TLR4-Dependent NF-κB Activation and Mitogen- and Stress-Activated Protein Kinase 1-Triggered Phosphorylation Events Are Central to Helicobacter pylori Peptidyl Prolyl This information is current as cis-, trans-Isomerase (HP0175)-Mediated of June 26, 2015. Induction of IL-6 Release from Sushil Kumar Pathak, Sanchita Basu, Asima Bhattacharyya, Shresh Pathak, Anirban Banerjee, Joyoti Basu and Manikuntala Kundu Downloaded from J Immunol 2006; 177:7950-7958; ; doi: 10.4049/jimmunol.177.11.7950 http://www.jimmunol.org/content/177/11/7950 http://www.jimmunol.org/ References This article cites 46 articles, 24 of which you can access for free at: http://www.jimmunol.org/content/177/11/7950.full#ref-list-1 Subscriptions Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscriptions Permissions Submit copyright permission requests at:

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 9650 Rockville Pike, Bethesda, MD 20814-3994. Copyright © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

TLR4-Dependent NF-␬B Activation and Mitogen- and Stress-Activated Protein Kinase 1-Triggered Phosphorylation Events Are Central to Helicobacter pylori Peptidyl Prolyl cis-, trans-Isomerase (HP0175)-Mediated Induction of IL-6 Release from Macrophages1

Sushil Kumar Pathak, Sanchita Basu, Asima Bhattacharyya, Shresh Pathak, Anirban Banerjee, Joyoti Basu, and Manikuntala Kundu2

Helicobacter pylori infection is associated with the local production of chemokines and , of which IL-6 is overexpressed at the margin of gastric ulcer in H. pylori-positive gastritis. Cells of the monocytic lineage are the major sources of IL-6, and Downloaded from mononuclear cell infiltration in the lamina propria is characteristic of H. pylori-induced chronic infection. Our study shows for the first time that a secreted peptidyl prolyl cis-, trans-isomerase, HP0175 elicits IL-6 gene expression and IL-6 release from macro- phages. An isogenic strain inactivated in the HP0175 gene (knockout) was attenuated in its IL-6-inducing ability, which was restored after complementation with the HP0175 gene. The specificity of the HP0175-induced effect was confirmed by the fact that rHP0175 purified from HEK293 cells could also induce IL-6 release, ruling out the possibility that the observed effect was due to

bacterial contaminants. HP0175 was capable of interacting directly with the extracellular domain of TLR4. HP0175-induced IL-6 http://www.jimmunol.org/ gene expression was critically dependent on TLR4-dependent NF-␬B and MAPK activation. TLR4/PI3K-dependent ERK1/2 and p38 MAPK signaling converged upon activation of mitogen- and stress-activated protein kinase 1 (MSK1). The central role of MSK1 was borne out by the fact that silencing of MSK1 expression abrogated HP0175-mediated NF-␬B-dependent IL-6 gene transcription. MSK1 regulated the recruitment of p65 and phopho-Ser10-histone H3 to the IL-6 promoter. HP0175 therefore regulated IL-6 gene transcription through chromatin modification at the IL-6 promoter. The Journal of Immunology, 2006, 177: 7950–7958.

