneumoniae-infected BMM{phi}.

Consistent with the importance of the Janus kinase-STAT pathway in mediating the actions of IFN-{alpha}{beta}, mice lacking either STAT1 (30, 31) or STAT2 (57) have impaired IFN-{alpha}{beta}-regulated responses and are highly sensitive to viral infection. However, IFN-{alpha}{beta} can also control cellular functions in a STAT1-independent fashion (26, 58, 59, 60, 61): STAT1–/– mice are thus more resistant to virus infection than mice lacking expression of both IFN-{alpha}{beta}R and IFN-{gamma}R (26, 58, 59, 60, 61). We found that STAT1 was critical for the control of C. pneumoniae by BMM{phi}. IFN-{alpha}{beta}R signaling fully accounted for C. pneumoniae-induced STAT1 phosphorylation. Moreover, IFN-{alpha}{beta}-dependent STAT1 signaling was necessary for IFN-{gamma} secretion. To our knowledge, our report is the first indicating the latter. Paradoxically, STAT4 activation was shown to be the critical intermediary in the induction of IFN-{gamma} in IFN-{alpha}{beta}-stimulated T cells (24), while STAT1 was found to be a negative regulator of IFN-{gamma} in the same system (62).

IL-15 is strongly induced by C. pneumoniae infection in WT or IFN-{gamma}R–/–, but absent in IFN-{alpha}{beta}R–/– or STAT1–/– BMM{phi}. This is also occurring after influenza virus infection or LPS stimulation (26). Chlamydia-infected RAG-1–/–/{gamma}cR–/– BMM{phi}, which lack IL-15 responses, showed enhanced bacterial growth and decreased IFN-{gamma}, but normal IFN-{alpha} mRNA accumulation.

PKR, responsible for IFN-{alpha}{beta}-dependent antiviral effects, also functions as a signal transducer in the proinflammatory response to many agents (11). Our results suggest that PKR is activated in C. pneumoniae-infected BMM{phi} in an IFN-{alpha}{beta}-dependent manner and that it plays a role in IFN-{gamma} expression and control of infection. However, such a protective role(s) of PKR (and IFN-{alpha}{beta}) does not depend on the NF-{kappa}B-activating properties of the enzyme. Whether PKR mediate control of chlamydial infection via MAPK activation remains to be explored.

In summary, our results indicate that TLR4-MyD88-dependent and TLR4-MyD88-independent signaling are both critical and complementary for IFN-{gamma} expression in macrophages after intracellular bacterial infection. Our results indicate the simultaneous presence of high diversity and nonredundancy in the signals required for this process. A novel pathway of IFN-{gamma} induction mediated by STAT1 activation is also demonstrated by our data.


   Acknowledgments
 
We thank Berit Olsson for excellent technical assistance. STAT1–/– mice were kindly provided by Dr. D. Levy (New York University, New York, NY). IFN-{gamma}R–/– were originally provided by Dr. M. Aguet (Swiss Institute of Experimental Cancer Research, Epalinges, Switzerland). MyD88–/– and the various TLR–/– mice were kindly provided by Dr. S. Akira (Osaka University, Osaka, Japan) to the Microbiology and Tumor Biology Center, Karolinska Institute animal house facility.


   Footnotes
 
1 This work was supported by the European Community QLK2-CT-2002-00846 Grant, the Karolinska Institute, the Swedish Health Insurance Company Arbetsförsäkringsbolaget, the Swedish Cancer Society, and the Swedish Research Council. Back

2 Address correspondence and reprint requests to Dr. Martin E. Rottenberg, Microbiology and Tumorbiology Center, Karolinska Institute, 171 77 Stockholm, Sweden. E-mail address: Martin.Rottenberg{at}mtc.ki.se Back

3 Abbreviations used in this paper: TLR, Toll-like receptor; 2-AP, 2-aminopurine; BMM{phi}, bone marrow-derived macrophage; DC, dendritic cell; {gamma}cR, common cytokine receptor {gamma}{alpha}µµ{alpha}-chain; ICE, IL-1-converting enzyme; IFU, inclusion-forming unit; iNOS, inducible NO synthase; MAPK, mitogen-activated protein kinase; MyD88, myeloid differentiation factor 88; 2'5'OAS, 2'-5'-oligoadenylate synthetase; PKR, RNA-dependent protein kinase; RAG, recombination-activating gene; SPG, sucrose-phosphate-glutamate; WT, wild type. Back

