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Originally published online as doi:10.1189/jlb.0508304 on September 17, 2008

Published online before print September 17, 2008
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(Journal of Leukocyte Biology. 2008;84:1585-1593.)
© 2008 by Society for Leukocyte Biology

Induction of iNOS by Chlamydophila pneumoniae requires MyD88-dependent activation of JNK

Nuria Rodriguez, Roland Lang, Nina Wantia, Christine Cirl, Tanja Ertl, Susanne Dürr, Hermann Wagner and Thomas Miethke1

Institut für Medizinische Mikrobiologie, Immunologie und Hygiene, Technische Universität München, München, Germany

1 Correspondence: Institute of Medical Microbiology, Immunology and Hygiene, Technical University of Munich, Trogerstr. 30, 81675 Munich, Germany. E-mail: thomas.miethke{at}lrz.tum.de


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ABSTRACT
 
Innate immune cells produce NO via inducible NO synthase (iNOS) in response to certain infections or upon stimulation with cytokines such as IFN-{gamma} and TNF. NO plays an important role in host defense against intracellular bacteria including Chlamydophila pneumoniae as a result of its microbicidal activity. In MyD88-deficient mice, which succumb to C. pneumoniae infection, iNOS induction is impaired 6 days postinfection, although pulmonary levels of IFN-{gamma} and TNF are elevated as in wild-type mice at this time-point. Here, we demonstrate that induction of iNOS in macrophages upon C. pneumoniae infection is controlled by MyD88 via two pathways: NF-{kappa}B activation and phosphorylation of the MAPK JNK, which leads to the nuclear translocation of c-Jun, one of the two components of the AP-1 complex. In addition, phosphorylation of STAT1 and expression of IFN regulatory factor 1 (IRF-1) were delayed in the absence of MyD88 after C. pneumoniae infection but not after IFN-{gamma} stimulation. Taken together, our data show that for optimal induction of iNOS during C. pneumoniae infection, the concerted action of the MyD88-dependent transcription factors NF-{kappa}B and AP-1 and of the MyD88-independent transcription factors phosphorylated STAT1 and IRF-1 is required.

Key Words: bacterial infection • immune response • IFN-{gamma} • Toll-like receptors • MAPK


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INTRODUCTION
 
One of the fastest and most effective defense mechanisms against infections is the production of the free radical NO by the immune system, which is mediated by the regulated expression of inducible NO synthase (iNOS) [1 ]. NO functions as an intercellular and intracellular signaling molecule, and as a result of its chemical properties, it is able to diffuse easily across cell membranes and between cells. In contrast to the other isoforms producing NO, iNOS expression is inducible and Ca2+-calmodulin-independent [2 , 3 ]. The enzyme produces high levels of NO in a wide variety of cell types and is regulated primarily at the transcriptional level [4 5 6 7 ].

The murine iNOS promoter has been studied extensively. It contains several binding sites for transcription factors involved in its regulation through mechanisms that require strict control, as an overproduction of NO might cause abnormal inflammation and cellular damage. So far, two NF-{kappa}B-binding sites have been identified, one of which is involved directly in iNOS induction after LPS stimulation, and deletion of this site abolished iNOS transcription [8 ]. Additionally, IFN-{gamma} synergistically enhances the induction of iNOS by LPS or other stimuli [5 , 9 ]. Similarly, TNF and IFN-{gamma} activate iNOS in a synergistic manner [10 ]. This is driven by the interaction of IFN regulatory factor 1 (IRF-1) and phosphorylated (p)-STAT1, with their corresponding binding sites IFN-stimulated response element and {gamma}-activated sequence, respectively, both located in the enhancer region of the promoter [11 ]. An AP-1-binding site was described to be highly relevant in the regulation of a human [12 , 13 ] and murine iNOS promoter [14 ], as pretreatment with MAPK inhibitors reduced iNOS transcription dramatically [12 , 15 ].

Chlamydophila pneumoniae is an obligate intracellular bacterium that causes pneumonia and bronchitis in humans and mice [16 17 18 ]. Infection with Chlamydia induces a strong Th1 response marked by the secretion of cytokines such as IL-12, TNF, and IFN-{gamma} [18 19 20 21 ]. In vitro, it has been shown that TNF and IFN-{gamma} act in synergism to inhibit C. pneumoniae replication [22 ], and these cytokines are involved in controlling acute lung infection [23 ]. In particular, IFN-{gamma} plays an important role in defense against acute infection by this microorganism, as IFN-{gamma}–/–- or IFN-{gamma}R–/–-infected mice displayed higher levels of bacterial burden in the lungs in comparison with controls [24 ]. IFN-{gamma} induces iNOS in vivo and also in bone marrow-derived macrophages (BMDM) after chlamydial infection [24 , 25 ]. The effects of IFN-{gamma} leading to inhibition of chlamydial growth are not restricted to induction of iNOS [26 ] but also include induction of indoleamine 2,3-dioxygenase, which limits the availability of tryptophan [27 ] and deprivation of an intracellular iron reservoir [28 ]. The importance of iNOS to restrict replication of C. pneumoniae in vivo was shown by infecting iNOS-deficient mice [24 ]. Thus, IFN-{gamma}R–/– mice or iNOS–/– showed higher pulmonary levels of chlamydial mRNA in comparison with infection of wild-type (WT) mice, although the phenotype was stronger in IFN-{gamma}R–/– mice, underlining the pleiotropic effects of IFN-{gamma}.

