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Previous Article | Next Article 
Infection and Immunity, March 2005, p. 1523-1531, Vol. 73, No. 3
0019-9567/05/$08.00+0 doi:10.1128/IAI.73.3.1523-1531.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
and
Joanna B. Goldberg1*,
Department of Microbiology, University of Virginia Health Sciences Center, Charlottesville, Virginia1
Received 20 September 2004/ Returned for modification 27 October 2004/ Accepted 17 November 2004
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ABSTRACT |
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B. Here, we evaluated the contribution of mitogen-activated protein kinase signaling pathways to TLR2-dependent and TLR5-dependent secretion of IL-8. Secretion of IL-8 from H. pylori-infected HEK293 cells was augmented by the expression of TLR2 or TLR5. While H. pylori infection resulted in the activation of ERK, JNK, and p38, the enhanced IL-8 secretion from TLR2- and TLR5-expressing cells coincided with increased p38 activation and phosphorylation of the transcription factor ATF2. When p38 activity was inhibited in TLR2- or TLR5-expressing cells, H. pylori-dependent IL-8 secretion returned to the level observed in infected parental HEK293 cells that did not express TLR2 or TLR5; inhibition of p38 had no effect on IL-8 secretion from infected parental HEK cells. In contrast, inhibition of JNK and/or ERK resulted in substantially less IL-8 secretion from infected cells, independent of TLR2 or TLR5 expression. Based on these data, we propose that H. pylori induces IL-8 secretion through a dual mechanism that includes a TLR2/5-independent component involving the activities of JNK and ERK and a TLR2/5-dependent component that requires p38 activity. |
INTRODUCTION |
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Infection by Helicobacter pylori can cause gastritis and is also highly associated with gastric ulcers, gastric adenocarcinomas, and mucosa-associated lymphoid tissue lymphomas (6, 14). Upon H. pylori infection, gastric epithelial cells respond to H. pylori by activating many signaling cascades. These lead to cytokine and chemokine secretion, which recruit innate and adaptive immune cells to the site of infection. Despite a vigorous host immune response, H. pylori infection is persistent and can be lifelong without medical intervention.
Interleukin-8 (IL-8) is an important chemokine in mediating the inflammatory response to H. pylori. Aihara et al. demonstrated that both the NF-B and activating protein 1 (AP-1) DNA binding sites within the IL-8 promoter are required for optimal transcription in response to infection of gastric epithelial cells by H. pylori (3). During H. pylori infections, both NF-B and members of the mitogen-activated protein kinase (MAPK) family become activated (11, 12, 15, 18). Activated MAPKs then phosphorylate AP-1 complexes, which results in increased AP-1-dependent transcription. As such, signaling pathways that activate NF-B and/or AP-1 could result in increased IL-8 secretion.
In an in vivo H. pylori infection, gastric epithelial cells may be the first cells to induce innate immune signaling pathways. These cells can express TLR2 and TLR5, among other TLRs (11). Our previous work demonstrated that H. pylori lipopolysaccharide (LPS) and flagellin are TLR2 and TLR5 agonists, respectively, and that expression of TLR2 or TLR5 results in enhanced NF-B activation upon in vitro H. pylori infection of gastric epithelial cells. We also noted variability in TLR expression within gastric epithelial cell lines. In addition, IL-8 mRNA levels were found to be elevated in TLR2-expressing epithelial cells upon H. pylori infection (20). Based on these initial findings, we hypothesized that, in addition to activating NF-B, TLRs might be important for increased IL-8 secretion from H. pylori-infected cells through their ability to enhance AP-1 activation. Here, we evaluated the contribution of TLR2 and TLR5 expression to IL-8 secretion from H. pylori-infected cells, using HEK293 cells or the same cells expressing TLR2 or TLR5 as a model system, and tested the role of MAPK signaling pathways in the TLR-mediated responses to H. pylori infection.
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MATERIALS AND METHODS |
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Cell and H. pylori culture. Human embryonic kidney cells of the HEK293 line (HEK) were obtained from American Type Culture Collection (Manassas, Va.). HEK cells were routinely cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Gibco, Carlsbad, Calif.) and 1x penicillin-streptomycin (Gibco). HEK293 cells stably transfected with human TLR2, (HEK-hTLR2; TLR2) or with human TLR5 (HEK-hTLR5; TLR5) were purchased from InvivoGen. TLR2 and TLR5 cells were cultured in DMEM supplemented with 10% FBS, 1% penicillin-streptomycin, and 10 of blasticidin (InvivoGen)/ml at 37°C in 7.5% CO2.
