Proteasomal degradation of Nod2 protein mediates tolerance to bacterial cell wall components

Abstract

The innate immune system serves as the first line of defense by detecting microbes and initiating inflammatory responses. Although both Toll-like receptor (TLR) and nucleotide binding domain and leucine-rich repeat (NLR) proteins are important for this process, their excessive activation is hazardous to hosts; thus, tight regulation is required. Endotoxin tolerance is refractory to repeated lipopolysaccharide (LPS) stimulation and serves as a host defense mechanism against septic shock caused by an excessive TLR4 response during gram-negative bacterial infection. Gram-positive bacteria as well as their cell wall components also induce shock. However, the mechanism underlying tolerance is not understood. Here, we show that activation of Nod2 by its ligand, muramyl dipeptide (MDP) in the bacterial cell wall, induces rapid degradation of Nod2, which confers MDP tolerance in vitro and in vivo. Nod2 is constitutively associated with a chaperone protein, Hsp90, which is required for Nod2 stability and protects Nod2 from degradation. Upon MDP stimulation, Hsp90 rapidly dissociates from Nod2, which subsequently undergoes ubiquitination and proteasomal degradation. The SOCS-3 protein induced by Nod2 activation further facilitates this degradation process. Therefore, Nod2 protein stability is a key factor in determining responsiveness to MDP stimulation. This indicates that TLRs and NLRs induce a tolerant state through distinct molecular mechanisms that protect the host from septic shock.

FIGURE 1.

Pretreatment with MDP inhibits proinflammatory responses to subsequent MDP treatment. A and C, RAW264.7 cells were pretreated with MDP (100 μg/ml) for 4 h, washed, and restimulated with MDP (100 μg/ml), LPS (10 ng/ml), CpG-B (1 μm), Pam3CSK4 (10 μg/ml), or flagellin (3 μg/ml) for 24 h. B, BMDM were stimulated with LPS (0.5 ng/ml) for 6 h and then incubated with MDP (100 μg/ml) for 4 h in the absence of LPS. After several washes, the cells were restimulated with MDP (100 μg/ml) for 24 h. Supernatants were subjected to ELISA for TNF-α. D and E, wild-type BMDM or Rip2-deficient BMDM were pretreated with LPS (0.1 ng/ml) for 6 h and stimulated with MDP for 4 h in the absence of LPS. The cells were washed and restimulated with MDP for 30 min. D, cell extracts were subjected to Western blot analysis for IκBα, p-ERK, p-JNK, and actin. E, cells were fixed and permeabilized for 5 min. Immunofluorescent staining for p65 was performed using anti-p65 Ab followed by Alexa Fluor 594 detection antibody. Cells were analyzed under a fluorescence microscope. Error bars represent the mean ± S.D. of triplicates. **, p < 0.005. Data are representative of three independent experiments with similar results. n.s., nonspecific; N.D., not detected.

FIGURE 2.

MDP treatment induces degradation of Nod2 and Rip2 via the proteasomal pathway. A, RAW264.7 cells were treated with MDP (100 μg/ml) for the indicated times or with LPS (10 ng/ml) for 6 h. B, RAW264.7 cells were pretreated with IFN-γ (100 units/ml) for 6 h and then stimulated with MDP for the indicated times. C, BMDM were co-treated with MDP (100 μg/ml) in the presence or absence of LPS (0.5 ng/ml) for the indicated times. D, SW480 cells were treated with MDP for the indicated times. Cells were transfected with pcDNA3 or HA-human Nod2 plasmid vectors. E, RAW264.7 cells were stimulated with LPS (10 ng/ml), CpG-B (1 μm), Pam3CSK4 (10 μg/ml), or flagellin (1 μg/ml) for 6 h. Cell extracts were subjected to Western blot analysis for Nod2, Rip2, and actin. n.s., nonspecific; ctrl, control.

FIGURE 3.

