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J. Biol. Chem., Vol. 279, Issue 35, 36426-36432, August 27, 2004

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Nucleotide-binding Oligomerization Domain Proteins Are Innate Immune Receptors for Internalized Streptococcus pneumoniae^*

Bastian Opitz, Anja Püschel, Bernd Schmeck, Andreas C. Hocke, Simone Rosseau, Sven Hammerschmidt, Ralf R. Schumann¶, Norbert Suttorp, and Stefan Hippenstiel||

From the Department of Internal Medicine/Infectious Diseases, Charité University Medicine Berlin, 1 Augustenburger Platz, 13353 Berlin, Germany, Research Center for Infectious Diseases, University of Würzburg, 11 Röntgenring, 97070 Würzburg, Germany, and ^¶Department of Microbiology and Hygiene, Charité University Medicine Berlin, 96 Dorotheenstrasse, 10117 Berlin, Germany

Received for publication, April 7, 2004

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Streptococcus pneumoniae, the major cause of community-acquired pneumonia and bacterial meningitis, has been shown to transiently invade epithelial and endothelial cells. Innate immune receptors including Toll-like receptors recognize various pathogens, such as S. pneumoniae, by identifying conserved pathogen-associated molecular patterns. Recently, two members of a novel class of pattern recognition receptors, the cytosolic proteins nucleotide-binding oligomerization domain 1 (Nod1)/CARD4 and Nod2/CARD15, have been found to detect cell wall peptidoglycans. Here we tested the hypothesis that Nod proteins are involved in the intracellular recognition of pneumococci. Data indicate that pneumococci invade HEK293 cells. Genetic complementation studies in these cells demonstrate that NF-B activation induced by S. pneumoniae depends on Nod2. Moreover, intracellular transfection of inactivated pneumococci yielded similar effects, confirming the Nod2 dependence of NF-B activation by pneumococci in HEK293 cells. By dominant negative overexpression and small interfering RNA experiments, we show for the first time that interleukin-1 receptor-associated kinase participates in Nod2-dependent NF-B activation. Additionally, dominant negative interleukin-1 receptor-associated kinase 2, tumor necrosis factor receptor-associated factor 6, NF-B-inducing kinase, transforming growth factor--activated kinase-binding protein 2, and transforming growth factor--activated kinase 1 also inhibited Nod2-dependent NF-B activation. We finally demonstrate that in C57BL/6 mouse lung tissue in vivo as well as in the bronchial epithelial cell line BEAS-2B, Nod1 and Nod2 mRNA expressions were up-regulated after pneumococcal infection. Data presented suggest that Nod proteins contribute to innate immune recognition of S. pneumoniae. Furthermore, Rip-2 and members of the Toll-like receptor-signaling cascade are involved in the Nod2-dependent activation of NF-B induced by pneumococci.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Streptococcus pneumoniae, a human pathogen that frequently colonizes the upper respiratory tract, is the leading bacterial cause of community-acquired pneumonia and meningitis (1, 2). In the United States alone, an estimated 40,000 deaths per year are caused by pneumococcal pneumoniae or meningitis (3). In order to infect different organs, S. pneumoniae crosses blood-tissue barriers by invading and transmigrating epithelial and endothelial cells (4, 5). Hereby, the cell surface protein platelet-activating factor (PAF)¹ receptor as well as phosphorylcholin components of the pneumococcal cell wall are involved (4).

