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University of Michigan Medical School, Department of Pathology and Comprehensive Cancer Center, Ann Arbor, MI 48109
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Abstract |
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-D-glutamyl-meso-diaminopimelic acid was intact in MyD88 deficient mice but abolished in RICK-null mice. Stimulation of macrophages with muramyl dipeptide, the Nod2 activator, enhanced immune responses induced by LPS, IFN-, and heat-killed Listeria in wild-type but not in RICK- or Nod2-deficient macrophages. Finally, we show that the absence of RICK or double deficiency of Nod1 and Nod2 was associated with reduced cytokine production in Listeria-infected macrophages. These results demonstrate that RICK functions in innate immunity by mediating Nod1 and Nod2 signaling but not TLR-mediated immune responses. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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B transcription factor and the MAPK signaling pathways (4, 5, 6, 7, 8).
RICK (also called RIP2 and CARDIAK) is a caspase-recruitment domain (CARD)-containing kinase that has been implicated in Nod1 and Nod2 signaling. Mice deficient in RICK exhibit increased susceptibility to systemic infection with Listeria monocytogenes (9, 10), indicating that RICK plays a role in host defense against intracellular bacteria. However, the mechanism by which RICK regulates Listeria infection remains poorly understood. Stimulation of Nod1 or Nod2 by their specific bacterial activators causes the recruitment of RICK and the association of RICK with Nod1 or Nod2 via CARD-CARD interactions (11, 12). RICK directly binds to IB kinase (IKK) and activates the IKK complex by promoting the ubiquitinylation of IKK and stimulating the kinase activity of the two other components of the IKK complex, IKK
and IKK
(11, 13). Transfection of plasmids expressing Nod1 or Nod2 do not activate NF-B in mouse embryo fibroblasts derived from mice deficient in RICK, suggesting that RICK is critical for signaling via Nod1 and Nod2 (10). However, the role of RICK in mediating Nod1 and Nod2 responses under more physiological conditions such as those induced by microbial stimuli in macrophages or in the animal remains to be investigated. In addition, RICK-deficient macrophages exhibited reduced responses when stimulated with lipoteichoic acid (LTA; TLR2 agonist), LPS (TLR4 agonist), and polyinosinic- polycitidylic acid (poly(I:C); a TLR3 agonist), but not CpG DNA (TLR9 agonist) (9, 10, 14). These studies suggested that RICK is involved in TLR signaling. However, many preparations of bacterial components contain Nod1- and Nod2-stimulating molecules (4, 5, 7, 15, 16) and the presence of such contaminants could also explain reduced TLR signaling in RICK-deficient macrophages. In the present report, we have studied the role of RICK in TLR signaling in macrophages and in the animal. In addition, we examined the contribution of RICK to immune responses induced by Nod1 and Nod2 activation and Listeria infection.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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RICK knockout (KO), Nod1 KO, and Nod2 KO have been described (5, 8, 10). RICK KO, Nod1 KO, and Nod2 KO mice were in a C57BL/6 background. MyD88 KO mice in C57BL/6 background were a gift from S. Akira (Osaka University, Osaka, Japan). Mice deficient in Nod1 and Nod2 were generated by crossing Nod1 KO and Nod2 KO mice and intercrossing the F1 generation. C57BL/6J mice were purchased from The Jackson Laboratory. Mice were housed in specific pathogen-free facility. Animal studies were approved by the University of Michigan Committee on Use and Care of Animals (Ann Arbor, Michigan).
Reagents and bacterial culture
Ultrapure LPS from Escherichia coli 0111:B4 and S. minnesota, monophosphoryl lipid A, LTA, poly I:C, and imiquimod were purchased from Invivogen. LPS preparations denoted as A (from E. coli 055:B5), B (from Salmonella typhimurium), and C (from Klebsiella pneumoniae) were purified by TCA extraction and purchased from Sigma-Aldrich. Lipid A extracted from S. minnesota and E. coli were purchased from Sigma-Aldrich. Synthetic lipid A was a gift from Dr. K. Fukase (Osaka University, Osaka, Japan). MDP (Ac-(6-O-stearoyl)-muramyl-Ala-D-Glu-NH2) was purchased from Bachem. N-myristoyl (C14) -D-glutamyl-meso-diaminopimelic acid (iE-DAP; in a modified form denoted KF1B), a Nod1 activator, has been described (17). The amount of Nod1 and Nod2 stimulatory activity in LPS preparations was determined as described (15, 18) and given as units per microgram of weight. One unit of the Nod1 and Nod2 stimulatory activity is equivalent to that of 1 ng of synthetic iE-DAP and MDP, respectively. The L. monocytogenes strain 10403S was a gift from Dr. M. O’Riordan (University of Michigan, Ann Arbor, MI). Single colonies were inoculated into 5 ml of brain-heart infusion medium and grown overnight at 37°C with shaking. A 1/10 dilution of the overnight culture was prepared and allowed to grow at 37°C with shaking to A600 = 0.5, which corresponds to 