MDP treatment induces proteasomal degradation of Nod2 and Rip2. A and C, upper, RAW264.7 cells were pretreated with IFN-γ for 6 h and then stimulated with MDP for the indicated times. A, the expression of Nod2 was examined by quantitative real time PCR. Data were normalized to the expression of the β-actin gene. Data represent the mean ± S.D. of triplicates. B, cells were pretreated with IFN-γ for 6 h and then stimulated with MDP for the indicated times in the presence or absence of MG-132 (10 μm). Cell extracts were subjected to Western blot analysis for Nod2, Rip2, and actin. *: slowly migrating Nod2. C, upper, cell extracts were immunoprecipitated (IP) with anti-Nod2 antibody. The level of ubiquitination on Nod2 was analyzed. C, lower, HEK293T cells were transfected with FLAG-Nod2 and HA-WT-Ub plasmid vectors. Forty-eight hours after transfection, cells were treated with MG-132 (10 μm) for 2 h. Cell extracts were immunoprecipitated with anti-Nod2 antibody. The level of Nod2 ubiquitination was analyzed. Error bars represent the mean ± S.D. of triplicates. Data are representative of three independent experiments with similar results. L.E., long exposure; n.s., nonspecific.

FIGURE 4.

MDP-induced degradation of Nod2 and Rip2 is specific to MDP stimulation. A, RAW264.7 cells were pretreated with LPS (0.1 ng/ml) or IFN-γ (100 units/ml) for 6 h and then stimulated with LPS, CpG-B, or MDP for the indicated times. B, wild-type, Nod2-deficient, or Rip2-deficient BMDM were pretreated with LPS (0.2 ng/ml) for 6 h and then stimulated with MDP (100 μg/ml) for the indicated times. C, RAW264.7 cells were pretreated with LPS (1 ng/ml) for 6 h and then incubated with MDP (MDP-ld) or biologically inactive MDP-ll (100 μg/ml) for 3 h. D, wild-type or MyD88-deficient BMDM were stimulated with LPS for 6 h and then infected with heat-killed B. subtilis (5 × 10⁶ or 20 × 10⁶ cells/ml) in the presence of cycloheximide (CHX; 20 μg/ml) for 4 h. Cell extracts were subjected to Western blot analysis for Nod2, Rip2, and actin. Data are representative of three independent experiments with similar results. S.E., short exposure; L.E., long exposure; n.s., nonspecific.

FIGURE 5.

MDP treatment induces rapid dissociation of the Nod2·Hsp90 complex. A, RAW264.7 cells were treated with MDP for the indicated times. B, cells were pretreated with IFN-γ for 6 h and then stimulated with MDP for the indicated times. Cell lysates were immunoprecipitated (IP) with anti-Hsp90 antibody, and associated Nod2 was analyzed by Western blotting. C, the expression vectors for Nod2 deletion mutants used in the study. D, HEK293T cells were transfected with the indicated plasmids. Forty-eight hours after transfection, total cellular proteins were extracted. Mutant Nod2 proteins in the lysates were immunoprecipitated, and associated endogenous Hsp90 protein was analyzed by Western blotting. Data are representative of three independent experiments with similar results. S.E., short exposure; L.E., long exposure; n.s., nonspecific; E.V., empty vector.

FIGURE 6.