Our previous work demonstrated that dendritic cells recognize C. pneumoniae via TLR2 and to a minor extent, via TLR4 [29 ]. In vivo, TLR2–/–/TLR4d/d and MyD88-deficient mice succumb to the infection, although pulmonary TNF and IFN-{gamma} levels are increased as in WT mice at Day 6 postinfection [20 , 21 ]. As expression of IFN-{gamma} is not sufficient for efficient control of chlamydial growth in MyD88–/– mice, we considered an impaired IFN-{gamma} responsiveness as causative. We therefore dissected the MyD88-dependent regulation of the critical effector protein iNOS in response to C. pneumoniae infection. This report analyzes in detail the MyD88- and IFN-{gamma}-dependent contribution for iNOS induction upon infection with C. pneumoniae.


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MATERIALS AND METHODS
 
Mice
C57BL/6 and C3H/HeN mice were purchased from Harlan Winkelmann GmbH (Borchen, Germany). Breeding pairs of MyD88–/– mice were kindly provided by Dr. Shizuo Akira (Department of Host Defense, Research Institute for Medical Disease, Osaka University, Osaka, Japan) and were backcrossed 10x to C57BL/6 mice. TLR2–/–/TLR4d/d mice were 6x backcrossed to C3H/HeN mice. The different strains were bred in the animal facility at the Institute of Medical Microbiology, Immunology and Hygiene, Technical University of Munich (Germany).

Reagents, antibodies, and recombinant cytokines
Calbiochem/Merck (Germany) provided the p38 inhibitor (SB203580), MEK1/2 inhibitor (U0126), and p-JNK inhibitor (SP600125). The peroxidase-conjugated AffiniPure F(ab')2 fragment donkey anti-rabbit IgG (H+L) and peroxidase-conjugated AffiniPure F(ab')2 fragment goat anti-mouse IgG + IgM (H+L) were purchased from Dianova (Germany), and the mAb to β-actin was provided by Sigma-Aldrich (Germany). The mAb to IRF-1 (M-20): sc-640, NF-{kappa}B p65 (C-20): sc-372, c-Jun (D): sc-45, and c-Fos (K-25): sc-253 were provided by Santa Cruz Biotechnology (Santa Cruz, CA, USA). p-STAT1 (Tyr701) antibody as well as antibodies specific for p-p38, p-ERK, and p-JNK were delivered by Cell Signaling Technology (Beverly, MA, USA), and the mAb specific for iNOS was purchased from Upstate Millipore (Germany). Murine IFN-{gamma} (315-05) and murine TNF (315-01A) were purchased from Peprotech Inc. (Rocky Hill, NJ, USA).

Multiplication and purification of C. pneumoniae
C. pneumoniae CM-1 (VR-1360; American Type Culture Collection, Manassas, VA, USA) were multiplied according to Maass and Harig [30 ]. Chlamydial elementary bodies were centrifuged (2000 g, 35 min, 35°C) on confluent monolayers of HEp-2 cells in the presence of cycloheximide (1 µg/ml) and 0% FBS. After 72 h of culture, the harvested cells were disrupted with glass beads, and chlamydial elementary bodies were purified with a sucrose urografin gradient (bottom layer, 50% w/v sucrose solution; top layer, 30% v/v urografin in 30 mM Tris-HCl buffer, pH 7.4) at 9000 g and 4°C for 60 min. After one wash step with 0.2 µm-filtered PBS (pH 7.4), purified elementary bodies were stored in 0.22 M sucrose, 8.6 mM Na2HPO4, 3.8 mM KH2PO4, 5 mM glutamic acid, 0.2 µm-filtered, pH 7.4 (SPG buffer), at –80°C until use. To quantify the number of elementary bodies, HEp-2 cells were infected and stained with the chlamydia-specific antibody (ACI-FITC, Progen Biotechnik GmbH, Heidelberg, Germany). The number of inclusion-forming units (IFU) was counted as determined by fluorescence microscopy (Carl Zeiss Jena, Göttingen, Germany) 48 h after infection. For control, noninfected HEp-2 cells were treated in the same way. Contamination with mycoplasma was excluded regularly by Mycoplasma-PCR using specific primers (MWG Biotech, Martinsried, Germany).