elicobacter pylori is a Gram-negative microaerophilic in stimulation of signaling pathways widely involving recruitment bacterium that causes chronic gastritis and also peptic of various adaptor molecules such as MyD88 (11–13), followed by by guest on June 26, 2015 H ulcer, gastric carcinoma, and gastric lymphoma (1). In- the serine/threonine kinase IL-1R-associated kinase 1 (IRAK1)3 fection is associated with the local production of chemokines and (14). IRAK1 becomes phosphorylated, dissociates from the com- cytokines, such as IL-1␤, IL-6, and IL-8 (2, 3). IL-6 is a pleiotropic plex, and associates with TNFR-associated factor 6 (15, 16), fi- with both pro- and anti-inflammatory properties (4) that is nally leading to the activation of MAPKs, transcription factors overexpressed at the margin of gastric ulcer in H. pylori-positive such as NF-␬B, and concomitant production of cytokines (17, 18). gastritis (5, 6). Its levels are high in H. pylori-infected early gastric The role of TLRs in the response of macrophages during H. pylori cancer and fall significantly after the cure of H. pylori (7). Con- infection has not been studied extensively. TLR4 expression has sidering that activated macrophages are the main sources of IL-6, been reported to be up-regulated in gastric biopsies obtained from it is necessary to understand the effectors of H. pylori driving IL-6 H. pylori-positive patients compared with uninfected controls (19). gene induction in macrophages. Inflammation-associated factors, H. pylori has a large repertoire of secreted proteins, including such as TNF-␣, platelet-derived growth factor, and bacterial en- the best studied virulence factors VacA and CagA. However, the dotoxins, all enhance IL-6 gene expression. clinical outcome of the disease does not necessarily correlate with TLRs play central roles in innate immunity by recognition and the absence or presence of VacA or CagA, making it important to discrimination of specific conserved patterns of molecules derived identify other factors that could modulate the clinical course of the from bacteria, fungi, or viruses (8–10). Activation of TLRs results disease. Till date, urease, and heat shock protein 60 of H. pylori have been reported to induce IL-6 production in macrophages (20, 21). By two-dimensional gel electrophoresis, followed by mass Department of Chemistry, Bose Institute, , India spectrometric analysis, Kim et al. (22) have demonstrated the Received for publication January 17, 2006. Accepted for publication September secretion of HP0175, a peptidyl prolyl cis-, trans-isomerase (PPIase), 6, 2006. in the supernatant when H. pylori is grown in vitro. HP0175 is one 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 of the highly and consistently reactive Ags recognized by the sera with 18 U.S.C. Section 1734 solely to indicate this fact. of H. pylori-infected patients (23, 24). It would therefore not be 1 This work was supported in part by grants from the Indian Council of Medical Research and the Department of Atomic Energy (to M.K.). S.K.P. was supported by a fellowship from the Council of Scientific and Industrial Research. 3 Abbreviations used in this paper: IRAK1, IL-1R-associated kinase 1; ChIP, chro- 2 Address correspondence and reprint requests to Dr. Manikuntala Kundu, Depart- matin immunoprecipitation; dn, dominant-negative; ECD, extracellular domain; KO, ment of Chemistry, Bose Institute, 93/1 Acharya Prafulla, Chandra Road, Kolkata knockout; MSK1, mitogen- and stress-activated protein kinase 1; PPIase, peptidyl 700009, India. E-mail address: [email protected] prolyl cis-, trans-isomerase; siRNA, small interfering RNA.

Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00 The Journal of Immunology 7951

unlikely for this Ag to have a role in the pathogenesis of lease of IL-6 from THP-1 cells. For the expression of His-HP0175 in H. pylori-associated disease. Our previous studies have identified HEK293 cells, the gene encoding HP0175 was cloned between the XbaI HP0175 as a TLR4-interacting protein (25). Because TLR4 is one and EcoRI sites of pcDNA myc-His, followed by transfection in HEK293 cells. HP0175 was purified from the cell lysate by chromatography on of the best studied receptors driving the innate , Ni2ϩ-NTA agarose. we reasoned that it would be worthwhile to explore the effects of HP0175 on cytokine induction. The study described Complementation of HP0175 in this work provides evidence that H. pylori HP0175 induces the HP0175 was amplified by PCR using the primer pairs 5Ј-GGGGTACCATG release of IL-6 from human macrophages in a TLR4-/MAPK- AAAAAAAATATCTTAAA-3Ј (sense) and 5Ј-GAAGATCTTTACTTGT dependent manner by activating NF-␬B-driven IL-6 gene tran- TGATAACAATT-3Ј (antisense). The resulting PCR product was cloned scription. The contribution of HP0175 in the release of IL-6 was between the KpnI and BglII sites of the shuttle vector pHel2 (a gift from R. Haas, Max von Pettenkofer-Institut fu¨r Hygiene und Medizinische Mikro- confirmed by the observation that an isogenic mutant of H. pylori biologie, Munich, Germany) (28). The shuttle plasmid was introduced into 26695 disrupted in the HP0175 gene was impaired in its ability to the knockout (KO) strain (described in Ref. 25) by natural transformation. release IL-6. Immunodepletion of HP0175 from the aqueous ex- Colonies were selected on plates containing 4 ␮g/ml chloramphenicol ␮ tract of H. pylori also led to the inhibition of IL-6-releasing ability and 10 g/ml kanamycin. and further confirmed the novel role of this protein in the induction Cell culture of cytokine production from macrophages. Chromatin remodeling represents a determining factor control- THP-1 and HEK293 cells were obtained from the National Centre for Cell Science (Pune, India). THP-1 was maintained in RPMI 1640 medium and ling binding of transcription factors and the formation of preini- treated with PMA to induce maturation of the monocytes to a macrophage- tiation complexes. It therefore dictates selective induction of sub- like adherent phenotype, as described (27). Blood was drawn from healthy

