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Nod1-Mediated Endothelial Cell Activation by Chlamydophila pneumoniae

Originally publishedhttps://doi.org/10.1161/01.RES.0000155721.83594.2cCirculation Research. 2005;96:319–326

Abstract

Seroepidemiological and animal studies, as well as demonstration of viable bacteria in atherosclerotic plaques, have linked Chlamydophila pneumoniae infection to development of chronic vascular lesions and coronary heart disease. Inflammation and immune responses are dependent on host recognition of invading pathogens. The recently identified cytosolic Nod proteins are candidates for intracellular recognition of bacteria, such as the obligate intracellular chlamydia. In the present study, mechanisms of endothelial cell activation by C. pneumoniae via Nod proteins were examined. Viable, but not heat-inactivated, chlamydia activated human endothelial cells, suggesting that invasion of these cells is necessary for their profound activation. Endothelial cells express Nod1. Nod1 gene silencing by small interfering RNA reduced C pneumoniae–induced IL-8 release markedly. Moreover, in HEK293 cells, overexpressed Nod1 or Nod2 amplified the capacity of C pneumoniae to induce nuclear factor κB (NF-κB) activation. Interestingly, heat-inactivated bacteria were still able to induced a NF-κB reporter gene activity via Nod proteins when transfected intracellularly, but not when provided from the extracellular side. In contrast, TLR2 sensed extracellular heat-inactivated chlamydia. In conclusion, we demonstrated that C pneumoniae induced a Nod1-mediated and Nod2-mediated NF-κB activation in HEK293 cells. In endothelial cells, Nod1 played a dominant role in triggering a chlamydia-mediated inflammatory process.

Chlamydia are Gram-negative bacteria that are obligate intracellular parasites of eukaryotic cells. Chronic or recurrent infections with Chlamydophila (Chlamydia) pneumoniae have been associated with the development of vascular lesions and atherosclerosis.1–4 Chronic infection of susceptible target cells (monocytes, macrophages, endothelial cells, fibroblasts, and smooth muscle cells), as well as direct activation of endothelial cells, may initiate and perpetuate a local inflammation by inducing cytokine release (eg, IL-6, IL-8, etc), and increased expression of adhesion molecules (P-selectin/E-selectin, ICAM [intercellular adhesion molecule]-1, VCAM [vascular cell adhesion molecule]-1), which subsequently results in increased adhesion of leukocytes or platelets to the endothelium.5,6 Although chlamydiae may reside and replicate in different cell types and induce a chronic immune activation, little is known about the mechanisms of C pneumoniae–induced target cell alteration. The field is troubled by the “hen versus egg” problem and causative proof is difficult because there are no good markers to differentiate among new versus old (IgM versus IgG), as well as acute versus chronic persistent (IgM versus IgA), C pneumoniae infection. Chlamydia are able to persist in infected target cells. These persistent bacteria are resistant against almost all of the known chlamydia-targeting antibiotics (macrolides, tetracycline).7 Therefore, all studies (like WIZARD or PROVE-IT) to evaluate the importance of chlamydial infections for the development of vascular diseases via the use of antibiotics are unlikely to obtain useful results.

The innate immune system relies on surveillance proteins to recognize pathogens by sensing pathogen-associated molecular patterns. A well-studied group of pattern recognition receptors are the Toll-like receptors (TLRs), which are mainly expressed on the surface of a broad diversity of cells. TLR2 and TLR4 have been demonstrated to mediate host defense against C pneumoniae or chlamydial components.8–11 The recently identified nucleotide-binding oligomerization domain (Nod) proteins, also called caspase recruitment domain-containing proteins, are molecules that have been implicated in intracellular pattern recognition.12,13 More than 20 proteins that are homologues to Nod1 have been identified in the human genome, but only a few members of this growing family are functionally characterized.13 So far, Nod1 has been described to mediate the activation of nuclear factor κB (NF-κB) induced by peptidoglycans containing meso-diaminopimelate acid found mainly in Gram-negative bacteria,14,15 whereas Nod2 (caspase recruitment domain 15) mediates responsiveness to the muramyldipeptide MurNAc-l-Ala-d-isoGln conserved in peptidoglycans of basically all bacteria.16,17 Little is known about the Nod-dependent signaling cascade activated by ligand-binding. There is evidence that oligomerization of Nod1 (and Nod2) induces the recruitment of its interacting partner Rip2 kinase (RICK or CARDIAK). Subsequent activation of NF-κB therefore relies on activation of downstream effectors of RICK, like the inhibitor of NF-κB kinase complex.18–20

Because persistent intracellular infection of endothelial cells with C pneumoniae has been associated with development of cardiovascular diseases, we examined the importance of cytosolic immune receptors in this context. We demonstrate that Nod proteins mediate NF-κB activation by C pneumoniae and suggest that Nod1 plays a major role in endothelial cell immunity.

Materials and Methods

Chlamydophila Strains

C pneumoniae strain TW-183 (VR2282; ATCC, Rockville, Md) was cultured and purified as described by Maass et al.2 Briefly, TW-183 was grown to high titers in cyclohexamide-treated HEp-2 cells. Infected monolayers were harvested from culture flasks and sonicated for 30 seconds. Cellular debris was removed by centrifugation at 500g for 10 minutes at 4°C. Aliquots of purified elementary bodies were stored at −75°C until use. For some experiments, C pneumoniae were inactivated by heat treatment (90°C, 30 minutes).