Isolation and differentiation of BMDM
BMDM were generated according to Rutschman et al. [31 ]. Briefly, femora and tibiae of mice were rinsed with cell culture medium applied through a 26-gauge syringe. BM cells were cultured in petri dishes at a density of 5 x 106 cells/dish in the presence of L cell-conditioned medium as a source of M-CSF-1. The medium used was low endotoxin DMEM (PAA Laboratories GmbH, Austria) supplemented with 10% FBS (Biochrom AG, Germany), 2-ME (50 µM; Life Technologies, Germany), and the antibiotics vancomycin and gentamicin, both provided by Sigma-Aldrich. Cells were washed vigorously, and only adherent macrophages were used 6–7 days after plating. FACS analysis showed that these BMDM were CD45+, F4/80+, CD11b+, and CD11c– as described previously [20 ].

Isolation and infection of pulmonary cells with C. pneumoniae
Pulmonary cells were isolated by collagen digestion as described previously [21 ]. The cells were incubated in DMEM 10% FBS for 24 h at 37ºC. Thereafter, cells were washed with DMEM 0% FBS, and adherent cells were infected or mock-infected with C. pneumoniae at a multiplicity of infection (MOI) of 10 by centrifugation (3000 rpm, 35 min, 35°C). After 24 h, cells were stimulated or not with 10 ng/ml IFN-{gamma} for another 24 h.

C. pneumoniae infection and cytokine stimulation of BMDM
BMDM were plated in M-12 cell culture dishes at a density of 0.75 x 106 cells/well in 1 ml fresh medium containing 10% FBS. One day later, BMDM were stimulated with 30 ng/ml murine TNF or not and infected or mock-infected with C. pneumoniae at a MOI of 10 in DMEM 0% FBS for 24 h at 37°C. Twenty-four hours later, TNF-stimulated cells received a second dose of the cytokine. If cells were stimulated with murine IFN-{gamma} (10 ng/ml), the cytokine was added 24 h postinfection or addition of the first TNF dose.

Cell lysis, SDS-PAGE, and Western blotting
Cell extracts were prepared for Western blotting using radioimmunoprecipitation assay buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 1 mM EDTA, 0.1% SDS), which was supplemented with sodium Orto-Vanadat (Sigma-Aldrich) and a protease inhibitor cocktail (Roche Diagnostics GmbH, Germany). Cell lysates were clarified by sonication and centrifugation before electrophoresis. Nucleic lysates were prepared using a commercial kit (Nuclei EZ Prep nuclei isolation kit, Sigma-Aldrich) following the instructions of the manufacturers. Proteins were separated by SDS-PAGE (10% acrylamid) in TANK buffer (25 mM Tris, 0.2 M glycine, 0.1% SDS) using Laemmli buffer (62.5 mM Tris, 50% glycerol, 2% SDS, 2 mM EDTA) for sample loading. After transfer to the nitrocellulose membrane by semidry electroblotting for 1.5 h (2 mA/cm2) in transfer buffer (25 mM Tris, 250 mM glycine, 20% methanol, 0.35% SDS), membranes were blocked in TBST (2.4 g/l Tris, 8 g/l NaCl, 0.1% Tween, pH 7.6, containing 5% milk powder, 2 h, room temperature). Thereafter, membranes were incubated with antibodies specific for iNOS (diluted 1:1000), p-STAT1 (diluted 1:800), IRF-1 (diluted 1:500), p-p38, anti-p-ERK, or p-JNK (the last three were diluted 1:1000). All primary antibodies were incubated overnight at 4°C. After three washing steps with TBST, the secondary antibody was added (diluted 1:8000 in TBST containing 5% milk powder, 2 h, room temperature). The blot was washed again three times with TBST and visualized using the Western LightningTM chemiluminescence reagent (Perkin Elmer LAS Inc., North Billerica, MA, USA) as described by the manufacturer.

Infection of mice with C. pneumoniae
Mice were infected intranasally with 2.5 x 106 IFU of the microorganism as described before [21 ]. The lungs were removed after 3 and 6 days postinfection, and a total lysate was prepared as described above. The experiments were performed with the permission of local authorities (Regierung von Oberbayern, Germany; File Number 55.2-1-54-2531-59-06).

Nitrite measurement
Nitrite production was measured from WT and MyD88-deficient BMDM supernatants. Briefly, the cells were cultured in 12-well plates in 1 ml culture medium until confluence. The cells were treated with IFN-{gamma}, TNF or C. pneumoniae for the time indicated, and the culture supernatants were collected. Nitrite was measured by adding 50 µl Griess reagent (1% sulfanilamide and 0.1% naphthylethylenediamide in 5% phosphoric acid) to 50 µl samples of culture medium. The OD at 550 nm (OD550) was measured using a microplate reader and the nitrite concentration, calculated by comparison with the OD550 produced using standard solutions of sodium nitrite in the culture medium.


