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The Journal of Immunology, 2006, 176: 3796-3803.
Copyright © 2006 by The American Association of Immunologists

MyD88 and TNF Receptor-Associated Factor 6 Are Critical Signal Transducers in Helicobacter pylori-Infected Human Epithelial Cells1

Yoshihiro Hirata2, Tomoya Ohmae, Wataru Shibata, Shin Maeda, Keiji Ogura, Haruhiko Yoshida, Takao Kawabe and Masao Omata

Department of Gastroenterology, University of Tokyo, Tokyo, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Helicobacter pylori induces NF-{kappa}B activation, leading to mucosal inflammation via cag pathogenicity island. Although recent studies have implicated several candidate proteins of both H. pylori and host, the molecular mechanism by which H. pylori activates NF-{kappa}B remains unclear. The aim of this study was to analyze the mechanism of cag pathogenicity island-mediated NF-{kappa}B activation in epithelial cells. The responses of human cell lines and mouse embryonic fibroblasts to infection with wild-type H. pylori or cagE mutant were investigated. The effect of small interfering RNAs (siRNAs) for several NF-{kappa}B signaling intermediate molecules was evaluated in H. pylori-induced I{kappa}B{alpha} phosphorylation and IL-8 production. Protein interactions of exogenously expressed TNFR-associated factor 6 (TRAF6) and MyD88 or receptor-interacting protein 2 and nucleotide-binding oligomerization domain 1 or those of endogenous I{kappa}B kinase, TGF-beta-activated kinase 1 (TAK1), and TRAF6 were assessed by immunoprecipitation. Cag pathogenicity island-dependent NF-{kappa}B activation was observed in human cell lines, but not in mouse fibroblasts. In human epithelial cells, H. pylori-induced I{kappa}B{alpha} phosphorylation and IL-8 production were severely inhibited by siRNAs directed against TAK1, TRAF6, and MyD88. In contrast, siRNAs for TRAF2, IL-1R-associated kinases 1 and 4, and cell surface receptor proteins did not affect these responses. H. pylori infection greatly enhanced MyD88 and TRAF6 complex formation in a cag-dependent manner, but did not enhance Nod1 and receptor-interacting protein 2 complex formation. H. pylori also induced TAK1 and TRAF6 complexes. These results suggest that the cag pathogenicity island of H. pylori is a cell type-specific NF-{kappa}B activator. TAK1, TRAF6, and MyD88 are important signal transducers in H. pylori-infected human epithelial cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Helicobacter pylori is a causative agent of human gastric disease. Chronic infection with H. pylori is associated with gastroduodenal ulcer, MALT lymphoma, and gastric cancer. About half the world’s population is infected with this bacterium, although only a proportion of infected individuals develop symptomatic disease (reviewed in Refs.1, 2, 3, 4). Clinical investigations have revealed several bacterial virulence factors that are strongly associated with severe diseases (5, 6, 7, 8, 9, 10). One of the most important virulence factors of H. pylori is the cag pathogenicity island (PAI).3 The presence of cag PAI is strongly related to the development of gastric cancer and ulcer diseases (7, 8). Histological examinations have elucidated that cag PAI is associated with severe gastric inflammation (5, 11). Thus, the concept of sequential progression of gastric disease, i.e., cag-positive H. pylori infection, superficial gastritis, atrophic gastritis, intestinal metaplasia, and gastric cancer, is widely accepted (2, 3, 4).

The virulence of cag PAI-positive H. pylori has been investigated intensively for several years in an attempt to elucidate the pathogenesis of gastric disease. Recent advances in cell biology have shown that cag-positive H. pylori strains are markedly different from cag-negative strains in terms of their effects on epithelial cells. Initially, chemokine IL-8 production was reported to be dependent on the presence of cag PAI in an epithelial cell infection model (8, 12). This was in accordance with a clinical investigation that assessed the relationship between IL-8 secretion and the status of cag PAI in the human stomach (11, 13). Thereafter, attention was focused on the intracellular signaling pathways. In particular, the NF-{kappa}B and MAPK signaling pathways have been shown to be activated in epithelial cells infected with cag-positive H. pylori, whereas they are not activated by either cag-negative or mutant strains of H. pylori (12, 14, 15, 16).

NF-{kappa}B is an important transcriptional factor that regulates multiple cellular responses, such as inflammation, cell survival, and cell proliferation (reviewed in Refs.17, 18, 19). Several genes are known to be targets of NF-{kappa}B and are actually up-regulated by proinflammatory cytokines and bacterial components. Activation of NF-{kappa}B and subsequent gene expression are necessary to maintain normal cell physiology and prevent damage to the individual. However, hyperactivation or unregulated activation of NF-{kappa}B is associated with disease. Constitutive activation of NF-{kappa}B is observed in the tissue of patients with inflammatory diseases, such as rheumatoid arthritis, inflammatory bowel diseases, and asthma (18, 20). Furthermore, NF-{kappa}B activation is found in several malignant diseases (21, 22). Certain types of lymphoma with chromosomal translocations t(11,18)(q21;q21) or t(1,14)(p22;q32) show aberrant activation of NF-{kappa}B. In addition, NF-{kappa}B hyperactivity has been reported for epithelial cell carcinoma, such as breast cancer (23). Thus, detailed investigation of this signaling pathway will increase our understanding of the pathogenesis of malignancies and should be useful in mapping out therapeutic strategies.

Although many studies have reported that cag-positive H. pylori strains activate NF-{kappa}B, the mechanisms by which these bacteria activate the cellular signaling cascades remain unclear. Recently, peptidoglycan injected by H. pylori infection has been shown to interact with the cellular molecule nucleotide-binding oligomerization domain 1 (Nod1), leading to activation of the NF-{kappa}B signaling pathway (24). However, it remains unsolved whether Nod1 pathway is the main and only signaling pathway for NF-{kappa}B activation by H. pylori. To obtain a better understanding of H. pylori pathogenesis, we have examined the molecular pathway of NF-{kappa}B activation induced by cag-positive H. pylori.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cell lines and H. pylori strains

The AGS human gastric cancer cell line was maintained at 37°C in 5% CO2 in Ham’s F-12 medium that was supplemented with 10% FBS as described previously (25). HeLa cells and HEK293T cells were purchased from the Riken Cell Bank and maintained as described previously (26, 27). Mouse embryonic fibroblasts (MEFs) were obtained from 13.5-day-old embryos of C57BL/6 mice. The cells were cultured at 37°C in 5% CO2 in DMEM that contained 10% FBS supplemented with penicillin, streptomycin, L-glutamate, sodium pyruvate, and nonessential amino acids.