109 CFU/ml. Bacteria were diluted to the desired concentration and used in subsequent experiments.
Stimulation of BMDM with ligands and bacteria
Bone marrow derived macrophages (BMDMs) were prepared as previously described (19). Briefly, bone marrow cells were cultured for 5 days with IMDM supplemented with 30% L929 supernatant containing macrophage-stimulating factor, glutamine, sodium pyruvate, 10% heat-inactivated FBS (Invitrogen Life Technologies), and antibiotics. The cells were stimulated with various ligands singly or in different combinations at the concentrations described in the figures for 24 h. In addition, the cells were treated with heat-killed L. monocytogenes (HKLM) or live bacteria. Culture supernatants were collected and assayed for cytokine production.
Measurement of NO and cytokines
Nitrite and mouse cytokines were measured in culture supernatants using Griess reagent (20) and ELISA kits from R&D Systems, respectively.
Western blot
For analysis of phosphorylation of I-B, p38, ERK, and JNK, cells were stimulated with LPS or MDP, harvested, and lysed in buffer containing 1% Nonidet P-40 supplemented with complete protease inhibitor mixture (Roche) and 2 mM DTT. Lysates were resolved by SDS-PAGE, transferred to polyvinyldifluoride membranes, and immunoblotted with primary Abs. Abs against mouse I-B, p38, ERK1/2, and JNK (phosphorylated and unphosphorylated forms) were purchased from Cell Signaling. Proteins were detected by ECL.
In vivo induction of cytokines
Mice were injected i. p. with 20 µg of LPS or 50 µg of KF1B and blood samples were collected at 3, 6, and 24 h after injection. Sera were isolated by centrifugation at 3,000 rpm for 10 min and submitted to ELISA for analysis of keratinocyte-derived chemokine (KC) and IL-6.
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Results |
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To assess the contribution of RICK to TLR-mediated immune responses, macrophages from wild-type (WT) and RICK-deficient mice were stimulated with several preparations of LPS. We found the production of IL-6 was reduced in RICK-deficient macrophages in response to LPS containing relatively high amounts of Nod1 ligand, but not in response to LPS preparations with low levels of Nod1 and Nod2-stimulating activity (Fig. 1A). Highly purified LPS from E. coli and S. minnesota induced comparable levels of IL-6 and TNF- in WT and RICK-deficient macrophages (Fig. 1B). In contrast, the response elicited by LPS was abrogated in macrophages lacking TLR4 (Fig. 1C), indicating that such LPS preparations mediate cytokine responses through TLR4. To verify these results, we stimulated macrophages with monophosphoryl lipid A and synthetic lipid A, the essential component of LPS required for TLR4 signaling (1). In addition, we tested lipid A preparations from E. coli and S. minnesota. As we observed with LPS preparations, IL-6 production induced by natural and synthetic lipid A was unimpaired in RICK-deficient macrophages (Fig. 1, D and E). Moreover, secretion of IL-6 induced by LTA (TLR2 agonist), poly(I:C) (TLR3 agonist), and imiquimod (TLR7 agonist) was comparable in WT and RICK-deficient macrophages (Fig. 1F). These results indicate that RICK does not contribute to IL-6 and TNF production by macrophages in response to TLR agonists.
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B and MAPK activation in response to MDP but not LPS
LPS and MDP induce the secretion of proinflammatory cytokines through transcriptional gene responses via NF-B and MAPK activation (1, 4, 6). To determine the role of RICK in NF-B and MAPK activation, macrophage extracts were prepared at different times after LPS or MDP stimulation and immunoblotted with Abs that recognize activated forms of NF-B, ERK, JNK, and p38. By 15 min of stimulation, LPS induced phosphorylation and degradation of I-B as well as phosphorylation of p38, ERK, and JNK, which was unaffected in RICK-deficient macrophages (Fig. 2A). Such activation of NF-B and MAPKs in response to LPS was greatly inhibited or abolished in TLR4-null macrophages (Fig. 2A). In contrast, phosphorylation of I-B, p38, ERK, and JNK induced by MDP was abrogated in RICK-deficient macrophages (Fig. 2B). These results indicate that RICK is required for signaling induced through MDP, the Nod2 ligand, but not LPS.