The innate immune system recognizes pathogens by sensing so-called pathogen-associated molecular patterns via its pattern recognition receptors. The best studied innate immune receptors are the Toll-like receptors (TLRs), which also have been found to be involved in defense against S. pneumoniae (6–9). Additionally, we could recently show that lipopolysaccharide binding protein has an important role in defense against pneumococci (10). Another subset of pattern recognition receptors are the recently identified nucleotide-binding oligomerization domain (Nod) proteins, molecules that have been implicated in intracellular recognition of pathogens (11). Whereas Nod1, also called caspase recruitment domain (CARD)-4 protein, has been found to recognize peptidoglycans containing meso-diaminopimelate acid found mainly in Gram-negative bacteria (12, 13), Nod2 (CARD15) mediates responsiveness to the muramyldipeptide MurNAc-L-Ala-D-iso-Gln (MDP) conserved in peptidoglycans of basically all bacteria (14, 15). Nod1 and Nod2 are composed of one or two N-terminal CARDs, a centrally located Nod, and a C-terminal leucine-rich repeat domain (16–18). Nod1 is expressed in multiple tissues, whereas Nod2 expression is mainly restricted to leukocytes, dendritic cells, and epithelial cells (17, 19). Furthermore, Nod1 mRNA has been shown to be up-regulated by interferon- in intestinal epithelial cells, and Nod2 expression was increased in vitro by various inflammatory stimuli like TNF-, interferon-, or lipopolysaccharide (19–21). Currently, the Nod-dependent signaling cascade is poorly understood. After activation of Nod proteins, the Rip2 kinase (also called RICK or CARDIAK) interacts through homophilic CARD-CARD interactions and eventually leads to nuclear translocation of the transcription factor NF-B via IB release by the IB kinase complex (16–18). However, there might be intermediate signaling molecules leading to NF-B activation by Nod proteins that are still unknown.

Because pneumococci penetrate epithelial as well as endothelial cells and Nod proteins mediate intracellular detection of bacterial components, we tested the hypothesis that Nod proteins are involved in host recognition of S. pneumoniae. Overexpression experiments indicate that Nod2 recognizes S. pneumoniae. We furthermore show that Nod protein expression is up-regulated in vivo and in vitro by pneumococcal infection. Moreover, this study suggests that in addition to Rip2, the signal-transducing molecules IRAK, IRAK2, TRAF6, NIK, TAB2, and TAK1 are involved in NF-B activation by Nod proteins in pneumococci-stimulated cells.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Bacterial Strains—Capsulated S. pneumoniae serotype 3 (NCTC 7978) were used for the murine pneumonia model. Bacteria were incubated at 37 °C with 5% CO₂ on Columbia agar with 5% sheep blood at 37 °C. Single colonies were expanded by resuspension in Todd-Hewitt broth supplemented with 0.5% yeast extract and incubation at 37 °C for 3–4 h to midlogarithmic phase (A₆₀₀ = 0.2–0.4). Bacteria were harvested by centrifugation at 1500 x g for 15 min at 4 °C and were washed twice in sterile PBS. Bacteria were then resuspended in sterile PBS, at 2.5 x 10⁸ CFU/ml, as determined by plating serial 10-fold dilutions onto Columbia-agar plates.

The S. pneumoniae R6x is the unencapsulated derivate of serotype 2 strain D39 (22). A pneumolysin-negative mutant of R6x ply^– was generated by insertion-duplication mutagenesis using the pJDC9:ply construct. After transformation of the plasmid into S. pneumoniae strain R6x, erythromycin-resistant transformants were selected with 1 µg/ml erythromycin of Luria-Bertani agar containing 5% sheep blood. Insertion of the plasmid into the pneumolysin-encoding gene ply was confirmed by PCR analysis and DNA sequencing.

For cell culture stimulation studies, R6x or R6x ply^– pneumococci were expanded as described above, harvested by centrifugation, and resuspended in suitable cell culture medium or inactivated by heating at 56 °C for 30 min and sonication. The ethanol inactivation was performed as described before (7).

HEK293 Cell Invasion Assay and PAF Receptor Overexpression—The cDNA encoding the human PAF receptor (kindly provided by T. Shimizu, Tokyo, Japan) was subcloned into the human expression vector cDNA3.1 (Invitrogen). Human embryonic kidney HEK293 cells (ATCC) were cultured in 12-well plates with Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal calf serum. In some experiments, subconfluent cells were transfected with 0.3 µg of PAF receptor plasmid using Effectene transfection reagent (Qiagen, Hilden, Germany). Pneumococcal invasion of HEK293 was assessed by antibiotic protection assay as described previously for endothelial and epithelial cells (4, 5). Briefly, HEK293 cells were infected with 10⁶ CFU/ml S. pneumoniae strain R6x ply^–. After the indicated time intervals, cells were washed three times with PBS and incubated for 1 h with culture medium supplemented with penicillin G (10 µg/ml) and gentamicin (200 µg/ml) to kill extracellular bacteria. Subsequently, HEK293 cells were washed three times again with PBS and lysed by using ice-cold 0.025% Triton X-100, and 100-µl aliquots were plated on Columbia agar with 5% sheep blood to assess numbers of intracellular pneumococci. In a control, the antibiotics used were sufficient to kill 5 x 10⁷ CFU/ml S. pneumoniae in 1 h.

