Differential role for p120-catenin in regulation of TLR4 signaling in macrophages

Abstract

Activation of TLR signaling through recognition of pathogen-associated molecular patterns is essential for the innate immune response against bacterial and viral infections. We have shown that p120-catenin suppresses TLR4-mediated NF-κB signaling in LPS-challenged endothelial cells. Here we report that p120-catenin differentially regulates LPS/TLR4 signaling in mouse bone marrow-derived macrophages. We observed that p120-catenin inhibited MyD88-dependent NF-κB activation and release of TNF-α and IL-6, but enhanced TIR-domain-containing adapter-inducing interferon-β (TRIF)-dependent IRF3 activation and release of IFN-β upon LPS exposure. p120-catenin silencing diminished LPS-induced TLR4 internalization, whereas genetic and pharmacological inhibition of RhoA GTPase rescued the decrease in endocytosis of TLR4 and TLR4-MyD88 signaling and reversed the increase in TLR4-TRIF signaling induced by p120-catenin depletion. Furthermore, we demonstrated that altered p120-catenin expression in macrophages regulates the inflammatory phenotype of LPS-induced acute lung injury. These results indicate that p120-catenin functions as a differential regulator of TLR4 signaling pathways by facilitating TLR4 endocytic trafficking in macrophages and support a novel role for p120-catenin in influencing the macrophages in the lung inflammatory response to endotoxin.

Introduction

Activation of innate immune response plays a crucial role in the development of systemic inflammatory response syndrome (SIRS) or the sepsis. Pattern recognition receptors (PRR) can sense pathogen-associated molecular patterns (PAMP) on bacteria and endogenous stress signals termed danger-associated molecular patterns (DAMP) from injured cells to initiate and perpetuate immune and inflammatory responses. TLRs are the best characterized PRRs and importantly contribute to the innate immune response to invading pathogens. TLR4 is unique among TLRs in its ability to activate both MyD88-dependent induction of genes encoding proinflammatory molecules and TIR-domain-containing adapter-inducing interferon-β (TRIF)-dependent production of type I interferon (1). Bacterial LPS, the major structural component of the outer wall of all Gram-negative bacteria, engages TLR4 to induce a signaling cascade leading to the activation of inflammatory response. MyD88-mediated TLR4 signaling involves interleukin-1 receptor–associated kinase (IRAK) phosphorylation, association of TNF receptor-associated factor 6 (TRAF6), and subsequent activation of NF-κB, and MAPK which in turn induces release of proinflammatory cytokines such as TNF-α and IL-6 (2). In contrast, TRIF-mediated TLR4 signaling involves activation of IFN regulatory factor 3 (IRF3) and STAT1 (3) with release of IFN-β (4), IL-10 (3), and RANTES (3), as well as the late phase NF-κB activation. Recent studies have shown that the endocytosis of TLR4 is a critical control step in the signal transduction process. TLR4 localization has a key role in determining which of these signal transduction pathways are activated. MyD88-mediated TLR4 signaling pathway is first activated on the plasma membrane after TLR4 encounters LPS, which is followed by translocation of TLR4 into endosomes where activation of TRIF-dependent TLR4 signaling takes place (5,6). Endocytosis of plasma membrane-localized TLRs downregulates the MyD88 signaling pathway (5,7) whereas TLR4 delivery to endosomes upregulates the TRIF signaling pathway (6,8).

p120-catenin (p120) is the prototypic member of a subfamily of armadillo repeat domain proteins and has been shown to localize to the inner leaflet of the plasma membrane as well as cytosol and nuclei. It is widely expressed in endothelial and epithelial cells, fibroblasts, cardiomyocytes, neurons, and immune cells including macrophages (9). p120 binds directly to the cytoplasmic domain of cadherin and contributes to the regulation of cell–cell junctional integrity, cell–cell adhesion, embryonic development, cell proliferation and polarity, tumor cell migration, and cancer progression (10). Recent evidence indicates that p120 has a role in the anti-inflammatory responses in both infectious and non-infectious models (10). We recently showed that p120 attenuates TLR4 signaling which reduces LPS-induced lung inflammatory injury (11) at least in part, through inhibition of NF-κB activation in endothelial cells.

In the present study, we show, that paradoxically, p120 dampens MyD88-dependent NF-κB activation and stimulates TRIF-dependent IRF3 activation via facilitating translocation of TLR4 from plasma membrane to endosomes in LPS-stimulated macrophages. Mechanistically, inhibition of RhoA activation is required for p120-mediated TLR4 internalization. Our data indicate that p120 has a novel role in mediating macrophage involvement of inflammatory signaling.

Materials and Methods

Reagents

LPS (E. coli. 0111:B4) was from Calbiochem. Anti- TLR4 (M300), TLR2, MyD88, IκBα, early endosomal antigen 1 (EEA1), IRAK1, phosphor-IRAK1, TRAF6, RhoA, p120 and HRP-conjugated Abs were obtained from Santa Cruze Biotechnology. Anti- IRF3, pIRF3 (S396) and GAPDH Abs were from Cell Signaling. Anti-TRIF Ab was from Abcam. PE-conjugated anti-TLR4 Abs, anti-mouse CD16/CD32 Abs and PE-conjugated mouse IgG κ isotype control were from eBiosciences. Y27632, Lipoteichoic acid (LTA), and poly(I:C) were from Sigma-Aldrich. Cytokine ELISA kits were purchased from Biolegend and PBL Biomedical Laboratories. Rhotekin-RBD protein agarose beads were from Cytoskeleton. Amaxa cell line nucleofector kit V was obtained from Lonza. Cell surface protein isolation kit and RIPA buffer were from Pierce Biotechnology. Bicinchoninic acid kits and sample buffer were from Bio-rad. Alexa Fluor 568- and Alexa Fluor 488-conjugated Abs were from Invitrogen. DMEM/F12 and non-enzymatic cell dissociation solution were from Cellgro. BSA, FBS and heat inactivated FBS were from Gibco. Dominant negative mutant RhoA T19N adenovirus (DN RhoA) was a gift from Dr. Jingsong Xu (University of Illinois at Chicago).