H. pylori strain 26695 was routinely grown on sheep blood agar plates (BAP) (Remel, Lenexa, Kans.) in 10% CO2 at 37°C. For in vitro infections, H. pylori was cultured for 24 h on BAP, harvested with a sterile cotton swab, and resuspended in 1 ml of brucella broth per plate. The bacteria were pelleted at 3,000 x g for 5 min and then resuspended in 5 ml of brucella broth. This wash procedure was repeated, and the bacteria were resuspended in 2 ml of brucella broth. The number of viable bacteria was determined by serial dilution of the bacterial suspension, plating onto BAP, and incubation for 72 h at 37°C.
In vitro infections. For biochemical assays, 6 x 106 epithelial cells were plated in 60-mm-diameter dishes in normal growth medium. The next day, the cells were washed with phosphate-buffered saline, pH 7.4, and subsequently serum starved in serum-free DMEM for 4 h without antibiotics. The medium was then exchanged for fresh DMEM containing 2% FBS. The eukaryotic cells were then stimulated with either 100 ng of EGF/ml for 10 min, 10 µg of anisomycin/ml for 1 h, or H. pylori at a multiplicity of infection (MOI) of approximately 100:1 for 1 h. Following treatment, the cells were washed with cold DMEM and then lysed on ice with 1 ml of cold lysis buffer (50 mM Tris, pH 7.3, 150 mM NaCl, 1% Triton X-100, 0.5 mM EDTA, 0.5 mM EGTA, 10% glycerol) supplemented with protease and phosphatase inhibitors (100 µM leupeptin, 1 mM phenylmethylsulfonyl fluoride, 0.15 U of aprotinin/ml, and 1 mM sodium orthovanadate).
For enzyme-linked immunosorbent assays (ELISAs), 3 x 105 cells/well were plated into individual wells of a 24-well dish in normal growth medium without antibiotics. The following day, cells were treated in triplicate with Pam3CSK4, S. enterica serovar Typhimurium flagellin, or H. pylori at an MOI of 100:1. Twenty-four-hour culture supernatants were collected, clarified by centrifugation, and evaluated for IL-8 secretion by ELISA, as described below.
MAPK inhibitor studies. The kinase inhibitor studies were performed with the inhibitors SB202190 (p38 inhibitor), SP600125 (JNK inhibitor), and U0126 (MEK1/2 inhibitor) at a final concentration of 10 µM in dimethyl sulfoxide (DMSO) (Sigma-Aldrich). Cells were treated with the inhibitor(s) or DMSO for 1 h at 37°C prior to infection or treatment.
Immunoprecipitation and immunoblotting. Immunoprecipitations were performed by incubating 1 mg of cell lysate with 5 µl of anti-human TLR2, 5 µl of anti-human TLR5, or 4 µl of anti-ATF2 overnight at 4°C with rotation. The immune complexes were recovered by incubation with 60 µl of protein A-Sepharose bead slurry for a further 4 h at 4°C. The immunoprecipitates were washed twice in cold lysis buffer and twice in cold Tris-buffered saline (150 mM NaCl, 50 mM Tris, pH 7.5). The immune complexes were then resuspended in 2x Laemmli sample buffer and boiled for 10 min.
For immunoblots, cell lysates were boiled in Laemmli sample buffer for 10 min. Cell lysates and immunoprecipitates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; 10% polyacrylamide) and transferred to nitrocellulose. The membranes were blocked following manufacturer's recommendations. Membranes were incubated overnight with primary antibodies at 4°C as indicated followed by secondary anti-rabbit IgG or anti-mouse IgG conjugated to HRP (1:1,000) for 1 h at room temperature. Immunoreactive bands were detected by ECL and radiography. Films in the linear range of detection were used to analyze the relative activation of the MAPKs or ATF2. Bands were quantified by densitometry and normalized for total protein with a Molecular Dynamics densitometer and ImageQuant 5.0 software (Amersham Biosciences).
