β-PIX and Rac1 GTPase Mediate Trafficking and Negative Regulation of NOD2

Julia Eitel, Matthias Krüll, Andreas C. Hocke, Philippe Dje N′Guessan, Janine Zahlten, Bernd Schmeck, Hortense Slevogt, Stefan Hippenstiel, Norbert Suttorp and Bastian Opitz
J Immunol August 15, 2008, 181 (4) 2664-2671; DOI: https://doi.org/10.4049/jimmunol.181.4.2664

Abstract

The nucleotide-binding domain and leucine-rich repeat containing protein NOD2 serves as a cytoplasmic pattern recognition molecule sensing bacterial muramyl dipeptide (MDP), whereas TLR2 mediates cell surface recognition of bacterial lipopeptides. In this study, we show that NOD2 stimulation activated Rac1 in human THP-1 cells and primary human monocytes. Rac1 inhibition or knock-down, or actin cytoskeleton disruption increased MDP-stimulated IL-8 secretion and NF-κB activation, whereas TLR2-dependent cell activation was suppressed by Rac1 inhibition. p21-activated kinase [Pak]-interacting exchange factor (β-PIX) plays a role in this negative regulation, because knock-down of β-PIX also led to increased NOD2-mediated but not TLR2-mediated IL-8 secretion, and coimmunoprecipitation experiments demonstrated that NOD2 interacted with β-PIX as well as Rac1 upon MDP stimulation. Moreover, knock-down of β-PIX or Rac1 abrogated membrane recruitment of NOD2, and interaction of NOD2 with its negative regulator Erbin. Overall, our data indicate that β-PIX and Rac1 mediate trafficking and negative regulation of NOD2-dependent signaling which is different from Rac1’s positive regulatory role in TLR2 signaling.

The innate immune system builds the first line of host defense against invading microorganisms. It recognizes infectious organisms through so-called pattern recognition receptors (1) such as the transmembrane TLRs, the recently found RIG-like helicases, and the intracellular nucleotide-binding domain and leucine-rich repeat containing proteins (NLRs)3 (2, 3, 4, 5, 6). Among the 22 human NLRs, members of the NOD protein subgroup are composed of three domains: the N-terminal caspase activation and recruitment domain (CARD), an internal nucleotide-binding oligomerization domain (NOD), and a C-terminal leucine rich repeat (LRR) domain (7).

One of the first discovered NLRs was NOD2; while mutations in the Nod2 gene are strongly associated with the two chronic inflammatory disorders, Crohn’s disease and Blau syndrome (8, 9, 10, 11, 12, 13), the physiological function of NOD2 seems to be the intracellular recognition of bacterial infections (9, 14, 15, 16). For this purpose, NOD2 detects the muramyl dipeptide MurNAc-l-Ala-D-isoGln (MDP), the largest molecular motif common to peptidoglycans of Gram-negative and Gram-positive bacteria (17, 18). According to the current concept of NOD2 function, sensing of MDP is mediated through the LRR domain of NOD2, which leads to conformational change, membrane recruitment, and activation of downstream signaling by a homophilic interaction of the CARD of NOD2 with the CARD of receptor interacting protein 2 (RIP2) or with the CARD of another recently identified adapter molecule, CARD9 (19, 20, 21, 22, 23). RIP2 in turn triggers activation of NF-κB (9, 20, 22), while CARD9 seems to trigger NOD2-dependent activation of the downstream MAPKs p38 and JNK, finally leading to inflammatory gene expression (21). In addition, negative regulators of NOD2 signaling such as Erbin and Centaurin β 1 as well as an alternatively spliced inhibitory isoform of NOD2 (NOD2-S) have also been identified (24, 25, 26, 27).

The Rho family GTPase Rac1 is a key regulator of various cellular functions such as cytoskeletal reorganization, cellular growth, and apoptosis (28, 29, 30). Moreover, Rac1 is implicated in different aspects of antibacterial host defense, including leukocyte chemotaxis (28), TLR2-dependent modulation of monocyte adhesive activities (31), pathogen phagocytosis (32, 33), and has also been implicated in inflammatory signaling pathways leading to NF-κB activation including the TLR2-dependent signaling (34, 35, 36, 37, 38). In resting cells, most of Rac1 is in an inactive state (Rac1-GDI) (39). Upon cell stimulation, guanine nucleotide exchange factors, including the recently found p21-activated kinase [Pak]-interacting exchange factor (β-PIX) (40), become activated and associate with the membrane, while also the Rac1-GDI complex translocates to the plasma membrane and dissociates (41, 42). The GDI-free Rac1 thus represents the active GTP-bound state. Interestingly, β-PIX has recently been shown to directly bind Rac1, and thus to mediate both targeting and localized activation of Rac1 (40).

In this study, we describe a novel role of Rac1 and β-PIX as regulators of NOD2 trafficking and NOD2-dependent signal transduction.