Infection of Human Umbilical Vein Endothelial Cells, Human Aortic Endothelial Cells, and HEK293 Cells

Human aortic endothelial cells (HAECs) were obtained from Clonetics (San Diego, Calif), and human umbilical vein endothelial cells (HUVECs) were isolated from human umbilical cord veins. Both cell types were cultured as described previously.6 HUVECs, HAECs, or HEK293 cells were inoculated with C pneumoniae using a multiplicity of infection (MOI) of 0.5 to 5 in MCDB131, EGM-2, or DMEM, respectively. Plates were centrifuged at 800g at 37°C for 1 hour and subsequently incubated for 7 hours (luciferase assay) or overnight (IL-8 enzyme-linked immunosorbent assay [ELISA]). For some experiments, chlamydia or lipopolysaccharide (Sigma, Deisenhofen, Germany) were preincubated with polymyxin B (10 μg/mL; Sigma) to inactivate lipopolysaccharide. To investigate importance of TLR2 and Nod1 or Nod2 for intracellular activation of target cells, heat-inactivated C pneumoniae (MOI=5) were transfected intracellularly using the calcium phosphate method (Clonetech, Palo Alto, Calif) before transfection with different plasmids as it has been described for different stimuli.15,17 Briefly, immediately before DNA/calcium phosphate suspensions were added, cells were fed with culture medium containing bacterial components.

RNA Interference

Control nonsilencing small interfering RNA (siRNA) (sense, UUCUCCGAACGUGUCACGUtt; antisense, ACGUGACACGUUCGGAGGAGAAtt) and different siRNAs targeting Nod1 (sequence 1 sense, GGGUGAGACCAUCUUCAUCtt; antisense, GAUGAAGAUGGUCUCACCCtg; sequence 2 sense, GGCCAAAGUCUAUGAAGAUtt; antisense, AUCUUCAUAGACUUUGGCCtc) were purchased from Ambion (Huntingdon, UK). HUVECs and HAECs were transfected using an Amaxa Nucleofector (Amaxa, Cologne, Germany) according to the manufacturer’s protocol with 1.5 or 2 μg siRNA, respectively.

Expression Plasmids and HEK293 Cell Overexpression Experiments

Expression plasmids for Nod1 and Nod2 were kindly provided by G. Nuñez, (Ann Arbor, Mich). Dominant-negative Rip-2 were a generous gift from P. Dempsey (Los Angeles, Calif). The constructs encoding TLR2 and dominant-negative MyD88 were kindly donated by C. Kirschning (Munich, Germany) and Tularik Inc (San Francisco, Calif). Mal was generously provided by K. Fitzgerald (Worchester, Mass). HEK293 cells were cultured in 24-well plates with DMEM (Invitrogen, Paisley, UK) supplemented with 10% fetal calf serum. Subconfluent HEK293 cells were cotransfected using the calcium phosphate method (Clonetech, Palo Alto, Calif) with 0.05 μg NF-κB–dependent luciferase reporter and 0.3 ng Nod expression vectors or 0.05 μg TLR2. For some experiments, cells were cotransfected with dominant-negative mutants of signaling molecules Rip-2 or MyD88.

Luciferase Assay

Luciferase activity was measured using the Luciferase Reporter-Gene Assay (Promega, Mannheim, Germany) as described previously.6 The luminescence readings demonstrated were normalized to total protein and expressed as fold activation relative to unstimulated, mock-transfected cells±SE (Figures 2 and 3). In Figure 4, mock-transfected cells were not demonstrated; therefore, the relative luminescence readings were expressed as fold activation relative to unstimulated, Nod1-transfected or Nod2-transfected cells.

Figure 2. Nod1 and Nod2 mediated NF-κB activation in HEK293 cells by chlamydia. A, HEK293 cells were grown on Thermanox coverslips and stimulated with C pneumoniae (MOI=5) for 24 hours. Cells were then fixed, permeabilized, and stained with FITC-conjugated genus-specific monoclonal antibody to demonstrate the intracellular presence of chlamydia. Cells were analyzed by confocal laser scanning microscopy (magnification ×630). Z-stack analysis confirmed that in infected HEK293 cells, all visualized chlamydia were intracellular. B, HEK293 cells were transiently transfected with a control vector, Nod1 or Nod2 expression plasmids, and a NF-κB–dependent luciferase reporter. The next day, cells were infected with the indicated doses of C pneumoniae for 7 hours, and luciferase activity was measured. Data are shown as means±SE for one representative experiment of three, with each transfection performed in triplicate (**P<0.01).

Figure 3. Nods and TLR2 serve as receptors for chlamydia in different compartments. HEK293 cells were transiently transfected with NF-κB–dependent luciferase reporter and a control vector, Nod1, Nod2, or TLR2 expression plasmids as indicated. The next day, cells were stimulated with viable or heat-inactivated C pneumoniae for 7 hours. For experiments demonstrating intracellular stimulation of HEK293 cells, inactivated chlamydia were added to the cells during transfection (for details, see Materials and Methods). Luciferase activity was measured and indicated as fold induction. Data are shown as means±SE for one representative experiment of three, with each transfection performed in triplicate (**P<0.01).

Figure 4. Rip2, but not MyD88, is involved in NF-κB activation by chlamydia. HEK293 cells were cotransfected with NF-κB–dependent luciferase reporter and Nod1 (A and B), Nod2 (C and D), TLR2 (C), or Mal (D) expression plasmid as indicated. Additionally, an empty control vector or dominant-negative mutants of MyD88 or Rip2 was cotransfected. The next day, cells were infected with C pneumoniae for 7 hours; subsequently, luciferase activity was demonstrated by luciferase assay. Relative luciferase activity is shown as means of fold inductions±SE for one representative experiment of three, with transfection performed in triplicate (**P<0.01).