The type I H. pylori strains TN2 and 26695 were maintained under microaerophilic conditions in Brucella broth that was supplemented with 5% horse serum. TN2-{Delta}cagE, an isogenic mutant that lacks a functional type IV secretion system, was grown on kanamycin-containing plates (25 µg/ml) and maintained in Brucella broth without antibiotics (16, 26). These bacterial strains were washed with PBS, and the concentrations were estimated using OD560 = 0.1 as equivalent to 4 x 107 CFU H. pylori. The bacteria were resuspended in Ham’s F-12 or DMEM before infection.

Cultured cells were washed twice with PBS and supplemented with fresh medium without antibiotics, followed by infection with H. pylori for the indicated time periods at a multiplicity of infection of 100.

Small-interfering RNA (siRNA), plasmids, and transfection

RNA oligonucleotides for siRNA were synthesized (Qiagen) with the following sequences: for the nonsilencing control, 5'-UUCUCCGAACGUGUCACGU-3'; for TGF-beta-activated kinase 1 (TAK1), 5'-UGGCUUAUCUUACACUGGA-3' (28); for TNFR-associated factor 6 (TRAF6), 5'-AAGGAGAAACCUGUUGUGAUU-3'; for TRAF2, 5'-AGGAGCAUUGGCCUCAAGGA-3'; for MyD88, 5'-AAGGCAAUGAAGAAAGAGUUC-3'; and for IL-1R-associated kinase 1 (IRAK1), 5'-AAGUUGCCAUCCUCAGCCUCC-3' (29). The siGENOME mixture for Nod1, TLR2, TLR4, TLR9, and IL-1R accessory protein (AcP) was obtained from Dharmacon. Silencer-validated siRNA for receptor-interacting protein (RIP2; ID no. 456) was purchased from Ambion. All siRNA transfections were performed with LipofectAMINE 2000 reagent (Invitrogen Life Technologies). All siRNAs were tested and verified as reducing expression by >80% protein reduction in AGS cells by immunoblot analysis or reducing expression of >50% of mRNA by real-time PCR when appropriate Ab was not available. For the AGS cells, 100 nM of each siRNA was transfected 48 h before H. pylori infection.

The pNF-{kappa}B-luciferase (pNF-{kappa}B-Luc), pRL-thymidine kinase (pRL-TK), and Flag-TRAF6 expression vectors were used as described previously (16). The pTriEx-2 hemagglutinin (HA)-MyD88 and pcDNA3.1 Myc-RIP2 were generated by RT-PCR with cDNA obtained from AGS cells. HA-Nod1 was cloned by PCR from the cDNA for Nod1, which was purchased from OriGene Technologies. The pSilencer vector, which is a plasmid for generating short hairpin RNAs for gene silencing, was purchased from Ambion and was used to generate MyD88 knockdown cells. All clones were sequenced and transfected, followed by immunoblotting with anti-tag Ab and/or the indicated Ab. All plasmid transfection experiments were performed with the Effectene transfection reagent (Qiagen).

To establish stable cell lines that express different amounts of MyD88 protein, AGS cells were transfected with pTK-hygromycin (BD Clontech) for the control cells, pTK-hygromycin and pTriEx-2 HA-MyD88 for the MyD88-overexpressing cells, or pSilencer MyD88 for the MyD88-silencing cells. These transfectants were cultured with 200 µg/ml hygromycin or 400 µg/ml G418, and selected clones were subjected to immunoblot analysis with the anti-MyD88 Ab.

Abs and reagents

Human IL-1beta (10 ng/ml), human TNF-{alpha} (10 ng/ml; R&D Systems), and 10 µg/ml LPS (Escherichia coli O111:B4; Sigma-Aldrich) were used to stimulate the cells. The LPS used in this study has been shown to activate Nod1-dependent signaling pathways (30, 31). The polyclonal anti-phospho-I{kappa}B-{alpha} (Ser32), anti-phospho-JNK (Thr183/Tyr185), and anti-I{kappa}B kinase (IKK) {gamma} Abs were purchased from Cell Signaling Technology. The polyclonal anti-IRAK1 and anti-TRAF6 Abs and the monoclonal anti-TAK1, anti-HA, and anti-c-Myc Abs were purchased from Santa Cruz Biotechnology, and the polyclonal anti-IRAK4 and anti-p100/p52 Abs were obtained from Upstate Biotechnology. The monoclonal anti-TRAF2 and anti-JNK1/JNK2 Abs were purchased from BD Pharmingen. The polyclonal anti-TAK1, anti-MyD88, and anti-RIP2 Abs were obtained from StressGen Biotechnologies, and the monoclonal anti-actin and anti-Flag (M2) Abs were purchased from Sigma-Aldrich.

Immunoblot analysis

The cells were seeded in 12-well plates and treated with siRNAs for 48 h as indicated. The cells were then stimulated with H. pylori or reagents for different time periods, washed once with cold PBS, and lysed in ice-cold Triton X-100 buffer (50 mM Tris-HCl (pH 7.6), 1% Triton X-100, 5 mM EDTA, 1 mM Na3VO4, 10 mM sodium fluoride, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 1 mM PMSF). The cell lysates were centrifuged at 10,000 x g for 10 min at 4°C. Equal amounts of the cell lysates were separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane. The membrane was probed with the indicated primary Ab, followed by HRP-conjugated secondary Ab, and was developed using the ECL Plus kit (Amersham Biosciences). The membranes were stripped and reblotted to confirm equal loading of the samples. Each immunoblot experiment was performed more than twice, and a representative image is shown.

Immunoprecipitation

AGS cells were cultured in 6-cm diameter dishes and stimulated with H. pylori, IL-1beta, or TNF-{alpha} for the indicated periods of time. Where indicated, Flag-TRAF6 (0.5 µg) and HA-MyD88 (1 µg) or Myc-RIP2 (0.5 µg) and HA-Nod1 (1 µg) were transfected into the cells 24 h before stimulation. Aliquots (300 µl) of the cell lysates were collected as described above. The cell lysates were immunoprecipitated at 4°C with 5 µl of agarose-conjugated Flag or Myc Ab overnight or with polyclonal TAK1 Ab (2 µl) overnight, followed by a 1-h incubation with protein G-Sepharose beads (Amersham Biosciences). The precipitates were washed three times with ice-cold Triton X-100 buffer, boiled in SDS sample buffer for 5 min, and immunoblotted with the indicated Abs.