Hsp90 inhibition decreases endogenous and inducible levels of Nod2 and Rip2 and blocks MDP response. A and B, RAW264.7 cells were pretreated with 17AAG (2 or 5 μm) or RAD (1 or 2 μm) for 8 h and then stimulated with MDP (100 μg/ml) for the indicated times or IFN-γ (100 units/ml) for 6 h in the presence of Hsp90 inhibitors. C, RAW264.7 cells were transfected with siRNAs targeting Hsp90 or control siRNAs. Forty-eight hours after transfection, cells were stimulated with LPS (0.1 ng/ml) for 6 h. Cell extracts were subjected to Western blot analysis for Nod2, Rip2, Hsp90, and actin. D, RAW264.7 cells were pretreated with 17AAG (2 μm) and then stimulated with MDP for 5 h in the presence or absence of 17AAG. LPS (10 ng/ml; 5 h) was used as a positive control. E, HEK293T cells were co-transfected with pBVI-Luc reporter and pRLNull plasmids. Twenty-four hours after transfection, cells were co-treated with MDP and 17AAG (2 μm) for 20 h. NF-κB activity was measured by Dual-Luciferase assay. F and G, RAW264.7 cells were pretreated with 17AAG (2 μm) or RAD (1 μm) and then stimulated with MDP for 24 h. TNF-α in the media was measured by ELISA. Cell extracts were subjected to Western blot analysis for iNOS and actin. H, RAW264.7 cells were transfected as in C. Cells were stimulated with MDP for 24 h. The iNOS level was detected by Western blotting. Error bars represent the mean ± S.D. of triplicates. Data are representative of three independent experiments with similar results. L.E., long exposure; S.E., short exposure; n.s., nonspecific; Scr, scrambled.

FIGURE 7.

Knockdown of SOCS-3 suppresses MDP-mediated Nod2 degradation. A, wild-type, Nod2-deficient, or Rip2-deficient BMDM were pretreated with LPS (0.2 ng/ml) for 6 h and then stimulated with MDP (100 μg/ml) for the indicated times. Cell extracts were subjected to Western blot analysis for SOCS-3 and actin. B, RAW264.7 cells were pretreated with LPS or IFN-γ for 6 h and then incubated with MDP for the indicated times. Nod2 was immunoprecipitated (IP) with anti-Nod2 antibody. Nod2-associated SOCS-3 was detected by Western blotting. C, HEK293T cells were transfected with FLAG-Nod2 and various concentrations of FLAG-SOCS-3 plasmids. Forty-eight hours after transfection, total cellular proteins were extracted. Cell extracts were immunoprecipitated with anti-Nod2 antibody. Co-precipitated proteins were analyzed by Western blotting with anti-SOCS-3 antibody. D, HEK293T cells were transfected with the expression vectors for FLAG-SOCS-3 and HA-Nod2 deletion mutants. SOCS-3 was immunoprecipitated with anti-FLAG antibody. SOCS-3-associated Nod2 deletion mutants were detected by Western blotting using anti-HA antibody. E, RAW264.7 cells were transfected with siRNAs for SOCS-3 and control siRNAs. Forty-eight hours after transfection, cells were treated with LPS (0.5 ng/ml) for 6 h and then stimulated with MDP for the indicated times. Cell extracts were subjected to Western blot analysis for Nod2, Rip2, SOCS-3, and actin. Data are representative of three independent experiments with similar results. n.s., nonspecific; E.V., empty vector; Scr, scrambled.

FIGURE 8.

MDP tolerance is mediated by Nod2 and Rip2 down-regulation in vivo. Wild-type or Nod2-deficient mice were injected with 2 ml of autoclaved 4% thioglycollate intraperitoneally. Five days after thioglycollate treatment, MDP (35 mg/kg) was administered to the mice intraperitoneally for the indicated times, and then the mice were reinjected with MDP. Three hours after the second MDP injection, peritoneal fluid and macrophages were collected (n = 3 per group). A, IL-6 and TNF-α in peritoneal fluid were measured by ELISA. Mean value is indicated by a bar. B and C, cell extracts were subjected to Western blot analysis for TNF-α, Nod2, Rip2, Hsp90, and p38. RAW264.7 cells treated with IFN-γ (100 units/ml) were used as a positive control. D, model of MDP tolerance mediated by rapid Nod2 degradation. MDP stimulation causes rapid dissociation of the Nod2·Hsp90 complex (1). MDP-induced SOCS-3 binds to Nod2 (2). Nod2 undergoes polyubiquitination and proteasome-mediated degradation, resulting in reduced responsiveness to subsequent MDP. inj., injection.