Published online before print April 2, 2007
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,1,1,2
* Department of Immunology, The Scripps Research Institute, La Jolla, California, USA;
Ludwig Institute of Cancer Research and University of California at San Diego, La Jolla, California, USA;
Department of Cancer Immunology & AIDS, Dana-Farber Cancer Institute, Harvard School of Public Health, Boston, Massachusetts, USA;
Millennium Pharmaceuticals, Inc., Cambridge, Massachusetts, USA; and
|| Section of Immunobiology, Yale University School of Medicine, New Haven, Connecticut, USA
3 Correspondence: Department of Immunology, IMM12, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA. E-mail: ulevitch{at}scripps.edu
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: cytokine • inflammation • PAMP • NLR
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Genetic studies in man have linked mutations in NLR family members with various human diseases characterized by dysregulated, inflammatory responses. For example, mutations in Nod2 have been linked to Blau syndrome [6 ] or to increased susceptibility to Crohn’s disease [7 , 8 ]. Mutations in another NLR family member, CIAS1/NACHT domain, leucine-rich repeat, and pyrin domain-containing protein 3 (NALP3), are linked to three autoinflammatory disorders, collectively known as cryopyrinopathies, which include familial cold autoinflammatory syndrome, Muckle-Wells syndrome, and neonatal onset multisystem inflammatory disease [9 , 10 ]. Hyperproduction of IL-1ß has been suggested to be causal in at least some of these diseases. Studies of CIAS1/NALP3 have provided support for a multiprotein complex, the inflammasome, and its role in processing of IL-1ß [11 , 12 ]. Most recently, in studies with CIAS1/NALP3-deficient mice, a variety of proinflammatory agonists was shown to require CIAS1/NALP3 for caspase-1-dependent processing of pro-IL-1ß [13 14 15 16 ]. It has also been suggested that muramyl dipeptide (MDP)-induced IL-1ß requires CIAS1/NALP3 [17 ]. However, MDP was first shown to be a specific activator of another NLR family member, Nod2 [18 ]. Despite this, several recent reviews suggested that MDP induces IL-1ß release solely through a CIAS1/NALP3-dependent pathway [19 , 20 ] and surprisingly, do not consider any role for Nod2. At present, the relationships among Nod2, CIAS1/NALP3, and other proteins linked to Nod2 and CIAS1/NALP3 pathways have not been studied directly in the context of processing of pro-IL-1ß to mature IL-1ß. This has been a significant gap in our knowledge needing clarification because of the potential roles for Nod2 and CIAS1/NALP3 in human disease.
IL-ß is an important mediator of chronic inflammatory diseases including rheumatoid arthritis, psoriasis, and Crohn’s disease [21 ]. In contrast to many other cytokines, which are almost exclusively regulated at the transcriptional level, mature IL-1ß release is controlled by a key post-transcriptional event involving caspase-1-dependent proteolysis of pro-IL-ß to the mature form. As caspase-1 is present in cells in a precursor form, its activation must occur to process pro-IL-1ß. Here, we have used bone marrow-derived macrophages (BMDM) from wild-type and mice bearing specific gene deletions in members of the NLR family or in other genes thought to be linked to pathways involving NLR family members to clarify the requirements for MDP-induced IL-1ß release. Data from in vivo and in vitro studies support our contention that Nod2, together with CIAS1/NALP3, in conjunction with a number of additional proteins, including caspase-1, apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (Asc), and receptor-interacting protein-2 (Rip2), is required for MDP-induced, IL-1ß release. The totality of our data indicates that Nod2 and CIAS1/NALP3 have essential, nonredundant roles in processing pro-IL-1ß when there is a source of MDP present in the extracellular space.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Macrophage cell culture
BMDM were isolated and cultured as described [26
]. Briefly, BM was flushed from mouse femurs and cultured in BM macrophage growth media (DMEM containing 10% heat-inactivated FBS, 30% L929 cell-conditioned media, 2 mM L-glutamine, 100 µg ml–1 streptomycin, and 100 units ml–1 penicillin) for 7 days at 37°C to allow differentiation and maturation. The cells were observed to be >94% CD11b positive by FACS.