Materials and Methods

Cell culture and materials

Human monocytes were isolated from buffy coat preparations supplied by the German Red Cross. Blood was diluted 1/2 in RPMI 1640 EDTA medium (Life Technologies) and centrifuged over Ficoll (Amersham) for 25 min at 20°C and 800 × g, and cultured in RPMI 1640 supplemented with or without 10% heat-inactivated FCS, 100 IU penicillin, 100 μg/ml streptomycin, and 4.5 mM glutamine. After 2 h (pull-down assay and coimmunoprecipitation) or 3 days of culture (RNA interference (RNAi) experiment), cells were used for experiments. HEK293 and THP-1 cells were obtained from DSMZ and cultured in DMEM and RPMI 1640 (Life Technologies), respectively, supplemented with 10% heat-inactivated FCS. MDP-LD, MDP-DD, and Malp2 were purchased from Alexis Biochemicals and the Rac activation-specific inhibitor NSC23766 (43) was purchased from Calbiochem. All other chemicals used were of analytical grade and obtained from commercial sources.

RNAi in HEK293 cells and human monocytes

Control nonsilencing small interfering RNA (siRNA) (sense, UUCUCCGAACGUGUCACGUtt; antisense, ACGUGACACGUUCGGAGGAGAAtt), siRNA targeting Rac1 (sense, CACCACUGUCCCAACACUCtt; antisense, GAGUGUUGGGACAGUGGUGtt), and siRNA targeting β-PIX (sequence 1, sense, CGACAGGAAUGACAAUCACtt; antisense, GUGAUUGUCAUUCCUGUCGtt; sequence 2, sense, CCAGUGAGAAGUUAAGUUCtt; antisense, GAACUUAACUUCUCACUGGtc) were purchased from MWG-Biotech or Ambion. HEK293 cells were transfected by using RNAiFect (Qiagen) according to the manufacturer’s protocol with 1 μg siRNA per 2 × 105 cells. Primary human monocytes or THP-1 cells were transfected by using Amaxa Nucleofector (Amaxa) according to the manufacturer’s protocol (Human Monocyte Nucleofector Kit, Nucleofector program Y-01, or Nucleofector Solution V, Nucleofector program T-08, respectively) with 3 μg siRNA per 107 cells (primary monocytes) or 2 μg siRNA per 106 cells (THP-1). The efficiency of Rac1 silencing was analyzed 72 h after transfection by immunoblotting. Functional studies examining the role of Rac1 in cell activation by MDP were performed 72 h after siRNA transfection.

Expression plasmids and HEK293 cell overexpression experiments

The expression plasmids pCR3.V72-Met-VSV-RIP2 and pFlag-CMV-TLR2 were provided by J. Tschopp, University of Lausanne, Lausanne, Switzerland, and C. Kirschning, Technical University of Munich, Munich, Germany, respectively. pcDNA3 containing NOD2 was a gift of G. Nuñez, University of Michigan Medical School, Ann Arbor, Michigan, and was subcloned into pCMV-Tag 3C (HindIII, XhoI) to produce pMyc-NOD2. The pRK5-Rac1 expression vector and its mutant forms were generous gifts of A. Ridley, the RSV-β-galactosidase reporter plasmid was provided by W. Hallatschek, Charité-University Medicine, Berlin, Germany, and the IL-8 promoter luciferase reporter plasmid pUHC13-3-IL-8pr (nucleotides 1348–1527 of the IL-8 gene) was a kind gift of M. Kracht, Hannover Medical School, Hannover, Germany. For reporter gene assays, HEK293 cells cultured in 24-well plates were cotransfected using the calcium phosphate method (Clontech) with 0.05 μg NF-κB reporter or 0.05 μg IL-8 reporter, 0.05 μg RSV-β-galactosidase reporter, 0.2 ng of NOD2, 0.025 μg of TLR2, or 0.1 μg of RIP2 expression vectors and 0.1 μg of the different Rac1 expression vectors. For the coimmunoprecipitation experiments, HEK293 cells cultured in 6-well plates were transfected with 2.0 μg of either an empty control vector or Myc-NOD2 expression plasmids, respectively, together with 2 μg of Rac1 expression plasmid by using the Superfect reagent (Qiagen).

Rac1 activation assay

Rac1 activation in stimulated cells was determined with the Rac1 activation kit (Chemicon) according to the manufacturer’s instructions. In brief, THP-1 cells or primary monocytes were grown to ∼107 cells per sample, serum-starved for 3 h, and stimulated with MDP (0.1–10 μg/ml) for 5 to 60 min. Cells were washed twice with ice-cold PBS and resuspended in the assay buffer. Cell lysates were mixed with the PAK-1 PBD agarose slurry (50%) for 1 h at 4°C. Beads were collected by centrifugation, washed with the assay buffer, and resuspended in SDS sample buffer. Activated Rac1 in the cell lysates was visualized by Western blot using an anti-Rac1 mAb and a Cy5.5-labeled anti-mouse secondary Ab (Rockland). Proteins were detected using the Odyssey infrared imaging system (Li-Cor).

Luciferase assay

Luciferase activity was measured by using the luciferase reporter-gene assay (Promega), and results were normalized with values obtained by RSV-β-galactosidase as described previously (44).

IL-8 ELISA

IL-8 concentrations in the THP-1 or primary monocyte supernatants were quantified using a commercially available sandwich ELISA kit (R&D Systems).

