SEARCH

SEARCH BY CITATION

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Conflict of interests
  9. References

Receptor-interacting protein 2 (RIP2) is a caspase recruitment domain (CARD)-containing serine/threonine kinase that is activated by NOD1 or NOD2 recognition of their ligands and essential for the activation of NF-κB and mitogen-activated protein kinase (MAPK). RIP2 has been known to play an important role in innate immune responses against certain bacterial infection. However, the role and interplay of RIP2 with TLR signalling on cytokine production in macrophages against Yersinia enterocolitica infection remains poorly understood. In the present study, we examined whether RIP2 is essential for Yersinia-induced production of cytokines in macrophages. Our results showed that naïve RIP2-deficient macrophages produced similar level of IL-6, TNF-α and IL-10 upon Y. enterocolitica infection compared with wild-type macrophages. However, the production of IL-6, TNF-α and IL-10 by Y. enterocolitica was impaired in RIP2-deficient macrophages after lipopolysaccharide (LPS) pretreatment, a TLR4-tolerant condition. In addition, RIP2 inhibitors, SB203580, PP2, and gefitinib, reduced IL-6 production in TLR4-deficient macrophages in response to Y. enterocolitica, whereas they did not affect the cytokines production in WT cells. These results demonstrate that RIP2 may play an important role in proinflammatory cytokine production in macrophages at the absence of TLR signalling.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Conflict of interests
  9. References

Pattern recognition receptors (PRRs) on innate immune cells such as dendritic cells and macrophages recognize microbial moieties or damage signals and trigger host immune responses [1, 2]. PRRs include Toll-like receptors (TLRs), nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) and retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) [3, 4]. TLRs recognize lipopolysaccharide (LPS), lipoprotein and flagellin at the cell surface and microbial nucleic acids in endosomes [3]. In contrast, NLRs are responsible for cytosolic recognition of microbial molecules or danger signals and trigger various inflammatory signals including NF-κB and caspase-1 activation [4, 5]. The first identified NLRs, NOD1 and NOD2, sense bacterial peptidoglycan derivatives in the cytosol [4]. NOD1 recognizes meso-diaminopimelic acid (meso-DAP) that is produced by most gram-negative bacteria and certain gram-positive bacteria, and NOD2 recognizes muramyl dipeptide present in all types of peptidoglycan [6, 7]. The activation of NF-κB and mitogen-activated protein kinase (MAPK) by TLRs, NOD1 and NOD2 induces the production of proinflammatory cytokines and antimicrobial molecules [1, 8].

RIP2 is a caspase recruitment domain (CARD)-containing serine/threonine kinase that is activated by NOD1 and NOD2 recognition of their ligands and essential for the activation of NF-κB and MAPK [8]. RIP2 has an important role in host defence against certain gram-positive intracellular bacteria as well as gram-negative bacteria [9-11]. Mice deficient in RIP2 showed increased susceptibility to intravenously infected Listeria monocytogenes [11] and impaired bacterial clearance caused by reduced neutrophil recruitment against pulmonary E. coli infection [12]. RIP2 also has an essential role in type I interferon response to Mycobacterium tuberculosis [13] and chemokine response to Legionella pneumophila [14].

Yersinia enterocolitica, one of the human pathogenic Yersinia species, is facultative anaerobic pleomorphic gram-negative extracellular bacteria that colonize the small intestine. Infections are caused by the ingestion of contaminated food or drinking water and can cause gastrointestinal symptoms such as enteritis, diarrhoea and lymphadenitis [15, 16]. Although several reports showed the contribution of TLR2 and TLR4 in Y. enterocolitica infection [17, 18], the role of RIP2 in host immune response against Y. enterocolitica has not been explored. In this study, we evaluated the role of RIP2 and its interplay with TLR4 on cytokine production in macrophages against Y. enterocolitica infection.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Conflict of interests
  9. References

Mice

RIP2- and TLR4-deficient mice on C57BL/6 background were purchased from the Jackson Laboratories (Bar Harbor, ME, USA). Wild-type C57BL/6 mice were obtained from Koatech (Pyeongtaek, Korea). All animal studies were approved and followed by the regulations of the Institutional Animal Care and Use Committee in Konyang University (Daejeon, Korea).