EMSA
Preparation of nuclear extracts and EMSA were performed as described previously [27
]. Briefly, cells were lysed in buffer A (10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM PMSF) on ice. Nuclei were separated from cytosol by centrifugation and were resuspended in Buffer B (20 mM HEPES, pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 0.1 mM PMSF). The supernatants containing nuclear proteins after a second centrifugation were transferred to new vials. Aliquots of the nuclear extracts (2.5 µg) were incubated with 12 µl binding buffer for 10 min at room temperature. Then, 32P-labeled NF-
B oligonucleotide probe was added, and the mixture was incubated at room temperature for 10 min. The samples were analyzed on nondenaturing 5% acrylamide gels. Gel contents were transferred to Whatman DE-81 paper, dried, and exposed overnight at –80°C with an intensifying screen.
In vivo IL-1ß release assay
Mice (8–12 weeks old) were injected i.p. with 10 µg LPS from E. coli (Serotype 0111:B4, List Biological Laboratories). Three hours later, these mice received an additional injection of MDP (300 µg in 50 µl saline), ATP (50 µl 100 mM in saline), or saline (50 µl). Serum samples were collected by retro-orbital bleed 1.5 h after the second injection.
Macrophage infection with Salmonella typhimurium
BMDM were plated in a 96-well plate at 5 x 104 cells per well in BM macrophage growth media and primed with 0.5 ng/ml LPS for 6 h to induce intracellular pro-IL-1ß. Cells were washed with fresh media, which lacked antibiotics, and infected with wild-type S. typhimurium CS401 (kindly provided by Dr. Samuel Miller, University of Washington, Seattle, WA, USA) at a multiplicity of infection of 50/cell for 30 min.
Cytokine measurements
Culture supernatants from infected/stimulated macrophages or serum from activated mice were assayed for IL-1ß and IL-6 by ELISA (PharMingen, San Diego, CA, USA).
Detection of caspase-1 activation and IL-1ß procession
Ten milliliters of culture supernatants from MDP-activated or S. typhimurium-infected macrophages was precipitated with 25 µg rabbit anticaspase-1 (SC514, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or 15 µg goat antimouse IL-1ß (AF-401-NA, R&D Systems, Minneapolis, MN, USA) in the presence of protein-G beads. The complexes were resolved in 4–20% Tris-glycine gradient gels (Invitrogen) and transferred to Immobilon-P [polyvinylidene difluoride (PVDF)] membranes (Millipore, Bedford, MA, USA) by electroblotting. The membranes were immunoblotted with rabbit anticaspase-1 (SC514, Santa Cruz Biotechnology) and hamster antimouse IL-1ß (Clone B122, BD PharMingen, San Diego, CA, USA), respectively.
Western blotting
For Western blotting analysis, the lysates from activated macrophages were resolved in 12% SDS-PAGE gel, and the following antibodies were used for immunoblotting: rabbit anti-IB
(Santa Cruz Biotechnology), mouse anti-phospho-p38 (Cell Signaling Technology, Beverly, MA, USA), rabbit anti-p38 (Cell Signaling Technology).
Statistical analysis
The significance of experimental observations was evaluated by a two-tailed t test. A P value of less than 0.05 was considered significant.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B to the nucleus (Fig. 1A
), increased the extent and duration of MDP-induced p38 phosphorylation, and prevented the resynthesis of IB (Fig. 1B)
. It is most important that CHX enhanced MDP-induced release of mature IL-1ß to levels substantially above that induced by MDP alone but had no effect on release of mature IL-1ß induced by ATP, a known inflammasome activator (Fig. 1C)
. Moreover, we have observed enhanced cytokine production with the addition of low-dose CHX in a variety of cell types activated with MDP or with peptidic Nod1 activators containing diamino-pimelic acid but not with activators of TLRs such as LPS (R. J. Ulevitch, C. Fearns, Q. Pan, unpublished data). Although our understanding of how CHX increases the extent of MDP-induced cell activation is not complete, we have compared the combined effects of CHX and MDP with MDP alone in all of the in vitro studies presented here to enhance the pathways involved in IL-1ß production.
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Figure 1. CHX enhances MDP signaling in BMDM. (A) CHX enhances MDP-induced NF-B DNA-binding activity. C57BL/6 BMDM were primed with LPS (0.5 ng/ml) for 6 h, and then media were removed, and cells were stimulated with MDP (10 µg/ml), CHX (1 µg/ml), or MDP together with CHX for the indicated time-points. DNA-binding activity was determined by EMSA. Time 0 is set at the end of LPS priming. n.s., Nonspecific band. (B) CHX enhances MDP-induced p38 phosphorylation and IB degradation. BMDM were stimulated as described in A, and protein phosphorylation (p38) and degradation (IB) were detected by specific immunoblot. (C) CHX enhances MDP-induced IL-1ß release. LPS-primed BMDM were stimulated with MDP (10 µg/ml) or ATP (5 mM), alone or in the presence of CHX (1 µg/ml) for 18 h, and IL-1ß in cell supernatants was measured by ELISA. Data are representative of three independent experiments. Results represent the mean ± SD of triplicate wells.