Received for publication November 14, 2003. Accepted for publication March 4, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Takeda, K., T. Kaisho, S. Akira. 2003. Toll-like receptors. Annu. Rev. Immunol. 21:335.[Medline]
  2. Adachi, O., T. Kawai, K. Takeda, M. Matsumoto, H. Tsutsui, M. Sakagami, K. Nakanishi, S. Akira. 1998. Targeted disruption of the MyD88 gene results in loss of IL-1- and IL-18-mediated function. Immunity 9:143.[Medline]
  3. Hemmi, H., T. Kaisho, K. Takeda, S. Akira. 2003. The roles of Toll-like receptor 9, MyD88, and DNA-dependent protein kinase catalytic subunit in the effects of two distinct CpG DNAs on dendritic cell subsets. J. Immunol. 170:3059.[Abstract/Free Full Text]
  4. Hemmi, H., T. Kaisho, O. Takeuchi, S. Sato, H. Sanjo, K. Hoshino, T. Horiuchi, H. Tomizawa, K. Takeda, S. Akira. 2002. Small anti-viral compounds activate immune cells via the TLR7 MyD88-dependent signaling pathway. Nat. Immunol. 3:196.[Medline]
  5. Shinobu, N., T. Iwamura, M. Yoneyama, K. Yamaguchi, W. Suhara, Y. Fukuhara, F. Amano, T. Fujita. 2002. Involvement of TIRAP/MAL in signaling for the activation of interferon regulatory factor 3 by lipopolysaccharide. FEBS Lett. 517:251.[Medline]
  6. Toshchakov, V., B. W. Jones, P. Y. Perera, K. Thomas, M. J. Cody, S. Zhang, B. R. Williams, J. Major, T. A. Hamilton, M. J. Fenton, S. N. Vogel. 2002. TLR4, but not TLR2, mediates IFN-{beta}-induced STAT1{alpha}/{beta}-dependent gene expression in macrophages. Nat. Immunol. 3:392.[Medline]
  7. Karaghiosoff, M., R. Steinborn, P. Kovarik, G. Kriegshauser, M. Baccarini, B. Donabauer, U. Reichart, T. Kolbe, C. Bogdan, T. Leanderson, et al 2003. Central role for type I interferons and Tyk2 in lipopolysaccharide-induced endotoxin shock. Nat. Immunol. 4:471.[Medline]
  8. Oshiumi, H., M. Matsumoto, K. Funami, T. Akazawa, T. Seya. 2003. TICAM-1, an adaptor molecule that participates in Toll-like receptor 3-mediated interferon-{beta} induction. Nat. Immunol. 4:161.[Medline]
  9. Yamamoto, M., S. Sato, K. Mori, K. Hoshino, O. Takeuchi, K. Takeda, S. Akira. 2002. Cutting edge: a novel Toll/IL-1 receptor domain-containing adapter that preferentially activates the IFN-{beta} promoter in the Toll-like receptor signaling. J. Immunol. 169:6668.[Abstract/Free Full Text]
  10. Yamamoto, M., S. Sato, H. Hemmi, K. Hoshino, T. Kaisho, H. Sanjo, O. Takeuchi, M. Sugiyama, M. Okabe, K. Takeda, S. Akira. 2003. Role of adaptor TRIF in the MyD88-independent Toll-like receptor signaling pathway. Science 301:640.[Abstract/Free Full Text]
  11. Williams, B. R. 2001. Signal integration via PKR. Sci. STKE 2001:RE2.
  12. Levy, D. E., J. E. Darnell, Jr. 2002. Stats: transcriptional control and biological impact. Nat. Rev. Mol. Cell Biol. 3:651.[Medline]
  13. Boehm, U., T. Klamp, M. Groot, J. C. Howard. 1997. Cellular responses to interferon-{gamma}. Annu. Rev. Immunol. 15:749.[Medline]
  14. Kuo, C. C., L. A. Jackson, L. A. Campbell, J. T. Grayston. 1995. Chlamydia pneumoniae (TWAR). Clin. Microbiol. Rev. 8:451.[Abstract]
  15. Gupta, S., E. W. Leatham. 1997. The relation between Chlamydia pneumoniae and atherosclerosis. Heart 77:7.[Free Full Text]