Reagents and bacterial culture

Ultrapure LPS from Escherichia coli O111:B4 and poly (I:C) were purchased from InvivoGen (San Diego, CA, USA), and MTT [3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide] was purchased from Amresco (Solon, OH, USA). Y. enterocolitica strain 8081 was prepared to infect macrophages as described in our previous reports [19]. Single colonies were inoculated into 5 ml of Luria–Bertani (LB) broth and grown overnight at 28 °C in the shaking incubator. A 1/5 dilution of the overnight culture was prepared and allowed to grow at 37 °C with shaking to A600 = 0.6, which corresponds to ~109 CFU/ml. After washing twice with phosphate-buffered saline (PBS; pH 7.4), bacteria were diluted to the desired concentrations media and used in a further experiment.

Preparation of murine macrophages and bacterial infection

Bone marrow-derived macrophages (BMDMs) were prepared as previously described [20]. The cells were seeded in 48-well plates at the concentration of 2 × 105/well and incubated in a 5% CO2 incubator at 37 °C overnight. Subsequently, the cells were left uninfected or infected with Y. enterocolitica at the indicated multiplicity of infection (MOI) for 60 min, and extracellular bacterial growth was inhibited by gentamicin (50 μg/ml) treatment. For LPS pretreatment experiments, the day after plating, cells were left untreated or treated with LPS (100 ng/ml) for 24 h and then infected with Y. enterocolitica. Culture supernatant was collected at 18 h after infection for cytokines measurement.

Immunoblotting

The cells were stimulated with Y. enterocolitica, harvested and lysed in a buffer containing 1% Nonidet P-40 supplemented with a complete protease inhibitor ‘cocktail’ (Roche, Mannheim, Germany) and 2 mm dithiothreitol. Lysates were separated by 10% SDS-PAGE and were transferred to polyvinylidene fluoride (PVDF) membranes by electroblotting. The membranes were immunoblotted with primary antibodies, such as regular- and phospho-IκB-α, phospho-JNK (Cell signaling Technology, Beverly, MA, USA), and phospho-p38, regular- and phospho ERK (Santa Cruz biotechnology, Santa Cruz, CA, USA). After immunoblotting with secondary antibodies, proteins were detected with enhanced chemiluminescence (ECL) reagent (Intron Biotechnology, Seong-Nam, Korea).

Inhibitor assay

SB203580 (Selleckchem, Houston, TX, USA), PP2 (Calbiochem, La Jolla, CA, USA) and gefitinib (Selleckchem) were used as RIP2 inhibitors. Macrophages were left untreated or pretreated with various doses of each inhibitor 2 h before infection. The cells were then infected with Y. enterocolitica at MOI 1/10 at the presence of the inhibitors. At 18 h after infection, culture supernatant was collected for cytokines measurement.

Cytokines measurement

The concentrations of IL-6, TNF-α and IL-10 in collected culture supernatants were determined using a commercial enzyme-linked immunosorbent assay (ELISA) kit from R&D System, USA.

Cell viability analysis

Cell viability was evaluated by MTT assay. After treatment of Y. enterocolitica and inhibitors, the BMDMs were incubated with MTT solution (0.4 mg/ml) at 37 °C. At 4 h after treatment, the solution was removed and 150 μl of dimethyl sulphoxide (DMSO) was added into each well. The absorbance was measured at 570 nm on a 96-well microplate reader.