Expression Plasmids, HEK293 Cell Transfection, and Luciferase Assays—Expression plasmids for Nod1 and Nod2 were kindly provided by G. Nuñez (Ann Arbor, MI). Expression vectors encoding wild-type Myc-Rip2 and dominant negative (dn) Rip-2 were a generous gift from P. Dempsey (Los Angeles, CA). The constructs encoding dnMyD88, dnIRAK, dnIRAK2, dnTRAF6, and dnNIK were from C. Kirschning (Munich, Germany) and Tularik Inc. (San Francisco, CA); dnTAK1 and dnTAB2 were a kind gift from Tohru Ishitani (Nagoya, Japan). dnMal and dnTRIF were from K. Fitzgerald (Golenbock Laboratory, University of Massachusetts, Worchester, MA). Subconfluent HEK293 cells were cotransfected the following day using the calcium phosphate method according to the manufacturer's instructions (Clontech) with 0.1 µg of NF-B-dependent luciferase reporter (23), 0.1 µg of RSV -galactosidase plasmid, 0.1 µg of PAF receptor, and 0.3 ng of Nod or 0.5 µg of Rip2 expression vectors. In some experiments, 0.5 µg/ml MDP (Calbiochem) or 5 x 10⁷ CFU/ml inactivated S. pneumoniae were added to the cells prior to transfection to allow their entry into the cells. When indicated, cells were additionally transfected with 0.1 µg of dominant negative mutants of several signaling molecules. The experiment shown in Fig. 4B was performed using 5, 25, and 125 ng of dnIRAK or dnIRAK2. In some experiments, cells were incubated with S. pneumoniae strain R6x ply^– for 5 h on the following day. Luciferase activity was measured by using the Luciferase Reporter-Gene Assay (Promega, Mannheim, Germany), and results were normalized with values obtained by RSV--galactosidase or protein concentrations (Fig. 1A).

View larger version (26K): [in this window] [in a new window]   FIG. 4. Inhibition of Nod2-dependent activation of NF-B by dominant negative mutants or siRNA targeting IRAK as well as dominant negative IRAK2, TRAF6, NIK, TAB2, and TAK1. HEK293 cells were cotransfected with Nod2 expression plasmid, NF-B-dependent luciferase reporter, and RSV -galactosidase plasmid in the presence of 5 x 107 CFU/ml heat-inactivated pneumococci (S.p.) or 0.5 µg/ml MDP. Additionally, 0.1 µg of dn mutants of IRAK, IRAK2, TRAF6, NIK, TAB2, and TAK1 (A) or different amounts as indicated of dnIRAK and dnIRAK2 (B) were cotransfected. Luciferase activity was obtained by luciferase assay and normalized with -galactosidase activity. Relative luciferase activities are shown as means of -fold inductions ± S.E. for one representative experiment of two with transfection performed in triplicate (A), and titration of dominant negative constructs was performed in duplicate (B). C, HEK293 cells were transfected with nonsilencing siRNA (c) or different sequences of siRNA targeting IRAK (S1–S3) for 24 h. After 72 h, cells were lysed, and Western blots (WB) using antibodies recognizing IRAK or p38 MAP kinase (as a control) were performed. D, HEK293 cells were transfected with nonsilencing siRNA or siRNA targeting IRAK (final concentration 100 nM) for 24 h. After further 24 h, cells were transfected with an NF-B-dependent luciferase reporter, RSV -galactosidase plasmid, and Nod2 expression plasmid where indicated. Stimulation with 10 ng/ml TNF- was used as a control. siRNA experiments were performed three times with similar results, each transfection performed in duplicate (D).

View larger version (13K): [in this window] [in a new window]   FIG. 1. Internalization of HEK293 cells by S. pneumoniae and effects of Nod1 and Nod2 overexpression on NF-B activation. A, HEK293 cells were transiently transfected with a control vector, Nod1 or Nod2 expression plasmids, and plasmids encoding PAF receptor and an NF-B-dependent luciferase reporter. The next day, cells were infected with the indicated doses of S. pneumoniae for 5 h. Relative luciferase activities are shown as means of -fold inductions ± S.E. for one representative experiment of three, each transfection performed in triplicate. B, HEK293 cells overexpressing PAF receptor were infected with S. pneumoniae for the indicated time intervals, and an antibiotic protection assay was performed. The number of internalized pneumococci was determined by assessing CFU surviving antibiotic treatment.