IL-8 ELISA. Clarified culture supernatants from 24-h infections or treatments were diluted 1:2 to 1:10, as needed. Supernatants were analyzed for IL-8 protein with a Quantikine human IL-8 ELISA kit (R&D Systems, Minneapolis, Minn.), as directed by the manufacturer.
Statistical analyses. All ELISA experiments were performed in triplicate and were repeated a minimum of three times. Data from the repeated experiments were pooled, and the natural log of the values was taken. The data set was analyzed for statistical significance by analysis of variance (ANOVA). P values reported are derived from the combined data from all repetitions of each experiment.
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RESULTS |
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B activation upon infection by H. pylori (20). In light of these data and the fact that IL-8 mRNA levels were also shown to be elevated in TLR2-expressing cells, we hypothesized that TLR expression might result in enhanced IL-8 secretion from infected cells. To test this hypothesis, we took advantage of HEK293 cell lines that stably express TLR2 or TLR5 and HEK293 cells that do not express these receptors (Fig. 1A). To assess the contribution of TLR2 or TLR5 expression to IL-8 secretion, we measured the IL-8 protein levels of culture supernatants from 24-h H. pylori infections of HEK, TLR2, or TLR5 cells by ELISA. As expected, HEK cells were observed to secrete IL-8 in response to H. pylori infection (P 
0.0001) (Fig. 1B). The level of IL-8 secreted from infected TLR2 or TLR5 cells was significantly higher than that secreted by parental HEK cells (P 0.0001).
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TLR2- and TLR5-expressing cells show increased p38 activation in response to infection by H. pylori.
Since both the AP-1 and NF-B sites are required for optimal expression of the IL-8 gene following H. pylori infection (3) and NF-B activation was enhanced by TLR2 or TLR5 expression (20), we hypothesized that TLR2 or TLR5 expression also might increase MAPK signaling upstream of AP-1 and thus contribute to the enhanced IL-8 secretion. To investigate the effect of TLR2 or TLR5 expression on the H. pylori-dependent activation of MAPK family members, we measured activation of the MAPKs in cell lysates from infected HEK, TLR2, and TLR5 cells using phosphorylation-specific antibodies. In HEK cells, H. pylori infection induced the activation of p38, JNK, and ERK (Fig. 2A, upper panels, compare lanes 3 and 4). Infected TLR2 cells also showed an H. pylori-dependent activation of all three MAPKs (upper panels, compare lanes 7 and 8). However, while JNK and ERK were activated to equivalent levels in HEK and TLR2 cells, p38 activation in response to H. pylori infection was consistently fourfold to sixfold greater in TLR2 than in HEK cells, as determined by densitometry (upper panel, compare lanes 4 and 8). All three MAPK family members responded equivalently to conventional agonists (EGF for ERK and anisomycin for JNK and p38; compare lanes 1 and 2 with lanes 5 and 6), indicating that there are no intrinsic differences between the HEK and TLR2 cell lines in their ability to activate the MAPKs.
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p38 plays a key role in TLR2- and TLR5-dependent secretion of IL-8 from H. pylori-infected cells.
The importance of p38 activity on H. pylori-induced IL-8 secretion was tested with the selective p38 inhibitor SB202190. This inhibitor had no effect on H. pylori-dependent IL-8 secretion in HEK cells (Fig. 3A, black bar). However, treatment of TLR2 or TLR5 cells with SB202190 significantly reduced IL-8 secretion in response to H. pylori infection to the level observed in infected HEK cells (P 0.02). This suggests that the TLR2- and TLR5-dependent augmentation in IL-8 secretion following H. pylori infection is dependent on p38 activity. However, p38 does not appear to be essential for the induction of IL-8 by HEK cells following H. pylori infection, nor is it required to elicit the residual IL-8 response in infected TLR2 and TLR5 cells that remains following treatment with SB202190. The activity and specificity of this inhibitor were verified by examining the phosphorylation state of ATF2 to measure p38 activity, c-Jun to measure JNK activity, and Elk-1 to measure ERK activity in EGF- or anisomycin-treated cells following a 24-h incubation with SB202190. Whereas p38 activity was reduced in all three cell lines in the presence of the inhibitor (Fig. 3B, top panel), the kinase activities of JNK and ERK were not affected (middle and bottom panels).