Immunoblotting

Cell extracts were separated by SDS-PAGE and blotted on Hybond-ECL membranes (Amersham Biosciences). Membranes were exposed to Abs specific to Rac1 (Transduction Laboratories), NOD2 (ProSci), Nalp3 (Biozol), β-Pix, c-Myc, Erbin, or ERK2 (Santa Cruz Biotechnology), respectively. Subsequently, membranes were incubated with secondary Abs (IRDye 800-labeled anti-rabbit or Cy5.5-labeled anti-mouse, Rockland). Proteins were detected by using an Odyssey infrared imaging system (Li-Cor).

Immunoprecipitation

For immunoprecipitation, cells were lysed in RIPA buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP40, 0.5% sodiumdeoxycholat) containing protease and phosphatase inhibitors. Lysates were cleared for 15 min at 14,000 g at 4°C. Protein lysates (500 μg/500 μl) were precleared with 0.25 μg isotype specific Ab and Protein-G-Agarose (Upstate Biotechnology) for 1 h at 4°C and the supernatants were recovered. Subsequently, 2 μg of primary Ab was added and incubated on an “end-to-end” shaker for 1.5 h at 4°C. Protein G-Agarose slurry (50 μl) was added and incubated overnight at 4°C. The beads were precipitated by centrifugation steps and washed five times in RIPA buffer before SDS loading buffer was added.

Confocal laser scanning microscopy

For indirect immunofluorescence microscopy, HEK293 were seeded on coverslips, transfected with NOD2 and Rac1 expression vectors using the Superfect reagent (Qiagen) as described above, fixed in 3% paraformaldehyde in PBS, and permeabilized with 1% Triton X-100 for 15 min at room temperature. Cells were incubated in 5% goat serum and staining was done by subsequent incubation of primary and secondary Abs in 1% goat serum. Primary Abs were rabbit anti-NOD2 (1/500; ProSci) and mouse anti-Rac1 (1/500; Transduction Laboratories). Primary Abs were detected with Alexa 488-conjugated goat anti-rabbit IgG and Alexa 546-conjugated goat anti-mouse IgG (1/8000; Invitrogen Molecular Probes) secondary Abs, respectively. Fluorescent images were acquired using an Axioskop 2 mot (objective: PlanNeoFluar 60×, NA 1.4) confocal laser scanning microscope (Zeiss).

Cell fractionation (membrane/cytosol) experiments

THP-1 cells or primary monocytes were seeded on 6-well plates at a density of ∼107 cells/3 ml and serum-starved for 3 h. Subsequently, MDP was added for 30 to 120 min. Protein extraction was conducted as previously described (19). In brief, cells were pelleted for 5 min at 800 × g, medium was removed and 200 μl of lysis buffer (1% Triton X-100, 0.1 M NaCl, 10 mM HEPES (pH 5.6), 2 mM EDTA, 4 mM Na3VO4, and 40 mM NaF), supplemented with protease inhibitors, was added. Lysates were harvested and passed through a 21-gauge needle ten times. The cytosolic fraction was obtained by centrifugation at 10,000 × g for 30 min at 4°C. The pellet was resuspended in 150 μl of lysis buffer containing 1% SDS and was sonicated twice for 20 s. After centrifugation for 5 min at 10,000 × g, the resulting supernatant was collected. The protein concentration was determined by using the Bio-Rad Protein Assay (Bio-Rad).

Statistical analysis

Data are shown as means ± SD of three independent experiments. A one-way ANOVA was used for data of Figs. 2, B–D; 3, B–G; and 6, E and F. Main effects were then compared by a Newman-Keul‘s posttest. Throughout the figures, p < 0.01 is indicated by double asterisks.

Results

NOD2 stimulation by MDP activates Rac1

Two considerations led us to examine the role of Rac1 in NOD2 signaling: the fact that Rac1 has been implicated in different NF-κB-regulating pathways, and secondly its involvement in actin cytoskeletal reorganization which might contribute to the membrane recruitment of NOD2 (19, 24). A possible activation of Rac1 in NOD2-mediated signal transduction was determined by pull-down assays using PAK-1 agarose to detect the activated form of Rac1, Rac1-GTP. Stimulation of THP-1 monocytes (Fig. 1⇓, A and C) and primary human monocytes (Fig. 1⇓B) with the NOD2 agonist MDP-LD (hereafter referred to as MDP) resulted in activation of endogenous Rac1 in a time- and dose-dependent manner. In contrast, stimulation of THP-1 cells with the inactive control compound MDP-DD did not activate Rac1 (Fig. 1⇓A), suggesting that Rac1 activation was not simply related to unspecific uptake of the components used.

FIGURE 1.
FIGURE 1.

Activation of Rac1 by the NOD2 agonist MDP in THP-1 cells and primary human monocytes. THP-1 cells (A) were stimulated with either the NOD2 agonist MDP-LD (10 μg/ml) or with the inactive compound MDP-DD (10 μg/ml), or primary human monocytes (B) were treated with MDP-LD for the indicated time intervals, and Rac1 activation was determined by the amount bound to the GST-PAK Rac1 interaction binding site (PD, pulldown). C, THP-1 cells were stimulated with MDP-LD in concentrations as indicated for 30 min and Rac1 activation was determined by Rac1 pulldown assay. Activated GTPase levels were normalized to the amount of total Rac1 in cell lysates (IB, immunoblot) as analyzed by Western blotting. The intensity of the bands was quantified and the values are the mean ± SD (error bars) of three independent experiments normalized to the response of untreated cells.

