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Departments of*
Medicine, Gastroenterology, and Tumor Immunology, Radboud University Medical Center Nijmegen, and Nijmegen University Center for Infectious Diseases, Nijmegen, The Netherlands; and ¶Unité de Pathogénie Microbienne Moléculaire, Institut Pasteur, Paris, France.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Initially, NOD2 was suggested to be an intracellular pattern recognition receptor for LPS (4), similar to the NOD1 (6), but later studies have demonstrated that NOD2 is the intracellular receptor for the muramyl dipeptide (MDP) component of peptidoglycan (PGN) (7, 8). However, other studies have shown that PGN stimulates cytokine production through TLR2, and it has been unclear whether NOD2 and TLR2 pathways are independent or interact with each other. The latter possibility has been suggested in patients with Crohn’s disease homozygous for the 3020insC mutation of the NOD2 gene, who were found to have a defective release of cytokines not only after stimulation with MDP and PGN, but also after other TLR2 stimuli such as tripalmitoyl-S-glycerylcysteine (Pam3Cys) (9). Moreover, the interaction between the NOD2 and TLR2 pathways is sustained by recent data from mice deficient in NOD2, which show down-regulation of TLR2-mediated Th1 responses by MDP (10).
In the present study we investigated the possible interaction between NOD2 and TLR pathways in human cells by studying the modulation of TLR-induced cytokine production by NOD2 signals activated by MDP. We assessed 1) the differential role of NOD2 and TLR2 for the induction of cytokines by Staphylococcus aureus PGN; 2) the synergistic effects of MDP on the cytokine production stimulated by the TLR2 ligands Pam3Cys and macrophage-activating lipopeptide 2 from Mycoplasma fermentation (MALP2); and 3) the functional consequences for the NOD2/TLR2 synergism of the NOD2 3020insC frameshift mutation in patients with Crohn’s disease. In addition, we have investigated the possible interaction of NOD2 with other TLRs, namely the synergism of MDP/NOD2 with LPS (TLR4 signals), flagellin (TLR5), polyinosinic-polycytidylic (poly(I:C); TLR3), unmethylated CpG sequences of bacterial DNA (TLR9), and loxoribin (TLR7).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Blood was collected from 74 patients with Crohn’s disease and 10 healthy volunteers. PCR amplification of NOD2 gene fragments containing the polymorphic site 3020insC was performed in 50-µl reaction volumes containing 100–200 ng of genomic DNA, as previously described (9). The 3020insC polymorphism was analyzed by Genescan analysis on an ABI PRISM 3100 Genetic Analyzer according to the protocol of the manufacturer (Applied Biosystems).
Four patients with Crohn’s disease were found to be homozygous for the 3020insC mutation, and they were further investigated in the cytokine studies. As control groups, five patients with Crohn’s disease heterozygous for the 3020 insC NOD2 mutation, five patients with Crohn’s disease bearing the wild-type allele, and five healthy volunteers homozygous for the wild-type NOD2 allele were included.
Isolation of mononuclear cells and stimulation of cytokine production
After informed consent, venous blood was drawn from the cubital vein of patients and healthy volunteers into three 10-ml EDTA tubes (Monoject; s-Hertogenbosch). Isolation of mononuclear cells (MNC) was performed as described previously (11) with minor modifications. The MNC fraction was obtained by density centrifugation of blood diluted 1/1 in pyrogen-free saline over Ficoll-Paque (Pharmacia Biotech). Cells were washed twice in saline and suspended in culture medium (RPMI 1640 DM) supplemented with 10 µg/ml gentamicin, 10 mM L-glutamine, and 10 mM pyruvate. The cells were counted in a Coulter counter (Coulter Electronics), and the number was adjusted to 5 x 106 cells/ml.
MNC (5 x 105) in a 100-µl volume were added to round-bottom, 96-well plates (Greiner) and incubated with either 100 µl of culture medium (negative control) or the various stimuli (MDP (10 µg/ml; Sigma-Aldrich), commercial staphylococcal peptidoglycan (1 µg/ml; Sigma-Aldrich), purified peptidoglycan (gift from Dr. S. Girardin, Institute Pasteur, Paris, France), or synthetic Pam3Cys or MALP2 lipopeptides (1 µg/ml; EMC Microcollections)).