Animals

Seventy-four male C57BL/6J mice (25–30 g) were used in this study. Mice were housed in microisolator cages under specific pathogen-free conditions, fed with autoclaved food, and used in experiments at 8–12 wk of age. Animal protocols received institutional review and committee approval, and all studies were conducted under anesthesia using either inhaled isoflurane or i.p.-injected ketamine (60 mg/kg).

Isolation and culture of mouse bone marrow-derived macrophages

Murine bone marrow-derived macrophages (BMDMs) were generated from femurs of C57/BL6J mice as described previously (12). Briefly, mice were sacrificed by rapid cervical dislocation. Bone marrow was flushed from femurs with PBS (without Ca²⁺ and Mg²⁺). Wash medium (2–5 ml) was used in each femur. Collected bone marrow cell suspensions were then centrifuged for 10 min at 500g at room temperature. Cell pellets were resuspended in macrophage complete medium (DMEM/F12 with 10% FBS, 20% L-929 cells conditioned medium, 10 mM L-glutamine, 100 IU/ml penicillin, and 100 μg/ml streptomycin). Cells (4 × 10⁵) were then added to each sterile plastic petri dish in 10 ml macrophage complete medium and incubated at 37°C and 5% CO₂. On day 3, another 5 ml of macrophage complete medium was added to each dish. After 7 d in culture, the adherent cells were approximately 95% pure macrophages as evidenced by their expression of cell surface markers HLA-DR (BioLegend), CD11b and CD206, and these cells were used in subsequent experiments.

p120 knockdown in macrophages

p120 small interfering RNA (siRNA), which is a pool of 3 target-specific 20–25 nt siRNAs (Dharmacon) at a concentration of 50 nM, was added to 2×10⁶ BMDMs in 6-well plates to deplete p120 using the protocol provided by the manufacturer. Successful downregulation of p120 in BMDMs in all experiments was confirmed by Western blot analysis. All experiments were performed 48 h posttransfection.

Electrotransfection

Transient transfection of BMDMs was performed according to the manufacturer’s instructions (Dharmacon). Briefly, 2×10⁶ BMDMs were resuspended in 20 μl of nucleofector solution, mixed with 2 μg cDNA or 10 pM of control nontargeting or gene-specific ON-TARGETplus SMARTpool siRNAs. Nucleofection was performed using program D-032. Cells were rapidly transferred to pre-equilibrated culture medium and incubated for 24–96 h at 37°C. Viability of cells was evaluated by vital dye exclusion. Successful transfection was confirmed by Western blot analysis.

Flow cytometry

Adherent BMDMs were incubated with or without LPS (100 ng/ml) in medium for 30 min and detached with non-enzymatic cell dissociation solution. Cell suspensions were centrifuged for 5 min at 200g, and washed twice with cold FCM buffer (PBS with 1% BSA and without Ca²⁺ and Mg²⁺). The pellets were resuspended in ice-cold FCM buffer at a final cell concentration of 2×10⁷/ml. Cells (1×10⁶ at 50 μl) were added to each tube and incubated with 0.5 μg of anti-mouse CD16/CD32 for 20 min on ice. BMDMs were then incubated with PE-conjugated anti-TLR4 Abs or mouse IgG κ isotype control (0.5 μg per tube) for 45 min on ice, washed and analyzed by flow cytometry (Becton Dickinson LSR I).

RhoA-GTP pull-down assay

RhoA pull-down assays were performed using Rhotekin-RBD Protein GST Beads (Cytoskeleton Inc.) according to the manufacturer’s instructions. Cells were washed with ice-cold PBS and lysed with cell lysis buffer (25 mM Tris, pH 7.5, 10 mM MgCl₂, 300 mM NaCl, 2% IGEPAL and protease inhibitor). Lysates were centrifuged at 10,000g for 5 min, and the supernatant was incubated with 60 μg of Rhotekin-RBD Protein GST Beads for 1 h at 4°C on a roller system. The beads were then collected by centrifugation at 5000g, 4°C for 1 min, and washed twice with wash buffer (25 mM Tris, pH 7.5, 30 mM MgCl₂, 40 mM NaCl). Beads were finally resuspended in Laemmli sample buffer (30 μl) and boiled for immunoblotting with anti-RhoA Ab. The total level of RhoA was measured by Western blot analysis of cell lysates.

Immunofluorescence

Immunofluorescence was performed as previously described (13). Cells plated on coverslips were fixed with 2% paraformaldehyde for 15 min, washed 3 times with 100 mM glycine in HBSS for 10 min followed by washing once with HBSS for 10 min. Cells were permeabilized with 0.1% Triton X-100 in HBSS for 30 min and then incubated with anti-TLR4 (1:100) or anti-EEA1 (1:200) Ab for 1 h, followed by incubation with Alexa Fluor 568-, or Alexa Fluor 488-conjugated secondary Abs (1:800) for 1 h. Cell nuclei were stained with 4′,6-diamidino-2-phenyl indole dihydrochloride (DAPI, 1:2000) for 15 min. The cells were rinsed 3 times, and finally mounted on glass slides using ProLong antifade mounting medium (Molecular Probes). Confocal images were acquired with a laser-scanning confocal microscope (Zeiss LSM 510 META) using Hg lamp and UV-filter set to detect DAPI [band pass (BP) 385–470 nm emission], 488 nm excitation laser line to Alexa 488 (BP505-550 nm emission) and 568 nm excitation laser line to Alexa Fluor 568 (excitation/emission ~578/603 nm). Optical sections had a thickness of < 1 μm (pinhole set to achieve 1 Airy unit).