Confocal Laser Scanning Microscopy Analysis of HUVECs and HEK293 Cell Infection

HUVECs or HEK293 cells grown on Thermanox slides (Falcon Culture Slide; Becton Dickinson, Rutherford, NJ) were infected with C pneumoniae (MOI=5) for 24 hours, and cells were fixed with 3% paraformaldehyde for 20 minutes and permeabilized with 1% Triton for 15 minutes. For chlamydia staining, the primary Ab (a genus-specific monoclonal antibody, “chlamydia culture conformation system;” Sanofi Diagnostics Pasteur, Freiburg, Germany) was incubated overnight at 4°C. Bound antibodies were detected with ALEXA-488–conjugated goat anti-rabbit mAb (4°C, overnight; Molecular Probes, Eugene, Ore). Endothelial f-actin was counterstained using ALEXA-546–conjugated phalloidin (Molecular Probes). Cells were analyzed using a Pascal 5 confocal laser scanning microscope (Zeiss, Jena Germany).

IL-8 ELISA

IL-8 concentrations in the supernatants of infected HUVECs or HAECs were quantified using a commercially available sandwich ELISA Kit (R&D Systems, Wiesbaden, Germany).

Reverse-Transcription Polymerase Chain Reaction Analysis

Total RNA from different cell lines were isolated with the RNeasy Mini kit (Qiagen, Hilden, Germany) and reverse-transcribed using AMV reverse-transcriptase (Promega). The generated cDNA was amplified by polymerase chain reaction (PCR) using specific primers (Nod1-sense 5′-AAGCGAAGAGCTGACCAAAT-3′; Nod1-antisense 5′-TTCATAGACTTTGGCCTCCTC-3′, Nod2-sense, 5′-AGCCATTGTCAGGAGGCTC-3′; Nod2-antisense, 5′-CGTCTCTGCTCCATCATAGG-3′; Tib MolBiol, Berlin, Germany). After 30 amplification cycles, the PCR products were analyzed on 1.5% agarose gels, stained with ethidium bromide, and subsequently visualized. To confirm equal amounts of RNA in each experiment, all samples were checked for GAPDH mRNA expression.

Statistics

Stimulatory effects of C pneumoniae and inhibitory effects of dominant-negative signaling molecule mutants or siRNAs were statistically evaluated using Student t test. Throughout the figure, P<0.05 is indicated by one asterisk; P<0.01 is indicated by double asterisks.

Results

Viable but Not Heat-Inactivated C pneumoniae Infect Human Endothelial Cells and Induce IL-8 Expression

Infection of endothelial cells by C pneumoniae was demonstrated by confocal laser scanning microscopy of HUVECs 24 hours after addition of bacteria. ALEXA-546 phalloidin was used to counterstain endothelial F-actin; Figure 1A represents uninfected endothelial cells. C pneumoniae (Figure 1B) were inoculated onto HUVEC monolayer using an MOI of 5. This resulted in bacterial adhesion after the first minutes of cell contact and subsequent internalization. After 24 hours of exposure, only few bacteria were adherent to the cell surface; instead, a perinuclear formation of internalized bacteria was observed.

Figure 1. Infection of endothelial cells by Chlamydophila pneumoniae release of endothelial IL-8. Infection was demonstrated by confocal laser scanning microscopy. HUVECs were grown on glass slides and exposed to chlamydia using a MOI of 5. The 24-hour postinfection cells were fixed with paraformaldehyde, permeabilized with Triton, and labeled with phalloidin as indicated in Materials and Methods. A, Uninfected control cells. Z-stack analysis confirmed that in infected HUVECs, all visualized chlamydia were intracellular (B). Note that internalized bacteria have started to decondense and form typical perinuclear-located inclusion body-like structures. Representative pictures (of three independent experiments; magnification ×630) are presented. HUVECs (C) or HAECs (D) were stimulated with either viable or heat-inactivated C pneumoniae. E, Before HUVEC stimulation, C pneumonaie or lipopolysaccharide was pretreated with polymyxin B (10 μg/mL, 15 minutes). IL-8 in the supernatant was determined by ELISA. Data from one of three representative experiments with similar results are shown as mean±SE (*P<0.05; **P<0.01). F, Nod1 expression in different cell lines was assessed by RT-PCR analysis.

C pneumoniae infection of human endothelial cells induces a NF-κB–dependent pro-inflammatory phenotype as indicated by enhanced expression of cytokines/chemokines and adhesion molecules.6,21,22 To further characterize the chlamydial stimulation of endothelial cells, we incubated HUVECs or HAECs with viable or heat-inactivated C pneumoniae and assessed IL-8 secretion in the supernatant. Viable, but not heat-inactivated, chlamydia were able to induce IL-8 expression in HUVECs (Figure 1C) or HAECs (Figure 1D) 24 hours after infection. Preparations of uninfected HEp-2 cells were without effect on HUVEC-related IL-8 secretion (data not shown). Preincubation of C pneumoniae with polymyxin B to inactivate lipopolysaccharides did not significantly reduce IL-8 expression (Figure 1E). The fact that intracellular infection of endothelial cells appeared to be essential for IL-8 induction raised the question if intracellular immune receptors might be involved. In line with this hypothesis, we were able to detect Nod1 mRNA in HUVECs and HAECs by reverse-transcription PCR (RT-PCR) (Figure 1F). In addition, analyzing different epithelial, monocytic, and lymphocytic cell lines, we found that human endothelial cells seemed to express the highest levels of Nod1. In contrast, Nod2 mRNA was barely found in HUVECs but was slightly detectable in the bronchoepithelial cell line BEAS-2B.