In separate experiments, stimulation of MNC with other TLR agonists was performed: TLR3 with poly(I:C) (50 µg/ml), TLR4 with purified Escherichia coli LPS (1 ng/ml), TLR5 with bacterial DNA (5 µg/ml), TLR5 with flagellin (10 ng/ml), TLR7 with loxoribin (5 µg/ml), and TLR9 with CpG (5 µg/ml). The synergism between NOD2 and TLR pathways was assessed using combinations of MDP with the various TLR stimuli. Submaximal concentrations of the TLR agonists were used in the experiments to allow better distinction of the synergistic effects with MDP among the various groups. The submaximal concentrations described above were determined in pilot experiments (not shown). All TLR agonists were checked for contamination with LPS in the Limulus amebocyte lysate assay and were found to be negative.
Cytokine production by murine peritoneal macrophages
Resident peritoneal macrophages from either ScCr (TLR4-defective) or C57BL/10J (TLR4 control) mice and from either TLR2–/– or control TLR2+/+ mice (provided by Dr. S. Akira, Research Institute for Microbiol Diseases, Osaka University, Osaka, Japan) (12) were harvested by injection of 4 ml of sterile PBS containing 0.38% sodium citrate (13). After centrifugation and washing, the cells were resuspended in RPMI 1640 containing 1 mM pyruvate, 2 mM L-glutamine, 100 µg/ml gentamicin, and 2% fresh mouse plasma. Cells were cultured in 96-well microtiter plates (Greiner) at 1 x 105 cells/well in a volume of 100 µl. The cells were stimulated with either purified staphylococcal peptidoglycan (1 µg/ml; commercial or purified) or a combination of MDP with Pam3Cys (both 1 µg/ml). After 24-h incubation at 37°C, the supernatants were collected and stored at –70°C until cytokine assays were performed.
Cytokine measurements
Human and murine TNF-
and IL-1
concentrations were determined by specific RIAs as previously described (14, 15). IL-10 was measured by a commercial ELISA kit (Pelikine Compact; CLB) according to the instructions of the manufacturer. IL-12 concentrations were measured using two ELISA kits from R&D Systems and Pierce.
PGN signaling through human TLR2 in transfected cell lines
Chinese hamster ovary (CHO) fibroblasts stably transfected with human CD14 and TLR2 (3E10-TLR2) or TLR4 (3E10-TLR4), were a gift from Dr. R. Ingalls (Boston University, Boston, MA; Ref. 16). Cell lines express inducible membrane CD25 under control of a region from the human E-selectin (ELAM-1) promoter containing NF-
B binding sites. Cells were maintained at 37°C and 5% CO2 in Ham’s F-12 medium (Invitrogen Life Technologies) supplemented with 10% FCS, 0.01% L-glutamine, 50 µg/ml gentamicin, 400 U/ml hygromycin, and 0.5 mg/ml G418 (for 3E10-TLR2) or 0.05 mg/ml puromycin (for 3E10-TLR4) as additional selection antibiotics. TLR2 and TLR4 expression was confirmed by flow cytometry (EPICS XL-MCL, Beckman Coulter) using PE-labeled anti-TLR2 (clone TL2.1) or anti-TLR4 (clone HTA125; Immunosource).
For stimulation experiments, 500 µl of cells in culture medium at a density of 1 x 105/ml were plated in 24-well culture plates. After an overnight incubation, cells were incubated with control medium, PGN (10 µg/ml), Pam3Cys (10 µg/ml), or LPS (1 µg/ml) for 20 h; thereafter, cells were harvested using trypsin/EDTA (Cambrex) and prepared for flow cytometry (Coulter FACScan). CD25 expression of the CHO cells was measured using FITC-labeled anti-CD25 (DakoCytomation).