Murine Model of Pneumococcal Pneumonia—Pathogen-free, female C57BL/6 mice were obtained from Charles River (Sulzfeld, Germany). The animals were kept with a 12-h light/dark cycle and were given free access to food and water. The study was approved by the Institutional Review Board for the care of animal subjects. Mice were challenged at 10 weeks of age and 19–20 g of weight.

Mice were lightly anesthetized by intraperitoneal injection of ketamine and xylazine and were inoculated intranasally with 20 µl of bacterial suspension (5 x 10⁶ CFU of S. pneumoniae serotype 3). Control mice were challenged with 20 µl of sterile PBS. Groups of three mice were sacrificed 6, 12, 24, or 48 h postinfection, and lungs were removed aseptically and immediately frozen in liquid nitrogen. Control mice were sacrificed 6 h postinfection (n = 3). Frozen lung tissue was crushed using a sterile, nitrogen-cooled high grade steel plate homogenizer. Total RNA was extracted using an RNeasy Mini Isolation kit (Quiagen) following the manufacturer's protocol. For cDNA synthesis, 2 µg of pooled RNA from each group was reversed transcribed using a Pro Stare First Strand RT-PCR kit (Stratagene Europe, Amsterdam, The Netherlands) following the manufacturer's protocol by using oligo(dT) primers. Murine glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as housekeeping gene. After 35 (Nod1) or 40 (Nod2) PCR amplification cycles, samples were resolved on a 1.2% agarose gel. The PCR primers for Nod1 (expected size 469 bp) were 5'-CTTGCATTCAATGGCATCTC-3' and 5'-ACATCGGTGTGCACTGTGGA-3'; primers for Nod2 (expected size 515 bp) were 5'-AGCAGAACTTCTTGTCCCTGA-3' and 5'-TCACAACAAGAGTCTGGCGT-3'; and primers for GAPDH (expected size 496 bp) were 5'-TGATGGGTGTGAACCACGAG-3' and 5'-TCAGTGTAGCCCAAGATGCC-3'. Total RNA from lipopolysaccharide-stimulated murine macrophages (RAW 264.7; murine macrophage cell line) served as positive control.

Stimulation of BEAS-2B Cells, mRNA Isolation, and Reverse Transcription-PCR—The human bronchial epithelial cell line BEAS-2B (a generous gift of Dr. Curtis Harris, Bethesda, MD) was cultured in keratinocyte medium supplemented according to the manufacturer's instructions (ATCC, Manassas, VA). Cells were grown to confluence in 3.5-cm well plates, subsequently infected with 10⁶ CFU/ml S. pneumoniae strain R6x for different durations, stimulated with 5 x 10⁷ CFU/ml heat-inactivated pneumococci or 20 ng/ml TNF- (R&D Systems, Wiesbaden, Germany) for 24 h. Total RNA was isolated with the RNeasy minikit (Qiagen) and reverse transcribed using avian myeloblastosis virus reverse transcriptase (Promega, Heidelberg, Germany). The generated cDNA was amplified by PCR using specific primers (Nod1-sense, 5'-AAGCGAAGAGCTGACCAAAT-3'; Nod1-antisense, 5'-TTCATAGACTTTGGCCTCCTC-3'; Nod2-sense, 5'-AGCCATTGTCAGGAGGCTC-3'; Nod2-antisense, 5'-CGTCTCTGCTCCATCATAGG-3'). All primers were purchased from TIB MOLBIOL (Berlin, Germany). After 35 (Nod1) or 40 (Nod2) 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—Nod2-dependent stimulatory effects of active or inactive S. pneumoniae as well as inhibitory effects of dominant negative signaling molecule mutants were statistically evaluated employing Student's t test. Throughout the figures, p values of <0.05 are indicated by one asterisk, and p values of <0.01 are shown by double asterisks.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
S. pneumoniae Invade HEK293 Cells and Activate NF-B in a Nod2-dependent Manner—To explore a possible function of Nod proteins in host recognition of S. pneumoniae, we performed overexpression assays in HEK293 cells. This cell line has been described as not expressing TLR2, a well known receptor for Gram-positive patterns (24). Moreover, we could not detect any Nod2 mRNA and detected only very low levels of Nod1 mRNAs in these cells by RT-PCR analysis (data not shown). We transiently transfected the cells with an NF-B-dependent luciferase reporter construct and Nod1 or Nod2, respectively. Additionally, since the PAF receptor has been shown to be involved in internalization of pneumococci (4, 5), we cotransfected a plasmid encoding the PAF receptor. On the next day, cells were exposed to different concentrations of viable S. pneumoniae strain R6x ply^–. We used this pneumolysin-negative mutant for HEK cell experiments in order to avoid pneumolysin-induced damage of HEK293 cells. The bacteria failed to cause induction of reporter gene activity if only a control vector was transfected (Fig. 1A). After overexpressing of Nod1 or Nod2, we observed a constitutive reporter activity, which is in line with previously published results (14). Pneumococcal infection further enhanced the reporter gene activity in Nod2-transfected cells in a dose-dependent manner, thus indicating a role of Nod2 in NF-B activation by pneumococci. Nod2-dependent NF-B activation by pneumococci was less pronounced in cells that were transfected with an empty control vector instead of the PAF receptor plasmid, indicating the importance of cell invasion of pneumococci for Nod signaling (data not shown). Taken together, the data indicate that Nod2 is involved in recognition of internalized S. pneumoniae.