Quantification of chemokines by ELISA

The IL-8 levels in the culture supernatants were measured by ELISA as specified by the manufacturer (Techne). AGS or HeLa cells plated in 24-well plates were transfected with siRNAs for 48 h and infected with H. pylori for an additional 12 h. The culture supernatants were aspirated and stored at –70°C until assayed by ELISA. The concentration of IL-8 was determined using a standard curve obtained with rIL-8 protein. The values were determined in more than three independent experiments and are represented as the mean ± SD.

Statistical methods

Statistical analysis was performed using Student’s t test and two-sided or one-way ANOVA with Dunnett’s multiple comparison. Differences were considered statistically significant at p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
The cag PAI system is required for NF-{kappa}B activation in cells that do not respond to LPS stimulation

Initially, we investigated the specificity of cag PAI for NF-{kappa}B activation using various types of cultured cells. Consistent with previous reports (12, 32), 12-h stimulation with the cag-positive H. pylori strains activated NF-{kappa}B in AGS cells, which are representative of gastric cancer cell lines (Fig. 1A, bottom panel). In accordance with the levels of NF-{kappa}B activity measured in the reporter assay, wild-type H. pylori, TNF-{alpha}, and IL-1beta increased the levels of p100 protein, which is a known target gene of NF-{kappa}B, in 12 h (Fig. 1A, top panel). This cell line was unresponsive to stimulation with either LPS or the H. pylori cag mutant strain in terms of both NF-{kappa}B activation and production of p100 protein. We also tested the human epithelial cell lines HeLa and HEK293T and found that wild-type H. pylori, but neither cag mutant H. pylori nor LPS, activated NF-{kappa}B reporter and p100 protein production in these cells (data not shown). In contrast to these human epithelial cells, MEFs were highly responsive to both the wild-type and mutant H. pylori strains in terms of NF-{kappa}B activation and target protein production (Fig. 1B). MEFs were also sensitive to LPS.


Figure 1
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  		FIGURE 1. Influence of cag PAI of H. pylori on epithelial cell signaling. A and B, Cells were treated with IL-1beta (10 ng/ml), TNF-{alpha} (10 ng/ml), and LPS (10 µg/ml) or were infected with H. pylori (TN2 or TN2-{Delta}cagE) for 12 h. The level of p100 protein production was measured by immunoblotting (top panel). Actin was used as the loading control (middle panel). NF-{kappa}B activity was measured with the same stimulation of NF-{kappa}B reporter transfected cells. The values shown are the mean ± SD (n = 3; bottom panel). *, p < 0.05, by one-way ANOVA. A, AGS; B, MEFs. C and D, AGS (C) or MEF (D) cells were treated with H. pylori (TN2 or TN2-{Delta}cagE) or 10 µg/ml LPS for the indicated period of time. I{kappa}B{alpha} phosphorylation was analyzed by immunoblotting (top panel). Urease or actin was used as the loading control for H. pylori or the cell lysates, respectively (middle and bottom panels, respectively).

 
We evaluated in these cells the level of I{kappa}B{alpha} phosphorylation, which is a key signaling event for classical NF-{kappa}B activation. Once again, the wild-type H. pylori, but neither the cagE mutant nor LPS, induced I{kappa}B{alpha} phosphorylation in AGS (Fig. 1C) and HEK293 (data not shown) cells. However, in MEFs, all these stimuli strongly induced I{kappa}B{alpha} phosphorylation in a time-dependent manner (Fig. 1D). When we used lymphocytes from C57BL/6 mice or the human lymphocyte cell line IM-9, we observed similar results, i.e., both H. pylori mutant and LPS induced I{kappa}B{alpha} phosphorylation to the same extent as wild-type H. pylori (data not shown). These results suggest that the cag PAI system of H. pylori is a specialized bacterial component for inducing NF-{kappa}B activity in epithelial cells that are essentially unresponsive to LPS.

TAK1 and TRAF6 are required for cag PAI-mediated NF-{kappa}B activation

To address the mechanism of cag PAI-mediated NF-{kappa}B activation, we used siRNAs to block the signaling intermediates in this pathway. Because TAK1 is known to transduce signals from the IL-1R or TNFR to IKK (28, 33), we initially transfected siRNA for TAK1 into AGS cells and assessed the effect on H. pylori infection. As shown in Fig. 2A, TAK1 siRNA effectively blocked H. pylori-mediated I{kappa}B{alpha} phosphorylation. Furthermore, JNK activation was abolished in these cells. Thus, TAK1 is involved in two important cag PAI-induced cell responses, namely, the NF-{kappa}B and JNK signaling pathways.


Figure 2
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  		FIGURE 2. TAK1 and TRAF6 are required for H. pylori-induced NF-{kappa}B activation in human epithelial cells. AGS cells were treated with siRNA for TAK1 (A), TRAF6 (B), and TRAF2 (C), followed by stimulation with IL-1beta (A and B), TNF-{alpha} (C), and H. pylori (TN2) for the indicated periods of time. The cell lysates were subjected to immunoblot analysis for phospho-I{kappa}B{alpha}, phospho-JNK, or other proteins.

 
We have previously reported that dominant-negative TRAF2 and dominant-negative TRAF6 effectively inhibit NF-{kappa}B activation in gastric cancer MKN45 cells (16). TRAFs are now known to regulate the activity of IKK together with TAK1 (28, 34). Thus, we used siRNAs for TRAF2 and TRAF6. As shown in Fig. 2B, siRNA for TRAF6 inhibited the phosphorylation of both I{kappa}B{alpha} and JNK induced by H. pylori in AGS cells. The siRNA for TRAF6 had negligible effects on I{kappa}B{alpha} phosphorylation induced by TNF-{alpha} (data not shown). In contrast, TRAF2 silencing did not affect H. pylori infection-mediated signaling activation, although TNF-{alpha}-induced signaling was effectively down-regulated (Fig. 2C). The specificities of TRAF6-dependent and TRAF2-independent NF-{kappa}B and JNK signaling activation by H. pylori were also confirmed in HeLa cells (data not shown).

MyD88 is required for cag PAI-mediated NF-{kappa}B activation

TRAF6 is an important signal transducer in IL-1R signaling and TLR signaling, both of which pathways involve MyD88 as an adaptor molecule (reviewed in Refs.35 and 36). Therefore, we assessed the involvement of this adaptor molecule in H. pylori-infected AGS cells. As shown in Fig. 3A, MyD88 silencing effectively inhibited the phosphorylation of I{kappa}B{alpha} and JNK caused by either H. pylori infection or IL-1 stimulation. In contrast, silencing of TRADD, the adaptor molecule for TNFR signaling, did not affect H. pylori-mediated signaling activation (data not shown). We also confirmed that siRNA for MyD88 did not reduce TNF-mediated I{kappa}B{alpha} phosphorylation (data not shown). These results indicate that cag PAI-dependent NF-{kappa}B activation by H. pylori requires the MyD88 protein.