Rac1 inhibition by siRNA or NSC23766 increased the NOD2-mediated IL-8 production but suppressed the TLR2-dependent IL-8 secretion

To assess the impact of Rac1 in NOD2-dependent gene expression, first we made use of RNAi experiments in primary human monocytes. Rac1-specific siRNA but not an unspecific control siRNA reduced Rac1 expression in these cells (Fig. 2⇓A). Rac1 siRNA enhanced the NOD2-mediated IL-8 production but reduced IL-8 release induced by TLR2 stimulation (Fig. 2⇓B). Next, we made use of the Rac1 inhibitor NSC23766 and examined its effect on NOD2-dependent IL-8 production in THP-1 monocytes. The TLR2-dependent chemokine induction was tested in parallel as a control. In line with results shown above, Fig. 2⇓C shows that preincubation with the Rac1 inhibitor strongly increased the MDP-stimulated IL-8 secretion. In contrast to the NOD2-dependent IL-8 expression and consistent with published studies, IL-8 release stimulated by the TLR2 ligand Malp2 was inhibited by NSC23766 (Fig. 2⇓D). According to the hypothesis that Rac1 might influence the NOD2 pathway via actin cytoskeletal reorganization (30), disruption of actin cytoskeleton by cytochalasin D also led to an enhanced IL-8 secretion in human monocytes (data not shown). These results suggest that Rac1, via actin regulation, can function as a negative regulator of NOD2-dependent IL-8 production but positively contributes to TLR2-dependent gene expression.

FIGURE 2.
FIGURE 2.

Effect of Rac1 siRNA and the Rac1 inhibitor NSC23766 on NOD2- and TLR2-mediated IL-8 secretion. Primary human monocytes were transfected with control nonsilencing siRNA (c-siRNA) or siRNA targeting Rac1 (si-Rac1). After 72 h, cells were lysed and Western blots using anti-Rac1 Abs were performed (A). Membranes were simultaneously probed with anti-ERK2 Abs to confirm equal protein loading. B, Primary human monocytes were transfected with siRNAs as indicated, and after 72 h, stimulated with MDP or Malp2 for 16 h, and the supernatants were analyzed for IL-8 secretion by ELISA. THP-1 cells were untreated (none) or preincubated overnight with the Rac1 inhibitor NSC23766 (200 μM). Subsequently, the cells were either stimulated with MDP (C) or Malp2 (D) for 16 h, and the supernatants were analyzed for IL-8 secretion by ELISA. Data presented are mean ± SD of three different experiments performed in duplicates (∗∗, p < 0.01).

Rac1 inhibition enhanced IL-8 and NF-κB reporter activities mediated by NOD2, but reduced TLR2-dependent NF-κB reporter activation

Next, we more closely examined the involvement of Rac1 in NOD2-dependent IL-8 induction and also in NF-κB activation which is a hallmark event downstream of NOD2 and is crucial for IL-8 expression. Transfection of HEK293 cells with Rac1 siRNA specifically attenuated the Rac1 expression compared with untransfected cells or cells which were transfected with control siRNA as shown by immunoblot analysis (Fig. 3⇓A). Then, HEK293 cells were transfected with control or Rac1-specific siRNAs or left untreated, subsequently transfected with NOD2 and IL-8 or NF-κB luciferase reporters, and stimulated with MDP. Treatment with MDP stimulated reporter activity in untransfected and in control siRNA-transfected cells. Importantly, Rac1 siRNA clearly increased the NOD2-mediated IL-8 and NF-κB reporter activities (Fig. 3⇓, B and C). In contrast to the NOD2-dependent signaling, Rac1 inhibition by RNAi abrogated the NF-κB activation by Malp2 in TLR2-transfected HEK293 cells (Fig. 3⇓D), confirming results shown above and published studies (34). Moreover, cotransfection of wild-type Rac1 slightly and of constitutive active Rac1L61 markedly reduced the NOD2-mediated IL-8 promoter activation, whereas the dominant negative Rac1N17 mutant dramatically enhanced the IL-8 and NF-κB reporter activities in HEK293 cells stimulated with MDP (Fig. 3⇓, E and F). According to our RNAi data, dominant-negative Rac1 reduced the TLR2-mediated NF-κB activation as shown by us previously (37).

FIGURE 3.
FIGURE 3.

Influence of Rac1 on NOD2- or TLR2-mediated IL-8 and NF-κB activation. A, HEK293 cells were left untreated, or were transfected with control non-silencing siRNA (c-siRNA) or siRNA targeting Rac1 (si-Rac1). After 72 h, cells were lysed and Western blots using anti-Rac1 Abs were performed in duplicates. Membranes were simultaneously probed with anti-ERK2 Abs to confirm equal protein loading. B–D, HEK293 cells were left untreated (ctrl), or were transfected with control non-silencing siRNA (c-siRNA) or siRNA targeting Rac1 (si-Rac1). After 48 h, the cells were additionally cotransfected with NOD2 (B and C) or TLR2 (D) expression plasmids, together with an IL-8-reporter (B) or NF-κB reporter construct (C and D) and a β-galactosidase reporter plasmid. Cells were either left untreated (−) or stimulated with MDP (B and C) or Malp2 (D) and relative luciferase activities were obtained. E and F, HEK293 cells seeded in 24-well plates were transiently transfected with a control vector (ctrl) or NOD2 expression plasmid along with an IL-8 luciferase reporter plasmid (E) or a NF-κB-driven luciferase reporter (F), respectively, and a β-galactosidase reporter plasmid. Additionally, the cells were cotransfected with wild-type Rac1 (Rac1wt), dominant negative Rac1N17 or constitutively active Rac1L61. Cells were either stimulated with 10 μg/ml MDP (MDP) or left untreated (−), and relative luciferase activities were obtained the next day. G, HEK293 cells seeded in 24-well plates were transiently transfected with a control vector (ctrl), or a RIP2 expression plasmid along with a NF-κB-driven luciferase reporter and a β-galactosidase plasmid. The influence of Rac1 was tested by additionally introducing wild-type Rac1 (Rac1wt), dominant negative Rac1 (Rac1N17), or constitutive active Rac1 (Rac1L61) mean ± SD; ∗∗, p < 0.01.