Nod1- and Nod2-Mediated NF-κB Activation in C pneumoniae Infected HEK293 Cells

To investigate a possible Nod protein-mediated cell activation by chlamydia, we overexpressed Nod1 and Nod2 in HEK293 cells. These cells are particularly amenable for overexpression assays. C pneumoniae was able to infect HEK293 cells as demonstrated by CSLM experiments. Multiple inclusion bodies were found within HEK293 cells as early as 24 hours after infection (Figure 2A). Next, we cotransfected HEK293 cells with a NF-κB luciferase reporter and Nod1 or Nod2, respectively. Cells were infected with increasing concentrations of C pneumoniae as described in Materials and Methods. The bacteria caused a slight induction of reporter gene activity if a control vector (“mock”) was transfected instead of Nod1/2 (Figure 2B). In cells overexpressing Nod1 or Nod2, C pneumoniae induced a marked concentration-dependent NF-κB activation, and maximal effects were seen using a MOI of 5.

Nod1 and Nod2 Recognize Only Intracellular, Whereas TLR2 Detects Extracellular, Viable or Heat-Inactivated Chlamydia

Nod proteins are receptors for peptidoglycans. Because these molecules are heat-stable,23 we next assessed the ability of viable or heat-inactivated C pneumoniae to activate Nod1, Nod2, and TLR2 while applied extracellularly or intracellularly.

To allow intracellular stimulation with heated bacteria, heat-inactivated chlamydia were transfected into target cells as described in Materials and Methods. Activation of NF-κB was used as “read out.” The Nod1/2-mediated NF-κB activation was only initiated when C pneumoniae (viable or heat-killed) were provided intracellularly, either by infection with viable chlamydia or by intracellular transfection of heat-inactivated chlamydia (Figure 3). When applied extracellularly, the heat-inactivated chlamydia failed to initiate Nod-mediated signal transduction in HEK293 cells. Note that Nod1/2-overexpressing cells have a high constitutive activity of NF-κB. This observation is currently unexplained but clearly in line with several recently published studies.16,18 Viable and heat-inactivated C pneumoniae were very well able to activate NF-κB in a TLR2-dependent manner (Figure 3) when chlamydia were provided from the extracellular side. When performing intracellular stimulation with inactivated bacteria, some TLR2-related reporter gene activity could be observed. This phenomenon is most likely attributable to the fact that during transfection of inactivated chlamydia, some bacteria nevertheless stay outside of the cells and possibly signal via TLR2.

Importance of Rip2 but Not of MyD88 for Chlamydia-Mediated NF-κB Activation via Nod Proteins

Next, we investigated the involvement of the signaling molecules Rip2 or MyD88, which participate during NF-κB activation by Nod proteins or TLRs, respectively.24–26 Therefore, we cotransfected HEK293 cells with a NF-κB luciferase reporter, Nod1 or Nod2, as well as different amounts of dominant-negative mutants of Rip2 or MyD88, and stimulated these cells with viable C pneumoniae. TLR2-dependent NF-κB activation served as a positive control for dominant-negative MyD88, and overexpressed Mal (an adapter molecule of TLR2 and TLR427) was used as a negative control for dominant-negative Rip2. Dominant-negative MyD88 failed to exhibit inhibitory effects on Nod protein-mediated NF-κB activation by chlamydia but clearly reduced the TLR2-dependent cell activation in HEK293 cells (Figure 4A and 4C). Dominant-negative Rip2, however, inhibited the Nod protein-associated NF-κB reporter gene activity induced by chlamydia in a dose-dependent manner, indicating that Rip2 is involved in Nod-dependent NF-κB activation by C pneumoniae (Figure 4B and 4D). In contrast, NF-κB activity induced by overexpressed Mal was only slightly reduced by dominant-negative Rip2.

Nod1 Is Fundamental for Chlamydia-Induced IL-8 Production in Endothelial Cells

To confirm the critical involvement of Nod1 in endothelial cell activation by C pneumoniae, we used siRNA technology to inhibit the expression of endogenous Nod1. First, we evaluated three different siRNA duplexes targeting Nod1 for their ability to reduce Nod1 mRNA expression in endothelial cells. Nonsilencing siRNA served as a control. Only the Nod1-specific oligonucleotides inhibited Nod1 mRNA levels in HUVECs and HAECs (Figure 5A and data not shown). Importantly, these Nod1-specific siRNAs also blocked the IL-8 production induced by C pneumoniae in HUVECs and HAECs (Figure 5B and 5C). In contrast, the tumor necrosis factor-α–induced (10 ng/mL) cytokine production in HUVECs was not altered by any siRNA used (Figure 5B), thus demonstrating specific effects of the siRNAs in our experiment. These observations confirmed our results from the HEK293 cell studies and demonstrated that Nod1 is important for endothelial cell activation by C pneumoniae.