sets of genes. Our studies present evidence of HP0175 triggering adult volunteers, and PBMC were isolated, as described (27). Downloaded from mitogen- and stress-activated kinase 1 (MSK1)-dependent phos- For treatments with bacteria, H. pylori strains were grown, as described (25), and incubated with PMA-treated THP-1 cells at a multiplicity of phorylation of p65 (RelA) and histone H3. MSK1-mediated chro- infection 50–100 on culture plates. HEK293 cells were grown in MEM matin modifications most likely play a central role in the HP0175- supplemented with 10% FBS; 6 ϫ 105 cells/well were transfected with dependent transcriptional activity of NF-␬B and subsequent Polyfect complex, according to the manufacturer’s instructions. For THP-1 release of IL-6. cells (4 ϫ 105/well), transfection was conducted with 2 ␮g of plasmid (empty vector or recombinant) using Fugene 6 (Roche), according to the http://www.jimmunol.org/ Materials and Methods manufacturer’s protocol. Reagents Enzyme-linked immunoassay for IL-6 Wortmannin, SB203580, U0126, and PMA were products of EMD Bio- Transfected or untransfected cells were either left untreated or treated with sciences. Anti-FLAG Ab, polymyxin B, and CREBTIDE were products of different inhibitors, followed by incubation with recombinant purified pro- Sigma-Aldrich. Anti-p38 MAPK, anti-ERK1/2, and all phospho-specific tein HP0175 (1 ␮g/ml) (or with bacteria). The conditioned medium was Abs were from Technology. Anti-p85, anti-MSK1, and anti- removed and assayed for IL-6 by ELISA using the human IL-6 assay kit. TLR4 Abs were from Santa Cruz Biotechnology. The human IL-6 ELISA kit, anti-Ras, and anti-Rac1 were from BD Biosciences. The human TLR4- Western blotting neutralizing Ab and p65 ELISA kit were from Imgenex India. Antagonistic TLR2 Ab (T2.5) was from eBioscience. [␥-32P]ATP was from Jonaki. Proteins were separated by SDS-PAGE and then transferred electrophoreti- by guest on June 26, 2015 cally to polyvinylidene difluoride membranes. Western blotting was done, Plasmid constructs as described earlier (25). cDNAs for p65 and TLR2 were obtained by RT-PCR from a total RNA Analysis of the interaction between the ECD of TLR4 and preparation from THP-1 cells and cloned in pCDNA3.1 and pFLAG-CMV- HP0175 6a, respectively. For generating dominant-negative (dn) TLR2, a 15-aa deletion from the C terminus was conducted. p65(S276A) was generated Binding of HP0175 with TLR4-ECD was conducted by an ELISA-like by site-directed mutagenesis. TLR4 (1–643) (TLR4-dn) has been de- binding assay. ELISA plates were coated with increasing concentrations of scribed previously (25). The extracellular domain (ECD) of human TLR4 HP0175 purified from HEK293 cells. The plates were blocked with 1% 1 631 (amino acids Met to Lys ) (26) was cloned in pFLAG-CMV 6a or in BSA in PBS containing 1% Tween 20 for 1 h and incubated with FLAG- pET 28a. p1168 hu.IL-6-luc, a reporter construct that codes for the human TLR4- or FLAG-TLR2-ECD at a concentration of 1 ␮g/ml for 2 h. After IL-6 promoter-luciferase in pGL3-basic or mutants carrying mutations at three washes, anti-FLAG mAb (1:5000) was added and incubated for 1 h. the binding sites of NF-␬B, AP1, CRE, NF-IL-6, ETS, and C/EBP indi- Wells were again washed, incubated with HRP-linked anti-mouse Ab, fol- vidually were obtained from Laboratorium voor Moleculaire Biologie Plas- lowed by the chromogenic substrate tetramethylbenzidine. Reactions were miden. MyD88 (152–196) (MyD88-dn) was obtained from M. Muzio and stopped with H2SO4, and bound TLR-ECD was determined spectrophoto- A. Mantovani (Mario Negri Institute for Pharmacological Research, Milan, metrically