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To assess the requirement for RICK in TLR4 and Nod1 signaling in vivo, WT and RICK-deficient mice were administered LPS or KF1B, a modified iE-DAP dipeptide and the bacterial activator of Nod1 (17). Injection of LPS i. p. induced comparable levels of KC and IL-6 in WT and RICK-deficient mice (Fig. 3A), which is consistent with the results presented in Figs. 1 and 2. To determine the role of MyD88, a critical mediator of TLR activation, in Nod1-induced responses, WT and MyD88-null mice were injected with KF1B i. p. and the levels of KC were determined in serum at different times postinjection. We found no significant differences in the production of KC between WT and MyD88-deficient mice (Fig. 3B). In contrast, the production of KC induced by KF1B was abolished in RICK-deficient mice when compared with WT mice (Fig. 3C). Together, these studies indicate that RICK is required for Nod1-mediated signaling but does not contribute to the production of KC or IL-6 induced by LPS in vivo. Furthermore, MyD88 is dispensable for Nod1-mediated inflammatory responses in mice.
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RICK has been shown to be required for Nod1- and Nod2-induced NF-B activation in plasmid DNA transfection experiments using mouse embryonic fibroblasts (10). However the role of RICK under more physiological conditions such as those induced by Nod2 activation in macrophages is unclear. The stimulation of mouse macrophages with MDP alone does not induce the production of cytokines but enhances the response to low doses of LPS. To determine whether Nod2-dependent responses require RICK, macrophages from WT and RICK-deficient mice were costimulated with 10 ng/ml LPS in the presence and absence of MDP. Stimulation with MDP enhanced the production of IL-6 and TNF- in WT but not in RICK-deficient macrophages (Fig. 4A). To verify that Nod2 was mediating the MDP response, macrophages from Nod2-deficient were stimulated with LPS and MDP. As was observed with RICK-deficient macrophages, the enhancement of the LPS response induced by MDP was abolished in Nod2-deficient macrophages (Fig. 4B). MDP has been shown to induce NO production through Nod2 in macrophages (21). To assess the RICK requirement for NO production, WT and mutant macrophages were stimulated with IFN- in the presence or absence of MDP, and the production of nitrite was measured 48 h after stimulation. Although WT macrophages produced significant NO after stimulation with IFN- and MDP, this response was abrogated in RICK-deficient macrophages (Fig. 4C). These results indicate that RICK is required for MDP-mediated immune responses in macrophages.
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In initial studies we compared the response of WT and RICK-deficient macrophages to HKLM. We found that cytokine responses to HKLM were reduced in RICK-deficient macrophages when compared with WT cells (Fig. 5A). Furthermore, the contribution of RICK to the cytokine response was more pronounced at a low HKLM/macrophage ratio (Fig. 5A). To further assess the cooperation between RICK and TLR signaling, we stimulated macrophages with low number of HKLM to assess the enhancement of the response by MDP stimulation. We found that stimulation with MDP enhanced the production of IL-6 and TNF- in response to HKLM (Fig. 5, B and C). Notably, the enhancement in cytokine production induced by MDP was abolished in RICK-deficient macrophages (Fig. 5, B and C). To verify that the requirement for RICK was mediated via Nod2, we assessed cytokine production in macrophages deficient in Nod2 in response to HKLM. As it was observed with RICK-deficient macrophages, the enhancement of IL-6 production induced by MDP was abrogated in Nod2-deficient macrophages (Fig. 5D). These results indicate that MDP enhances the immune response to HKLM and that this response is mediated by RICK and Nod2.