Statistical analysis

The differences in mean values among different groups were tested, and the values were expressed as mean ± SD. All of the statistical calculations were performed by one- or two-way ANOVA with Bonferroni post-tests using graphpad prism, version 5.00. Values of *< 0.05, **< 0.01, ***< 0.001 were considered significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Conflict of interests
  9. References

RIP2 deficiency impairs the production of IL-6, TNF-α and IL-10 in Y. enterocolitica-infected macrophages after LPS pretreatment

First, we confirmed the phenotype of RIP2-deficient macrophages by measuring ability of the cells to produce cytokines in response to LPS and MDP. Compared with WT macrophages, RIP2-deficient macrophages produced a similar level of IL-6 and TNF-α in response to LPS (100 ng/ml) (Fig. 1A,B). However, cotreatment of LPS (10 ng/ml) and MDP led to increased production of IL-6 and TNF-α in WT macrophages but not in RIP2-deficient macrophages, indicating that RIP2 is essential for the cytokine response to MDP in macrophages (Fig. 1A,B).

image

Figure 1. RIP2 deficiency impairs the production of IL-6, TNF-α and IL-10 in Yersinia enterocolitica-infected macrophages after LPS pretreatment. WT and RIP2-deficient macrophages were stimulated with different doses of MDP (10 μg/ml) and LPS (10 and 100 ng/ml) or the combination of LPS (10 ng/ml) and MDP (10 μg/ml) for 18 h (A, B). WT and RIP2-deficient macrophages were left uninfected or infected with Y. enterocolitica at the indicated multiplicity of infection (MOI) with (F-H) or without (C-E) LPS pretreatment (100 ng/ml for 24 h). At 18 h after infection, the concentration of IL-6, TNF-α and IL-10 in culture supernatant was determined by enzyme-linked immunosorbent assay (ELISA). The results are from one representative experiment of three independent experiments (**< 0.01, ***< 0.001).

Download figure to PowerPoint

To determine the role of RIP2 on Y. enterocolitica-induced production of proinflammatory cytokines, macrophages derived from bone marrow of WT and RIP2-deficient mice were infected with Y. enterocolitica at various multiplicity of infection (MOI). Both WT and RIP2-deficient macrophages produced IL-6, TNF-α and IL-10 in a dose-dependent manner (Fig. 1C–E). And the production of IL-6, TNF-α and IL-10 between WT and RIP2-deficient macrophages was comparable at each indicated MOI (Fig. 1C–E). Next, we evaluated the role of RIP2 in LPS-pretreated macrophages in response to Y. enterocolitica. LPS pretreatment reduced the production of IL-6, TNF-α and IL-10 in both WT and RIP2-deficient macrophages compared with naïve macrophage upon Y. enterocolitica infection (Fig. 1C–H). However, RIP2-deficient macrophages showed the reduced production of IL-6, TNF-α and IL-10 compared with WT macrophages after LPS pretreatment (Fig. 1F–H). These results demonstrate that RIP2 may play a critical role in the production of cytokines in Yersinia-infected macrophages at TLR4-tolerant condition.

RIP2 deficiency impairs NF-κB and MAPK activation in Y. enterocolitica-infected macrophages after LPS pretreatment

The activation of NF-κB and MAPK by TLRs, NOD1 and NOD2 induces the production of cytokines. To determine whether RIP2 deficiency is associated with an altered activation of these molecules in Yersinia-infected macrophages after LPS pretreatment, we infected WT and RIP2-deficient macrophages with Y. enterocolitica at 1/20 (MOI), and protein extracts from infected cells were prepared at indicated time points. Y. enterocolitica infection induced comparable IκB-α degradation and phosphorylation of IκB-α, p38, JNK and ERK in both WT and RIP2-deficient macrophages at 30 min after infection (Fig. 2). However, activation of these molecules in response to Y. enterocolitica infection was diminished by LPS pretreatment in WT and RIP2-deficient macrophages (Fig. 2). In accordance with cytokine response shown above, RIP2-deficient macrophages showed reduced degradation of IκB-α and phosphorylation of IκB-α, p38, JNK and ERK compared with WT macrophages after LPS pretreatment (Fig. 2). These results demonstrate that RIP2 may play a critical role in the activation of IκB-α and MAPK in Yersinia-infected macrophages at TLR4-tolerant condition.

image

Figure 2. RIP2 deficiency alters the activation of NF-κB and mitogen-activated protein kinase (MAPK) in Yersinia enterocolitica-infected macrophages after LPS pretreatment. WT and RIP2-deficient macrophages were infected with Y. enterocolitica at the 1/20 multiplicity of infection (MOI) with or without LPS pretreatment (100 ng/ml for 24 h). The protein from infected macrophages was extracted at the indicated time points. IκB-a degradation and the phosphorylation of IκB-a, p38, JNK and ERK were examined by Western blotting. Primary antibody against the regular form of ERK was used to confirm the loaded protein doses. The results are from one representative experiment of two independent experiments.