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RESULTS
 
iNOS expression is impaired in MyD88–/– mice upon infection with C. pneumoniae
To test whether MyD88–/– mice are impaired to induce iNOS upon infection with C. pneumoniae, we analyzed the expression of the enzyme in whole lung lysates from WT and MyD88–/– mice by Western blot at different time-points postintranasal infection (Fig. 1 ). iNOS was induced slightly in WT lung lysates at Day 3 postinfection. However, at Day 6, a strong expression in the lungs of WT mice was noticed. In contrast, iNOS was not detectable in lungs of MyD88–/– mice on Day 3 but was induced after 6 days, although at a much lower level in comparison with WT lungs. These data correlated with NO levels obtained from the supernatant of minced lungs (data not shown).


Figure 1
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  		Figure 1. Induction of iNOS in lungs from infected MyD88–/– mice is delayed. WT and MyD88–/– mice were infected intranasally with 2.5 x 106 of C. pneumoniae. Lungs were taken after 3 and 6 days postinfection, and total lysates were performed to detect iNOS by Western blotting. β-Actin was used as a loading control. This experiment was done twice with the same result. The density of each band for iNOS was quantified by ImageJ 1.37v (National Institutes of Health, Bethesda, MD, USA; http://rsb.info.nih.gov/ij/). Values are reported as the ratio between the relative density units (RDU) of the iNOS band and the corresponding β-actin band.

Impaired induction of iNOS in MyD88–/–, TLR2–/–/TLR4d/d, and TLR2–/– but not in TLR4d/d BMDM after C. pneumoniae infection
To study the induction of iNOS in more detail and to reduce the complexity of the model system, we examined the expression of iNOS in BMDM after infection with C. pneumoniae. iNOS was induced in WT BMDM postinfection, as it has already been described [25 ]. However, it was severely impaired in TLR2–/– or TLR2–/–/TLR4d/d and abolished in MyD88–/– BMDM (Fig. 2A ). Similar findings were obtained in lung cells isolated from WT and MyD88–/– mice, although in these cells, infection with the bacterium alone was not sufficient to induce iNOS (Fig. 2C) . In contrast, TLR4d/d macrophages showed an increased iNOS expression after infection (Fig. 2A) . Only a marginal or no induction of iNOS was observed after 24 h of IFN-{gamma} stimulation in any of the BMDM tested. However, we observed a synergistic effect on iNOS expression after infection with C. pneumoniae and IFN-{gamma} stimulation in WT cells, although induction was still poor in TLR2–/–, TLR2–/–/TLR4d/d, and MyD88–/– BMDM under these conditions (Fig. 2A) . The amount of NO generated by the different BMDM mirrored essentially the induced iNOS levels (Fig. 2B) .


Figure 2
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  		Figure 2. Impaired iNOS expression and NO secretion after C. pneumoniae infection in the absence of TLR2-MyD88 signaling. (A) iNOS was detected by Western blot from lysates of WT BMDM or BMDM deficient for TLR4, TLR2, TLR2/TLR4, and MyD88. Cells were untreated (–), stimulated with 10 ng/ml IFN-{gamma} for 24 h, or infected for 48 h with C. pneumoniae (CP) at a multiplicity of infection (MOI) of 10. Additionally, cells were first infected with C. pneumoniae and after 24 h, treated for an additional 24 h with IFN-{gamma}. β-Actin was used as a loading control. (B) Supernatants of BMDM cultures treated identically as described in A were used to determine NO concentrations. Error bars represent SD of three individual cultures. (C) iNOS detection by Western blot from lysates of WT or MyD88–/– pulmonary cells, which were treated as described in A. β-Actin was used as a loading control. Ratios of RDU values of iNOS and corresponding β-actin bands were determined as described in Figure 1 .

MyD88 dependence of transcription factors involved in iNOS regulation
To understand the reasons why BMDM, deficient for TLR2, TLR2/TLR4, or MyD88, were not able to induce iNOS expression after C. pneumoniae infection, we first analyzed the role of NF-{kappa}B, and NF-{kappa}B activation was indirectly determined via I{kappa}B degradation. The protein was degraded in WT BMDM after 5–15 min of C. pneumoniae infection. However, no degradation was observed in MyD88–/– BMDM under these conditions (Fig. 3 ). Similar results were obtained from BMDM lacking TLR2 or TLR2 and TLR4 (data not shown). Consequently, an increase of NF-{kappa}B p65 in the nucleus was observed after 45 min of C. pneumoniae infection only in WT BMDM (see Go Go Fig. 6A ).


Figure 3
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  		Figure 3. Degradation of I{kappa}B in MyD88–/–-deficient macrophages after C. pneumoniae infection. BMDM from WT or MyD88–/–mice were infected with C. pneumoniae (MOI=10) in 0% FBS medium containing cycloheximide. Lysates were taken at different time-points, and I{kappa}B degradation was evaluated by Western blotting. β-Actin served as a loading control. Ratios of RDU values of I{kappa}B and corresponding β-actin bands were determined as described in Figure 1 .