Moreover, we evaluated the effect of Rac1 on NF-κB activation induced by RIP2, a key protein of the NOD2 signaling pathway. Overexpression of RIP2 in HEK293 cells activated the NF-κB reporter as shown before (16, 45). Cotransfection of dominant negative Rac1N17 strongly increased, whereas wild-type Rac1 or the constitutive active mutant Rac1L61 only slightly influenced the NF-κB activation induced by RIP2 (Fig. 3⇑G). In an analog experiment, IKKβ induced NF-κB activation was not enhanced but even moderately reduced by dominant negative Rac1 (data not shown), indicating that Rac1 interacts with the NOD2 pathway at a level upstream of IKKβ, possibly at the level of the NOD2/RIP2 complex. Taken together, Rac1 seems to play a differential role in NOD2- and TLR-dependent signaling: it negatively regulates NOD2-mediated cell activation but positively affects TLR2-induced NF-κB activation.

NOD2 and activated Rac1 form a signaling complex

To verify the hypothesis that Rac1 effects NOD2 signaling at the level of NOD2 itself, confocal microscopy to look for colocalization was performed. HEK293 cells were transiently transfected with NOD2 and (wild-type) Rac1 expression plasmids and the proteins were visualized by confocal microscopy using the indicated Abs. As shown in Fig. 4⇓A, both proteins are enriched close to the plasma membrane suggesting colocalization and possibly interaction. Although this membrane localization of overexpressed but otherwise unstimulated NOD2 seems somehow unexpected, it is in agreement with recent studies (17, 24) and is likely caused by the constitutive activation of ectopically expressed NOD2. Next, possible NOD2 and Rac1 interaction was analyzed. HEK293 cells were transiently transfected with either expression vectors encoding Myc-tagged NOD2 and Rac1 (wt) or Myc-NOD2 and a control plasmid. Overexpression of both NOD2 and Rac1 in HEK293 cells led to an association of the two proteins, as detected by immunoblotting using Ab to Rac1 after immunoprecipitation of Myc-tagged NOD2 (Fig. 4⇓B). In a vice versa approach, immunoprecipitation of Rac1 and immunoblotting using anti-Myc Ab also showed an interaction of the two proteins (Fig. 4⇓B). Control experiments using an unrelated IgG-Ab for immunoprecipitation yielded in negative results (data not shown).

FIGURE 4.
FIGURE 4.

Rac1 and NOD2 colocalized and interacted at the plasma membrane. A, Immunofluorescence micrographs of HEK293 cells cotransfected with NOD2 and Rac1 expression plasmids. Fixed cells were stained with anti-NOD2 and anti-Rac1 Abs. Signals obtained with the two Abs (left and middle panels) are presented and a merged image is shown in the right panel. B, Western blot analysis of coimmunoprecipitations from HEK293 cells are shown. Myc-NOD2 and Rac1 wild-type were ectopically expressed in HEK293 cells and lysates were precipitated using anti-c-myc Ab (IP) and coprecipitated Rac1 was detected using anti-Rac1 Ab (IB). Vice versa, lysates were precipitated with anti-Rac1 (IP) and coprecipitated myc-NOD2 was detected using an anti-c-myc Ab (IB). MDP-stimulated THP-1 cells (C) or primary monocytes (D) were lysed at different time points, immunoprecipitations of endogenous NOD2 or endogenous Rac1 with the respective Abs were performed, and immune complexes were probed for the presence of Rac1 or NOD2, respectively. Equal loading was confirmed by blotting whole cell lysates with anti-Rac1 Ab (Input). E, MDP-stimulated THP-1 cells were immunoprecipitated with Abs directed against Nalp3 and immune complexes were probed for the presence of Rac1 or Nalp3. All experiments were repeated three times.

The physical interaction of endogenous NOD2 and Rac1 was subsequently demonstrated in THP-1 cells and primary monocytes. In this more physiological situation, an association of endogenous NOD2 with Rac1 was detected which was further enhanced upon MDP stimulation with a maximum after ∼15–30 min, which represents a time interval similar to the time point in which maximum Rac1 activation occurred as seen above (Fig. 4⇑, C and D, see also Fig. 1⇑). In contrast to the NOD2-Rac1 interaction, the NLR protein Nalp3 did not co-precipitate with Rac1 (Fig. 4⇑E).