  16. Rottenberg, M. E., A. Gigliotti-Rothfuchs, H. Wigzell. 2002. The role of IFN-{gamma} in the outcome of chlamydial infection. Curr. Opin. Immunol. 14:444.[Medline]
  17. Frucht, D. M., T. Fukao, C. Bogdan, H. Schindler, J. J. O’Shea, S. Koyasu. 2001. IFN-{gamma} production by antigen-presenting cells: mechanisms emerge. Trends Immunol. 22:556.[Medline]
  18. Rothfuchs, A. G., D. Gigliotti, K. Palmblad, U. Andersson, H. Wigzell, M. E. Rottenberg. 2001. IFN-{alpha}{beta}-dependent, IFN-{gamma} secretion by bone marrow-derived macrophages controls an intracellular bacterial infection. J. Immunol. 167:6453.[Abstract/Free Full Text]
  19. Prebeck, S., C. Kirschning, S. Durr, C. da Costa, B. Donath, K. Brand, V. Redecke, H. Wagner, T. Miethke. 2001. Predominant role of Toll-like receptor 2 versus 4 in Chlamydia pneumoniae-induced activation of dendritic cells. J. Immunol. 167:3316.[Abstract/Free Full Text]
  20. Sasu, S., D. LaVerda, N. Qureshi, D. T. Golenbock, D. Beasley. 2001. Chlamydia pneumoniae and chlamydial heat shock protein 60 stimulate proliferation of human vascular smooth muscle cells via Toll-like receptor 4 and p44/p42 mitogen-activated protein kinase activation. Circ. Res. 89:244.[Abstract/Free Full Text]
  21. Bulut, Y., E. Faure, L. Thomas, H. Karahashi, K. S. Michelsen, O. Equils, S. G. Morrison, R. P. Morrison, M. Arditi. 2002. Chlamydial heat shock protein 60 activates macrophages and endothelial cells through Toll-like receptor 4 and MD2 in a MyD88-dependent pathway. J. Immunol. 168:1435.[Abstract/Free Full Text]
  22. Netea, M. G., B. J. Kullberg, J. M. Galama, A. F. Stalenhoef, C. A. Dinarello, J. W. Van der Meer. 2002. Non-LPS components of Chlamydia pneumoniae stimulate cytokine production through Toll-like receptor 2-dependent pathways. Eur. J. Immunol. 32:1188.[Medline]
  23. Freudenberg, M. A., T. Merlin, C. Kalis, Y. Chvatchko, H. Stubig, C. Galanos. 2002. Cutting edge: a murine, IL-12-independent pathway of IFN-{gamma} induction by Gram-negative bacteria based on STAT4 activation by type I IFN and IL-18 signaling. J. Immunol. 169:1665.[Abstract/Free Full Text]
  24. Nguyen, K. B., W. T. Watford, R. Salomon, S. R. Hofmann, G. C. Pien, A. Morinobu, M. Gadina, J. J. O’Shea, C. A. Biron. 2002. Critical role for STAT4 activation by type 1 interferons in the interferon-{gamma} response to viral infection. Science 297:2063.[Abstract/Free Full Text]
  25. Mattei, F., G. Schiavoni, F. Belardelli, D. F. Tough. 2001. IL-15 is expressed by dendritic cells in response to type I IFN, double-stranded RNA, or lipopolysaccharide and promotes dendritic cell activation. J. Immunol. 167:1179.[Abstract/Free Full Text]
  26. Durbin, J. E., A. Fernandez-Sesma, C.-K. Lee, T. D. Rao, A. B. Frey, T. M. Moran, S. Vukmanovic, A. Garcia-Sastre, D. E. Levy. 2000. Type I IFN modulates innate and specific antiviral immunity. J. Immunol. 164:4220.[Abstract/Free Full Text]
  27. Ohteki, T., K. Suzue, C. Maki, T. Ota, S. Koyasu. 2001. Critical role of IL-15-IL-15R for antigen-presenting cell functions in the innate immune response. Nat. Immunol. 2:1138.[Medline]