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The early recognition of Listeria involves activation of TLRs at the plasma membrane and endosomes as well as in the cytosol via NLRs and receptors involved in nucleic acid recognition (22, 23, 24). To assess the contribution of RICK in the cytokine response induced by live Listeria, WT and RICK-deficient macrophages were infected with the bacterium and the production of cytokines was determined 12 and 24 h postinfection. We found reduced levels of IL-6 and TNF- in RICK-deficient macrophages after Listeria infection when compared with WT macrophages (Fig. 6, A and B). Listeria produces PGN molecules containing both iE-DAP and MDP motifs, the activators of Nod1 and Nod2, respectively. We first tested the role of Nod1 in the response to Listeria by an analysis of Nod1-deficient macrophages. There was no significant contribution of Nod1 in the IL-6 response to Listeria infection (Fig. 6C). We next tested the production of cytokines in WT and mutant mice deficient in Nod2 or both Nod1 and Nod2. The results revealed that Nod1 and Nod2 contribute in a redundant manner to the immune response to Listeria in that the deficiency of both proteins, but not Nod1or Nod2 alone, resulted in a significant reduction in IL-6 production when compared with WT macrophages (Fig. 6, B and C). To compare the contribution of RICK and double Nod1/Nod2 deficiency, we infected macrophages lacking RICK, both Nod1 and Nod2, or MyD88 in the same experiment and assessed the IL-6 response. Consistent with the results shown in Fig. 6, B and C, there was a comparable reduction in IL-6 production in macrophages deficient in RICK and Nod1/Nod2 (Fig. 6D). The IL-6 response induced by Listeria was abolished in macrophages deficient in MyD88 (Fig. 6D). These results indicate that RICK pathways contribute to the production of IL-6 in response to Listeria, whereas TLR pathways are essential for their response.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Previous studies revealed that mice deficient in RICK are more susceptible to i.p. and i.v. infection with Listeria than control mice (9). Impaired bacterial clearance was observed by day 3 postinfection, suggesting a role for RICK in innate immunity against Listeria(9). However, the mechanism by which RICK contributes to the early response to the bacterium remains poorly understood. We found that RICK-deficient macrophages exhibit reduced production of cytokines in response to Listeria infection. These results suggest that RICK promotes host defense, at least in part, through the induction of proinflammatory cytokines during Listeria infection. Single deficiency Nod1 or Nod2 did not result in a significant alteration in the cytokine response to Listeria infection. However, macrophages lacking both Nod1 and Nod2 exhibited reduced production of cytokines in response to the live bacterium. These results indicate a redundant role for Nod1 and Nod2 in the response to Listeria and suggest that the impairment of the cytokine response observed in RICK-deficient macrophages is mediated by the loss of both Nod1 and Nod2 signaling. We also found that stimulation with MDP, the activator of Nod2, increases the production of cytokines induced by heat-killed Listeria, an activity that required both Nod2 and RICK. This finding is consistent with the observation that Nod2 and RICK cooperate with TLR signaling for optimal responses to TLR ligands. The innate immune response to Listeria, including cytokine production, also involves TLR signaling particularly through TLR2 (22, 24). Consistent with these finding, we observed that in the absence of RICK there was a considerable residual cytokine response induced by Listeria in macrophages. We found that the cytokine response induced by Listeria was abolished in MyD88-deficient macrophages, which is consistent with previous studies (22, 27). The latter results appear at odds with a model in which RICK and MyD88 function in parallel signaling pathways. However, the interpretation of the latter results is difficult given than MyD88–null macrophages express lower levels of many TLR-regulated genes, including NF-B subunits in the absence of stimulation (28). Although the molecular basis for the reduction of TLR-regulated genes in MyD88-null macrophages remains unclear, it has been suggested that the presence of trace amounts of endotoxin and/or other microbial products in the culture medium may explain these observations (29). Thus, the greatly diminished response of MyD88 KO macrophages to Listeria that appears to involve both TLR-MyD88 and RICK signaling pathways could be explained, at least in part, by a generalized low response of MyD88-deficient macrophages to microbial ligands, including to Nod1 and Nod2 ligands in vitro. Consistent with this thesis, we found no impairment of the chemokine responses induced by the Nod1 ligand in MyD88 KO mice in vivo (Fig. 3). Furthermore, it is well documented that the response of MyD88 KO macrophages is abolished or greatly diminished to Listeria in vitro but that MyD88 KO mice secrete abundant amounts of cytokines when challenged with Listeria in vivo (27). Although our in vivo results with the Nod1 ligand suggest that MyD88 is not involved in Nod1 signaling, we cannot formally rule out that MyD88 may contribute directly or indirectly to Nod1 and Nod2 signaling. Together, our results and those from several laboratories indicate that both TLR and RICK signaling pathways contribute to the production of proinflammatory cytokines in response to Listeria.
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Acknowledgments |
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Disclosures |
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Footnotes |
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1 This work was supported by National Institutes of Health Grants AI063331 and AI064748 (to G.N.) and GM-60421 (to N.I.). M.B.-M. was supported by a postdoctoral fellowship from University of Michigan Center for Genetics in Health and Medicine. C.M. was supported by a Career Development Award from the Crohn’s and Colitis Foundation of America. 
2 Address correspondence and reprint requests to Dr. Gabriel Núñez University of Michigan Medical School Department of Pathology and Comprehensive Cancer Center, 1500 East Medical Center Drive, Ann Arbor, MI 48109. E-mail address: bclx{at}umich.edu
3 Abbreviations used in this paper: NOD, nucleotide-binding oligomerization domain; NLR, nucleotide-binding oligomerization domain-like receptor; BMDM, bone marrow derived macrophage; CARD, caspase-recruitment domain; HKLM, heat-killed Listeria monocytogenes; iE-DAP, -D-glutamyl-meso-diaminopimelic acid; IKK, IB kinase; KC, keratinocyte-derived chemokine; KO, knockout; LTA, lipoteichoic acid; MDP, muramyl dipeptide; PGN, peptidoglycan; poly(I:C), polyinosinic-polycitidylic acid; WT, wild type.