Western blotting and immunoprecipitation

Cells were lysed in RIPA buffer supplemented with 1 mM PMSF, 1 mM Na₄VO₃, protease and phosphatase inhibitor cocktail. Lysates were sonicated and centrifuged at 10,000g for 10 min at 4°C. The concentration of proteins was measured with bicinchoninic acid kit (Pierce). Equal amounts of proteins were loaded for polyacrylamide gel electrophoresis (10–15%) and transferred onto nitrocellulose membranes. The membranes were blocked with 5% nonfat milk, and probed with primary Abs for 2 h at room temperature or overnight at 4°C and then incubated with HRP-conjugated secondary Abs at room temperature for 1 h. The protein bands were determined using the ECL reagent (Pierce). Relative band densities of the various proteins were measured from scanned films using ImageJ Software (NIH).

Immunoprecipitation analysis was performed as described previously (11,13). Cell lysates were pre-cleared with 1 μg of normal rabbit IgG and 20 μl Protein A+G Agarose beads for 2 h at 4°C, and then incubated overnight at 4°C with primary Ab, followed by addition of 25 ml Protein A/G PLUS-Agarose and further incubated at 4°C for 2 h. Equal amounts of protein were electrophoresed on SDS PAGE gels (10–12%) and subsequently transferred to 0.22-mm nitrocellulose membranes. The membranes were blocked with 5% nonfat milk and probed with the appropriate Abs.

IRF3 Dimerization Assay

Cell lysates were electrophoresed on 10% native PAGE gels. IRF3 monomers and dimers were detected by using Western blot analysis.

Cell surface biotinylation

Cell surface biotinylation was used for TLR4 endocytosis assay according to a method described in detail previously (14) with slight modifications. BMDMs were washed 3 times with ice-cold PBS and then incubated in Sulfo-NHS-SS-Biotin solution for 30 min at 4 °C. At this point in the protocol, biotinylated TLR4 proteins reside within the endosomal compartment. Subsequently, the cells were lysed, and the biotinylated proteins were isolated by streptavidin-agarose beads, eluted into SDS sample buffer, and separated by 10% SDS-PAGE. Biotinylated TLR4 was analyzed by Western blot analysis as described above.

Depletion of alveolar macrophages in mice

Depletion of alveolar macrophages (AMs) was performed as described previously (15). The clodronate liposome (Encapsula NanoSciences LLC, Nashville, TN) was delivered to anesthetized (ketamine 90 mg/kg, i.p.) mice by nebulization.

Murine model of LPS-induced acute lung injury

BMDMs were isolated and cultured using a standard protocol as previously described (16). BMDMs were transfected with a scrambled siRNA or p120 siRNA. AM-depleted mice were divided into three groups (n = 6 each). Saline or BMDMs transfected with a scrambled siRNA or p120 siRNA (2 × 10⁶ cells, 200 μl total volume each) were given to AM-depleted mice via a jugular venous cannula 30 min before LPS challenge. Mice were then nebulized and challenged with 1.5 mg/ml LPS for 1 h. At 4 h after LPS challenge, mice were sacrificed and lung injury was assessed by analysis of bronchoalveolar lavage (BAL) fluid, extravascular lung water measurement and biochemical/immunological analysis of lung tissue.

Determination of polymorphonuclear neutrophil (PMN) counts in BAL fluid

At the end of the experiments, BAL was performed by intratracheal injection of 1 ml PBS followed by gentle aspiration. The lavage was repeated three times. The pooled BAL fluid was centrifuged, and cell pellets were suspended in PBS. Cell suspensions were cytospun onto slides with a cytocentrifuge (Shandon, Southern Sewickley, PA). Slides were stained with Diff-Quick dye (Dade Behring, Newark, DE) and examined at a magnification of ×20 and ×40 by light microscopy. The percentage of PMNs was determined after counting 300 cells in randomly selected fields.

Assessment of pulmonary edema formation

Extravascular lung water (ELW) was used as an index of lung water content and edema (17).

Statistical Analysis

One-way analysis of variance and Student’s Newman-Keuls test for post hoc comparisons were used to determine differences between control and experimental groups. Parameter changes between different groups over time were evaluated by a two-way analysis of variance with repeated measures. Data are expressed as mean ± SEM. Differences were considered significant when p < 0.05.

Results

p120 suppresses MyD88-dependent TLR4 signaling in macrophages

To address the potential mechanisms by which p120 regulates MyD88-dependent TLR4 signaling, we first determined whether p120 associated with TLR4 and its downstream molecules. Surprisingly, we did not detect p120 association with TLR4, MyD88, IRAK1, and TRAF6 in either the absence or the presence of LPS stimulation (Supplemental Figure 1A). Recruitment of MyD88 to TLR4 is one of the earliest events of TLR4 signaling (1,2,6). We previously showed that p120 inhibited TLR4 signaling by attenuating the association of MyD88 and TLR4 in lung endothelial cells, and that p120 degradation in response to LPS challenge led to an increase in TLR4 signaling (11). In the present study we investigated the potential role of p120 in the regulation of TLR4 signaling in macrophages. As expected, LPS caused a transient increase in the association between TLR4 and MyD88. Remarkably, knockdown (90%) of p120 augmented the LPS-induced association of TLR4 and MyD88, whereas p120 siRNA alone had no effect on this interaction (Fig. 1A). LPS also induced IRAK-1 phosphorylation and degradation, consistent with previous studies (2), whereas these responses were exaggerated by p120 silencing (Supplemental Figure 1B). Furthermore, activation of NF-κB as measured by LPS-induced degradation of inhibitory IκB-α subunit was consistently increased in macrophages transfected with p120 siRNA (Fig. 1B). Next, we studied the impact of p120 depletion on production of the MyD88-dependent cytokine TNF-α and IL-6 (18,19). In response to LPS challenge, p120 knockdown led to substantially greater production of TNF-α (Fig. 1C) and IL-6 (Fig. 1D) compared to scrambled siRNA-treated macrophages, whereas p120 knockdown alone had no effect on the production of TNF-α and IL-6. To determine if p120 specifically inhibits TLR signaling, we utilized a non-TLR control curdlan (20) to stimulate macrophages. Curdlan dramatically caused the release of TNF-α and IL-6, whereas these responses were not altered by p120 knockdown (Supplemental Figure 2). These findings indicate that p120 is a potential negative regulator of MyD88-mediated TLR4 signaling in macrophages.