at 450 nm. In a separate set of experiments, plates were first Italy). FLAG-tagged JNK and its dn mutant JNK (T183A/Y185F) were coated with TLR4- or TLR2-ECD (20 ␮g/ml), followed by blocking, as obtained from R. Davis (University of Massachusetts Medical School, described above. Myc-tagged HP0175 was added at different concentra- Worcester, MA). dn (K63W) TAK1 was a gift from K. Matsumoto tions, and bound HP0175 was determined using anti-myc mAb, as de- (Nagoya University, Nagoya, Japan); wild-type and dn (D195A) MSK1 in scribed above. Each experiment was performed in triplicate. pCMV-FLAG were gifts from D. Alessi (University of Dundee, Dundee, U.K.). dn I␬B␣ (S32A, S36A) and dn Ras were purchased from BD RT-PCR Biosciences. A total of 100 ng of RNA was reverse transcribed using the Titanium Expression, purification, and immunodepletion of HP0175 One-Step RT-PCR Kit (BD Biosciences). The primers 5Ј-GTACC CCCAGGAGAAAGATTCC-3Ј (sense) and 5Ј-CAAACTGCATAGCCAC This was conducted by expressing His-tagged HP0175 in Escherichia coli TTTCC-3Ј (antisense) were used to amplify 819 bp of IL-6 mRNA. The BL21 (DE3), followed by purification of His-HP0175 from the lysates, as gapdh was amplified using the primers 5Ј-CCA TCA ATG ACC CCT TCA described earlier (25). Purified protein was used at a concentration of 1 TTG ACC-3Ј (sense) and 5Ј-GAA GGC CAT GCC AGT GAG CTT CC-3Ј ␮g/ml for treatments, unless otherwise stated. Constructs for expression of (antisense) to generate a 604-bp product. The PCR conditions for IL-6 truncated versions of HP0175 encompassing amino acid residues 1–154 mRNA were denaturation at 94°C for 30 s, annealing at 50°C for 1 min, and 154–299 were generated by PCR amplification of required portions of and extension at 68°C for 1 min, for 35 cycles. the HP0175 gene and cloning in pET28a. Aqueous extract of H. pylori was prepared, as described earlier (27). HP0175 was depleted from the aqueous Ras and Rac1 activity assays extract by incubation with anti-HP0175 Ab or with preimmune serum (as a control), followed by an additional incubation with protein A/G agarose Ras and Rac1 activities were measured in cell extracts by assaying for to immunoprecipitate HP0175. The supernatant was used to study the re- activated Ras or Rac1 bound to immobilized Ras binding domain of Raf-1 7952 H. pylori INDUCES TLR4-DEPENDENT IL-6 FROM MACROPHAGES Downloaded from FIGURE 1. HP0175 stimulates release of IL-6 from THP-1 cells. Cells were either left untreated or treated with different concentrations of HP0175 for 24h(A) or for different time periods (B). The last two bars in B represents results obtained using rHP0175 encompassing the N-terminal (M1-V154) or the C-terminal domain (V154-K299). In another set of experiments (A), HP0175 was subjected to boiling for 1 h (heat) or was treated with polymyxin B (PB)-agarose for1hat4°C(PB) before use. The last two bars in A represent results obtained with HP0175 purified from HEK293. C, THP-1 cells were cocultured with H. pylori (wild type (WT) or KO or KO complemented with HP0175 (COM)) at a multiplicity of infection of 100 for 24 h. D, THP-1 cells were treated with aqueous extracts from WT H. pylori or from the KO strain (KO) or from the KO strain complemented with HP0175 (COM). In separate experiments, THP-1 cells were treated with aqueous extracts from WT H. pylori (WT) treated either with preimmune sera or with anti-HP0175 Ab, as http://www.jimmunol.org/ indicated. In all cases, the release of IL-6 in the supernatant was measured using the BIOTRAK IL-6 ELISA kit, according to the manufacturer’s protocol. Results represent the means Ϯ SD of three separate experiments.