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B, as measured by p38 phosphorylation and IB degradation, when compared with the effects of LPS. The weak induction of NF-B may provide some insight into why MDP alone fails to up-regulate pro-IL-1ß (Fig. 2A)
. Finally, our data are in line with the view that multiple steps induced by distinct signals are required to produce mature IL-1ß.
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Figure 2. MDP activates caspase-1 and induces maturation of IL-1ß in the presence of CHX. (A) LPS, but not MDP, induces intracellular pro-IL-ß. C57BL/6 BMDM were stimulated with MDP (10 µg/ml) or LPS (1 ng/ml) for different time-points. Phosphorylation of p38, degradation of IB, and induction of pro-IL-1ß were monitored by immunoblotting of cell lysate. The sustained p38 activation seen at 6 h compared with that observed in Figure 1
may be a result of stimulation with a higher concentration of LPS. (B) Cells were primed with LPS (0.5 ng/ml) for 6 h and then activated by MDP (M; 10 µg/ml) and CHX (C; 1 µg/ml) with freshly added media for 18 h. As a positive control, cells were also infected with S. typhimurium for 30 min. Activated caspase-1 in the culture supernatants was immunoprecipitated and immunoblotted using a rabbit anticaspase-1 antibody. Secreted, mature IL-1ß was immunoprecipitated and immunoblotted by goat and hamster anti-IL-1ß antibodies, respectively.
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Identification of proteins required for MDP-induced IL-1ß release by BMDM
Studies from several laboratories have provided support for a model, whereby oligomerization of NLRs results in recruitment of adaptor proteins into a multiprotein complex, which catalyzes caspase-1 activation [25
, 32
]. The composition of a caspase-1-activating complex formed in responses to MDP is unknown. To address this gap in our knowledge, we used a series of murine BMDM derived from mice bearing specific gene deletions in the NLR family members Nod2 [23
, 33
], CIAS1/NALP3 [13
, 16
], and IPAF [34
, 35
]; in the kinase Rip2 [24
]; and in the adaptor protein Asc [22
]. First, we asked whether LPS-induced, pro-IL-1ß protein expression was changed when wild-type and BMDM bearing the previous group of gene deletions are compared. We observed that the absence of Nod2, Rip2, Asc, CIAS1/NALP3, caspase-1, or IPAF (Fig. 3A
3B
3C
3D
3E
3F
) did not prevent expression of LPS-induced, pro-IL-1ß protein. In contrast, BMDM from TLR4–/– mice failed to express pro-IL-1ß protein after addition of LPS (data not shown). It is not surprising that we did note a modest increase in pro-IL-1ß protein in wild-type cells when we added MDP to the LPS-pretreated BMDM from wild-type but not from Nod2 or Rip2–/– BMDM. Moreover, we observed that the addition of CHX did not reduce pro-IL-1ß protein expression in any of the LPS-pretreated BMDM.
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Figure 3. Intracellular pro-IL-1ß induction was not affected in Nod2–/–, Rip2–/–, Asc–/–, CIAS1/NALP3–/–, IPAF–/–, and caspase-1–/– cells. BMDM were primed with LPS (0.5 ng/ml) for 6 h, and then MDP (10 µg/ml) and CHX (1 µg/ml) were added to cells with fresh media. Cells were lysed with SDS-running buffer 6 h later. Pro-IL-1ß induction was assayed by immunoblot.
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Figure 4. MDP-induced IL-1ß release was abolished in Nod2–/–, Rip2–/–, Asc–/–, CIAS1/NALP3–/–, caspase-1–/–, but not IPAF-1–/– cells. BMDM were primed and stimulated as in Figure 2B
. Cytokine release was measured by ELISA. Data are representative of three independent experiments. Results represent the mean ± SD of triplicate wells. ND, Not detected. S, Saline. Brackets indicate P < 0.05.