Figure 3
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  		FIGURE 3. MyD88 is required for H. pylori-induced NF-{kappa}B activation in human epithelial cells. A, AGS cells were treated with siRNA for MyD88 and subsequently stimulated with IL-1beta and H. pylori (TN2) for the indicated periods of time. The cell lysates were analyzed by immunoblotting, as described in Fig. 2. B, Stable cell lines for control (AGS-C1), MyD88 overexpression (AGS-B4), and MyD88 knockdown (AGS-71) were infected with H. pylori (TN2), and I{kappa}B{alpha} phosphorylation was assayed (top panel). The MyD88 protein level is also shown (middle panel). The arrow and arrowhead indicate endogenous and HA-tagged MyD88 proteins, respectively.

 
We also established two stable AGS cell lines; AGS-B4 showed increased MyD88 protein due to exogenous CAG promoter-driven expression, whereas AGS-71 showed decreased MyD88 protein expression due to U6 promoter-driven MyD88 silencing. H. pylori induced a higher level of I{kappa}B{alpha} phosphorylation in AGS-B4 cells than in control (AGS-C1) cells (Fig. 3B). In contrast, H. pylori induced a lower level of phosphorylation in AGS-71 cells. This result represents additional evidence for the involvement of the MyD88 adaptor protein in cag PAI-mediated NF-{kappa}B activation in AGS cells.

Analysis of the signaling complex in H. pylori-infected epithelial cells

We next investigated the involvement of TAK1, TRAF6, and MyD88 in the H. pylori-mediated signalosome by assessing the intermolecular interactions. Initially, we transfected tagged MyD88 and TRAF6 into AGS cells and performed immunoprecipitation. As with IL-1 stimulation, H. pylori induced complex formation between HA-tagged MyD88 and Flag-tagged TRAF6 within 30 min of infection (Fig. 4A). Importantly, this interaction was induced only by the wild-type H. pylori and not by the cagE mutant (Fig. 4B). This suggests that these molecules are involved in cag PAI-mediated cell signaling.


Figure 4
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  		FIGURE 4. Analysis of the molecular interactions induced by H. pylori. A, AGS cells were cultured in a 6-cm diameter dish and transfected with 1 µg of HA-MyD88 and 0.5 µg of Flag-TRAF6 vector for 24 h. The cells were then treated with TNF-{alpha}, IL-1beta, and H. pylori (TN2) for the indicated periods of time. The cell lysates were immunoprecipitated with anti-Flag Ab. The immunoprecipitated proteins (left panel) and total cell lysates (right panel) were analyzed by immunoblotting with the indicated Ab. B, AGS cells, which were transfected with HA-MyD88 and Flag-TRAF6, were infected with H. pylori (TN2 or TN2-{Delta}cagE). Immunoprecipitation and immunoblotting were performed as described in A. C, AGS cells were cultured in a 6-cm diameter dish and infected with H. pylori for the indicated time periods. The cell lysates were immunoprecipitated with 2 µl of polyclonal anti-TAK1 Ab. The immunoprecipitated proteins and total cell lysates were immunoblotted with the anti-TRAF6 Ab.

 
We also investigated the role of TAK1 in the signalosome. As shown in Fig. 4C, endogenous TRAF6 formed a complex with TAK1 upon H. pylori infection. Furthermore, TAK1 immunoprecipitated with IKK{gamma}, which is a critical regulator of the IKK complex, in a H. pylori infection-dependent manner (data not shown). These results indicate that TAK1, TRAF6, and MyD88 are important molecules that link cag-positive H. pylori infection to the IKK complex in AGS cells.

TAK1, TRAF6, and MyD88 are important for IL-8 secretion from H. pylori-infected epithelial cells

Because the above siRNA experiments involved only short postinfection incubation periods of 30–90 min, we could not exclude the possibility that these molecules are independent of H. pylori-induced cell signaling at later time points. Therefore, we investigated the effects of these siRNAs on IL-8 chemokine induction, which usually requires several hours. These siRNAs did not reduce IL-8 secretion from noninfected cells (70–200 pg/ml in AGS cells; data not shown). As shown in Fig. 5, coculture with H. pylori for 12 h induced ~2500 pg/ml IL-8 in the control oligonucleotide-transfected cells, whereas it induced only 800-1400 pg/ml IL-8 in the TAK1-, TRAF6-, or MyD88 siRNA-treated cells (p < 0.01). TRAF2 silencing slightly reduced IL-8 secretion from H. pylori-infected AGS cells; however it was not statistically significant. We found similar IL-8 suppression by siRNA for TAK1, TRAF6, and MyD88 in H. pylori-infected HeLa cells (data not shown). These results indicate that TAK1, TRAF6, and MyD88 are necessary not only for transient phosphorylation of I{kappa}B{alpha} and JNK, but also for subsequent IL-8 production in AGS cells.


Figure 5
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  		FIGURE 5. TAK1, TRAF6, and MyD88 are required for IL-8 secretion by H. pylori-infected epithelial cells. AGS cells were treated with the indicated siRNAs for 48 h and subsequently infected with H. pylori (TN2) for 12 h. The culture supernatants were examined for IL-8 content by ELISA. The values shown are the mean ± SD from three independent experiments. *, p < 0.01, by Student’s t test.

 
Analysis of molecules that affect the MyD88-TRAF6 complex

MyD88 was established as a signal transducer of IL-1beta and TLR signaling pathways. IL-1R, TLRs, IRAK1, and IRAK4 are known to interact with MyD88 and transduce the signal (35, 36, 37). Therefore, we examined the involvement of these molecules in H. pylori-induced cell signaling. When we transfected siRNA for IRAK1 or IRAK4 into AGS cells, H. pylori induced levels of I{kappa}B{alpha} and JNK activation similar to those in control transfected cells (Fig. 6A), which indicates that these kinases are not necessary for H. pylori-induced NF-{kappa}B activation. We also investigated cell surface receptors, such as IL-1R and TLRs. However, siRNAs for IL-1R Acp, TLR4, TLR2, and TLR9 did not affect I{kappa}B{alpha} phosphorylation after H. pylori infection (data not shown). In addition, IL-8 secretion from these cells after 12 h of H. pylori infection was not reduced (Fig. 6B). These results indicate that H. pylori activates NF-{kappa}B via MyD88, albeit independently of IL-1R and TLR.