(29) or immobilized p21 binding domain of PAK1, respectively (30), ex- ples with salmon sperm DNA/protein A/G agarose slurry for 30 min at 4°C. pressed as GST fusion proteins. The supernatant was collected and incubated with appropriate primary Ab or no Ab overnight at 4°C. Salmon sperm/protein A/G agarose beads were Luciferase reporter assays added and incubated for1hat4°C, and the beads were washed succes-

sively with low salt, high salt, and LiCl immune complex wash buffers by guest on June 26, 2015 THP-1 cells were transfected with luciferase reporter plasmid along with (supplied by the manufacturer). Bound complexes were eluted with the ␤-galactosidase reporter construct, and promoter activation was analyzed above buffer containing 0.5% SDS at 60°C for 15 min. Eluates were de- by luciferase activity assays, as described (27). cross-linked by incubation at 65°C for 12 h and digested with proteinase K. MSK1 assay DNA was purified by phenol/chloroform extraction and ethanol precipita- tion in the presence of glycogen and analyzed for IL-6 by PCR using the MSK1 was immunoprecipitated from cell lysates by incubation with primers 5Ј-GGCAAACCTCTGGCACAAGAG-3Ј (sense) and 5Ј-AGGT anti-MSK1 Ab, and immunoprecipitates were incubated with 30 ␮M CGTCATTGAGGCTAGCG-3Ј (antisense) (32). substrate CREBTIDE and 0.1 mM [␥-32P]ATP (20,000 cpm/pmol). The incorporation of phosphates in CREBTIDE was determined using p81 phosphocellulose paper (31). Results Transfection of small interfering RNAs (siRNAs) HP0175 stimulates IL-6 release from THP-1 cells Cells were transfected with 5–25 nM MSK1 Kinase ShortCut siRNA mix Macrophage-derived cytokine production is strongly up-regulated or Lit28i polylinker control ShortCut siRNA mix (New England Biolabs) during H. pylori infection (33). Secreted Ags are candidates for using TransPass R2 SiRNA transfection reagent, following the manufac- stimulating cytokine induction from macrophages. We therefore turer’s protocol. Silencing of MSK1 was confirmed by Western blotting of cell lysates with anti-MSK1 Ab. evaluated the role of the secreted PPIase, HP0175, in modulating cytokine induction. ELISA showed that HP0175 elicited IL-6 re- Extraction of histones and analysis of histone phosphorylation lease from PMA-differentiated THP-1 cells in a dose- (Fig. 1A) After treatments, cells were washed with ice-cold PBS and lysed, as de- and time-dependent (Fig. 1B) manner. IL-6 release peaked at 24 h. scribed above. Extracts were centrifuged at 1,500 ϫ g and pellets were HP0175-induced IL-6 release was not affected by polymyxin B extracted with 0.4 N HCl for1honice. Extracts were centrifuged at treatment (Fig. 1A), ruling out the possibility of the effect being ϫ 10,000 g for 15 min at 4°C, and histones were precipitated with chilled due to LPS contamination. Heat treatment led to a loss of IL-6- acetone overnight at Ϫ20°C. Precipitated proteins were washed with ace- tone, dried, reconstituted, and separated by SDS-PAGE, followed by im- inducing ability of the recombinant protein (Fig. 1A), supporting munoblotting with Abs specific for phospho-Ser10 histone H3. the view that IL-6 induction was most likely due to the HP0175 protein. In addition, to rule out the contribution, if any, of bacterial Chromatin immunoprecipitation (ChIP) assay products in the observed IL-6-inducing ability of HP0175, the pro- ChIP assay was conducted using the ChIP assay kit from Upstate Biotech- tein was purified from HEK293 cells. HP0175 derived from this nology. Briefly, cells after treatments were fixed with 1% formaldehyde; nonmicrobial source was also able to drive IL-6 induction in PMA- washed with PBS; resuspended in 1% SDS, 50 mM Tris, and 10 mM A EDTA; and sonicated to generate DNA fragments with an average size of differentiated THP-1 (Fig. 1 ). To narrow down on the domain of 1 kb. After centrifugation at 13,000 rpm for 10 min, the cell supernatant HP0175 responsible for IL-6 induction, truncated versions of was diluted with ChIP dilution buffer, followed by preclearing of the sam- HP0175 were tested for IL-6-inducing ability. The data shown in The Journal of Immunology 7953