FIGURE 1. Silencing of p120 expression potentiates the LPS-initiated MyD88- mediated TLR4 signaling pathway in macrophages.

BMDMs were transfected with a scrambled (Sc) siRNA or p120 siRNA. After 48 h, the cells were treated with 100 ng/ml LPS for 12 h. (A) Effects of p120 knockdown on the association of TLR4 and MyD88. Immunoprecipitation of TLR4 and immunoblotting with Ab against MyD88 were performed. The association of MyD88 and TLR4 was augmented in p120 knockdown macrophages. (B) Effects of p120 knockdown on degradation of IκB-α following LPS challenge. (C) TNF-α level in supernatants was measured by ELISA. (D) IL-6 level in supernatants was measured by ELISA. Data are mean ± SEM of three independent experiments. *p < 0.05 compared to control group (without LPS treatment); †p < 0.05 compared to Sc siRNA group. ND, not detectable.

p120 augments TRIF-dependent TLR4 signaling in macrophages

To discern whether p120 expression regulates the TLR4-triggered TRIF pathway, we examined LPS-induced recruitment of TRIF to TLR4 and subsequent activation of the TRIF-IRF3-IFN-β signaling axis (2,6). LPS induced the recruitment of TRIF to TLR4 within 15–30 min in scrambled siRNA-treated macrophages. In contrast to the data shown above for the MyD88 pathway, p120 knockdown nearly abolished LPS-induced association between TLR4 and TRIF (Fig. 2A). In order to confirm these data, we interrogated the requirement of TRIF recruitment to TLR4 for activation of IRF3 that requires the phosphorylation and dimerization of the monomer form of IRF3 (21,22). LPS markedly induced the phosphorylation of IRF3 in macrophages transfected with a scrambled siRNA, whereas LPS-induced IRF3 phosphorylation was abolished in p120 siRNA-treated macrophages (Fig. 2B). Significantly, comparable intensities of IRF3 bands were detected (Fig. 2B), indicating that the differences in IRF3 phosphorylation were not due to variations in the total level of IRF3 expression. Consistently, IRF3 dimers were increased after LPS treatment while depletion of p120 inhibited LPS-induced IRF3 dimerization (Fig. 2C). In order to determine the impact of p120 depletion on IRF3 transactivation, LPS-mediated induction of TRIF-controlled, IRF3-dependent IFN-β production was analyzed. LPS treatment of macrophages transfected with a scrambled siRNA led to robust IFN-β production (Fig. 2D). Depletion of p120 with a specific siRNA significantly decreased LPS-driven production of IFN-β (Fig. 2D) compared with the responses observed in scrambled siRNA-transfected cells. p120 knockdown alone had no effect on IFN-β production (Fig. 2D). These data indicate that p120 increased TRIF-mediated TLR4 signaling in macrophages.

FIGURE 2. Silencing of p120 expression suppresses the LPS-initiated TRIF-mediated TLR4 signaling pathway in macrophages.

BMDMs were transfected with a scrambled (Sc) siRNA or p120 siRNA. After 48 h, the cells were treated with 100 ng/ml LPS for 4h. (A) Effects of p120 knockdown on the association of TLR4 and TRIF. Immunoprecipitation of TLR4 and immunoblotting with Ab against TRIF were performed. The association of MyD88 and TRIF was diminished in p120 knockdown macrophages. (B) Effects of p120 knockdown on IRF3 phosphorylation following LPS challenge. (C) Effects of p120 knockdown on IRF3 dimerization following LPS challenge. Cells lysates were subjected to native PAGE, and monomeric and dimeric IRF3 were detected with anti-IRF-3 mAb. (D) IFN-β level in supernatants was measured by ELISA. Data are mean ± SEM of three independent experiments. *p < 0.05 compared to control group (without LPS treatment); †p < 0.05 compared to Sc siRNA group. ND, not detectable.

p120 is unable to regulate TLR2/3 signaling

To further determine whether the differential effect of p120 on MyD88 and TRIF signaling was specific for TLR4, we examined the effects of p120 depletion on TLR2/TLR3 signaling as measured by the production of TLR-induced cytokines, including TNF-α, IL-6, and IFN-β. As shown in Fig. 3A, p120 protein was decreased 90% by transfection of p120-specific siRNA, as measured by immunoblotting. However, although TNF-α, IL-6, and IFN-β secretion increased significantly after treatment of BMDMs with TLR2 ligand LTA, p120 knockdown had no effects on the production of TNF-α, IL-6, and IFN-β in response to LTA stimulation (Fig. 3B–D). Similarly, the release of TNF-α, IL-6, and IFN-β upon stimulation of TLR3 with the synthetic ligand poly(I:C) in scrambled siRNA-treated macrophages was not significantly affected by p120 siRNA transfection (Fig. 3E–G).

FIGURE 3. Knockdown of p120 has no effects on TLR2/3-mediated cytokine production.

(A) BMDMs were transfected with a scrambled (Sc) or p120 siRNA. After 48 h, effective knockdown of p120 expression was confirmed by Western blotting. (B–G) BMDMs transfected with Sc or p120 siRNA were stimulated with 5 μg/ml LTA (B–D) or 20 mg/ml poly(I:C) (E–G) for 12 h. TNF-α, IL-6, and IFN-β in the supernatants were measured by ELISA. ND, not detectable.