Download figure to PowerPoint

TLR4 deficiency impairs IL-6 production in Y. enterocolitica-infected macrophages

In the Fig. 1, we showed that LPS pretreatment reduced the production IL-6, TNF-α and IL-10 in both WT and RIP2-deficient macrophages against Y. enterocolitica infection. Because LPS pretreatment is known to make macrophages tolerant to TLR4 signalling [9, 21], these results made us speculate that TLR4 signalling is important in cytokine production of Y. enterocolitica-infected macrophages. To test this, we compared the production of IL-6, a well known cytokine produced by macrophages in WT and TLR4-deficient macrophages against Y. enterocolitica infection. First, we confirmed phenotype of TLR4-deficient macrophages by measuring cytokine production by LPS or poly (I:C) treatment. As expected, IL-6 production in TLR4-deficient macrophages was abolished compared with WT macrophages by LPS treatment but not poly (I:C) (Fig. 3). And Yersinia-induced IL-6 production was also impaired in TLR4-deficient macrophages (Fig. 3), suggesting that TLR4 signalling is important in the production of IL-6 in macrophages in response to Y.enterocolitica.

image

Figure 3. TLR4 deficiency impairs IL-6 production in Yersinia enterocolitica-infected macrophages. WT and TLR4-deficient macrophages were left untreated or treated with lipopolysaccharide (LPS) (1 μg/ml), poly (I:C) (100 μg/ml) or infected with Y. enterocolitica at the indicated multiplicity of infection (MOI). At 18 h after infection, the concentration of IL-6 in culture supernatant was determined by enzyme-linked immunosorbent assay (ELISA). The results are from one representative experiment of three independent experiments (**< 0.01, ***< 0.001).

Download figure to PowerPoint

RIP2 inhibitors impair IL-6 production upon Y. enterocolitica infection in TLR4-deficient, but not wild-type macrophages

Because RIP2 was dispensable in the production of IL-6, TNF-α and IL-10 upon Y. enterocolitica infection in naïve macrophages but not in LPS-pretreated macrophages, we hypothesized that RIP2 may be important in IL-6 production by Y. enterocolitica infection in TLR4-deficient macrophages. To test our hypothesis, we compared the production of IL-6 upon Y. enterocolitica infection in WT and TLR4-deficient macrophages after RIP2 inhibitor (SB203580, PP2 and Gefitinib) treatment [22, 23]. RIP2 inhibitors did not have any effect on IL-6 production in the Y. enterocolitica-infected WT and RIP2-deficient macrophages, whereas they impaired IL-6 production in TLR4-deficient macrophages against Y. enterocolitica infection in a dose-dependent manner (Fig. 4A–F). Because RIP2 inhibitors treatment did not have any effect on cell viability in Y. enterocolitica-infected WT and TLR4-deficient macrophages (Fig. 5A,B), such impaired IL-6 production in TLR4-deficient macrophages against Y. enterocolitica infection is not due to RIP2 inhibitor-induced effect on cell viability. These results indicate that RIP2 signalling may have an essential role on cytokines production in macrophages in response to Y. enterocolitica at TLR4-deficient condition.

image

Figure 4. RIP2 inhibitors impair IL-6 production upon Yersinia enterocolitica infection in TLR4-deficient, but not wild-type macrophages. WT, TLR4- and RIP2-deficient macrophages were left untreated or pretreated with various doses of RIP inhibitors, SB203580 (A, B), PP2 (C, D) or gefitinib (E, F) 2 h before infection. The cells were then infected with Y. enterocolitica at multiplicity of infection (MOI) 1/10. At 18 h after infection, the concentration of IL-6 in culture supernatant was determined by enzyme-linked immunosorbent assay (ELISA). The results are from one representative experiment of three independent experiments. The results are from one representative experiment of three independent experiments (**< 0.01, ***< 0.001).