FIGURE 2. HP0175 stimulates TLR4-dependent IL-6 release. THP-1 cells were either left untreated (control) or treated with isotype IgG or with

neutralizing anti-TLR4 or anti-TLR2 Ab (10 ␮g/ml) for 1 h, followed by Downloaded from treatment with HP0175 (A) of with H. pylori at a multiplicity of infection FIGURE 3. A, Increasing concentrations of HP0175 were initially ab- of 100 (D). B and E, Cells were transfected with empty vector (pcDNA3.1 sorbed on ELISA plates, and purified soluble FLAG-tagged ECD of TLR4 or pCMV) or with the indicated dn constructs, followed by treatment with (FLAG-TLR4) or TLR2 (FLAG-TLR2) was added at a concentration of 2 HP0175. C, HEK293 cells were transfected with empty vector or wild-type ␮g/ml. After binding, wells were washed and bound FLAG-tagged protein TLR2, or wild-type TLR4 along with MD2, followed by treatment with was quantitated using anti-Flag Ab, followed by color development using HP0175. The released IL-6 was measured using the BIOTRAK IL-6 secondary anti-HRP-lined Ab, as described in Materials and Methods. Ab- Ϯ http://www.jimmunol.org/ ELISA kit. Results represent the means SD of three separate experiments. sorbance was recorded at 450 nm. B, ELISA plates were coated with FLAG-TLR4 or FLAG-TLR2 (20 ␮g/ml), and increasing concentrations of Myc-tagged HP0175 were added. TLR-bound proteins were quantitated using anti-Myc Ab and HRP-linked secondary Ab, as described in A. Data Fig. 1B suggest the importance of the C-terminal domain (V154- represent ϮSD of three independent experiments. K299) in IL-6 induction. This domain also showed PPIase activity, whereas the N-terminal domain did not (data not shown). An isogenic mutant (KO) of H. pylori 26695 inactivated in the Binding of HP0175 to TLR4 HP0175 gene was attenuated in its ability to affect IL-6 release We have reported previously that immobilized HP0175 is capable from THP-1 cells (Fig. 1C). Complementation of the KO strain of immunoprecipitating TLR4 from lysates of AGS cells (25). To by guest on June 26, 2015 with the shuttle vector pHel2 harboring the HP0175 gene resulted establish that HP0175 and TLR4 interact directly without the need in restoration of the IL-6-inducing ability (Fig. 1C). An aqueous for any accessory molecule(s), the binding between the ECD of extract (HPE) of the wild-type strain (containing shed factors of TLR4 and HP0175 was analyzed. ELISA plates were coated with the bacterium) was capable of driving IL-6 release from PMA- increasing concentrations of purified HP0175. TLR4-ECD could differentiated THP-1 (Fig. 1D). IL-6-inducing ability of the HPE bind to E. coli-derived HP0175-coated plates, and the binding in- was attenuated in the KO strain (Fig. 1D), suggesting that HP0175 creased with increasing concentrations of HP0175 (Fig. 3A). In was one of the major components responsible for inducing IL-6 contrast, the TLR2-ECD did not bind to HP0175-coated wells, release. In harmony with this, IL-6-inducing ability was compro- supporting the specificity of the HP0175-TLR4 interaction. The mised in HP0175-immunodepleted extracts of the wild type (Fig. ECD of TLR4 did not bind to