Statistical analysis
The human experiments were performed in triplicate with blood obtained from patients and volunteers. The mouse experiments were performed twice in 10 mice/group, and the data are presented as the cumulative results of all experiments performed. The differences between groups were analyzed by Mann-Whitney U test and, where appropriate, by Kruskal-Wallis ANOVA. The level of significance between groups was set at p < 0.05. The data are given as the mean ± SD.
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Results |
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To investigate the involvement of TLR2 and NOD2 in the recognition of PGN, we stimulated cells isolated from mice deficient in TLR2 or from patients homozygous for the 3020insC mutation. TLR2–/– macrophages released significantly less TNF and IL-10 after stimulation with commercial PGN than control TLR2+/+ mice, whereas the production of TNF and IL-10 by TLR4-defective ScCr mice was intact (Fig. 1A). In contrast, stimulation with purified PGN, although less than that of commercial PGN, was independent of both TLR2 and TLR4 (Fig. 1B). In contrast, MNC isolated from patients homozygous for the 3020insC NOD2 mutation showed deficient production of TNF and IL-10 after stimulation with purified PGN (Fig. 2).
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NOD2 synergizes with TLR2 for production of cytokines
The data showing the requirement for both NOD2 and TLR2 for the production of cytokines by commercial PGN suggest an interaction between these two pathways. In line with this hypothesis, the specific NOD2 ligand MDP was found to have a synergistic effect on the induction of TNF, IL-1, and IL-10 upon costimulation with the specific TLR2 agonists Pam3Cys (Fig. 3A) and MALP2 (Fig. 3B). In contrast, no IL-12p70 production was measured with any of the stimuli tested in both ELISAs used.
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Microorganisms display complex combinations of pathogen-associated molecular patterns (PAMPs) involved in simultaneous stimulations of more TLR pathways. We have investigated whether NOD2 modulates signals induced by various TLR agonists. Costimulation with the specific NOD2 ligand MDP potentiated the induction of TNF induced by LPS (TLR4 stimulation; Fig. 6A) and poly(I:C) (TLR3; Fig. 6C), whereas no effect was observed after stimulation with CpG (TLR9; Fig. 6B), flagellin (TLR5; Fig. 6D), or loxoribin (TLR7; not shown). The loss of synergistic activity in patients homozygous for the 3020insC NOD2 allele demonstrates that NOD2 is crucial for the potentiation of TLR4 and TLR3 signals by MDP (Fig. 6).
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Discussion |
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There has been both confusion and controversy about whether recognition of PGN by the cells of the innate immune system occurs by surface TLR2 or by intracellular NOD2. Based on the studies performed in TLR2–/– mice, which are unresponsive to PGN and display enhanced susceptibility to Gram-positive microorganisms (17), it has initially been proposed that TLR2 is the cellular receptor for PGN. This assumption has later been challenged by the discovery of NOD2 as an intracellular receptor for the MDP component of PGN (18). NOD2 mediates recognition of MDP and induces NF-B activation after MDP challenge (7, 8), and mice deficient in NOD2 fail to respond to a challenge with MDP (19). In addition, mutations in NOD2 have been implicated in the pathogenesis of Crohn’s disease (3, 4), and patients homozygous for the 3020insC mutated allele have defective cellular responses to MDP and PGN (9). The apparent discrepancy between the studies implicating either TLR2 or NOD2 for the recognition of PGN could be explained in two ways: firstly, by the contamination of commercial PGN by TLR2 ligands such as lipoteichoic acid, and secondly, by the cooperation between NOD2 and TLR2 pathways for the induction of cytokines. In the present study we demonstrate that although NOD2 recognizes purified PGN, both TLR2 and NOD2 are required for the efficient induction of proinflammatory cytokines by commercial PGN preparations, demonstrating that there is interaction between the TLR2 and NOD2 pathways.