Figure 4
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  		Figure 4. Induction of the transcription factors p-STAT1 and IRF-1 in WT and MyD88–/– BMDM after IFN-{gamma} stimulation and/or C. pneumoniae infection. (A) WT or MyD88–/– BMDM were untreated (Mock) or infected with C. pneumoniae (MOI=10) or stimulated with IFN-{gamma} (10 ng/ml) at different time-points. p-STAT1 and IRF-1 were detected by Western blotting. β-Actin was used as a loading control. (B) WT or MyD88–/– BMDM were untreated (–), stimulated with 10 ng/ml IFN-{gamma} for 24 h, or infected for 48 h with C. pneumoniae at a MOI of 10. Additionally, cells were first infected with C. pneumoniae and after 24 h, treated for an additional 24 h with IFN-{gamma}. β-Actin was used as a loading control. Ratios of RDU values of IRF-1 or p-STAT1 and corresponding β-actin bands were determined as described in Figure 1 .


Figure 5
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  		Figure 5. iNOS expression is impaired in IRF-1–/– BMDM after C. pneumoniae infection. WT or IRF-1–/– BMDM were untreated (–), stimulated with 10 ng/ml IFN-{gamma} for 24 h, or infected for 48 h with C. pneumoniae (MOI=10). Additionally, cells were first infected with C. pneumoniae and after 24 h, treated for an additional 24 h with IFN-{gamma}. iNOS was detected by Western blotting, and β-actin was used as control. Ratios of RDU values of iNOS and corresponding β-actin bands were determined as described in Figure 1 .


Figure 6
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  		Figure 6. Nuclear levels of NF-{kappa}B and induction of iNOS in WT and MyD88–/– BMDM after C. pneumoniae infection and TNF or IFN-{gamma} stimulation. (A) Nuclear extracts were prepared from untreated cells or cells stimulated or costimulated with TNF (30 ng/ml) and IFN-{gamma} (10 ng/ml) or infected with C. pneumoniae (MOI=10) for 45 min. Western blotting was used to detect intranuclear levels of NF-{kappa}B p65. Histone detection was used as a loading control. Ratios of RDU values of NF-{kappa}B and corresponding histone bands were determined as described in Figure 1 . (B) Costimulation of TNF with IFN-{gamma} but not with C. pneumoniae induces iNOS expression in MyD88–/– BMDM. Cells were untreated (–) or stimulated with 10 ng/ml TNF (*) or 30 ng/ml TNF or 10 ng/ml IFN-{gamma} or infected with C. pneumoniae (MOI=10) or stimulated with combinations as indicated in the graph (+). TNF was added to the culture once every 24 h during 48 h. Cells were infected with C. pneumoniae for 48 h, and IFN-{gamma} was added for the last 24 h. iNOS expression was detected by Western blotting, and β-actin detection was used as a loading control. Ratios of RDU values of iNOS and corresponding β-actin bands were determined as described in Figure 1 . (C) NO secretion by WT or MyD88–/– BMDM after C. pneumoniae infection or TNF or IFN-{gamma} stimulation. Supernatants of cultures that were treated as described in B were tested for the presence of NO. Error bars represent SD of three individual cultures.

Besides NF-{kappa}B, other transcription factors such as AP-1, p-STAT1, and IRF-1 have been described to be involved in iNOS induction [15 , 32 ]. To test whether levels of p-STAT1 and IRF-1 differed in WT and MyD88–/– BMDM after infection with C. pneumoniae or IFN-{gamma} stimulation, we analyzed these proteins by immunoblot at different time-points. We observed that p-STAT1 was weakly induced in WT cells after 3 h of infection but not in MyD88–/– cells (Fig. 4A ). Forty-eight hours postinfection, p-STAT1 levels were clearly elevated in WT BMDM but again, weaker in MyD88–/– BMDM (Fig. 4B) . No substantial differences were observed in p-STAT1 expression after IFN-{gamma} stimulation in both groups of macrophages (Fig. 4 A and B) , similar to what has been described elsewhere [33 , 34 ].

IRF-1 expression was delayed in MyD88–/– BMDM within the first 3 h of infection with C. pneumoniae in comparison with WT macrophages (Fig. 4A) . However, later on, IRF-1 was detectable at comparable levels in WT and MyD88–/– BMDM (Fig. 4B) . With the possible exception of the earliest time-point tested (30 min), IFN-{gamma} stimulation induced approximately equal amounts of IRF-1 in both genotypes (Fig. 4 A and B) . We also verified that IRF-1 was translocated to the nucleus after C. pneumoniae infection and/or IFN-{gamma} stimulation (data not shown), concluding that the IFN-{gamma} signaling pathway was not affected in MyD88–/– cells.

To define the relevance of IRF-1 for the induction of iNOS upon infection with C. pneumoniae, IRF-1–/– BMDM were analyzed. As shown in Figure 5 , IRF-1–/– BMDM were not able to induce the enzyme after infection or were impaired severely upon additional IFN-{gamma} stimulation, indicating that IRF-1 is required for the induction of iNOS as described earlier [35 ].