Received for publication September 7, 2006. Accepted for publication November 21, 2006.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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B activation in the Nod1/RICK and RIP signaling pathways. J. Biol. Chem. 275: 27823-27831. B and JNK activation by invasive Shigella flexneri. EMBO Rep. 2: 736-742. [Medline]-interferon receptor that regulates macrophage tumoricidal activity. J. Exp. Med. 160: 55-74. enhances production of nitric oxide from macrophages via a mechanism that depends on nucleotide oligomerization domain-2. J. Immunol. 176: 4804-4810. /IL-18 secretion in response to Listeria monocytogenes. J. Immunol. 176: 4337-4342. B activation. J. Biol. Chem. 277: 41701-41705. yet mediates few responses to Mycobacterium tuberculosis. J. Exp. Med. 198: 987-997. This article has been cited by other articles:
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  Y.-G. Kim, J.-H. Park, S. Daignault, K. Fukase, and G. Nunez Cross-Tolerization between Nod1 and Nod2 Signaling Results in Reduced Refractoriness to Bacterial Infection in Nod2-Deficient Macrophages J. Immunol., September 15, 2008; 181(6): 4340 - 4346. [Abstract] [Full Text] [PDF]
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N. Marina-Garcia, L. Franchi, Y.-G. Kim, D. Miller, C. McDonald, G.-J. Boons, and G. Nunez Pannexin-1-Mediated Intracellular Delivery of Muramyl Dipeptide Induces Caspase-1 Activation via Cryopyrin/NLRP3 Independently of Nod2 J. Immunol., March 15, 2008; 180(6): 4050 - 4057. [Abstract] [Full Text] [PDF]
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 A. Dufner, G. S. Duncan, A. Wakeham, A. R. Elford, H. T. Hall, P. S. Ohashi, and T. W. Mak CARD6 Is Interferon Inducible but Not Involved in Nucleotide-Binding Oligomerization Domain Protein Signaling Leading to NF-{kappa}B Activation Mol. Cell. Biol., March 1, 2008; 28(5): 1541 - 1552. [Abstract] [Full Text] [PDF]
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Y. Yang, C. Yin, A. Pandey, D. Abbott, C. Sassetti, and M. A. Kelliher NOD2 Pathway Activation by MDP or Mycobacterium tuberculosis Infection Involves the Stable Polyubiquitination of Rip2 J. Biol. Chem., December 14, 2007; 282(50): 36223 - 36229. [Abstract] [Full Text] [PDF]
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 M. Hedl, J. Li, J. H. Cho, and C. Abraham Chronic stimulation of Nod2 mediates tolerance to bacterial products PNAS, December 4, 2007; 104(49): 19440 - 19445. [Abstract] [Full Text] [PDF]
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D. W. Abbott, Y. Yang, J. E. Hutti, S. Madhavarapu, M. A. Kelliher, and L. C. Cantley Coordinated Regulation of Toll-Like Receptor and NOD2 Signaling by K63-Linked Polyubiquitin Chains Mol. Cell. Biol., September 1, 2007; 27(17): 6012 - 6025. [Abstract] [Full Text] [PDF]
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 K. Brandl, G. Plitas, B. Schnabl, R. P. DeMatteo, and E. G. Pamer MyD88-mediated signals induce the bactericidal lectin RegIII{gamma} and protect mice against intestinal Listeria monocytogenes infection J. Exp. Med., August 6, 2007; 204(8): 1891 - 1900. [Abstract] [Full Text] [PDF]
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J.-H. Park, Y.-G. Kim, M. Shaw, T.-D. Kanneganti, Y. Fujimoto, K. Fukase, N. Inohara, and G. Nunez Nod1/RICK and TLR Signaling Regulate Chemokine and Antimicrobial Innate Immune Responses in Mesothelial Cells J. Immunol., July 1, 2007; 179(1): 514 - 521. [Abstract] [Full Text] [PDF]
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M. Hasegawa, A. Kawasaki, K. Yang, Y. Fujimoto, J. Masumoto, E. Breukink, G. Nunez, K. Fukase, and N. Inohara A Role of Lipophilic Peptidoglycan-related Molecules in Induction of Nod1-mediated Immune Responses J. Biol. Chem., April 20, 2007; 282(16): 11757 - 11764. [Abstract] [Full Text] [PDF]
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