A possible interaction between TLR2 and NOD2 pathways was also suggested by our observation that patients homozygous for the loss of function 3020insC NOD2 mutation are defective for the release of cytokines not only after PGN stimulation, but also after challenge with other TLR2 stimuli, such as Pam3Cys (9). In the present study we demonstrate a synergism between NOD2- and TLR2-mediated signals, as shown by the synergistic effect of MDP on both Pam3Cys- and MALP2-induced TNF, IL-1, and IL-10 production. The need for both receptors for this effect is demonstrated by the absence of synergism in patients bearing the 3020insC NOD2 mutation as well as in TLR2–/– mice. Our data regarding the interaction of the TLR2 and NOD2 pathways in human MNC are supported by earlier studies showing synergistic effects of MDP with PGN and lipoteichoic acid (20, 21) and are reinforced by the findings of Watanabe et al. (10), who reported an interaction between NOD2 and TLR2 signals in murine macrophages. Interestingly, stimulation of the anti-inflammatory cytokine IL-10 by Pam3Cys, a specific TLR2 ligand, also seems to be decreased in patients with the NOD2 3020insC mutation. We observed this effect in a previous study (9), and this suggests that NOD2 might exert some of its actions through directly mediating TLR2 signals. No such defects were observed when cells of the patients bearing the NOD2 mutation were stimulated with other TLR2 ligands.
Watanabe et al. (10) reported inhibitory effects of murine NOD2 signals on TLR2-induced Th1-type responses, as measured by IL-12 production. In our experiments using human NOD2-defective and control cells, IL-12 production was under the detection limit despite the use of stimulus concentrations as high as 10 µg/ml. We cannot confirm the inhibitory effect of NOD2 signals on Th1 responses found by Watanabe et al. (10) in murine cells, but a note of caution about their data should be mentioned, because the effects seen in their study were observed mainly at an MDP concentrations of 100 µg/ml, which is hardly relevant for the in vivo situations. Another source of differences between our study and that of Watanabe et al. (10) is that they did not find differences in IL-10 release after PGN stimulation of murine NOD2–/– and NOD2+/+ cells (10). This is an important observation, because it may underline important differences between the function of NOD2 in murine vs human cells and may explain the striking observation of the lack of intestinal inflammation in NOD2–/– mice (19) despite the crucial role of NOD2 in the development of Crohn’s disease in humans. Alternatively, the observed differences between studies performed in human and murine cells may be caused by the presence of a defective NOD2 in patients, whereas a complete deletion of NOD2 was present in the knockout mice. Despite these differences, the synergistic interaction between NOD2 and TLR2 signals for the production of IL-10 in human MNC as well as the negative regulation of IL-12 production by NOD2, as shown by Watanabe et al. (10), with a shift toward protective Th2-type responses are probably important pathogenetic mechanisms in Crohn’s disease.
The impact of NOD2 loss of function seems, therefore, to be related to the Th1/Th2 balance, rather than to the absolute defect in one of these cytokines. Thus, although production of the proinflammatory cytokines TNF and IL-1 is lower in Crohn’s disease patients with the NOD2 3020 insC mutation, this effect seems to be over-run by the lack of protective Th2-type responses. This does not contradict the important role of TNF, because patients with Crohn’s disease have been shown to have higher TNF production capacity and to be effectively treated by anti-TNF Abs (22). An alternative explanation for the decreased proinflammatory cytokine production in patients with the mutation could be represented by defective antibacterial defense, leading to bacterial overgrowth and inflammation, as also suggested by others (23).
In addition to the interaction of NOD2-induced signals with TLR2, we demonstrate synergism of NOD2 with TLR4- and TLR3-mediated signals. Intestinal microorganisms are complex pathogens, with PAMPs requiring recognition by diverse TLRs, such as TLR2, TLR4, TLR5, and TLR9. From this perspective, interaction of the NOD2 pathway with other TLRs can be of considerable importance. In line with this idea, spontaneous enterocolitis associated with IL-12 overproduction in STAT3 knockout mice does not occur in the absence of TLR4 (24), and TLR9 stimulation by CpG motifs increases the severity of dextran sodium sulfate-induced colitis (25).
Synergism between either PGN or MDP and bacterial LPS has been previously reported (20, 21, 26). By showing the loss of MDP/LPS synergism in patients with the 3020insC NOD2 mutation, we demonstrate that this effect is mediated by the interaction between NOD2 and TLR4 pathways. In addition, we report synergism between NOD2 and TLR3; this interaction needs to be explored in models of intestinal inflammation.