Figure 6
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  		FIGURE 6. Analysis of MyD88-related proteins in H. pylori-induced epithelial cell signaling. A, AGS cells were transfected with control, IRAK1, or IRAK4 siRNA for 48 h, then infected with H. pylori for the indicated time periods. Immunoblotting was performed with the indicated Abs. B, AGS cells were transfected with the indicated siRNAs, then infected with H. pylori for 12 h. The culture supernatants from three independent experiments were analyzed by ELISA as described in Fig. 5.

 
Assessment of the involvement of Nod1

During the course of our study, Viala et al. (24) described Nod1 as a critical regulator of H. pylori cag PAI-mediated signaling. Therefore, we evaluated the contribution of Nod1 to H. pylori-induced cell signaling. We used siRNAs for Nod1 and RIP2, which is an important kinase that links the Nod protein to the IKK complex (30, 38). When we infected cells with H. pylori TN2, we did not observe a decrease in I{kappa}B{alpha} phosphorylation compared with the control oligonucleotide-transfected cells (Fig. 7A). The levels of Nod1 transcripts were decreased in RT-PCR (data not shown). We also assessed the Nod1-RIP2 interaction by immunoprecipitation analysis. In contrast to the MyD88 and TRAF6 transfection experiment, the HA-Nod1 protein bound to Myc-RIP2 in untreated cells. However, this interaction was not enhanced by H. pylori infection (Fig. 7B). We also measured the levels of IL-8 secretion from these cells (Fig. 7C). H. pylori infection induced ~2500 pg/ml IL-8 in control-transfected AGS cells. Similar levels of IL-8 were observed in Nod1 siRNA-treated cells. H. pylori induced higher levels of IL-8 (3700 pg/ml) in RIP2 siRNA-transfected cells (p < 0.05).


Figure 7
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  		FIGURE 7. Analysis of Nod1 involvement in H. pylori-mediated cell signaling. A, AGS cells were treated with the indicated siRNAs for 48 h, then infected with H. pylori. Immunoblotting was performed as described in Fig. 2. B, AGS cells were transfected with 1 µg of HA-Nod1 and 0.5 µg of Myc-RIP2 vector for 24 h, then infected with H. pylori (TN2). The cell lysates were immunoprecipitated with anti-Myc Ab, and immunoblotting was performed. C, AGS cells were transfected with control, Nod1, or RIP2 siRNAs for 48 h, then infected with H. pylori for 12 h. IL-8 secretion was analyzed by ELISA in three independent experiments. *, p < 0.05, by Student’s t test. D, AGS cells were transfected with the indicated siRNAs, then infected with H. pylori strain 26695. Cell lysates were analyzed with anti-phospho-I{kappa}B{alpha} Ab.

 
Because Nod1-dependent signaling was shown using the H. pylori 26695 strain (24), we used the 26695 strain as another cag PAI-positive H. pylori strain, to exclude strain specificity. We found that strain 26695-induced I{kappa}B{alpha} phosphorylation was severely inhibited by the siRNA for MyD88, but not by the siRNA for Nod1 (Fig. 7D). Thus, I{kappa}B{alpha} phosphorylation induced by H. pylori seems to be independent of Nod1 expression. Collectively, these results suggest that the Nod1-RIP2 pathway is not the principal signaling transducer in H. pylori-infected epithelial cells, at least under our experimental conditions.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Since the discovery of the CagA protein and the cag PAI genes, they have become recognized as important markers of H. pylori virulence (5, 7, 8). Furthermore, strains that carry cag PAI have been revealed to activate various cellular signaling pathways. However, the mechanism by which cag PAI induces intracellular signaling activation has not been fully elucidated. In this report we describe the critical roles of MyD88 and TRAF6 in cag PAI-mediated signal transduction in epithelial cells.

Recently several receptors, such as, TLRs and Nod proteins, have been shown to play a central role in sensing bacterial components in the immune system (reviewed in Refs.35 , 36 , 39 , and 40). However the functionality and the importance of these bacterial receptors depend on cell types, and those in epithelial cells are as yet relatively undefined (41, 42). Therefore, we used several cell lines to assess cag PAI-mediated signaling transduction and found that cag PAI was especially significant in human epithelial cells. Importantly, LPS treatment had negligible effects on NF-{kappa}B activation in these cells. In contrast, in MEFs, the cag mutant H. pylori, as well as LPS activated NF-{kappa}B significantly. This result is similar to that of our previous studies with macrophages or lymphocytes, which revealed cag-independent NF-{kappa}B activation via TLR4 (27). Thus, it seems likely that the cag PAI system is required specifically for NF-{kappa}B activation in cells that are basically unresponsive to bacterial LPS.

Previously, several reports analyzed the signaling molecules involved in H. pylori-mediated NF-{kappa}B activation. These contained IKK complex (15, 16), TRAFs (16), NF-{kappa}B-inducing kinase (NIK) (15, 16), p21-activated kinase 1 (15), and Nod1 (24). Most experiments were based on overexpression of dominant-negative molecules, which sometimes leads to nonphysiological suppression of cell signaling. For example, dominant-negative NIK has been reported to suppress NF-{kappa}B activation induced by IL-1 and TNF-{alpha} (43). However, NIK knockout cells displayed normal NF-{kappa}B activation by IL-1 and TNF-{alpha} (44), indicating that the effect of dominant-negative NIK may not be physiological. Generally, knockout mice or cells derived from knockout mice are good tools for the evaluation of signaling pathways. However, in the H. pylori-infected C57BL/6 mouse model, we did not observe any difference between infection with wild-type (TN2) and cag mutant (TN2-{Delta}cagE) H. pylori strains, because both infected groups developed only mild gastritis (our unpublished observation). This scenario differs significantly from that of the Mongolian gerbil infection model, in which a variety of gastric diseases develops only when the animals are infected with cag-positive H. pylori (45, 46). We also found that MEFs are not useful for investigations of NF-{kappa}B activation mediated by H. pylori, because these cells are highly responsive to cag mutant H. pylori (Fig. 1).

Therefore, we used siRNA methods to examine the key molecules for pathogenesis in human epithelial cells. We found that siRNA for TAK1, TRAF6, and MyD88 significantly reduced H. pylori-induced I{kappa}B{alpha} phosphorylation and IL-8 secretion in human epithelial cells. Furthermore, we found that these molecules form a complex upon cag-positive H. pylori infection by immunoprecipitation analyses. Thus, these molecules seem to play important roles especially in H. pylori-infected human epithelial cell lines. In contrast to these molecules, TRAF2 was not directly involved in H. pylori-mediated NF-{kappa}B activation, because TRAF2 siRNA did not affect I{kappa}B{alpha} phosphorylation. The reason why TRAF2 siRNA slightly reduced IL-8 secretion is not clear (Fig. 5). It is possible that cytokines stimulated by H. pylori infection additively induce IL-8 via TRAF2.