wells coated with an irrelevant 1D). Taken together, these results suggested that HP0175 is a po- His-tagged protein (data not shown). In a second ELISA-like as- tent IL-6 inducer. say, wells were coated with the ECD of TLR4 (or TLR2), and the binding of HP0175 to the coated wells was studied. HP0175 could TLR4 signaling in HP0175-stimulated IL-6 production in THP-1 bind to wells coated with TLR4 in a dose-dependent manner (Fig. cells 3B), but not to wells coated with TLR2. These ELISA-like assays The TLR signaling pathway plays a role in regulation of cytokine confirmed that HP0175 interacts with the ECD of TLR4 without production through a signaling cascade leading to the activation of the need for an accessory molecule. Similar results were obtained NF-␬B (12, 13). Pretreatment of cells with neutralizing Abs using HP0175 purified from HEK293 (data not shown), ruling out against TLR4 (but not against TLR2) before challenge with the possibility of microbial products serving as intermediary mol- HP0175 blocked IL-6 release (Fig. 2A). Transfection of cells with ecules in this interaction. dn TLR4 (but not with dn TLR2) also inhibited HP0175-induced ␬ IL-6 release (Fig. 2B). Furthermore, HP0175 could induce IL-6 HP0175-induced IL-6 release depends on NF- B activation release from HEK293 cells transfected with TLR4, but not from In the canonical pathway of NF-␬B activation, TLR4 signals along cells transfected with either vector or with TLR2 only (Fig. 2C). the MyD88/IRAK/TNFR-associated factor 6 axis, leading to the We evaluated the role of HP0175-dependent TLR4 signaling phosphorylation of I␬B␣, its degradation by the proteasome, re- in IL-6 induction in the context of the bacterium as a whole. lease of NF-␬B, and movement of NF-␬B into the nucleus. Trans- H. pylori-mediated IL-6 induction was attenuated in cells treated fection with dn constructs of MyD88, or the superrepressor I␬B␣ with neutralizing Abs against TLR4 (but not TLR2) (Fig. 2D). This (S32A, S36A) (I␬B␣ (dn)), led to an inhibition of HP0175-induced suggested that TLR4 signaling plays a major role in H. pylori- IL-6 release, confirming the likely role of the canonical pathway of mediated IL-6 induction in macrophages. NF-␬B activation in HP0175-mediated IL-6 release (Fig. 2E). 7954 H. pylori INDUCES TLR4-DEPENDENT IL-6 FROM MACROPHAGES

PBMCs (Fig. 4). A combination of U0126 and SB203580 led to a complete inhibition of IL-6 release (Fig. 4), suggesting a syner- gistic role of ERK and p38 MAPKs in HP0175-triggered IL-6 release. Neither dn JNK nor the JNK inhibitor SP600125 inhibited HP0175-induced IL-6 release (data not shown), suggesting that JNK signaling was not involved in IL-6 release. Besides their abil- ity to phosphorylate and activate transcription factors, both ERK and p38 MAPKs transduce signals by activating downstream ki- nases such as MSK1 (34), which in turn phosphorylates transcrip- tion factors. PBMCs or THP-1 cells pretreated with H89 (a phar- macological inhibitor of MSK1 at the specific dose used in this study) led to a complete inhibition of IL-6 release, suggesting a role of MSK1 in IL-6 release (Fig. 4).