To further confirm internalization of pneumococci into HEK293 cells, we performed an antibiotic protection assay as described previously (4, 5). As seen in Fig. 1B, S. pneumoniae strain R6x ply^– was internalized into HEK293 cells overexpressing PAF receptor in a time-dependent manner.

Nod2 Mediates NF-B Activation by Inactivated S. pneumoniae—To further characterize the role of Nod proteins in the recognition of pneumococci, we used heat-inactivated, sonicated, or ethanol-inactivated pneumococci; MDP served as a positive control. In contrast to viable bacteria, inactivated pneumococci as well as MDP failed to induce NF-B activation in Nod-transfected cells when extracellular stimulation was performed (data not shown), an observation compatible with the notion that Nod2 detects only intracellularly located pathogen-derived material. We therefore assessed Nod-dependent NF-B activity after allowing the internalization of bacterial components into the cells (as described under "Experimental Procedures"). Under these conditions, Nod2 conferred a dose-dependent responsiveness to inactivated pneumococci and MDP (Fig. 2, A and B). Hereby, the ethanol-inactivated pneumococci were more potent then the heat-inactivated pneumococci, displaying Nod2 activity already at a concentration of 5 x 10⁵ CFU/ml. Again, we observed a certain constitutive activity caused by overexpression of Nod1 and Nod2. The different quantities in these background activities may be caused by slightly variable transfection conditions.

View larger version (22K): [in this window] [in a new window]   FIG. 2. Nod2 mediates NF-B activation by inactivated pneumococci when intracellularly challenged. HEK293 cells were transiently transfected with a control vector, Nod1 or Nod2 expression plasmids, an NF-B-dependent luciferase reporter, and an RSV -galactosidase plasmid. Moreover, heat-inactivated (A) or ethanol-inactivated (B) pneumococci (inact. S.p.) as well as MDP were added to the cells during transfection. Luciferase activities were obtained as described under "Experimental Procedures." Data are shown as means of -fold inductions ± S.E. for one representative experiment of three, each transfection performed in triplicate (A). The transfection experiment shown in B was performed in duplicate.

View larger version (22K): [in this window] [in a new window]   FIG. 3. Involvement of Rip2, but not of the TLR-adapter proteins in NF-B activation by inactivated pneumococci and MDP. HEK293 cells were cotransfected with Nod2 expression plasmid, NF-B-dependent luciferase reporter, RSV -galactosidase plasmid, and dn mutants of MyD88, Mal, TRIF, and Rip2 in the presence of 5 x 107 CFU/ml heat-inactivated pneumococci (S.p.) or 0.5 µg/ml MDP. After 24 h, luciferase activity was obtained by luciferase assay and normalized with -galactosidase activity. Relative luciferase activities are shown as means of -fold inductions ± S.E. for one representative experiment of two with transfection performed in triplicate.