TLRs and IL-1Rs are known to recruit MyD88 to transduce the extracellular signal (35, 36, 37). Although there have been many reports of the role of TLRs in H. pylori infection, this topic remains controversial (47). In our experiments, we could not find any involvement of TLRs or IL-1Rs. These results are consistent with our previous report in which we excluded the major role for TLRs in epithelial cells (27). Therefore, we propose another mechanism of NF-{kappa}B activation via MyD88. In this study, siRNAs for IRAK1 and IRAK4 had no effect on H. pylori-induced NF-{kappa}B activation. Furthermore, we observed degradation of IRAK1 by H. pylori infection only in MEFs (data not shown), not in human epithelial cells (Fig. 6A). Because IRAK1 degradation or IRAK4 kinase activity is required for IL-1R- or TLR4-mediated cell signaling (48, 49, 50, 51), our observation that H. pylori activates NF-{kappa}B without requiring IRAKs suggests a mode of MyD88 activation different from the receptor-mediated mechanism. The fact that cag PAI is a bacterial molecular transporter may support this idea. Thus, we speculate that H. pylori protein is translocated into epithelial cells via cag PAI to bind with MyD88, leading to subsequent activation of NF-{kappa}B in a receptor-independent manner. Because we could not find direct interaction between MyD88 and CagA protein, a reported substrate for the cag PAI secretion system, other bacterial molecules responsible for this phenomenon should be examined in future studies.

We also examined the role of Nod1, because Viala et al. (24) have reported that peptidoglycan from cag PAI-positive strains activates this intracellular protein. However, we could not find any evidence to support the involvement of Nod1 in the present study. Because there are some differences in the experimental conditions, we do not exclude the possibility of Nod1 involvement. It is noteworthy that Nod1-deficient mice have a heightened susceptibility to cag-positive H. pylori, indicating its importance in host defense (24). Our results also showed that RIP2, a downstream kinase that links Nod proteins to the IKK complex (30, 38), did not suppress H. pylori-induced cell responses. On the contrary, RIP2 siRNA increased IL-8 secretion by H. pylori, suggesting the inhibitory role of RIP2 in MyD88-TRAF6 signaling. Therefore, we speculate that these molecules are not the main targets of cag PAI in AGS cells. However, the linkage between the MyD88-TRAF6 pathway and the Nod1 or PAK1-NIK pathway should be investigated in future studies.

In our analysis, epithelial cells and fibroblasts were markedly different in sensing the cag-negative strain and LPS. It is reasonable that cells in bacteria-free interstitial space (such as fibroblasts and monocytes) are more sensitive to the presence of bacterial components than epithelial cells to protect against systemic infection. In contrast, epithelial cells, which are often exposed to bacteria, may respond exclusively to cag-positive virulent strains. Thus, we hypothesize that cag transportation system or its substrate is targeted by the host defense system, including the MyD88-TRAF6 and NF-{kappa}B signaling pathways of epithelial cells. This epithelial defense system may protect the host from virulent H. pylori invasion of the interstitial space by inducing proinflammatory cytokines, and it may protect the epithelial cell itself from cell death by inducing antiapoptotic genes. These responses result in gastritis, and when hyperactivated, they may lead to malignant transformation.

In conclusion, we have revealed that epithelial cells are different from other cell types with respect to responses to wild-type and cag mutant H. pylori. Epithelial cells activate NF-{kappa}B signaling via MyD88-TRAF6 complex formation, but only when infected with cag-positive H. pylori. Thus, these molecules appear to be the critical signal transducers of cag-positive H. pylori infection and important molecules in gastric diseases.


   Acknowledgments
 
We thank Mitsuko Tsubouchi for her excellent technical assistance.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
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 a grant from the Sankyo Foundation of Life Science. Back

2 Address correspondence and reprint requests to Dr. Yoshihiro Hirata, Department of Gastroenterology, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. E-mail address: yohirata-tky{at}umin.ac.jp Back

3 Abbreviations used in this paper: PAI, pathogenicity island; Nod1, nucleotide-binding oligomerization domain 1; TAK1, TGF-beta-activated kinase 1; AcP, accessory protein; IKK, I{kappa}B kinase; NIK, NF-{kappa}B-inducing kinase; HA, hemagglutinin; IRAK, IL-1R-associated kinase; Luc, luciferase; MEF, mouse embryonic fibroblast; RIP, receptor-interacting protein; siRNA, small-interfering RNA; TK, thymidine kinase; TRAF, TNFR-associated factor. Back