Rac1 inhibition abrogates membrane recruitment of NOD2

As both NOD2 and Rac1 were shown to be associated at the cell membrane after stimulation with MDP, we hypothesized that Rac1 might mediate recruitment of the NOD2/RIP2 complex to the membrane. To test this, THP-1 cells were left untreated or stimulated with MDP for 40 min. The membrane fractions were separated and subsequently immunoblotted with Rac1 and NOD2 Abs. After stimulation with MDP, recruitment of both NOD2 and Rac1 to the plasma membrane was observed (Fig. 5⇓A). Preincubation with the Rac1 inhibitor NSC23766 clearly decreased membrane association of NOD2 and Rac1 compared with untreated cells. We also observed recruitment of Rip2 to the membrane, which was abrogated by the Rac1 inhibitor (data not shown). Next, levels of Rac1 in primary human monocytes were reduced using siRNA, and membrane and cytosol fractions were separated and immunoblotted with NOD2 Abs. Rac1 knock-down led to reduced membrane association of NOD2 (Fig. 5⇓B), whereas in the cytosolic fraction, no major differences in NOD2 levels were observed. Moreover, cytochalasin D also abrogated membrane recruitment of Rac1 in human monocytes (data not shown). Overall, these data indicate that Rac1, possibly via actin organization, is involved in the recruitment of NOD2 to the plasma membrane.

FIGURE 5.
FIGURE 5.

Influence of Rac1 inhibition on recruitment of NOD2 and Rac1 to the plasma membrane. A, THP-1 cells were left untreated or preincubated with the Rac1 inhibitor NSC23766 (NSC) overnight. The next day, the cells were left untreated (−) or stimulated with 10 μg/ml MDP (MDP) for 40 min. Membrane fractions were separated and immunoblotted with anti-NOD2 or anti-Rac1 Abs. The intensity of the bands was quantified and the values are the mean ± SD (error bars) of three independent experiments normalized to untreated cells and indicated as fold activation. B, Primary human monocytes were mock-transfected (none), transfected with control non-silencing siRNA (c-siRNA) or siRNA targeting Rac1 (si-Rac1) and, after 72 h, stimulated with MDP for 40 min. Membrane and cytosol fractions were separated and immunoblotted with anti-NOD2 Abs. All experiments were repeated three times.

β-PIX is involved in regulation of NOD2 signaling by Rac1

β-PIX is a Rac/Cdc42GEF that has been shown to directly bind Rac1, thereby mediating Rac1 translocation to the plasma membrane as well as Rac1 activation (40). To test if β-PIX plays a role in Rac1’s regulatory role on NOD2 signaling, we first tested whether endogenous β-PIX binds to endogenous NOD2 upon stimulation with MDP. Our coimmunoprecipitation experiments in THP-1 cells and primary monocytes demonstrated interaction of NOD2 and β-PIX after MDP stimulation in a time dependent manner (Fig. 6⇓B and data not shown). According to the recent finding of Hordijk and colleagues (40), we also observed interaction of β-PIX and Rac1, which was enhanced after MDP stimulation (Fig. 6⇓A). Next, we conducted β-PIX gene silencing experiments. Human THP-1 monocytes were transfected with control siRNA or β-PIX-specific siRNA, which led to a reduced level of endogenous β-PIX expression (Fig. 6⇓C). Moreover, β-PIX knock-down cells showed reduced membrane translocation of NOD2 after MDP treatment (Fig. 6⇓D), suggesting that the NOD2-Rac1 complex can be targeted to the membrane by β-PIX. Most importantly, β-PIX siRNA enhanced the NOD2-mediated IL-8 production but had no effect on IL-8 release induced by TLR2 stimulation (Fig. 6⇓, E and F). Thus, β-PIX appears to play a role in Rac1-mediated negative regulation of NOD2 signaling.

FIGURE 6.
FIGURE 6.

Involvement of β-PIX in NOD2-mediated signaling. MDP-stimulated primary monocytes or THP-1 cells (A and B) were lysed at different time points, immunoprecipitations of endogenous Rac1 (A) or endogenous NOD2 (B) with the respective Abs were performed, and immune complexes were probed for the presence of β-Pix. Equal protein amounts in the lysates were confirmed by blotting total cell lysates with an ERK2 Ab (Input). All experiments were repeated three times. C, THP-1 cells were transfected with control non-silencing siRNA (c-siRNA) or siRNA targeting β-Pix (si-β-Pix_S1 (sequence 1), si-Pix_S2 (sequence 2)). After 72 h, cells were lysed and Western blots using anti-β-Pix Abs were performed. Western blots were simultaneously probed with anti-ERK2 Abs to confirm equal protein loading. D, THP-1 cells were transfected as indicated, incubated for 72 h, and stimulated with MDP (10 μg/ml) for 40 min. Membrane and cytosol fractions were separated and immunoblotted with anti-NOD2 Abs. E and F, THP-1 cells were transfected with siRNAs as indicated, and after 72 h, stimulated with MDP (E) or Malp2 (F) for 16 h, and the supernatants were analyzed for IL-8 secretion by ELISA. Data presented are mean ± SD of three different experiments performed in duplicates (∗∗, p < 0.01).