p120 facilitates LPS-induced TLR4 internalization

TLR4 internalization in response to LPS stimulation has been shown to regulate LPS-induced proinflammatory signaling (5,6,23). Therefore, we tested whether p120 regulates TLR4 internalization in macrophages. Western blot analysis of surface biotinylated TLR4 indicated that LPS caused a gradual increase in TLR4 internalization within 30 min in scrambled siRNA-treated macrophages. However, p120 knockdown significantly reduced LPS-induced TLR4 internalization (Figs. 4A, 4B). These data were confirmed by surface staining of macrophages and analysis by flow cytometry using a PE-conjugated anti-TLR4 Ab. Consistent with other reports (5,6,23), within 30 min of LPS treatment, TLR4 cell surface staining decreased in macrophages transfected with a scrambled siRNA, indicating that TLR4 is internalized after ligand binding. In contrast, depletion of p120 gene expression with a specific siRNA prevented the LPS-induced internalization of TLR4 (Figs. 4C, 4D). Furthermore, the localization of TLR4 to early endosomes was confirmed by staining fixed cells with an Ab to the early endosomal marker EEA1 (Fig. 4E). In unstimulated cells, TLR4 was present on small EEA1-positive endosomes that grew significantly larger in size after LPS stimulation (Fig. 4E). However, p120 knockdown significantly reduced the amount of TLR4 in EEA1-positive endosomes compared to scrambled siRNA-treated cells (Fig. 4F). To determine whether p120 also regulates TLR2 internalization in macrophages, we observed the effects of p120 knockdown on TLR2 endocytosis in response to LTA. As expected, LTA induced a gradual increase in TLR2 internalization within 30 min in scrambled siRNA-treated macrophages, whereas p120 knockdown did not alter LTA-induced TLR2 internalization (Supplemental Figure 3). Taken all together, our data show that p120 specifically facilitates TLR4 trafficking from the cell surfaces to early endosomes.

FIGURE 4. Silencing of p120 expression inhibits LPS-induced endocytosis of TLR4.

BMDMs were transfected with a scrambled (Sc) siRNA or p120 siRNA. After 48 h, the cells were treated with 100 ng/ml LPS for the indicated times. (A) Cell surface biotinylation of TLR4 in BMDMs. BMDMs were labeled with Sulfo-NHS-SS-Biotin. Cells were lysed with RIPA buffer and the cell lysates were either used directly for Western blot analysis or precipitated with streptavidin-conjugated agarose for biotinylated cell surface proteins and analyzed with Western blotting with TLR4 specific antibodies. Results are representative of 3 independent experiments. (B) Relative densities of the bands of TLR4 protein expression. The density of TLR4 protein in the control Sc group (Biotin) was used as a standard (1 arbitrary unit) to compare relative densities in the other groups. (C) Representative histograms of flow cytometry experiments demonstrating the effects of p120 silencing on cell surface expression of TLR4 protein in response to LPS stimulation. Cell surface expression of TLR4 protein was evaluated using phycoerythrin (PE)-conjugated MTS510 Ab and fluorescence-activated cell sorting analysis. Black lines depict staining with irrelevant IgG2a; blue lines for untreated cells; red lines for LPS-treated cells. (D) Quantitative data showing changes in mean fluorescent intensity (MFI) of PE-TLR4 following LPS stimulation (n = 3). (E, F) Accumulation of TLR4 in the endosomal compartment of macrophages. After labeling plasma membrane TLR4, cells were incubated at 37°C in the presence of LPS for the indicated times (E) or 20 min (F). Internalized TLR4 was detected by immunofluorescence for EEA-1 (green) and TLR4 (red) in control (E), Sc or p120 siRNA-treated macrophages (F) following LPS stimulation. Scale bar = 50 μm. *p < 0.05 compared to corresponding Sc siRNA+LPS groups (B) or No LPS group (C); †p < 0.05 compared to Sc siRNA+LPS group (C).

RhoA mediates p120-induced TLR4 internalization following LPS stimulation

Endocytosis of TLR4 following LPS stimulation is dependent on dynamin and clathrin (5). Actin polymerization and depolymerization are required to regulate endocytosis in mammalian cell lines (24,25). Rho inhibits clathrin-mediated endocytosis of membrane proteins (26). We therefore examined whether the regulatory role for p120 in LPS-induced TLR4 internalization is mediated via the small GTPase RhoA, which is known to be critical for active reorganization of cellular actin (27). Previous study has indicated that p120 negatively regulates RhoA activation via its direct or indirect interaction with RhoA–GDP and subsequent inhibition of GDP dissociation (27,28). In the present study, we showed that p120 associated with RhoA in macrophages and this association was significantly attenuated following LPS challenge (Fig. 5A). LPS caused RhoA activation in scrambled siRNA-treated BMDMs, whereas this response was significantly increased in p120 siRNA-treated cells (Fig. 5B). We next infected p120 siRNA-treated BMDMs with adenoviral vectors expressing DN RhoA. The activity of RhoA was nearly abrogated by transfection of the dominant negative form of the GTPase (Fig. 5C). Importantly, in p120-depleted macrophages, exogenous expression of empty vector or DN RhoA had no effect on the surface expression of TLR4, whereas impaired internalization of TLR4 by depletion of p120 in the presence of LPS could be rescued by expression of DN RhoA (Fig. 5D, 5E). This finding was confirmed in cell surface TLR4 biotinylation and internalization studies (Fig. 5F, 5G).

FIGURE 5. Genetic and pharmacological inhibition of RhoA rescues LPS-induced endocytosis of TLR4 impaired by p120 depletion.