Figure 5. siRNAs targeting Nod1 inhibited C pneumoniae-induced IL-8 production in HUVECs and HAECs. A, HUVECs were left unaffected, transfected with three different siRNAs targeting Nod1 (S1 to S3, sequences 1 to 3), or transfected with a control nonsilencing siRNA (c). After 24 hours, gene-silencing ability of the RNA duplexes used was assessed by RT-PCR analysis using specific primers against Nod1. HUVECs (B) or HAECs (C) were transfected with siRNAs targeting Nod1, as well as with the control siRNA. After 72 hours, C pneumoniae were added to HUVEC or HAEC monolayers by centrifugation at 800g. The next day, IL-8 concentration in the supernatant was analyzed by ELISA. Data represent mean±SE of two independent experiments out of four experiments with similar results (B); data in (C) represents mean±SE of one of three representative experiments performed in duplicate (*P<0.05; **P<0.1)

Discussion

The study presented demonstrates that Nod1 is indispensable for intracellular sensing of C pneumoniae in endothelial cells. Nod1 gene silencing by siRNA blocked the IL-8 production induced by C pneumoniae in HUVECs and HAECs. In addition, we demonstrated that C pneumoniae activated a Nod1-mediated and Nod2-mediated signal transduction pathway in HEK293 cells involving Rip2, but not MyD88. Heat-stable chlamydial components are responsible for the demonstrated effects. TLR2 acted as an extracellular receptor for initial chlamydia-induced target cell stimulation. These observations can improve our understanding how C pneumoniae infection may contribute to chronic inflammatory events associated with atherosclerosis.

After initial attachment, chlamydial elementary bodies are internalized and dissociate from the endocytotic pathway by actively modifying the vacuole to become fusogenic with exocytic vesicles.28 Interaction with this secretory pathway appears to provide a pathogenic mechanism that allows chlamydia to establish themselves in a site that is not destined to fuse with lysosomes. Almost nothing is known about the relationship between distinct steps of this chlamydial development cycle and initiation of host cell signaling pathways.

Nod proteins have so far been associated with recognition of different types of peptidoglycans.14–17 Although recent studies suggest a functional peptidoglycan pathway in chlamydia,29,30 clear-cut biochemical evidence for the synthesis of peptidoglycans in chlamydia is missing.31,32 Chlamydia, however, are sensitive to antibiotics that inhibit peptidoglycan synthesis.33 This phenomenon has been referred to the “chlamydial anomaly.” Our finding that C pneumoniae induced a Nod-mediated endothelial cell activation via heat-stable components does not prove, but may lend support to, the existence of peptidoglycan-like structures in chlamydia.

Recognition of chlamydia by Nod proteins could thus be interpreted in at least two different ways. First, the chlamydial cell wall contains peptidoglycan or peptidoglycan-like structures. This hypothesis is in accordance with several studies suggesting the expression of peptidoglycan-like structures not on the surface of elementary bodies but—after invasion of the target cells—on the subsequently developed reticular bodies.34 Second, Nod proteins act as receptors for molecules other than peptidoglycans. Proteins such as GroEL-1 or MOMPs could be involved in Nod activation because the minimal motif recognized by Nod1 is a dipeptide containing diaminopimelic acid,14 and chlamydia could possibly synthesize this dipeptide in a peptidoglycan-independent way. In line with this hypothesis are preliminary data suggesting the recognition of recombinant GroEL-1 by Nod proteins in HEK293 cells (data not shown). However, additional studies using purified components from C pneumoniae are necessary to further analyze the importance of different chlamydial virulence factors for Nod-mediated signal transduction.

Nod1 has been found in multiple tissues, whereas the expression of Nod2 is mainly restricted to leukocytes, dendritic cells, and epithelial cells.12,13 Analyzing endothelial cells and different epithelial, monocytic, and lymphocytic cell lines, we found that human endothelial cells seemed to express the highest levels of Nod1 mRNA, suggesting that Nod1 might play a key role in endothelial cells.

Little is known about the different functions of Nod1 and Nod2 proteins as receptors for intracellular pathogens like chlamydia. Because no differences in the downstream signaling of Nod1 and Nod2 were observed so far, both receptors might substitute for each other in some cases for intracellular recognition of bacteria in varying tissues.12,13

Several recent studies demonstrated the involvement of TLR2 and TLR4 in initiation of target cell activation by C pneumoniae or chlamydial components.8–11,35–37 TLR2 was suggested to be more important than TLR4 for recognition by and activation of innate immune cells.10 In our system of TLR2-overexpressing HEK293 cells, we could demonstrate that viable and heat-inactivated C pneumoniae were able to induce NF-κB-activation on cell contact when “applied” from the extracellular side. Heat-killed chlamydia, however, failed to activate NF-κB in Nod1 or Nod2-overexpressing HEK293 cells on extracellular challenge, indicating that Nod proteins serve as intracellular receptors. These considerations are in line with our observation that viable, but not heat-inactivated, chlamydia were able to induce a marked release of IL-8 from infected endothelial cells, because HUVECs express Nod1 but barely express TLR2.38 Thus, Nod proteins rather than TLR2 appear to contribute to C pneumoniae–mediated endothelial cell activation. Recently, a report was published showing a MyD88-dependent and TLR4-dependent interferon-α/γ induction by C pneumoniae in macrophages.37 In contrast, NF-κB activation and tumor necrosis factor-α, IL-1, and IL-6 secretion were MyD88-independent and TLR-4-independent. These data support the hypothesis of a TLR-independent but Nod-dependent mechanism of target cell activation by C pneumoniae.