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Figure 5. Impaired IL-1ß release in Nod2–/– and CIAS1/NALP3–/– mice following treatment with MDP. Groups of mice (n=8 per group for Nod2+/+ and Nod2–/– in A, or n=5 per group for CIAS1/NALP3+/+ and CIAS1/NALP3–/– for B) were injected i.p. with 10 µg LPS (O111:B4); 3 h later, these mice received an additional injection of 50 µl 100 mM ATP, 300 µg MDP, or saline, 1.5 h before bleeding. Cytokine induction was monitored by ELISA. Data are representative of two independent experiments. Results represent the mean ± SD. Brackets indicate P < 0.05.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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These results suggests that Nod2 and Rip2 may also be a part of the NALP3 inflammmasome, a multiprotein complex comprised of NALP3, Asc, and caspase-1, which leads to activation of caspase-1 and release of mature IL-1ß. We have some evidence from overexpression systems, which begins to address this. Our studies show that CIAS/NALP3 binds to Nod2 in a CARD-independent manner (data not shown). However, further studies are required to determine whether endogenous Nod2 and Rip2 are indeed part of a complex with CIAS/NALP3 and Asc. Alternately, Nod2 may not be part of the inflammasome but affect other aspects of IL-1ß homeostasis, such as pro-IL-1ß synthesis or stability or the secretion of mature IL-1ß. Whatever the mechanism of action is, Nod2 is absolutely required for MDP-induced release of IL-1ß.
Mutations in Nod2 and/or CIAS1/NALP3 have been linked to human diseases, which are characterized as autoinflammatory diseases and include Blau syndrome and the cryopyrinopathies. Hyperproduction of IL-1ß is thought to be a central event leading to symptoms in these diseases, as IL-1-targeted therapy has been used successfully for treatment. Moreover, mutations in Nod2 have also been linked to increased susceptibility to Crohn’s disease, and recently, it was suggested that the Crohn’s-associated Nod2 mutations result in gain-of-function and increased IL-1ß production [36
]. Here, we show an absolute dependence on CIAS1/NALP3 and Nod2 when MDP is used to activate BMDM to release mature IL-1ß. We also showed how MDP alone does not lead to induction of pro-IL-1ß, but rather, cells require priming by TLR agonists such as the prototypic TLR4 activator, LPS, or other TLR agonists (data not shown). MDP is a weak activator of NF-B and as a consequence, a weak inducer of cytokines when compared with other proinflammatory stimuli, such as LPS. Our studies suggest a major function of MDP is to actively trigger the maturation and release of IL-1ß, provided that the cell has been preactivated by stronger proinflammatory stimuli such as LPS. Also surprising is the importance of Rip2. Studies are currently underway to evaluate whether Rip2 requires its kinase function to participate in the events leading to mature IL-1ß release. It is possible that the primary function of Rip2 is to stabilize the caspase-1-activating complex formed in response to MDP rather than to phosphorylate as-yet-unidentified proteins.
At present, we can only speculate about the source or mechanism of cell entry of MDP or related muropeptides, which activate Nod2. One source might be from phagocytosed bacteria, which have undergone degradation within a phagocytic vacuole [37 , 38 ]. However, it is important to recall that more than two decades ago, studies of Martin et al. [39 ] provided evidence for accumulation of muropeptides, which could be derived only from endogenous peptidoglycan. Although the source(s) or mechanisms associated with this accumulation were not determined, it appears there may be unique pathways involving gut bacteria and release of peptidoglycan into the systemic circulation. Future studies of such pathways may be fruitful in the quest to identify endogenous activators of IL-1ß, which occur in the absence of infection. Finally, our data suggest that investigations into the genetics of autoinflammatory syndromes in man should include studies of Nod2, CIAS1/NALP3, Asc, and Rip2, as each of these proteins makes an essential contribution to MDP-dependent, IL-1ß release.
2 Current address: Medimmune, Gaithersburg, MD 20878, USA.
Received October 10, 2006; revised February 8, 2007; accepted February 22, 2007.
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, interleukin-1 ß, and interleukin-6 by morphologically normal intestinal biopsies from patients with Crohn’s disease Gut 39,684-689B-inducing kinase regulates selected gene expression in the Nod2 signaling pathway Infect. Immun. 74,2121-2127 B activation through a G protein-coupled pathway J. Biol. Chem. 270,14928-14934B activity and IL-1ß processing Science 307,734-738This article has been cited by other articles:
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