Received for publication November 8, 2005. Accepted for publication January 9, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Covacci, A., J. L. Telford, G. Del Giudice, J. Parsonnet, R. Rappuoli. 1999. Helicobacter pylori virulence and genetic geography. Science 284: 1328-1333. [Abstract/Free Full Text]
  2. Peek, R. M., Jr, M. J. Blaser. 2002. Helicobacter pylori and gastrointestinal tract adenocarcinomas. Nat. Rev. Cancer 2: 28-37. [Medline]
  3. Blaser, M. J., D. E. Berg. 2001. Helicobacter pylori genetic diversity and risk of human disease. J. Clin. Invest. 107: 767-773. [Medline]
  4. Correa, P.. 1996. Helicobacter pylori and gastric cancer: state of the art. Cancer Epidemiol. Biomarkers Prev. 5: 477-481. [Medline]
  5. Crabtree, J. E., J. D. Taylor, J. I. Wyatt, R. V. Heatley, T. M. Shallcross, D. S. Tompkins, B. J. Rathbone. 1991. Mucosal IgA recognition of Helicobacter pylori 120 kDa protein, peptic ulceration, and gastric pathology. Lancet 338: 332-335. [Medline]
  6. Atherton, J. C., P. Cao, R. M. Peek, Jr, M. K. Tummuru, M. J. Blaser, T. L. Cover. 1995. Mosaicism in vacuolating cytotoxin alleles of Helicobacter pylori: association of specific vacA types with cytotoxin production and peptic ulceration. J. Biol. Chem. 270: 17771-17777. [Abstract/Free Full Text]
  7. Blaser, M. J., G. I. Perez-Perez, H. Kleanthous, T. L. Cover, R. M. Peek, P. H. Chyou, G. N. Stemmermann, A. Nomura. 1995. Infection with Helicobacter pylori strains possessing cagA is associated with an increased risk of developing adenocarcinoma of the stomach. Cancer Res. 55: 2111-2115. [Abstract/Free Full Text]
  8. Censini, S., C. Lange, Z. Xiang, J. E. Crabtree, P. Ghiara, M. Borodovsky, R. Rappuoli, A. Covacci. 1996. cag, a pathogenicity island of Helicobacter pylori, encodes type I-specific and disease-associated virulence factors. Proc. Natl. Acad. Sci. USA 93: 14648-14653. [Abstract/Free Full Text]
  9. Gerhard, M., N. Lehn, N. Neumayer, T. Boren, R. Rad, W. Schepp, S. Miehlke, M. Classen, C. Prinz. 1999. Clinical relevance of the Helicobacter pylori gene for blood-group antigen-binding adhesin. Proc. Natl. Acad. Sci. USA 96: 12778-12783. [Abstract/Free Full Text]
  10. Yamaoka, Y., S. Kikuchi, H. M. el-Zimaity, O. Gutierrez, M. S. Osato, D. Y. Graham. 2002. Importance of Helicobacter pylori oipA in clinical presentation, gastric inflammation, and mucosal interleukin 8 production. Gastroenterology 123: 414-424.
  11. Peek, R. M., Jr, G. G. Miller, K. T. Tham, G. I. Perez-Perez, X. Zhao, J. C. Atherton, M. J. Blaser. 1995. Heightened inflammatory response and cytokine expression in vivo to cagA+ Helicobacter pylori strains. Lab. Invest. 73: 760-770. [Medline]
  12. Sharma, S. A., M. K. Tummuru, M. J. Blaser, L. D. Kerr. 1998. Activation of IL-8 gene expression by Helicobacter pylori is regulated by transcription factor nuclear factor-{kappa}B in gastric epithelial cells. J. Immunol. 160: 2401-2407. [Abstract/Free Full Text]
  13. Yamaoka, Y., M. Kita, T. Kodama, N. Sawai, N. Imanishi. 1996. Helicobacter pylori cagA gene and expression of cytokine messenger RNA in gastric mucosa. Gastroenterology 110: 1744-1752. [Medline]
  14. Keates, S., A. C. Keates, M. Warny, R. M. Peek, Jr, P. G. Murray, C. P. Kelly. 1999. Differential activation of mitogen-activated protein kinases in AGS gastric epithelial cells by cag+ and cag– Helicobacter pylori. J. Immunol. 163: 5552-5559. [Abstract/Free Full Text]
  15. Foryst-Ludwig, A., M. Naumann. 2000. p21-activated kinase 1 activates the nuclear factor {kappa}B (NF-{kappa}B)-inducing kinase-I{kappa}B kinases NF-{kappa}B pathway and proinflammatory cytokines in Helicobacter pylori infection. J. Biol. Chem. 275: 39779-39785. [Abstract/Free Full Text]
  16. Maeda, S., H. Yoshida, K. Ogura, Y. Mitsuno, Y. Hirata, Y. Yamaji, M. Akanuma, Y. Shiratori, M. Omata. 2000. Helicobacter pylori activates NF-{kappa}B through a signaling pathway involving I{kappa}B kinases, NF-{kappa}B-inducing kinase, TRAF2, and TRAF6 in gastric cancer cells. Gastroenterology 119: 97-108. [Medline]
  17. Karin, M., Y. Ben-Neriah. 2000. Phosphorylation meets ubiquitination: the control of NF-{kappa}B activity. Annu. Rev. Immunol. 18: 621-663. [Medline]
  18. Li, Q., I. M. Verma. 2002. NF-{kappa}B regulation in the immune system. Nat. Rev. Immunol. 2: 725-734. [Medline]
  19. Hayden, M. S., S. Ghosh. 2004. Signaling to NF-{kappa}B. Genes Dev. 18: 2195-2224. [Abstract/Free Full Text]
  20. Barnes, P. J., M. Karin. 1997. Nuclear factor-{kappa}B: a pivotal transcription factor in chronic inflammatory diseases. N. Engl. J. Med. 336: 1066-1071. [Free Full Text]
  21. Richmond, A.. 2002. NF-{kappa}B, chemokine gene transcription and tumour growth. Nat. Rev. Immunol. 2: 664-674. [Medline]
  22. Karin, M., Y. Cao, F. R. Greten, and Z. W. Li. 2002. NF-{kappa}B in cancer: from innocent bystander to major culprit. Nat. Rev. Cancer 2: 301–310.
  23. Sovak, M. A., R. E. Bellas, D. W. Kim, G. J. Zanieski, A. E. Rogers, A. M. Traish, G. E. Sonenshein. 1997. Aberrant nuclear factor-{kappa}B/Rel expression and the pathogenesis of breast cancer. J. Clin. Invest. 100: 2952-2960. [Medline]
  24. Viala, J., C. Chaput, I. G. Boneca, A. Cardona, S. E. Girardin, A. P. Moran, R. Athman, S. Memet, M. R. Huerre, A. J. Coyle, et al 2004. Nod1 responds to peptidoglycan delivered by the Helicobacter pylori cag pathogenicity island. Nat. Immunol. 5: 1166-1174. [Medline]
  25. Brandt, S., T. Kwok, R. Hartig, W. Konig, S. Backert. 2005. NF-{kappa}B activation and potentiation of proinflammatory responses by the Helicobacter pylori CagA protein. Proc. Natl. Acad. Sci. USA 102: 9300-9305. [Abstract/Free Full Text]