The association between chronic/persistent chlamydial infection and development of atherosclerosis is still controversially discussed. The idea was supported by: (1) experimental studies in animal models demonstrating enhanced development of atherosclerotic lesions after infection with C pneumoniae; (2) detection of C pneumoniae in atherosclerotic plaques (electron microscopy, immunocytochemistry, PCR); and (3) serological association between C pneumoniae infection and coronary heart disease.39–41 However, many epidemiological studies failed to consider the various confounding risks of atherosclerosis, such as cigarette smoking and hyperlipidemia. In addition, it was reported that mice that were held in a germ-free environment revealed no differences concerning atherosclerotic disease when compared with mice that were kept in normal environment.42 Finally, most of the clinical trials trying to demonstrate a direct link between antichlamydial antibiotic therapy and improved outcome of atherosclerosis failed.43 However, intracellularly persistent chlamydia are antibiotic-resistant,7 and therefore the interventional studies are not helpful to prove the concept. Thus, it still remains uncertain whether C pneumoniae represents a causative role in atherosclerosis or is just a concomitant phenomenon. In contrast, the role of the TLRs in atherosclerosis is firmly established.44,45 It is thus conceivable that other innate immune receptors such as Nod proteins might be involved in this inflammatory process. The recently established Nod1 and Nod2 knockout mice will be of outstanding value for future studies.14,15,46

Taken together we provide evidence that Nod1 is a potent innate immune receptor for C pneumoniae in endothelial cells. Whereas TLR2 might be important for initial cell activation on chlamydial contact in some cell types, Nod proteins are crucial for an intracellularly triggered prolonged and profound activation of target cells by intracellular chlamydia. The data presented may improve our understanding of (chronic) inflammatory processes in the endothelium.

Original received August 10, 2004; revision received December 27, 2004; accepted January 3, 2005.

Supported by the Deutsche Forschungsgemeinschaft (Kr 2197/1-2 to M.K. and N.S. and Ma 2070/4-2 to M.M.) and by the Bundesministerium für Bildung und Forschung (BMBF) (BMBF-NBL3 and -CAPNetz to N.S.). Parts of this work will be included in the MD thesis of S.F.

Footnotes

Correspondence to Matthias Krüll, MD, Department of Internal Medicine/Infectious Diseases, Charité University Medicine Berlin, Augustenburger Platz 1, 13353 Berlin, Germany. E-mail