Download figure to PowerPoint

image

Figure 5. RIP2 inhibitors do not have any effects on macrophages viability. WT and TLR4-deficient macrophages were left untreated or pretreated with various doses of RIP inhibitors, SB203580 (A) or PP2 (B) 2 h before infection. The cells were then infected with Yersinia enterocolitica at multiplicity of infection (MOI) 1/10. At 18 h after infection, cell viability was determined MTT assay. The results are from one representative experiment of three independent experiments.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Conflict of interests
  9. References

In most cases, PRRs recognition of microbes in dendritic cells and macrophages triggers robust proinflammatory cytokine production that is important for inflammatory response and elimination of pathogenic bacteria [24, 25]. However, overproduced proinflammatory cytokines by lipopolysaccharide (LPS) and other TLR ligands can induce host tissue damage and lead to septic shock and death. To avoid these cytokine-induced immunopathology, LPS-exposed macrophages become tolerant to subsequent LPS challenge [9, 21]. In the intestinal tract, macrophages and epithelial cells are constantly exposed to TLR ligands because there are large numbers of commensal flora with TLR activity. In fact, intestinal mononuclear phagocytes that normally reside in the intestinal lamina propria are hyporesponsive to TLR ligands and microbial stimulation [26, 27]. However, they still have NLR response against pathogenic bacteria [9, 27]. All these reports demonstrate the importance of NLR signalling on host immune response against intestinal infection.

Our results showed the important role of RIP2 signalling in the production of proinflammatory cytokines in Y. enterocolitica-infected macrophages in the TLR4-tolerant condition. This role of RIP2 in TLR4-tolerant macrophages was shown against several gram-negative bacteria like Pseudomonas aeruginosa and Escherichia coli [10]. Although we did not show the role of RIP2 signalling on the host response against Y. enterocolitica in the intestinal infection system, previous studies have revealed an important role of NOD1, NOD2 and RIP2 in intestinal immune response [28-30]. SipA-proficient Salmonella typhimurium strain exhibited marked intestinal inflammation in WT mice, which was absent in NOD1/NOD2-deficient mice [29]. Similarly, NOD2-deficient mice challenged with Listeria monocytogenes via intragastric dosing were more susceptible to infection and showed significantly greater number of bacteria recovered from both liver and spleen than did WT mice [28], and NOD1-deficient mice challenged with Helicobacter pylori showed increased bacterial loads in the stomach than WT mice [30].

We could not see any reduction in the production of IL-6 and TNF-α in Y. enterocolitica-infected RIP2-deficient macrophages compared with WT macrophages without TLR4 tolerization. However, RIP2 deficiency significantly reduced the production of IL-6 and TNF-α in Y. enterocolitica-infected macrophages with TLR4 tolerization (Fig. 1). It could be explained by recent microarray study showing that the level and number of genes induced by MDP are greatly increased in TLR-tolerant macrophages compared with naïve macrophages via TLR agonist-induced RIP2 expression [9].

In the TLR4-tolerant macrophages with LPS pretreatment, RIP2 deficiency impaired the production of IL-6 and TNF-α in Y. enterocolitica-infected macrophages. However, there were still low level of IL-6 and TNF-α at high MOI infection (Fig. 1C–D). It could explain by the recognition of bacterial molecules via another PRR such as RLRs.

Recently, we showed that NOD2 is dispensable with respect to the innate immune response against Y. enterocolitica in macrophage [19]. However, in vivo situation, especially intestinal tract, macrophages show hyporesponsiveness to TLR ligands and reduced TLR signalling [4, 26]. Thus, it was important to clarify the role of NLR in the Yersinia infection in TLR-tolerant condition. In this work, we show that RIP2 has important role on proinflammatory cytokine production in Y. enterocolitica-infected macrophages in TLR4-tolerant condition.

Acknowledgment

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Conflict of interests
  9. References

This study was supported by a grant from Agency for Defense Development (No. ADD-12-70-06-01).

Conflict of interests

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Conflict of interests
  9. References

The authors declare no financial or commercial conflict of interest.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Conflict of interests
  9. References