The effects of NOD2 on TLR pathways seem to be selective, because we found no interaction with TLR5, TLR9, or TLR7. Despite the fact that TLR9 has been implicated in the pathogenesis of Crohn’s disease based on experimental data (25, 27) as well as the association of Crohn’s disease with a TLR9 polymorphism (28), we could not find an interaction of NOD2 with the TLR9 pathway. Although the TLR5 agonist flagellin is an important PAMP of intestinal microorganisms, intestinal microvascular cells express TLR5, and bacterial flagellin has been suggested to be a dominant Ag in Crohn’s disease (29, 30), no interaction between NOD2 and TLR5 was detected. Likewise, there was no interaction between NOD2 and TLR7.
In conclusion, we report in this study that NOD2 enhances the signaling by TLR2, TLR4, and TLR3, whereas it does not influence the signals mediated by TLR5, TLR9, and TLR7. The interaction between NOD2 and TLRs is likely to be involved in the pathogenesis of Crohn’s disease, because the lack of synergism in patients with loss of function mutations of NOD2 results in defective IL-10 production and a Th1 bias, effects that seem to over-ride the lower production of TNF and IL-1. Studies are ongoing in our laboratory to decipher the precise intracellular mechanisms responsible for the NOD2/TLR synergism. Other studies have suggested the involvement of receptor-interacting protein 2 in the transduction of signals to both NOD2 and TLRs (31). In addition, Wolfert et al. (20) proposed that the synergistic effect of MDP and LPS is exerted at the translational level; MDP is effective in transcribing DNA information in mRNA, but little translation of proteins takes place. Costimulation with specific (but not all) TLR ligands provides the signals able to translate the mRNA pool, leading to synergistic effects at the protein level. It remains to be demonstrated which of these mechanisms is responsible for the synergistic effects of these two pathways.
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Disclosures |
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Footnotes |
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1 M.G.N. was supported by a ViDi grant from the Netherlands Organization for Scientific Research. 
2 Address correspondence and reprint requests to Dr. Mihai G. Netea, Department of Medicine (541), Radboud University Medical Center, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands. E-mail address: m.netea{at}aig.umcn.nl
3 Abbreviations used in this paper: NOD2, nucleotide-binding oligomerization domain-2; CHO, Chinese hamster ovary; MDP, muramyl dipeptide; MNC, mononuclear cell; PGN, peptidoglycan; poly(I:C), polyinosinic-polycytidylic acid; Pam3Cys, tripalmitoyl-S-glycerylcysteine; MALP2, macrophage-activating lipopeptide 2 from Mycoplasma fermentation; PAMP, pathogen-associated molecular pattern.
Received for publication September 30, 2004. Accepted for publication February 17, 2005.
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B. D. Rudd, J. J. Smit, R. A. Flavell, L. Alexopoulou, M. A. Schaller, A. Gruber, A. A. Berlin, and N. W. Lukacs Deletion of TLR3 Alters the Pulmonary Immune Environment and Mucus Production during Respiratory Syncytial Virus Infection J. Immunol., February 1, 2006; 176(3): 1937 - 1942. [Abstract] [Full Text] [PDF]
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M. G. Netea, G. Ferwerda, D. J. de Jong, C. Werts, I. G. Boneca, M. Jehanno, J. W. M. Van Der Meer, D. Mengin-Lecreulx, P. J. Sansonetti, D. J. Philpott, et al. The Frameshift Mutation in Nod2 Results in Unresponsiveness Not Only to Nod2- but Also Nod1-activating Peptidoglycan Agonists J. Biol. Chem., October 28, 2005; 280(43): 35859 - 35867. [Abstract] [Full Text] [PDF]
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) or mice deficient in TLR2 (
) or TLR4 (
) were stimulated with 1 µg/ml commercial PGN (A) or purified PGN (B) for 24 h at 37°C. TNF and IL-10 were measured by specific RIA and ELISA, respectively. Data are presented as the mean ± SD (n = 10/group) and were compared by the Mann-Whitney U test (*, p < 0.05).