Rac1 inhibition abrogated the interaction of NOD2 with its negative regulator Erbin

The fact that NOD2’s negative regulator Erbin has been shown to interact with NOD2 at the plasma membrane (24, 25), together with our observations that β-PIX-Rac1 influenced the membrane recruitment of NOD2 and negatively regulated the NOD2 signal cascade prompted us to the hypothesis that Rac1 somehow effects the interaction of NOD2 with Erbin. To test this, coimmunoprecipitation experiments with NOD2 and Erbin Abs were performed in THP-1 cells and primary monocytes, which were pretreated with or without the Rac1 inhibitor and subsequently stimulated with MDP. After stimulation with MDP, interaction of NOD2 and Erbin was observed in the cells which were not treated with the inhibitor (Fig. 7⇓A), consistent with published results (24, 25). Interestingly, preincubation with the Rac1 inhibitor NSC23766 abrogated the interaction of NOD2 with its negative regulator in THP-1 (data not shown) and primary monocytes (Fig. 7⇓A). Experiments using Rac1 RNAi confirmed these data, showing that Rac1 knock-down reduced NOD2- and Erbin-interaction in THP-1 (data not shown) and primary monocytes (Fig. 7⇓B). Furthermore, β-PIX knock-down also diminished the NOD2 and Erbin, as well as Rac1 and Erbin coimmunoprecipitations (Fig. 7⇓C). Overall, these findings might explain the aforementioned results demonstrating that Rac1 together with β-PIX negatively regulate NOD2 signaling.

FIGURE 7.
FIGURE 7.

Rac1 and β-PIX siRNAs as well as the Rac1 inhibitor NSC23766 inhibit interaction of NOD2 with Erbin. Primary monocytes (A and B) or THP-1 cells (C) were either preincubated with the Rac1 inhibitor NSC23766 (NSC) or were transfected with Rac1 siRNA or β-PIX siRNA, as indicated, and were stimulated with 10 μg/ml MDP (MDP) for 40 min. Subsequently, immunoprecipitations with an Erbin Ab and subsequent immunoblots with NOD2 and ERK2 Abs (A) or NOD2, Rac1, and Erbin Abs (B and C) were performed. One representative Western blot out of three is shown.

Discussion

In the present study, we demonstrated that β-PIX and Rac1 negatively regulated NOD2-mediated NF-κB-dependent IL-8 production. We showed that NOD2 stimulation led to interaction with and activation of β-PIX and Rac1, and that inhibition of β-PIX, Rac1, and actin polymerization strongly enhanced NOD2-mediated IL-8 production or NF-κB activation. By elucidating the underlying mechanisms, we found that β-PIX and Rac1 influenced membrane recruitment of NOD2, and that Rac1 and β-PIX inhibition reduced interaction of NOD2 with its negative regulator Erbin. Overall, our data suggest a model in which β-PIX and Rac1 mediate NOD2’s recruitment to the plasma membrane after a first recognition of MDP/bacteria by NOD2. At the plasma membrane, NOD2 might be further stimulated by MDP/bacteria being there, but it is at the same time negatively regulated by Erbin which prevents overwhelming activation of the NOD2-mediated signaling cascade (Fig. 8⇓). In light of the inflammatory diseases associated with NOD2 mutations, positive and negative regulation of its activation might be of special importance. The β-PIX/Rac1-mediated trafficking mechanisms described in this study might also be relevant to other members of the growing family of intracellular pattern recognition receptors.

FIGURE 8.
FIGURE 8.

Schema of the molecular association among NOD2, β-PIX, Rac1, and Erbin as discussed in the text.

By demonstrating that membrane recruitment of NOD2 occurs after activation, our study is consistent with two recent studies (19, 24). In one of these publications, however, some NOD2 variants with mutated LRR failed to recruit to the plasma membrane, which led the authors to hypothesize that NOD2’s membrane recruitment was crucial for NF-κB activation (19). In our opinion, this recent finding did not necessarily prove that NOD2’s membrane recruitment was indeed crucial for NF-κB activation. Alternatively, the impaired ability of mutated NOD2 to recognize MDP and to mediate signaling (perhaps via β-PIX and Rac1) might be responsible for the reduced membrane translocation of the mutated NOD2 variants, an assumption which appears in line with a recent study (46). Our data showing that β-PIX or Rac1 inhibition as well as disruption of actin cytoskeleton by cytochalasin D reduced NOD2’s membrane recruitment, NOD2’s interaction with Erbin, and NOD2-mediated signaling thus suggesting a modification of the recent model. We propose that recruitment of activated NOD2 to the cell surface membrane does not necessarily lead to positive regulation of NF-κB activation, but might lead to fine-tuning of NOD2 signaling perhaps by bringing NOD2 together with Erbin, and possibly other negative regulators of NOD2. It is however obvious that NOD2 activation also depends on the amount of MDP/bacteria located in proximity to NOD2 at the membrane, and with the overall activation level of NOD2 might be the sum of activating and inhibiting signals that both can get in touch with NOD2 near the plasma membrane. Although our data clearly demonstrate that Rac1 together with β-PIX can act as negative regulators of NOD2, additional work is needed to further clarify the impact of NOD2’s membrane recruitment on its activation level especially under different situations such as high-dose vs low-dose bacterial infection/MDP stimulation.