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0.0001 and P 0.005, respectively). Moreover, the augmentation of IL-8 production observed in TLR2- and TLR5-infected cells was ablated in the presence of the JNK and MEK inhibitors. The specificity of the inhibitors was confirmed (Fig. 4C and D) as described above. These data indicate that JNK and ERK play key roles in H. pylori-dependent secretion of IL-8 from epithelial cells and that the engagement of TLR2 or TLR5 by H. pylori does not overcome the requirement for JNK or ERK in the production of IL-8 in response to H. pylori infection.
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0.05; Fig. 5, compare hatched bars to black and gray bars). The greater inhibition observed in cells treated with both inhibitors suggests that JNK and ERK appear to each play a critical role in promoting IL-8 secretion from H. pylori-infected cells. However, treatment with both inhibitors did not fully reduce H. pylori-dependent IL-8 secretion to the levels observed for uninfected cells. The residual IL-8 secretion observed under these conditions may indicate that there is a JNK/ERK-independent pathway that remains active in the presence of both inhibitors or that the inhibitors did not fully block these signaling pathways under the conditions of the assay.
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While phosphorylation of ATF2 in response to anisomycin was readily apparent in all three cell lines (Fig. 6, lanes 1, 4, and 7, top panels and slower-migrating species in bottom panels), there was no detectable phosphorylation of ATF2 in response to H. pylori infection of HEK cells (compare lanes 2 and 3). In contrast, a slight but reproducible H. pylori-dependent increase in ATF2 phosphorylation was evident in TLR2 and TLR5 cells (compare lanes 6 and 9 with lanes 5 and 8). These data suggest that the elevated p38 activation that is evident in H. pylori-infected TLR2- or TLR5-expressing cells might lead to ATF2 phosphorylation, which could then result in more activated AP-1 complexes and increased IL-8 secretion from these cells.
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DISCUSSION |
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In addition to the in vitro experiments discussed above, two studies support a role for p38 in H. pylori-induced inflammation in animal models of infection and inflammation. Slomiany et al. reported that intragastric application of H. pylori LPS induced a strong inflammatory response and mucosal ulceration in rats; however, inflammation and ulceration were reduced more than 60% when the rats were given H. pylori LPS and a p38 inhibitor. This was in contrast to treatment with an ERK inhibitor, which resulted in a more modest reduction in inflammation (19). This suggests that p38 may play a more significant role than ERK in H. pylori LPS-induced inflammation in animals. Since H. pylori LPS is a TLR2 agonist (20), we would propose from our data that the p38-mediated inflammation observed in the Slomiany study could be due to TLR2 signaling through p38. In the second study, Takahashi et al. reported that administration of a p38 inhibitor to Mongolian gerbils markedly reduced IL-8 and other chemokine secretion, neutrophil infiltration, and mucosal injury observed in the H. pylori-infected animals (22). Since H. pylori disease pathology is caused by inflammation, any factor enhancing inflammation contributes to H. pylori disease. As such, our data and the results of Slominay and Takahashi suggest that TLR-dependent activation of p38 results in more severe disease in H. pylori infection.
Our data suggest that one of the possible mechanisms by which increased p38 activation in TLR2- and TLR5-expressing cells might result in elevated IL-8 production is through increased phosphorylation of ATF2. ATF2 phosphorylation can also be mediated through JNK and phosphatidlyinositol 3-kinase (PI 3-kinase) pathways. Since both of these molecules can be activated in response to H. pylori infection (16, 21), they may also contribute to ATF2 phosphorylation and activity. However, JNK activation by H. pylori was not found to be enhanced in TLR2 or TLR5 cells (Fig. 2A and B), and PI3 kinase activity, as measured by phosphorylation of AKT, was similarly unaffected by TLR2 or TLR5 expression (data not shown). Thus, we propose that ATF2 becomes phosphorylated in H. pylori-infected TLR2 and TLR5 cells through the activity of p38 and not by the JNK or PI 3-kinase pathways.
We demonstrated that H. pylori infection stimulated ATF2 phosphorylation in TLR2 and TLR5, but not HEK cells. The lack of phosphorylated ATF2 in infected HEK cells was unexpected, since p38 was activated in those cells. It is possible that H. pylori activation of the TLR2 or TLR5 pathways may provide specificity for p38-dependent phosphorylation of ATF2. Alternatively, there may be a threshold of p38 activation that is required in order to promote phosphorylation of ATF2. Finally, ATF2 may be phosphorylated in HEK cells in response to H. pylori infection, but the amount may be below the level of detection. This possibility is currently being tested with more sensitive chromatin immunoprecipitation assays.