Costimulation of MyD88–/– BMDM with TNF and IFN-{gamma} partially restores iNOS induction
C. pneumoniae efficiently induced nuclear recruitment of NF-{kappa}B in a MyD88-dependent manner (Fig. 6A ). To understand whether the main reason for the impaired iNOS expression in MyD88–/– BMDM was a lack of NF-{kappa}B activation upon C. pneumoniae infection, we treated the cells with TNF to activate NF-{kappa}B in a MyD88-independent manner. As expected, TNF stimulation of WT and MyD88–/– BMDM translocated NF-{kappa}B p65 to the nucleus (Fig. 6A) . However, TNF alone was not sufficient to induce iNOS (Fig. 6B) . Costimulation of MyD88–/– macrophages with TNF and IFN-{gamma} allowed the expression of iNOS and the production of NO as efficiently as in WT cells (Fig. 6 B and C) , indicating that the abrogated nuclear translocation of NF-{kappa}B is critical for the lack of iNOS expression in MyD88–/– BMDM. However, costimulation of C. pneumoniae-infected WT cells with IFN-{gamma} and TNF produced much higher NO levels than IFN-{gamma} and TNF alone, and this level could not be achieved by the same treatment in MyD88–/– BMDM (Fig. 6C) , although p-STAT1 and IRF-1 were induced, and the levels did not differ too much from WT cells (Fig. 4B) . Also, infected MyD88–/– macrophages costimulated with TNF failed to induce iNOS or to secrete NO (Fig. 6 B and C) . These observations guided us to evaluate further possible candidates involved in iNOS transcription upon infection with C. pneumoniae.

MyD88-dependent C. pneumoniae-mediated activation of MAPK in BMDM
Activation of the MAPK pathway in macrophages has been associated with pathogen-associated molecular pattern stimulation, resulting in the production of numerous proinflammatory molecules [36 , 37 ]. C. pneumoniae has been shown to induce MAPK activation in endothelial cells [38 ] and also to stimulate phosphorylation of ERK1/2 in mouse RAW macrophages [39 ]. To address the role of C. pneumoniae in the induction of MAPK in BMDM in the absence of MyD88, we measured by Western blotting the presence of p-p38, p-ERK1/2, and p-JNK in cells that were infected with C. pneumoniae or costimulated with IFN-{gamma} or TNF at different time-points.

p-p38, p-ERK1/2, and p-JNK were strongly induced in WT macrophages 1 h postinfection with C. pneumoniae, and the phosphorylation of these three MAPKs was impaired severely in MyD88–/– macrophages (Fig. 7 ). Costimulation with TNF did not change MAPK phosphorylation in infected cells (Fig. 7) . To define which MAPK participates in the regulation of C. pneumoniae-mediated iNOS induction, WT or MyD88–/– BMDM were treated with different MAPK inhibitors just before the infection, and 48 h later, iNOS induction and NO secretion were analyzed. Surprisingly, the addition of the MEK1/2 (U0126, which inhibits phosphorylation of ERK) or p38 MAPK (SB203580) inhibitors to the culture did not impede iNOS induction and NO secretion by WT macrophages in response to C. pneumoniae infection (Fig. 8 A and B ). Rather, the ERK1/2 inhibitor increased NO levels slightly (Fig. 8B) . However, treatment of WT BMDM with the JNK inhibitor SP600125 fully blocked the induction of iNOS and NO secretion (Fig. 8 A and B) , suggesting a main role for this MAPK in iNOS induction upon infection with C. pneumoniae. JNK phosphorylates c-Jun, allowing its translocation to the nucleus. We therefore tested nuclear levels of c-Fos and c-Jun, the two components of the heterodimer AP-1. We observed that both proteins increased after C. pneumoniae infection in WT but not in MyD88–/– BMDM (Fig. 9 , Lanes 5 and 9). We also analyzed whether TNF would enhance the nuclear amount of c-Fos and c-Jun. Although stimulation with TNF alone was sufficient to increase intranuclear expression of c-Fos in WT and MyD88–/– BMDM (Fig. 9 , Lanes 2 and 8), it did not alter nuclear levels of c-Jun in both groups of macrophages (Fig. 9 , Lanes 2 and 8). Additional stimulation with IFN-{gamma} or infection with C. pneumoniae did not substantially change TNF-induced nuclear c-Fos levels (Fig. 9 , Lanes 3 and 9 and 6 and 12).


Figure 7
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  		Figure 7. MAPK induction after IFN-{gamma} or TNF stimulation and/or C. pneumoniae infection in WT and MyD88–/– BMDM. WT or MyD88–/– BMDM were not stimulated (Mock) or stimulated with 30 ng/ml TNF or with 10 ng/ml IFN-{gamma} or with a combination of both cytokines or infected with C. pneumoniae alone or in combination with 30 ng/ml TNF during 1 h. Western blotting from a total cell lysate was used to detect the MAPKs p-ERK, p-JNK, and p-p38. β-Actin was used as a loading control. Ratios of RDU values of p-ERK, p-JNK, or p-p38 and corresponding β-actin bands were determined as described in Figure 1 .