  28. Muller, U., U. Steinhoff, L. F. Reis, S. Hemmi, J. Pavlovic, R. M. Zinkernagel, M. Aguet. 1994. Functional role of type I and type II interferons in antiviral defense. Science 264:1918.[Abstract/Free Full Text]
  29. Huang, S., W. Hendricks, A. Althage, S. Hemmi, H. Bluethmann, R. Kamijo, J. Vilcek, R. Zinkernagel, M. Aguet. 1993. Immune response in mice that lack the interferon-{gamma} receptor. Science 259:1742.[Abstract/Free Full Text]
  30. Meraz, M. A., J. M. White, K. C. Sheehan, E. A. Bach, S. J. Rodig, A. S. Dighe, D. H. Kaplan, J. K. Riley, A. C. Greenlund, D. Campbell, et al 1996. Targeted disruption of the Stat1 gene in mice reveals unexpected physiologic specificity in the JAK-STAT signaling pathway. Cell 84:431.[Medline]
  31. Durbin, J. E., R. Hackenmiller, M. C. Simon, D. E. Levy. 1996. Targeted disruption of the mouse Stat1 gene results in compromised innate immunity to viral disease. Cell 84:443.[Medline]
  32. Kawai, T., O. Adachi, T. Ogawa, K. Takeda, S. Akira. 1999. Unresponsiveness of MyD88-deficient mice to endotoxin. Immunity 11:115.[Medline]
  33. Takeuchi, O., K. Hoshino, T. Kawai, H. Sanjo, H. Takada, T. Ogawa, K. Takeda, S. Akira. 1999. Differential roles of TLR2 and TLR4 in recognition of Gram-negative and Gram-positive bacterial cell wall components. Immunity 11:443.[Medline]
  34. Hoshino, K., O. Takeuchi, T. Kawai, H. Sanjo, T. Ogawa, Y. Takeda, K. Takeda, S. Akira. 1999. Cutting edge: Toll-like receptor 4 (TLR4)-deficient mice are hyporesponsive to lipopolysaccharide: evidence for TLR4 as the Lps gene product. J. Immunol. 162:3749.[Abstract/Free Full Text]
  35. Takeuchi, O., T. Kawai, P. F. Muhlradt, M. Morr, J. D. Radolf, A. Zychlinsky, K. Takeda, S. Akira. 2001. Discrimination of bacterial lipoproteins by Toll-like receptor 6. Int. Immunol. 13:933.[Abstract/Free Full Text]
  36. Hemmi, H., O. Takeuchi, T. Kawai, T. Kaisho, S. Sato, H. Sanjo, M. Matsumoto, K. Hoshino, H. Wagner, K. Takeda, S. Akira. 2000. A Toll-like receptor recognizes bacterial DNA. Nature 408:740.[Medline]
  37. Kuida, K., J. A. Lippke, G. Ku, M. W. Harding, D. J. Livingston, M. S. Su, R. A. Flavell. 1995. Altered cytokine export and apoptosis in mice deficient in interleukin-1{beta} converting enzyme. Science 267:2000.[Abstract/Free Full Text]
  38. Cao, X., E. W. Shores, J. Hu-Li, M. R. Anver, B. L. Kelsall, S. M. Russell, J. Drago, M. Noguchi, A. Grinberg, E. T. Bloom, et al 1995. Defective lymphoid development in mice lacking expression of the common cytokine receptor {gamma} chain. Immunity 2:223.[Medline]
  39. Mombaerts, P., J. Iacomini, R. Johnson, K. Herrup, S. Tonegawa, V. Papaioannou. 1992. RAG-1 deficient mice have no mature T and B lymphocytes. Cell 68:869.[Medline]
  40. Eriksson, E., L. Dons, A. G. Rothfuchs, P. Heldin, H. Wigzell, M. E. Rottenberg. 2003. CD44-regulated intracellular proliferation of Listeria monocytogenes. Infect. Immun. 71:4102.[Abstract/Free Full Text]
  41. Ekman, M. R., J. T. Grayston, R. Visakorpi, M. Kleemola, C. C. Kuo, P. Saikku. 1993. An epidemic of infections due to Chlamydia pneumoniae in military conscripts. Clin. Infect. Dis. 17:420.[Medline]