References

  • 1 Fong IW, Chiu B, Viira E, Jang D, Mahony JB. De Novo induction of atherosclerosis by Chlamydia pneumoniae in a rabbit model. Infect Immun. 1999; 67: 6048–6055.CrossrefMedlineGoogle Scholar
  • 2 Maass M, Bartels C, Engel PM, Mamat U, Sievers HH. Endovascular presence of viable Chlamydia pneumoniae is a common phenomenon in coronary artery disease. J Am Coll Cardiol. 1998; 31: 827–832.CrossrefMedlineGoogle Scholar
  • 3 Saikku P, Leinonen M, Tenkanen L, Linnanmaki E, Ekman MR, Manninen V, Manttari M, Frick MH, Huttunen JK. Chronic Chlamydia pneumoniae infection as a risk factor for coronary heart disease in the Helsinki Heart Study. Ann Intern Med. 1992; 116: 273–278.CrossrefMedlineGoogle Scholar
  • 4 Shor A, Kuo CC, Patton DL. Detection of Chlamydia pneumoniae in coronary arterial fatty streaks and atheromatous plaques. S Afr Med J. 1992; 82: 158–161.MedlineGoogle Scholar
  • 5 Dechend R, Maass M, Gieffers J, Dietz R, Scheidereit C, Leutz A, Gulba DC. Chlamydia pneumoniae infection of vascular smooth muscle and endothelial cells activates NF-kappaB and induces tissue factor and PAI-1 expression: a potential link to accelerated arteriosclerosis. Circulation. 1999; 100: 1369–1373.CrossrefMedlineGoogle Scholar
  • 6 Krüll M, Klucken AC, Wuppermann FN, Fuhrmann O, Magerl C, Seybold J, Hippenstiel S, Hegemann JH, Jantos CA, Suttorp N. Signal transduction pathways activated in endothelial cells following infection with Chlamydia pneumoniae. J Immunol. 1999; 162: 4834–4841.CrossrefMedlineGoogle Scholar
  • 7 Gieffers J, Fullgraf H, Jahn J, Klinger M, Dalhoff K, Katus HA, Solbach W, Maass M. Chlamydia pneumoniae infection in circulating human monocytes is refractory to antibiotic treatment. Circulation. 2001; 103: 351–356.CrossrefMedlineGoogle Scholar
  • 8 Bulut Y, Faure E, Thomas L, Karahashi H, Michelsen KS, Equils O, Morrison SG, Morrison RP, Arditi M. Chlamydial heat shock protein 60 activates macrophages and endothelial cells through Toll-like receptor 4 and MD2 in a MyD88-dependent pathway. J Immunol. 2002; 168: 1435–1440.CrossrefMedlineGoogle Scholar
  • 9 Netea MG, Kullberg BJ, Galama JM, Stalenhoef AF, Dinarello CA, Van der Meer JW. Non-LPS components of Chlamydia pneumoniae stimulate cytokine production through Toll-like receptor 2-dependent pathways. Eur J Immunol. 2002; 32: 1188–1195.CrossrefMedlineGoogle Scholar
  • 10 Prebeck S, Kirschning C, Durr S, da Costa C, Donath B, Brand K, Redecke V, Wagner H, Miethke T. Predominant role of toll-like receptor 2 versus 4 in Chlamydia pneumoniae-induced activation of dendritic cells. J Immunol. 2001; 167: 3316–3323.CrossrefMedlineGoogle Scholar
  • 11 Sasu S, LaVerda D, Qureshi N, Golenbock DT, Beasley D. 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. 2001; 89: 244–250.CrossrefMedlineGoogle Scholar
  • 12 Chamaillard M, Girardin SE, Viala J, Philpott DJ. Nods, Nalps and Naip: intracellular regulators of bacterial-induced inflammation. Cell Microbiol. 2003; 5: 581–592.CrossrefMedlineGoogle Scholar
  • 13 Inohara N, Nunez G. NODs: intracellular proteins involved in inflammation and apoptosis. Nat Rev Immunol. 2003; 3: 371–382.CrossrefMedlineGoogle Scholar
  • 14 Chamaillard M, Hashimoto M, Horie Y, Masumoto J, Qiu S, Saab L, Ogura Y, Kawasaki A, Fukase K, Kusumoto S, Valvano MA, Foster SJ, Mak TW, Nunez G, Inohara N. An essential role for NOD1 in host recognition of bacterial peptidoglycan containing diaminopimelic acid. Nat Immunol. 2003; 4: 702–707.CrossrefMedlineGoogle Scholar
  • 15 Girardin SE, Boneca IG, Carneiro LA, Antignac A, Jehanno M, Viala J, Tedin K, Taha MK, Labigne A, Zähringer U, Coyle AJ, DiStefano PS, Bertin J, Sansonetti PJ, Philpott DJ. Nod1 detects a unique muropeptide from gram-negative bacterial peptidoglycan. Science. 2003; 300: 1584–1587.CrossrefMedlineGoogle Scholar
  • 16 Girardin SE, Boneca IG, Viala J, Chamaillard M, Labigne A, Thomas G, Philpott DJ, Sansonetti PJ. Nod2 is a general sensor of peptidoglycan through muramyl dipeptide (MDP) detection. J Biol Chem. 2003; 278: 8869–8872.CrossrefMedlineGoogle Scholar
  • 17 Inohara N, Ogura Y, Fontalba A, Gutierrez O, Pons F, Crespo J, Fukase K, Inamura S, Kusumoto S, Hashimoto M, Foster SJ, Moran AP, Fernandez-Luna JL, Nunez G. Host recognition of bacterial muramyl dipeptide mediated through NOD2. Implications for Crohn’s disease. J Biol Chem. 2003; 278: 5509–5512.CrossrefMedlineGoogle Scholar
  • 18 Bertin J, Nir WJ, Fischer CM, Tayber OV, Errada PR, Grant JR, Keilty JJ, Gosselin ML, Robison KE, Wong GH, Glucksmann MA, DiStefano PS. Human CARD4 protein is a novel CED-4/Apaf-1 cell death family member that activates NF-kappaB. J Biol Chem. 1999; 274: 12955–12958.CrossrefMedlineGoogle Scholar
  • 19 Inohara N, Koseki T, del Peso L, Hu Y, Yee C, Chen S, Carrio R, Merino J, Liu D, Ni J, Nunez G. Nod1, an Apaf-1-like activator of caspase-9 and nuclear factor-kappaB. J Biol Chem. 1999; 274: 14560–14567.CrossrefMedlineGoogle Scholar
  • 20 Ogura Y, Inohara N, Benito A, Chen FF, Yamaoka S, Nunez G. Nod2, a Nod1/Apaf-1 family member that is restricted to monocytes and activates NF-kappaB. J Biol Chem. 2001; 276: 4812–4818.CrossrefMedlineGoogle Scholar
  • 21 Gaydos CA, Summersgill JT, Sahney NN, Ramirez JA, Quinn TC. Replication of Chlamydia pneumoniae in vitro in human macrophages, endothelial cells, and aortic artery smooth muscle cells. Infect Immun. 1996; 64: 1614–1620.CrossrefMedlineGoogle Scholar
  • 22 Godzik KL, O’Brien ER, Wang SK, Kuo CC. In vitro susceptibility of human vascular wall cells to infection with Chlamydia pneumoniae. J Clin Microbiol. 1995; 33: 2411–2414.CrossrefMedlineGoogle Scholar