Previously, Naumann et al. and Meyer-ter-Vehn et al. reported that c-Jun and c-Fos, but not ATF2, were components of the AP-1 dimer that were induced in AGS cells by H. pylori infection (12, 15). On the other hand, Yamada et al. reported p38-dependent phosphorylation of ATF2 in MKN cells infected by H. pylori (25). These differing results can potentially be reconciled by our findings and those we reported previously (20). Since AGS cells do not contain detectable TLR2 mRNA (20), these cells may not undergo a hyperactivation of p38 in response to H. pylori and thus would not be expected to contain phosphorylated ATF2. In contrast, the MKN cells used by Yamada et al. and the TLR2 cells used here both express TLR2 mRNA (20) and as such promote TLR2-dependent p38 phosphorylation of ATF2 in H. pylori-infected cells.
We propose that phosphorylation of ATF2 by p38 in TLR2 and TLR5 cells may contribute to the enhanced IL-8 secretion observed upon H. pylori infection (Fig. 7B). Exactly how this occurs is not currently known. We suggest that increased levels of phosphorylated ATF2 could lead to larger pools of activated AP-1 complexes in TLR-expressing cells (ATF2/ATF2, ATF2/c-Jun, and c-Fos/c-Jun). This could result in increased AP-1 occupancy at the IL-8 promoter and increased IL-8 transcription in these cells (Fig. 7B, bold and dashed arrows). Alternatively, there could be a qualitative effect of ATF2-containing AP-1 complexes on IL-8 transcription. For example, ATF2/ATF2 or ATF2/c-Jun could replace c-Fos/c-Jun complexes at the IL-8 promoter. These ATF2-containing AP-1 complexes could have greater transactivation potential than the c-Fos/c-Jun AP-1 complexes, thus resulting in increased IL-8 transcription. These possibilities are currently being tested with chromatin immunoprecipitation assays.
Another possible mechanism by which p38 might enhance IL-8 secretion is by increasing NF-B transcriptional activity. Carter et al. and Goebeler et al. reported that p38 is able to regulate the transcriptional activity of NF-B at the monocyte chemoattractant protein 1 promoter. In these studies, p38 activity was found to enhance NF-B transcriptional activity by modulating the assembly and activity of the transcriptional complex. Pharmacological inhibition of p38 did not affect the activation of NF-B, but instead inhibited the recruitment of TATA-binding protein and blocked the enhancing effect of the transcriptional coactivator, p300 (5, 7).
Finally, it is possible that the enhanced p38 activation and phosphorylation of ATF2 that are observed in TLR2 and TLR5 cells following H. pylori infection could result in transcription at other promoters whose products could then stimulate IL-8 transcription and/or mRNA stability. For example, MAPKAP K2 (MK2), which can be phosphorylated by p38, has been shown to extend the half-life of IL-8 mRNAs by binding to AU-rich elements in IL-8 mRNA (24). Although the role of MK2 in H. pylori infection has not been assessed, it is possible that TLR2/5-dependent activation of p38 may lead to increased IL-8 secretion via MK2 stabilization of IL-8 mRNA. One argument against this possibility is that Yu et al. reported that IL-8 mRNA transcription was only slightly reduced and the half-life of IL-8 mRNA was unaffected by inhibition of p38 in flagellin-stimulated cells, even though IL-8 secretion was significantly decreased; the mechanism responsible for these observations was not proposed (26).
While it is clear that H. pylori can induce inflammation, most H. pylori infections of humans are chronic and can be lifelong without medical intervention. A pertinent question is why is the immune response ineffective against H. pylori? Why can other bacterial infections, such as those caused by Salmonella, be cleared by the immune response but those caused by H. pylori cannot? By studying the signaling pathways activated in H. pylori infection of human cells, it may be possible to identify bacterial virulence factors and/or host molecules or pathways unique to H. pylori infection that induce a host response that is inflammatory but functionally ineffective.
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ACKNOWLEDGMENTS |
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We would like to thank our colleagues in the lab for helpful discussions and Mark Smolkin for advice on the statistical analyses.
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FOOTNOTES |
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A.H.B. and J.B.G. contributed equally to this work.
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