  42. Rottenberg, M., E. Castaños-Velez, R. Mesquita, O. Goñi Laguardia, P. Biberfeld, A. Örn. 1996. Intracellular colocalization of inducible nitric oxide synthase and Trypanosoma cruzi: evidence for a dual pathway of iNOS induction. Eur. J. Immunol. 26:3203.[Medline]
  43. Siebert, P. D., J. W. Larrick. 1992. Competitive PCR. Nature 359:557.[Medline]
  44. Fantuzzi, G., A. J. Puren, M. W. Harding, D. J. Livingston, C. A. Dinarello. 1998. Interleukin-18 regulation of interferon {gamma} production and cell proliferation as shown in interleukin-1{beta}-converting enzyme (caspase-1)-deficient mice. Blood 91:2118.[Abstract/Free Full Text]
  45. Finco, T. S., A. A. Beg, A. S. Baldwin, Jr. 1994. Inducible phosphorylation of I{kappa}B{alpha} is not sufficient for its dissociation from NF-{kappa}B and is inhibited by protease inhibitors. Proc. Natl. Acad. Sci. USA 91:11884.[Abstract/Free Full Text]
  46. Baeuerle, P. A., D. Baltimore. 1988. I{kappa}B: a specific inhibitor of the NF-{kappa}B transcription factor. Science 242:540.[Abstract/Free Full Text]
  47. Hu, Y., T. W. Conway. 1993. 2-Aminopurine inhibits the double-stranded RNA-dependent protein kinase both in vitro and in vivo. J. Interferon Res. 13:323.[Medline]
  48. Pierce, J. W., R. Schoenleber, G. Jesmok, J. Best, S. A. Moore, T. Collins, M. E. Gerritsen. 1997. Novel inhibitors of cytokine-induced I{kappa}B-{alpha} phosphorylation and endothelial cell adhesion molecule expression show anti-inflammatory effects in vivo. J. Biol. Chem. 272:21096.[Abstract/Free Full Text]
  49. Takeuchi, O., K. Takeda, K. Hoshino, O. Adachi, T. Ogawa, S. Akira. 2000. Cellular responses to bacterial cell wall components are mediated through MyD88-dependent signaling cascades. Int. Immunol. 12:113.[Abstract/Free Full Text]
  50. Darville, T., J. M. O’Neill, C. W. Andrews, Jr, U. M. Nagarajan, L. Stahl, D. M. Ojcius. 2003. Toll-like receptor-2, but not Toll-like receptor-4, is essential for development of oviduct pathology in chlamydial genital tract infection. J. Immunol. 171:6187.[Abstract/Free Full Text]
  51. Sica, A., L. Dorman, V. Viggiano, M. Cippitelli, P. Ghosh, N. Rice, H. A. Young. 1997. Interaction of NF-{kappa}B and NFAT with the interferon-{gamma} promoter. J. Biol. Chem. 272:30412.[Abstract/Free Full Text]
  52. Kojima, H., Y. Aizawa, Y. Yanai, K. Nagaoka, M. Takeuchi, T. Ohta, H. Ikegami, M. Ikeda, M. Kurimoto. 1999. An essential role for NF-{kappa}B in IL-18-induced IFN-{gamma} expression in KG-1 cells. J. Immunol. 162:5063.[Abstract/Free Full Text]
  53. Inohara, N., G. Nunez. 2003. NODs: intracellular proteins involved in inflammation and apoptosis. Nat. Rev. Immunol. 3:371.[Medline]
  54. Girardin, S. E., I. G. Boneca, L. A. Carneiro, A. Antignac, M. Jehanno, J. Viala, K. Tedin, M. K. Taha, A. Labigne, U. Zathringer, et al 2003. Nod1 detects a unique muropeptide from Gram-negative bacterial peptidoglycan. Science 300:1584.[Abstract/Free Full Text]
  55. Chamaillard, M., M. Hashimoto, Y. Horie, J. Masumoto, S. Qiu, L. Saab, Y. Ogura, A. Kawasaki, K. Fukase, S. Kusumoto, et al 2003. An essential role for NOD1 in host recognition of bacterial peptidoglycan containing diaminopimelic acid. Nat. Immunol. 4:702.[Medline]