  • 23 Henneke P, Takeuchi O, van Strijp JA, Guttormsen HK, Smith JA, Schromm AB, Espevik TA, Akira S, Nizet V, Kasper DL, Golenbock DT. Novel engagement of CD14 and multiple toll-like receptors by group B streptococci. J Immunol. 2001; 167: 7069–7076.CrossrefMedlineGoogle Scholar
  • 24 Chin AI, Dempsey PW, Bruhn K, Miller JF, Xu Y, Cheng G. Involvement of receptor-interacting protein 2 in innate and adaptive immune responses. Nature. 2002; 416: 190–194.CrossrefMedlineGoogle Scholar
  • 25 Kawai T, Adachi O, Ogawa T, Takeda K, Akira S. Unresponsiveness of MyD88-deficient mice to endotoxin. Immunity. 1999; 11: 115–122.CrossrefMedlineGoogle Scholar
  • 26 Kobayashi K, Inohara N, Hernandez LD, Galan JE, Nunez G, Janeway CA, Medzhitov R, Flavell RA. RICK/Rip2/CARDIAK mediates signalling for receptors of the innate and adaptive immune systems. Nature. 2002; 416: 194–199.CrossrefMedlineGoogle Scholar
  • 27 O’Neill LA, Fitzgerald KA, Bowie AG. The Toll-IL-1 receptor adaptor family grows to five members. Trends Immunol. 2003; 24: 286–290.CrossrefMedlineGoogle Scholar
  • 28 Hackstadt T, Fischer ER, Scidmore MA, Rockey DD, Heinzen RA. Origins and functions of the chlamydial inclusion. Trends Microbiol. 1997; 5: 288–293.CrossrefMedlineGoogle Scholar
  • 29 Hesse L, Bostock J, Dementin S, Blanot D, Mengin-Lecreulx D, Chopra I. Functional and biochemical analysis of Chlamydia trachomatis MurC, an enzyme displaying UDP-N-acetylmuramate:amino acid ligase activity. J Bacteriol. 2003; 185: 6507–6512.CrossrefMedlineGoogle Scholar
  • 30 McCoy AJ, Sandlin RC, Maurelli AT. In vitro and in vivo functional activity of Chlamydia MurA, a UDP-N-acetylglucosamine enolpyruvyl transferase involved in peptidoglycan synthesis and fosfomycin resistance. J Bacteriol. 2003; 185: 1218–1228.CrossrefMedlineGoogle Scholar
  • 31 Chopra I, Storey C, Falla TJ, Pearce JH. Antibiotics, peptidoglycan synthesis and genomics: the chlamydial anomaly revisited. Microbiology. 1998; 144 (Pt 10): 2673–2678.CrossrefMedlineGoogle Scholar
  • 32 Fox A, Rogers JC, Gilbart J, Morgan S, Davis CH, Knight S, Wyrick PB. Muramic acid is not detectable in Chlamydia psittaci or Chlamydia trachomatis by gas chromatography-mass spectrometry. Infect Immun. 1990; 58: 835–837.CrossrefMedlineGoogle Scholar
  • 33 Moulder JW. Why is Chlamydia sensitive to penicillin in the absence of peptidoglycan? Infect Agents Dis. 1993; 2: 87–99.MedlineGoogle Scholar
  • 34 Bavoil PM, Hsia R, Ojcius DM. Closing in on Chlamydia and its intracellular bag of tricks. Microbiology. 2000; 146 (Pt 11): 2723–2731.CrossrefMedlineGoogle Scholar
  • 35 Bea F, Puolakkainen MH, McMillen T, Hudson FN, Mackman N, Kuo CC, Campbell LA, Rosenfeld ME. Chlamydia pneumoniae induces tissue factor expression in mouse macrophages via activation of Egr-1 and the MEK-ERK1/2 pathway. Circ Res. 2003; 92: 394–401.LinkGoogle Scholar
  • 36 Costa CP, Kirschning CJ, Busch D, Durr S, Jennen L, Heinzmann U, Prebeck S, Wagner H, Miethke T. Role of chlamydial heat shock protein 60 in the stimulation of innate immune cells by Chlamydia pneumoniae. Eur J Immunol. 2002; 32: 2460–2470.CrossrefMedlineGoogle Scholar
  • 37 Rothfuchs AG, Trumstedt C, Wigzell H, Rottenberg ME. Intracellular bacterial infection-induced IFN-gamma is critically but not solely dependent on toll-like receptor 4-myeloid differentiation factor 88-IFN-alphabeta-STAT1 signaling. J Immunol. 2004; 172: 6345–6353.CrossrefMedlineGoogle Scholar
  • 38 Faure E, Equils O, Sieling PA, Thomas L, Zhang FX, Kirschning CJ, Polentarutti N, Muzio M, Arditi M. Bacterial lipopolysaccharide activates NF-kappaB through toll-like receptor 4 (TLR-4) in cultured human dermal endothelial cells. Differential expression of TLR-4 and TLR-2 in endothelial cells. J Biol Chem. 2000; 275: 11058–11063.CrossrefMedlineGoogle Scholar
  • 39 Blessing E, Campbell LA, Rosenfeld ME, Chough N, Kuo CC. Chlamydia pneumoniae infection accelerates hyperlipidemia induced atherosclerotic lesion development in C57BL/6J mice. Atherosclerosis. 2001; 158: 13–17.CrossrefMedlineGoogle Scholar
  • 40 Hu H, Pierce GN, Zhong G. The atherogenic effects of chlamydia are dependent on serum cholesterol and specific to Chlamydia pneumoniae. J Clin Invest. 1999; 103: 747–753.CrossrefMedlineGoogle Scholar
  • 41 Muhlestein JB, Anderson JL, Hammond EH, Zhao L, Trehan S, Schwobe EP, Carlquist JF. Infection with Chlamydia pneumoniae accelerates the development of atherosclerosis and treatment with azithromycin prevents it in a rabbit model. Circulation. 1998; 97: 633–636.CrossrefMedlineGoogle Scholar
  • 42 Wright SD, Burton C, Hernandez M, Hassing H, Montenegro J, Mundt S, Patel S, Card DJ, Hermanowski-Vosatka A, Bergstrom JD, Sparrow CP, Detmers PA, Chao YS. Infectious agents are not necessary for murine atherogenesis. J Exp Med. 2000; 191: 1437–1442.CrossrefMedlineGoogle Scholar
  • 43 Grayston JT. Antibiotic treatment of atherosclerotic cardiovascular disease. Circulation. 2003; 107: 1228–1230.LinkGoogle Scholar
  • 44 Bjorkbacka H, Kunjathoor VV, Moore KJ, Koehn S, Ordija CM, Lee MA, Means T, Halmen K, Luster AD, Golenbock DT, Freeman MW. Reduced atherosclerosis in MyD88-null mice links elevated serum cholesterol levels to activation of innate immunity signaling pathways. Nat Med. 2004; 10: 416–421.CrossrefMedlineGoogle Scholar
  • 45 Michelsen KS, Wong MH, Shah PK, Zhang W, Yano J, Doherty TM, Akira S, Rajavashisth TB, Arditi M. Lack of Toll-like receptor 4 or myeloid differentiation factor 88 reduces atherosclerosis and alters plaque phenotype in mice deficient in apolipoprotein E. Proc Natl Acad Sci U S A. 2004; 101: 10679–10684.CrossrefMedlineGoogle Scholar
  • 46 Pauleau AL, Murray PJ. Role of nod2 in the response of macrophages to toll-like receptor agonists. Mol Cell Biol. 2003; 23: 7531–7539.CrossrefMedlineGoogle Scholar

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