  26. Hirata, Y., S. Maeda, Y. Mitsuno, K. Tateishi, A. Yanai, M. Akanuma, H. Yoshida, T. Kawabe, Y. Shiratori, M. Omata. 2002. Helicobacter pylori CagA protein activates serum response element-driven transcription independently of tyrosine phosphorylation. Gastroenterology 123: 1962-1971.
  27. Maeda, S., M. Akanuma, Y. Mitsuno, Y. Hirata, K. Ogura, H. Yoshida, Y. Shiratori, M. Omata. 2001. Distinct mechanism of Helicobacter pylori-mediated NF-{kappa}B activation between gastric cancer cells and monocytic cells. J. Biol. Chem. 276: 44856-44864. [Abstract/Free Full Text]
  28. Takaesu, G., R. M. Surabhi, K. J. Park, J. Ninomiya-Tsuji, K. Matsumoto, R. B. Gaynor. 2003. TAK1 is critical for I{kappa}B kinase-mediated activation of the NF-{kappa}B pathway. J. Mol. Biol. 326: 105-115. [Medline]
  29. Wan, J., L. Sun, J. W. Mendoza, Y. L. Chui, D. P. Huang, Z. J. Chen, N. Suzuki, S. Suzuki, W. C. Yeh, S. Akira, et al 2004. Elucidation of the c-Jun N-terminal kinase pathway mediated by Epstein-Barr virus-encoded latent membrane protein 1. Mol. Cell. Biol. 24: 192-199. [Abstract/Free Full Text]
  30. Girardin, S. E., R. Tournebize, M. Mavris, A. L. Page, X. Li, G. R. Stark, J. Bertin, P. S. DiStefano, M. Yaniv, P. J. Sansonetti, et al 2001. CARD4/Nod1 mediates NF-{kappa}B and JNK activation by invasive Shigella flexneri. EMBO Rep. 2: 736-742. [Medline]
  31. Girardin, S. E., I. G. Boneca, L. A. Carneiro, A. Antignac, M. Jehanno, J. Viala, K. Tedin, M. K. Taha, A. Labigne, U. Zahringer, et al 2003. Nod1 detects a unique muropeptide from Gram-negative bacterial peptidoglycan. Science 300: 1584-1587. [Abstract/Free Full Text]
  32. Keates, S., Y. S. Hitti, M. Upton, C. P. Kelly. 1997. Helicobacter pylori infection activates NF-{kappa}B in gastric epithelial cells. Gastroenterology 113: 1099-1109. [Medline]
  33. Ninomiya-Tsuji, J., K. Kishimoto, A. Hiyama, J. Inoue, Z. Cao, K. Matsumoto. 1999. The kinase TAK1 can activate the NIK-I {kappa}B as well as the MAP kinase cascade in the IL-1 signalling pathway. Nature 398: 252-256. [Medline]
  34. Sun, L. L. Deng, C. K. Ea, Z. P. Xia, and Z. J. Chen. 2004. The TRAF6 ubiquitin ligase and TAK1 kinase mediate IKK activation by BCL10 and MALT1 in T lymphocytes. Mol. Cell 14: 289–301.
  35. Takeda, K., S. Akira. 2004. TLR signaling pathways. Semin. Immunol. 16: 3-9. [Medline]
  36. Akira, S., K. Takeda. 2004. Toll-like receptor signalling. Nat. Rev. Immunol. 4: 499-511. [Medline]
  37. Muzio, M., J. Ni, P. Feng, V. M. Dixit. 1997. IRAK (Pelle) family member IRAK-2 and MyD88 as proximal mediators of IL-1 signaling. Science 278: 1612-1615. [Abstract/Free Full Text]
  38. Inohara, N., T. Koseki, J. Lin, L. del Peso, P. C. Lucas, F. F. Chen, Y. Ogura, G. Nunez. 2000. An induced proximity model for NF-{kappa}B activation in the Nod1/RICK and RIP signaling pathways. J. Biol. Chem. 275: 27823-27831. [Abstract/Free Full Text]
  39. Medzhitov, R.. 2001. Toll-like receptors and innate immunity. Nat. Rev. Immunol. 1: 135-145. [Medline]
  40. Inohara, N., M. Chamaillard, C. McDonald, G. Nunez. 2005. NOD-LRR proteins: role in host-microbial interactions and inflammatory disease. Annu. Rev. Biochem. 74: 355-383. [Medline]
  41. Cario, E., I. M. Rosenberg, S. L. Brandwein, P. L. Beck, H. C. Reinecker, D. K. Podolsky. 2000. Lipopolysaccharide activates distinct signaling pathways in intestinal epithelial cell lines expressing Toll-like receptors. J. Immunol. 164: 966-972. [Abstract/Free Full Text]
  42. Melmed, G., L. S. Thomas, N. Lee, S. Y. Tesfay, K. Lukasek, K. S. Michelsen, Y. Zhou, B. Hu, M. Arditi, M. T. Abreu. 2003. Human intestinal epithelial cells are broadly unresponsive to Toll-like receptor 2-dependent bacterial ligands: implications for host-microbial interactions in the gut. J. Immunol. 170: 1406-1415. [Abstract/Free Full Text]
  43. Malinin, N. L., M. P. Boldin, A. V. Kovalenko, D. Wallach. 1997. MAP3K-related kinase involved in NF-{kappa}B induction by TNF, CD95 and IL-1. Nature 385: 540-544. [Medline]
  44. Yin, L., L. Wu, H. Wesche, C. D. Arthur, J. M. White, D. V. Goeddel, R. D. Schreiber. 2001. Defective lymphotoxin-beta receptor-induced NF-{kappa}B transcriptional activity in NIK-deficient mice. Science 291: 2162-2165. [Abstract/Free Full Text]
  45. Ogura, K., S. Maeda, M. Nakao, T. Watanabe, M. Tada, T. Kyutoku, H. Yoshida, Y. Shiratori, M. Omata. 2000. Virulence factors of Helicobacter pylori responsible for gastric diseases in Mongolian gerbil. J. Exp. Med. 192: 1601-1610. [Abstract/Free Full Text]
  46. Israel, D. A., N. Salama, C. N. Arnold, S. F. Moss, T. Ando, H. P. Wirth, K. T. Tham, M. Camorlinga, M. J. Blaser, S. Falkow, et al 2001. Helicobacter pylori strain-specific differences in genetic content, identified by microarray, influence host inflammatory responses. J. Clin. Invest. 107: 611-620. [Medline]
  47. Ferrero, R. L.. 2005. Innate immune recognition of the extracellular mucosal pathogen, Helicobacter pylori. Mol. Immunol. 42: 879-885. [Medline]
  48. Yamin, T. T., D. K. Miller. 1997. The interleukin-1 receptor-associated kinase is degraded by proteasomes following its phosphorylation. J. Biol. Chem. 272: 21540-21547. [Abstract/Free Full Text]
  49. Swantek, J. L., M. F. Tsen, M. H. Cobb, J. A. Thomas. 2000. IL-1 receptor-associated kinase modulates host responsiveness to endotoxin. J. Immunol. 164: 4301-4306. [Abstract/Free Full Text]
  50. Suzuki, N., S. Suzuki, G. S. Duncan, D. G. Millar, T. Wada, C. Mirtsos, H. Takada, A. Wakeham, A. Itie, S. Li, et al 2002. Severe impairment of interleukin-1 and Toll-like receptor signalling in mice lacking IRAK-4. Nature 416: 750-756. [Medline]
  51. Janssens, S., R. Beyaert. 2003. Functional diversity and regulation of different interleukin-1 receptor-associated kinase (IRAK) family members. Mol. Cell 11: 293-302. [Medline]



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