BMDMs were transfected with a scrambled (Sc, B) or p120 siRNA (B–K) or a DN RhoA cDNA or vector (Vec) (C–G). Transfected BMDMs were exposed to 100ng/ml of LPS for 15 min. The activation of RhoA was measured by RhoA-GTP pull-down assay (B, C). In some experiments, after p120 depletion, BMDMs pretreated with 10μM of Y27632 or vehicle for 30 min were exposed to100 ng/ml of LPS for the indicated times (H–K). (A) Association of p120 and RhoA in macrophages in the absence and presence of LPS. (B) Effects of LPS on RhoA activation in BMDMs transfected with a Sc or p120 siRNA. (C) Effects of exogenous expression of DN RhoA on LPS-induced RhoA activation. (D) Effects of exogenous expression of DN RhoA on cell surface biotinylation of TLR4 in p120 silencing BMDMs. Representative histograms of flow cytometry experiments demonstrating the effects of RhoA activity on cell surface expression of TLR4 protein in p120 silencing cells in response to LPS stimulation. Cell surface expression of TLR4 protein was evaluated using phycoerythrin (PE)-conjugated MTS510 Ab and fluorescence-activated cell sorting analysis. (E) Quantitative data showing changes in mean fluorescent intensity (MFI) of PE-TLR4 (n = 3). (F) Effects of RhoA activity on cell surface biotinylation of TLR4 in p120 silencing BMDMs. BMDMs were labeled with Sulfo-NHS-SS-Biotin. Cells were lysed with RIPA buffer and the cell lysates were either used directly for Western blot analysis or precipitated with streptavidin-conjugated agarose for biotinylated cell surface proteins and analyzed with Western blotting with TLR4 specific antibodies. Results are representative of 3 independent experiments. (G) Relative densities of the bands of TLR4 protein expression (F). The density of TLR4 protein in the control Vec group (Biotin) was used as a standard (1 arbitrary unit) to compare relative densities in the other groups. (H) Effects of Y27632 on cell surface biotinylation of TLR4 in p120 silencing BMDMs. Representative flow cytometry data showing TLR4 expression on the surface of cells. (I) Quantitative data showing changes in mean fluorescent intensity (MFI) of PE-TLR4 (n = 3). (J) Effects of Y27632 on cell surface biotinylation of TLR4 in p120 silencing BMDMs. Results are representative of 3 independent experiments. (K) Relative densities of the bands of TLR4 protein expression (J). *p < 0.05 compared to Vec group (E), corresponding p120 siRNA+Vec group (G), No LPS group (I), or corresponding p120 siRNA alone group (K); †p < 0.05 compared to LPS+Vec group (E) or LPS group (I). CON, control; DRA, DN RhoA; B, biotin.

Because RhoA is known to activate Rho kinase, we assessed the effect of the Rho kinase inhibitor Y-27632 on TLR4 endocytosis upon LPS exposure. Inhibition of the activity of downstream RhoA effectors Rho-associated kinases I and II (ROCK I and II) with Y27632 led to a restoration of impaired TLR4 internalization in p120 knockdown macrophages following LPS stimulation (Fig. 5H–K), validating the finding that RhoA may regulate p120-mediated TLR4 endocytosis in macrophages following LPS stimulation. Taken all together, these results indicate that p120 regulates LPS-induced TLR4 endocytosis via modulation of RhoA GTPase activity.

RhoA contributes to the regulatory role of p120 in TLR4 signaling

To investigate whether RhoA is involved in p120-dependent regulation of TLR4 signaling following LPS stimulation, we examined the effect of RhoA inhibition on the association of TLR4 with its adaptor proteins MyD88 and TRIF in p120 siRNA-treated macrophages. Effective transient siRNA knockdown of p120 (90%) and exogenous expression of DN RhoA were confirmed by Western blot analysis (Data not shown). As shown in Fig. 1A, LPS caused a robust increase in the association between TLR4 and MyD88 in p120-depleted BMDMs expressing empty vector, whereas expression of DN RhoA reduced LPS-induced association between TLR4 and MyD88 (Fig. 6A). Furthermore, increased degradation of inhibitory IκB-α subunit upon LPS exposure was consistently abolished by expression of DN RhoA in macrophages transfected with p120 siRNA (Fig. 6B) and the increased TNF-α production in response to LPS challenge in p120 siRNA-treated BMDMs expressing empty vector was also reversed by expression of DN RhoA (Fig. 6C). We also observed that treatment with a pharmacological inhibitor of RhoA reversed increased association of TLR4 and MyD88 (Fig. 6D), IκB-α degradation (Fig. 6E), and TNF-α production (Fig. 6F) following LPS stimulation in p120 siRNA-treated BMDMs. In contrast to the role of RhoA in MyD88-mediated TLR4 signaling, genetic and pharmacological inhibition of RhoA blocked the decrease in the association of TLR4 and TRIF (Fig. 7A, 7B), IRF3 phosphorylation (Fig. 7C, 7D), and IFN-β production (Fig. 7E, 7F) following LPS stimulation in p120 siRNA-treated BMDMs. Collectively, these findings suggest that the inactivation of RhoA rescues LPS-induced TLR4 signaling impaired by p120 depletion, suggesting a central role in mediating the disparate effects of p120 on the MyD88 and TRIF signaling pathways.

FIGURE 6. Genetic and pharmacological inhibition of RhoA dampens MyD88-dependent pathway potentiated by p120 knockdown.

BMDMs were transfected with a scrambled (Sc) or p120 siRNA (A–F) or a combination of p120 siRNA and DN RhoA cDNA or vector (Vec) (A–C). Transfected BMDMs were exposed to 100 ng/ml of LPS for the indicated times (A–C). Cell lysates were collected for co-immunoprecipitation (A, D) or Western blot (B, E). TNF-α in supernatants was assayed by ELISA (C, F). After p120 depletion, BMDMs pretreated with 10μM of Y27632 or vehicle for 30 min were exposed to100 ng/ml of LPS for the indicated times (D–F). (A) Effects of RhoA activation on the association of MyD88 and TLR4 in p120 silencing BMDMs. (B) Effects of RhoA activation on IκB-α protein expression in p120 silencing BMDMs. (C) Effects of RhoA activation on TNF-α production in p120 silencing BMDMs. (D) Effects of Y27632 on the association of MyD88 and TLR4 in p120 silencing BMDMs. (E) Effects of Y27632 on IκB-α protein expression in p120 silencing BMDMs. (F) Effects of Y27632 on TNF-α production in p120 silencing BMDMs. After p120 depletion, BMDMs pretreated with 10μM of Y27632 or vehicle for 30 min were exposed to100 ng/ml of LPS for 12 h. *p < 0.05 compared to control group (without LPS treatment); †p < 0.05 compared to p120 siRNA+Vec group (C) or p120 siRNA (F) group. ND, not detectable

FIGURE 7. Genetic and pharmacological inhibition of RhoA rescues TRIF-dependent pathway impaired by p120 knock down.