Figure 8
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  		Figure 8. Expression of iNOS and NO secretion induced after C. pneumoniae infection in BMDM is abolished after inhibition of p-JNK. (A) WT BMDM were untreated (Mock) or infected with C. pneumoniae (MOI=10) in the presence of different MAPK inhibitors: the p38 inhibitor SB203580, p-JNK inhibitor SP600125, or MEK1/2 inhibitor U0126. Western blot was used to detect iNOS from total cellular lysates. β-Actin was used as a loading control. Ratios of RDU values of iNOS and corresponding β-actin bands were determined as described in Figure 1 . (B) NO secretion was determined in the culture supernatants from the experiment described in A. Error bars represent SD of three individual cultures.


Figure 9
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  		Figure 9. A MyD88-dependent increase of c-Fos and c-Jun in the nuclei after C. pneumoniae infection. Nuclear extracts were prepared from WT or MyD88–/– BMDM, which were untreated (Mock) or stimulated with 30 ng/ml TNF or with 10 ng/ml IFN-{gamma} or with a combination of both cytokines or infected with C. pneumoniae alone or in combination with 30 ng/ml TNF during 1 h. Western blotting was used to detect c-Fos and c-Jun. Histone was used as a loading control. Ratios of RDU values of c-Fos or c-Jun and corresponding histone bands were determined as described in Figure 1 .


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DISCUSSION
 
This study shows that TLR2–/–, TLR2–/–/TLR4d/d, and MyD88–/– BMDM are impaired to induce iNOS upon infection with C. pneumoniae. For maximal expression of iNOS, four different transcription factors are required. Two of them, NF-{kappa}B and AP-1, depend on MyD88, and IRF-1 and p-STAT1 are induced MyD88-independently, although their expression is delayed in MyD88–/– cells upon infection with C. pneumoniae. Costimulation of uninfected or infected, MyD88-deficient cells with TNF and IFN-{gamma} allows the induction of iNOS but only at a submaximal level. For maximal expression of iNOS and NO secretion upon infection with C. pneumoniae, additional activation of JNK is required.

Rottenberg et al. [24 ] showed the relevance of iNOS in pulmonary infection caused by C. pneumoniae. This group published that C. pneumoniae replicates to higher levels in iNOS–/– mice compared with WT mice. iNOS was also relevant in a genital tract infection model with Chlamydia trachomatis, as iNOS–/– mice were sicker than WT mice, although the recovery of culturable chlamydia did not differ between both mouse strains [40 ].

We have shown previously that MyD88–/– and TLR2–/–/TLR4d/d mice are unable to confine replication of C. pneumoniae and die, despite displaying increased pulmonary levels of IFN-{gamma} [20 , 21 ]. As this cytokine is required to control replication of C. pneumoniae in vivo [41 ], we speculated that IFN-{gamma}-mediated effects in MyD88–/– or TLR2–/–/TLR4d/d cells were impaired. According to a recent study, MyD88 is needed for the stabilization of IFN-{gamma}-induced mRNA transcripts such as TNF and IFN-inducible protein 10 (IP-10) [33 ]. In accordance with these observations, we found that after 3 days of C. pneumoniae infection, mRNA levels of IP-10 in the lungs of MyD88–/– mice were reduced significantly in comparison with WT mice [42 ]. In addition, TNF mRNA and protein levels were decreased in lungs from MyD88–/– mice after 3 days of infection [20 , 42 ]. However, IFN-{gamma} induced phosphorylation of STAT1 and expression of IRF-1 in MyD88–/– BMDM (Fig. 4A) , indicating that proximal IFN-{gamma} signaling and downstream gene expression are not crucially impaired.

Based on the fact that iNOS reduces disease severity caused by chlamydial infections, we studied the transcriptional regulation of this IFN-{gamma}-dependent gene in detail in MyD88–/– and WT BMDM. In contrast to WT BMDM, MyD88–/– but also TLR2–/– and TLR2–/–/TLR4d/d BMDM were impaired severely to induce iNOS upon infection with C. pneumoniae. The addition of IFN-{gamma} only slightly increased the ability of the gene-deficient cells to up-regulate iNOS expression (Fig. 2A) , although the cytokine efficiently induced the phosphorylation of STAT1 and the expression of IRF-1 in MyD88–/– BMDM (Fig. 4 A and B) . In infected WT pulmonary cells, IFN-{gamma} was required for the expression of iNOS (Fig. 2C) , but the cytokine was again ineffective in MyD88–/– pulmonary cells. Taken together, these findings indicate that the TLR signaling pathway contributes significantly to the regulation of iNOS expression. Our further studies revealed that the degradation of I{kappa}B in BMDM is impaired severely upon infection with C. pneumoniae if the cells lack TLR2 or MyD88. Stimulation of MyD88–/– BMDM with TNF successfully restored nuclear translocation of NF-{kappa}B but on its own, was still not sufficient to increase iNOS expression. Only costimulation of the cells with TNF and IFN-{gamma} allowed expression of iNOS and secretion of NO, indicating that NF-{kappa}B is required together with IRF-1 and p-STAT1. However, NO levels were never as high as the ones induced by infection with C. pneumoniae and costimulation with IFN-{gamma}. Thus, for a basal induction of iNOS, the transcription factors NF-{kappa}B, p-STAT1, and IRF-1 are required. As MyD88–/– BMDM were impaired severely to activate the MAPKs ERK1/2, JNK, and p38 upon infection with C. pneumoniae and as the promoter of iNOS contains binding sites for AP-1 complexes [43 ], we speculated that the activation of MAPK was additionally required for optimal iNOS induction. Indeed, we observed that treatment of the infected cells with the JNK inhibitor SP600125 completely abrogated the induction of iNOS and NO secretion (Fig. 8) . These findings indicate that induction of JNK is the second MyD88-dependent signal besides NF-{kappa}B, which is required for high-level NO secretion.