To further confirm the unexpected role of IRAK in Nod2 signaling, gene silencing experiments using siRNA were performed. To assess gene silencing activities, HEK293 cells were transfected with different siRNAs targeting IRAK as well as nonsilencing siRNA for 24 h, and Western blot analyses using anti-IRAK antibodies were performed 72 h after transfection. Sequence 3 was most effective in gene silencing (Fig. 4C) and was therefore used in subsequent reporter gene assays. As can be seen in Fig. 4D, siRNA targeting IRAK inhibited the NF-B activation brought about by overexpression of Nod2. In contrast, IRAK siRNA duplexes had no inhibitory effect on TNF- response.

IRAK, IRAK2, TRAF6, NIK, TAB2, and TAK1 Are Located Downstream of Nod2 and Rip2—To examine whether IRAK, IRAK2, NIK, TAB2 and TAK1 lie upstream or downstream of Rip2, plasmids encoding wild-type RIP2, an NF-B reporter, and the dominant negative mutants of signaling molecules examined were cotransfected in HEK293 cells. Overexpression of Rip2 leads to a strong NF-B activation (Fig. 5). In line with the above mentioned studies, the reporter gene activation could not be suppressed by dnMyD88, dnMal, and dnTRIF. Furthermore, dnIRAK, dnIRAK2, dnTRAF6, dnNIK, dnTAB2, and dnTAK1 inhibited Rip2-induced NF-B activation. Thus, IRAK, IRAK2, TRAF6, NIK, TAB2, and TAK1 seem to be located downstream of Rip2 and play essential roles in transducing information from Nod2 to NF-B activation.

View larger version (19K): [in this window] [in a new window]   FIG. 5. IRAK, IRAK2, TRAF6, NIK, TAB2, and TAK1 are located downstream of RIP2. HEK293 cells were transfected with empty control vector or wild-type Rip2, together with an NF-B-dependent luciferase reporter and RSV -galactosidase plasmid. Additionally, dominant negative mutants were cotransfected as indicated. After 24 h, luciferase activity was obtained by luciferase assay and normalized with -galactosidase activity. Relative luciferase activities are shown as means of -fold inductions ± S.E. for one representative experiment of two with transfection performed in triplicate.

View larger version (30K): [in this window] [in a new window]   FIG. 6. S. pneumoniae induces Nod1 and Nod2 mRNA levels in vivo and in vitro. A, C57BL/6 mice were inoculated intranasally with capsulated S. pneumoniae serotype 3 for different durations, total RNA of lung tissue was isolated, and RT-PCR for Nod1, Nod2, and GAPDH was performed. B, BEAS-2B cells were infected with S. pneumoniae or stimulated with heat-inactivated pneumococci (HIP) and TNF-. After the indicated time intervals, total RNA was isolated, and expression of Nod1 and Nod2 was analyzed by RT-PCR. GAPDH mRNA was used as an amplification control. The experiments were performed twice with independent samples.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
In the presented study, we show for the first time a role of Nod proteins in host recognition of S. pneumoniae. Overexpression experiments indicate that Nod2 mediates NF-B activation by viable S. pneumoniae or when inactivated pneumococci were transfected within the cells. Moreover, Rip2, IRAK, IRAK2, TRAF6, NIK, TAB2, and TAK1 seemed to be involved in Nod2-dependent NF-B activation. We finally demonstrate that Nod1 and Nod2 expression was up-regulated after pneumococcal infection in vivo and in vitro, suggesting a physiological role of Nod proteins in immune response against pneumococci in pulmonary tissue.

TLR2 and TLR4 have been associated with host recognition of pneumococci (6–9). Whereas these TLRs are likely to serve as the first line receptors for S. pneumoniae, the Nod proteins might play a major role in a subsequent phase of infection. Since TLRs mediate NF-B activation and NF-B binding sites have been identified in the Nod2 promoter (19, 21), recognition of pneumococci by the TLRs might cause the up-regulation of Nod2 (and probably even Nod1) and thereby facilitate the immune response of the host against this pathogen. In line with this hypothesis, the penetration of epithelial and endothelial cells by S. pneumoniae is initiated during the first hours after infection, and it is most pronounced after 4–6 h (5). mRNA levels of Nod1 and Nod2 increased within a similar time frame, suggesting that Nod-mediated NF-B activation might play an important role in this subsequent phase of host responses against S. pneumoniae.