BMDMs were transfected with a scrambled (Sc) siRNA, a combination of p120 siRNA and DN RhoA cDNA or vector (Vec). Transfected BMDMs were exposed to 100 ng/ml of LPS for the indicated times (A, C, E). In some experiments, after p120 depletion, BMDMs pretreated with 10μM of Y27632 or vehicle for 30 min were exposed to100 ng/ml of LPS for the indicated times (B, D, F). Cell lysates were collected for co-immunoprecipitation (A, B) or Western blot (C, D). IFN-β in supernatants was assayed by ELISA (E, F). (A) Effects of RhoA activation on the association of TRIF and TLR4 in p120 silencing BMDMs. (B) Effects of Y27632 on the association of TRIF and TLR4 in p120 silencing BMDMs. (C) Effects of RhoA activation on IRF3 phosphorylation in p120 silencing BMDMs. (D) Effects of Y27632 on IRF3 phosphorylation in p120 silencing BMDMs. (E) Effects of RhoA activation on IFN-β production in p120 silencing BMDMs. (F) Effects of Y27632 on IFN-β production in p120 silencing BMDMs. *p < 0.05 compared to control group (without LPS treatment); †p < 0.05 compared to p120 siRNA+Vec group (E) or p120 siRNA (F) group. ND, not detectable.

Reduced expression of p120 in macrophages enhances LPS-induced acute lung injury

Our results thus far indicated that p120 knockdown in macrophages caused increased MyD88-dependent and decreased TRIF-dependent cytokine responses activated by TLR4. These findings raise the possibility that the intentional lowering of p120 levels in lung macrophages would result in accentuated acute lung inflammation and secondary injury through impacting TLR4 signaling (11,29). We therefore determined whether a change in surface expression of TLR4 controlled by p120 could influence the inflammatory response using an in vivo model. Mice were depleted of AMs by using a liposomal clodronate technique (15). To ensure that clodronate reduced macrophage numbers, we performed BAL on mice administered clodronate as well as PBS and counted a minimum of 500 cells per slide. Lavageable AM count was reduced by 75% at 4 d with a 10-ml aerosolized dose of 20 mg/ml clodronate liposome solution. p120 expression was down-regulated approximately 90% in BMDMs transfected with a specific p120 siRNA (Fig. 8A). In vivo injection of BMDMs treated with a scrambled siRNA or p120-catenin siRNA resulted in comparable reconstitution of macrophages in the lung (Supplemental Figure 4). The total neutrophil count in BAL fluid and ELW increased approximately 8-fold and 5-fold following administration of LPS in control mice (no AM depletion) and AM-depleted mice receiving scrambled siRNA-transfected BMDMs, respectively. Mice depleted of AMs prior to LPS challenge showed less PMN infiltration and lung edema formation. AM-depleted mice receiving p120 siRNA-transfected BMDMs showed a significant increase in ELW (Fig. 8B) and the neutrophil counts in BAL fluid (Fig. 8C) compared with mice receiving scrambled siRNA-transfected BMDMs.

FIGURE 8. Repletion of BMDMs with decreased p120 aggravates lung inflammatory injury in AM-depleted mice.

BMDMs (MΦ) isolated from donor mice were transfected with a scramble siRNA (Sc) or p120 siRNA. After 48 h, the efficiency of transfection was evaluated by Western blot. Depletion of AMs and induction of acute lung injury in mice were performed as described in Materials and Methods. BMDMs treated with a Sc siRNA or p120 siRNA were intravenously injected into AM-depleted mice. (A) Representative data of Western blot showing the depletion of p120 protein expression from BMDMs cell lysate. (B) Pulmonary edema formation measured by extravascular lung water (ELW). (C) Neutrophils from BAL fluid were enumerated to evaluate lung airspace inflammation. (D–G) Levels of TNF-a (D), IL-6 (E), KC (F) and IFN-β (G) in BAL fluid were measured by ELISA. *p < 0.05 compared with control group; †p < 0.05 compared with LPS alone group; ^‡p < 0.05 compared with Sc siRNA group. n = 6/each group. CLOD, clodronate liposome. N.D, not detectable.

Proinflammatory cytokines were also measured in the BAL fluid collected from these mice. We found that TNF-α, IL-6 and keratinocyte-derived cytokine (KC) were elevated in the BAL fluid in response to LPS challenge in control mice (no AM depletion). However, the levels of TNF-α, IL-6 and KC after LPS challenge were significantly attenuated in AM-depleted mice (Fig. 8D–F). AM-depleted mice receiving scrambled siRNA-transfected BMDMs showed variably elevated levels of proinflammatory cytokines, whereas AM-depleted mice receiving p120-silenced BMDMs dramatically enhanced the levels of TNF-α, IL-6 and KC (Fig. 8D–F). In contrast, AM-depleted mice receiving p120-silenced BMDMs had a remarkably reduced level of IFN-β compared with those mice receiving scrambled siRNA-transfected BMDMs (Fig. 8G).

Discussions

Prolonged and uncontrolled inflammation in sever sepsis is a major contributor to morbidity and mortality. Tight regulation of TLR4 signaling is a pivotal mechanism to maintain the auspicious balance between pro- and anti-inflammatory immune responses, which determines the intensity and duration of the inflammatory response which influences the progression and clinical outcome of severe sepsis. To date, many regulators that reduce or terminate the activation of TLR4 signaling pathways have been identified that operate via various mechanisms that include dissociation of adaptor complexes, degradation and competition of signal proteins, and regulation of transcription factors (30,31). Our study shows a relatively simple but novel mechanism of TLR4 regulation that p120 differentially and specifically regulates TLR4 signaling in macrophages via modulation of TLR4 internalization. p120 inhibits MyD88-mediated NF-κB activation and generation of proinflammatory cytokine TNF-α, but enhances TRIF-mediated IRF3 activation and IFN-β production. Importantly, modification of cell surface TLR4 expression via p120 knockdown in macrophages exaggerates LPS-induced lung injury, indicating that p120 may be a potential therapeutic target for treatment of inflammatory diseases such as acute respiratory distress syndrome in humans.