The latter conclusion is also supported by our observation that infection with C. pneumoniae translocated c-Fos and c-Jun to the nucleus of WT but not of MyD88–/– BMDM. Furthermore, stimulation with TNF increased nuclear levels of c-Fos but not of c-Jun in both groups of macrophages. Although activation of JNK and p38 MAPK has been described after stimulation of BMDM with TNF [44 , 45 ], we were not able to detect activation of MAPK in WT or MyD88–/– BMDM after TNF stimulation during the first hour or later (Fig. 7 , and data not shown). Therefore, we conclude that upon infection with C. pneumoniae, only TLR signals are able to translocate c-Jun to the nucleus for maximal iNOS induction.

The relevance of IRF-1 in our model was shown by using IRF-1–/– BMDM. We confirmed that IRF-1 is crucial for iNOS transcription after IFN-{gamma} stimulation [35 ] but also after C. pneumoniae infection. In addition, it seems that the level of this transcription factor is critical to produce iNOS in the absence of the AP-1 complex. Induction of IRF-1 and p-STAT1 is much higher after stimulation with IFN-{gamma} compared with infection with C. pneumoniae (Fig. 4A) . Thus, stimulation of MyD88–/– BMDM with TNF and C. pneumoniae induces NF-{kappa}B but low levels of IRF-1 and p-STAT-1, which are not sufficient to induce iNOS. In contrast, costimulation of MyD88–/– BMDM with TNF and IFN-{gamma} also activates NF-{kappa}B but induces high levels of IRF-1 and p-STAT1, a condition that allows induction of iNOS (Fig. 6 B and C) .

p-STAT1 levels were slightly lower in infected MyD88–/– BMDM for reasons that are still unclear. As published recently, suppressor of cytokine signaling 1 (SOCS1) negatively regulates STAT1 phosphorylation and iNOS expression [46 ]. Whether increased expression of SOCS1 in C. pneumoniae-infected MyD88–/– BMDM explains delayed STAT1 phosphorylation remains to be seen. We also observed a delay of IRF-1 expression in MyD88–/– macrophages within the first 3 h upon infection with C. pneumoniae (Fig. 4A) , but at later time-points postinfection, these differences vanished (Fig. 4B) . The slight delay in IRF-1 expression in MyD88–/– macrophages might be explained by a lack of NF-{kappa}B, as the IRF-1 promoter also contains a binding site for this transcription factor (Fig. 4A) .

IFN-{gamma} is produced primarily by T and NK cells [47 ]. However, it has been reported that C. pneumoniae-induced IFN-{alpha}β enables macrophages to produce IFN-{gamma} in an autocrine/paracrine manner [25 ]. For unknown reasons, we could not confirm that C. pneumoniae-infected BMDM secreted detectable levels of IFN-{gamma} (data not shown), but others [48 ] questioned the concept of autocrine IFN-{gamma} secretion by macrophages. Furthermore, it was reported that IFN-{alpha}/β induces the expression of iNOS in an IFN-{gamma}-independent manner [25 ]. As type I IFNs are induced via the adaptor molecule, Toll/IL-1R-containing adaptor molecule-inducing IFN-β, secretion of these cytokines should be normal in MyD88–/– cells.

In summary, we have demonstrated that C. pneumoniae induces high-level expression of iNOS and NO secretion through the activation of four transcription factors: p-STAT1, IRF-1, AP-1, and NF-{kappa}B. In vitro, the activation of the former two transcription factors depends on IFN-{gamma} in a MyD88-independent manner, and the latter two are entirely MyD88-dependent. Finally, high-level induction of iNOS requires the activation of JNK.


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ACKNOWLEDGEMENTS
 
The authors thank Dr. S. Akira (Osaka, Japan) for providing MyD88–/– mice.

Received May 16, 2008; revised August 4, 2008; accepted August 20, 2008.


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