The downstream signaling cascade induced by Nod activation is currently poorly understood. By two different approaches, such as dominant negative overexpression and siRNA experiments, we demonstrated that IRAK participates in Nod2 signaling. Moreover, data presented suggest that molecules like IRAK2, TRAF6, NIK, TAB2, and TAK1 are additionally involved in NF-B activation and seemed to be located downstream of Nod2 and Rip2. Thus, Nod-mediated signaling might be partly homologous to TLR/MyD88 downstream signaling (25). Importantly, the TLR adapter proteins MyD88, Mal, and TRIF did not participate in NF-B activation by Nod2, indicating that Nod and TLR pathways merge down-stream of MyD88, Mal, and TRIF. In the Rip2 knockout mice, even TLR-dependent signal transduction was attenuated, showing that Rip2 is not exclusively involved in Nod-regulated cell activation (27, 28), and suggesting a model in which Rip2 could be a junction. Nevertheless, analyses using knockout mice and further siRNA experiments should be initiated to clarify differences and homologies of Nod and TLR signaling pathways.

The Nod-dependent NF-B activation by intact or inactive S. pneumoniae is most likely due to cell wall peptidoglycan. Nod2 has been found to mediate cell activation by a muramyl-dipeptide conserved in basically all kinds of peptidoglycans (14, 15), whereas meso-diaminopimelate acid is responsible for Nod1 utilization (12, 13). Our finding that pneumococci showed a clear Nod2 but no Nod1 activity is thus in line with the fact that lysine, and not meso-diaminopimelate acid, has been found as the third amino acid in peptidoglycans of various pneumococcal strains (29). The high constitutive activity of overexpressed Nod1 and Nod2 might be responsible for the limited effect of viable pneumococci on Nod-dependent NF-B activation (in contrast to the more distinct effects of intracellularly transfected pneumococci).

A recently published paper describes macrophages from Nod2 knockout mice as responding normally to different pathogen-associated molecules, leading these authors to challenge the role of Nod2 in host defenses (30). In this study, cells were stimulated with known TLR ligands from the extracellular side, and no viable pathogens were used. It remains to be determined how these Nod2-deficient cells would respond to living invasive bacteria. Thus, the Nod2-deficient mice as well as the recently generated Nod1 knockout mice will be of invaluable help to further elucidate the precise role of these proteins in host defense (12, 13, 30).

Overall, besides S. pneumoniae recognition by TLRs, Nod2 activation seems to play an important role in host cell activation by internalized pneumococci. Downstream of Nod2 and Rip2, signal-transducing molecules like IRAK, IRAK2, TRAF6, NIK, TAB2, and TAK1 might mediate NF-B-dependent cell activation. Knowledge about the molecular interaction of pneumococci with target cells may pave the way to innovative therapeutic strategies.

	FOOTNOTES

 
^* This work was supported in part by grants from the Bundesministerium für Bildung und Forschung NBL3 (to S. H.) and CAP-NETZ (to N. S., R. R. S., and S. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^|| To whom correspondence should be addressed. Tel.: 49-30-450-553052; Fax: 49-30-450-553911; E-mail: stefan.hippenstiel{at}charite.de.

¹ The abbreviations used are: PAF, platelet-activating factor; CARD, caspase recruitment domain; dn, dominant negative; IRAK, interleukin-1 receptor-associated kinase; MyD88, myeloid differentiation protein 88; Mal, MyD88 adapter-like; MDP, muramyl dipeptide; NIK, NF-B-inducing kinase; Nod, nucleotide-binding oligomerization domain; Rip2, receptor-interacting protein 2; TAB, transforming growth factor--activated kinase-binding protein; TAK, transforming growth factor--activated kinase; TLR, Toll-like receptors; TRAF, tumor necrosis factor receptor-associated factor; TRIF, TIR domain-containing adapter-inducing interferon-; TNF, tumor necrosis factor; PBS, phosphate-buffered saline; CFU, colony-forming units; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; siRNA, small interfering RNA; RT, reverse transcription.

	ACKNOWLEDGMENTS

 
We are grateful to G. Nuñez, P. Dempsey, K. Fitzgerald, C. Kirschning, Tularik Inc., T. Ishitani, and T. Shimizu for providing expression vectors. We further thank K. Mohr, V. Johnston, and S. Moderer for excellent technical assistance.

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TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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