Endocytosis of TLR4 and subsequent trafficking through the endosomal system is indispensable for the regulation of innate immunity and inflammatory response during sepsis. Upon LPS stimulation, TLR4 interacts with CD14 and MD2 on the plasma membrane where MyD88-mediated signaling is activated to initiate NF-κB activation and inflammatory cytokine expression, and then TLR4 is transported to endosomes where TRIF-mediated signaling can lead to IF3 activation and the expression of IFNs (22,32–34). Internalized TLR4 can be delivered to recycling endosomes and Golgi for recycling or to lysosomes for degradation (5,35–37). Inhibition of TLR4 endocytosis increased LPS-induced NF-κB activation and proinflammatory signaling, whereas disruption of TLR4 endocytosis was shown to inhibit TRIF-dependent TLR4 signaling upon LPS stimulation, indicating that the endocytosis of TLR4 is an important mechanism for termination of MyD88-mediated TLR4 signaling and activation of TRIF-mediated TLR4 signaling (6). Our results also show that p120 promotes the endocytosis of TLR4 in response to LPS stimulation, which in turn suppresses MyD88-mediated TLR4 signaling and release of the proinflammatory cytokine TNF-α. In contrast, p120 stimulates TRIF-mediated TLR4 signaling and IFN-β production via enhancement of endocytosis of TLR4. These data support the conclusion that p120 differentially regulates LPS-induced TLR4 signaling pathways via modulation of TLR4 internalization in macrophages.

The mechanism by which p120 promotes the endocytosis of TLR4 in response to LPS stimulation remains unclear. The endocytosis of TLR4 is dependent on dynamin and clathrin (5,6). Furthermore, clathrin-mediated endocytosis can be negatively regulated by Rho signaling (26,38), and p120 has been shown to inhibit RhoA activation (39). These findings raise the possibility that p120 facilitates TLR4 internalization via suppression of RhoA activation in macrophages. Our results indicate that p120 silencing significantly increases LPS-induced RhoA activation, verifying the inhibitory effect of p120 on RhoA activity in macrophages. It has been reported that in response to TLR2/3 stimulation, RhoA is required for NF-κB activation, but dispensable for type I IFN generation (40). Using genetic and pharmacological inhibitors, we demonstrated that inhibition of RhoA reversed p120 silencing -mediated increase in MyD88-mediated TLR4 signaling, NF-κB activation, and TNF-α production, as well as the decrease in TRIF-mediated TLR4 signaling, IRF3 activation, and IFN-β production. These data also indicate that RhoA serves as a negative regulator of TLR4 internalization in response to LPS stimulation in macrophages. RhoA can regulate the actin cytoskeleton through multiple signaling pathways, such as the ROCKI/myosin light chain/Myosin II pathway, ROCKI/LIM-kinase/cofilin pathway and mDia/Src pathway (41). This study showed that Y27632, a ROCKI inhibitor, rescued LPS-induced TLR4 endocytosis impaired by p120 knockdown, suggesting that p120 regulates the endocytosis of TLR4 in macrophages via the small GTPase RhoA and its downstream effector Rho kinase.

The role of p120 expression in macrophages during an inflammatory response such as that evoked by bacterial sepsis has not been reported. In the present study, we utilized a well-established mouse model of LPS-induced acute lung injury (42) to examine the effect of macrophages with varying levels of p120 expression on lung inflammatory injury. We found that intravenous administration of p120 siRNA-transfected BMDMs caused robust lung inflammation and edema in LPS-challenged mice. Our data indicate that p120 expression in macrophages plays an important role in LPS-induced lung inflammatory injury. These results, together with our previous findings from pulmonary endothelial cells (11), strongly highlight the fact that p120 expression in both endothelial cells and macrophages is important in the regulation of innate immunity and inflammation during sepsis.

Our observation that the cytokine (TNF-α, IL-6 and IFN-β) response to TLR2/TLR3 stimulation was normal in the p120 knockdown macrophages, suggests that the role of p120 in the regulation of MyD88- and TRIF-mediated TLR signaling is specific to TLR4. Like TLR4, TLR2 is found at the cell surface, whereas the TLR3 involved in the recognition of nucleic acids is localized within endolysosomal compartments (43). Interestingly, in contrast to TLR4, TLR2 internalization is clathrin-independent and lipid raft-dependent (44). Our data demonstrated that LTA-induced TLR2 internalization was not affected by silencing of p120, suggesting that p120 is unable to regulate TLR2 endocytosis and subsequent release of cytokines in response to LTA stimulation in macrophages. Taken together, p120 selectively regulates LPS/TLR4 signaling via RhoA-mediated TLR4 endocytosis in macrophages.

In summary, we demonstrate for the first time that p120 differentially and selectively regulates LPS-induced TLR4 signaling in macrophages. Our data support a model in which p120 promotes LPS-induced TLR4 internalization via inactivation of RhoA GTPase. By facilitating TLR4 internalization, p120 downregulates MyD88-mediated TLR4 signaling and proinflammatory cytokine production, while at the same time upregulates TRIF-mediated TLR4 signaling and IFN-β production (Fig. 9). These findings add to our understanding of molecular mechanisms mediating anti-inflammatory modulation of immune responses by p120. Thus, targeting p120, or its downstream effectors RhoA and Rho kinase, may be an effective strategy for preventing acute lung inflammatory injury.

FIGURE 9.

A proposed model for p120 in regulation of TLR signaling

Abbreviations used in this paper

AM

alveolar macrophage

BAL

bronchoalveolar lavage

BMDM

bone marrow-derived macrophages

DN

dominant negative

EEA1

early endosomal antigen 1

ELW

extravascular lung water

KC

keratinocyte-derived cytokine

LTA

lipoteichoic acid

p120

p120 catenin

PMN

polymorphonuclear neutrophil

ROCK

Rho-associated kinases

siRNA

small interfering RNA

TRIF

TIR-domain-containing adapter-inducing interferon-β