In a recently published study, it has been demonstrated that the interaction of Rac1 with β-PIX was necessary for Rac1 recruitment to membrane ruffles (40). This, together with recent data of Kufer et al. (2006) demonstrating enhanced colocalization of NOD2 and Erbin at bacterial entry sites and bacterially induced membrane ruffling (24) and our data, made it tempting to speculate that upon NOD2 stimulation, β-PIX mediated the activation of Rac1 which lead to recruitment of NOD2 and Rac1 to the plasma membrane and membrane ruffles where NOD2 associates with its negative regulator Erbin. To date, it is unknown, if the interaction of NOD2, Rac1, and β-PIX is direct or indirect via other factors but since NOD2 lacks a functional Rac1 binding domain, a direct interaction between NOD2 and Rac1 seems unlikely. Our data showing an (indirect) interaction of NOD2 with Rac1, and a negative regulation of membrane recruitment and signaling of NOD2 by Rac1 and actin cytoskeleton are supported by another study focusing on NOD2 signaling, which was published after completion of most of our work (47).

In contrast to the here-demonstrated role of Rac1 in limiting NOD2 activation, Rac1 has been shown to positively contribute to TLR2- and TLR4-mediated signaling (34, 37, 48). It was indicated that after activation of TLR2, Rac1 was recruited to the intracellular domain of TLR2 which via PI3K and Akt finally led to NF-κB transactivation (34). In our experiments using chemical inhibitors and siRNA, the positive regulatory role of Rac1 in TLR2 signaling could be confirmed. Nonetheless, the role of Rac1 in NOD2 signaling was different as a set of experiments using a chemical inhibitor, dominant negative overexpression, and RNAi application showed that Rac1 predominantly acts as a negative regulator of NOD2. Experiments using PI3K inhibitors, however, argue against a contribution of the PI3K-Akt cascade to the negative regulation of NOD2 signaling by Rac1 (data not shown). In contrast, PI3K might positively regulate NOD2-mediated signaling since inhibition of PI3K abrogated NOD2-stimulated IL-8 production. Overall, Rac1 seems to play differential roles in cell activation mediated by NOD2 and TLR2 which might be related to differences in trafficking of NOD2 (from cytosol to the plasma membrane; possibly dependent on its activation/signaling) and of TLR2 (from the cell surface to the Golgi apparatus; possibly independent of signaling but dependent on lipid rafts) (49).

Considering Rac1’s involvement in actin rearrangements involved also in phagocytosis, and in generating bactericidal reactive oxygen species via membrane-associated NADPH oxidase (50), it is tempting to speculate that NOD2- (and also TLR-) dependent recognition of bacterial cell wall components and subsequent Rac1 activation might influence not only inflammatory gene expression but somehow also ingestion and destruction of the bacteria. On the other hand, since Rac1 is also crucial for host cell invasion by intracellular bacteria, some bacteria might potentially “use” NOD2-mediated Rac1 activation to infect their target cells (35). Regardless of Rac1 contributing to innate defense or being exploited by some bacteria, the fact that certain bacteria additionally secrete virulence factors that influence the activation state of Rac1 to evade sufficient innate immune responses adds complexity (35, 51, 52). As Rac1 is modulated by those effectors, the mechanism described in our manuscript might be of importance considering that many bacteria negatively modulate Rac1 activity, which probably results in impaired TLR-mediated recognition of the bacteria when located extracellularly. In contrast, these bacterial effectors will lead to an enhanced innate immune response when the bacteria are recognized by NOD receptors after bacterial invasion.

In conclusion, we show that β-PIX and Rac1 negatively regulate NOD2 activation, which might be dependent on β-PIX- and Rac1-mediated interaction of NOD2 with Erbin at the plasma membrane. The fact, however, that Rac1 plays manifold functions suggests that the NOD2-β-PIX-Rac1 module might have additional impact on antibacterial innate defenses.

Acknowledgments

We thank P. Hordijk and A. Ridley for very helpful advice and discussions. We are grateful to A. Ridley, G. Núñez, and C. Kirschning for providing expression vectors. We further thank J. Hellwig, D. Stoll, and F. Schreiber for technical assistance.

Disclosures

The authors have no financial conflict of interest.

Footnotes

  • 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.

  • ↵1 This work was supported in part by grants given by the Deutsche Forschungsgemeinschaft to M.K. and N.S. (Kr 2197/1-2), and B.O. (OP-86/5-1), by the Bundesministerium für Bildung und Forschung-funded network PROGRESS to S.H. and N.S. (B3), and by the Deutsche Gesellschaft für Pneumologie und Beatmungsmedizin to B.O.

  • ↵2 Address correspondence and reprint requests to Dr. Bastian Opitz, Department of Internal Medicine/Infectious Diseases and Pulmonary Medicine, Charité-Universitätsmedizin Berlin, Augustenburger Platz 1, 13353 Berlin, Germany. E-mail address: bastian.opitz{at}charite.de

  • ↵3 Abbreviations used in this paper: NLR, nucleotide-binding domain and leucine-rich repeat containing protein; CARD, caspase activation and recruitment domain; NOD, nucleotide-binding oligomerization domain; LRR, leucine rich repeat; MDP, muramyl dipeptide; RIP2, receptor interacting protein 2; β-PIX; p21-activated kinase [Pak]-interacting exchange factor; RNAi, RNA interference; siRNA, small interfering RNA.

  • Received March 17, 2008.
  • Accepted June 4, 2008.

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