HP0175 stimulates IL-6 gene expression and stimulates IL-6 promoter activity in an NF-␬B-dependent manner Semiquantitative RT-PCR analysis for IL-6 mRNA expression showed that the steady state IL-6 mRNA levels increased in cells treated with HP0175 (Fig. 5A). HP0175-induced IL-6 gene expres- Downloaded from sion could be attenuated partially either by U0126 or by SB203580 alone, and inhibited almost completely by a combination of U0126 and SB203580, suggesting that IL-6 gene expression is coordi- FIGURE 4. Effect of inhibitors on HP0175-mediated IL-6 release from THP-1 (A) and PBMCs (B). Cells were either left untreated or treated with nately regulated by ERK and p38 MAPKs. different inhibitors at the indicated concentrations for 30 min before treat- THP-1 cells were transfected with different constructs of the ment with HP0175 (1 ␮g/ml) for 24 h (wort (wortmannin)). The release of human IL-6 promoter coupled with the luciferase reporter gene. http://www.jimmunol.org/ IL-6 in the supernatant was measured using the BIOTRAK IL-6 ELISA kit. Treatment of cells with HP0175 resulted in a ϳ20-fold increase Results represent the means Ϯ SD of three separate experiments. in luciferase activity in cells transiently expressing the IL-6 promoter (p1168 hu.IL-6-luc) (35) compared with untreated cells (Fig. 5B). THP-1 cells were also transfected with IL-6 HP0175-induced IL-6 release depends on ERK and p38 MAPKs promoter luciferase reporter constructs mutated individually at MAPKs are known to regulate the upstream signaling events that NF-␬B, AP-1, C/EBP/␤, CRE, and ETS sites (36), followed by control cytokine production. U0126 and SB203580, inhibitors of stimulation with HP0175 for 24 h. Mutation at only the NF-␬B the ERK and p38 MAPK signaling pathways, respectively, could site completely inhibited luciferase gene expression (Fig. 5B), ␬

inhibit HP0175-induced IL-6 release from THP-1 cells as well as suggesting that the NF- B element was necessary to drive by guest on June 26, 2015

FIGURE 5. HP0175 stimulates IL-6 gene expression and IL-6 promoter-driven luciferase reporter expression in THP-1 cells. A, Cells were pretreated either with DMSO or with inhibitors, followed by incubation without or with HP0175 for 12 h. Total RNA was isolated, and RT-PCR was performed using primers specific for the IL-6 or the gapdh genes. The products were visualized on agarose gels by ethidium bromide staining. B, Cells were transfected with IL-6 promoter luciferase reporter construct (IL6 WT) or mutant constructs along with ␤-galactosidase expression vector. C, In another set of experiments, cells after transfection were pretreated with different inhibitors. Cells were then left untreated or treated with HP0175 for 16 h. B and C, Luciferase reporter activities were measured in cell extracts using luciferin as a substrate in a luminometer, and the results were normalized for transfection efficiencies by assay of ␤-galactosidase activity. D, Cells were transfected with empty vector or with dn MSK1 along with IL-6 promoter luciferase reporter and ␤-galactosidase constructs before treatment without (Ϫ) or with (ϩ) HP0175. Luciferase activity was measured, as described. Results represent the means Ϯ SD of three separate experiments. The Journal of Immunology 7955

of p38 MAPK. Cotransfection with TAK1 K36W and dn Rac1 lead to a greater inhibition of HP0175-induced p38 MAPK acti- vation than observed using either construct alone (Fig. 6E), sug- gesting that distinct PI3K/Rac1 and TAK1 signaling pathways converged upon p38 MAPK activation.

HP0175 activates MSK1 The MAPKs phosphorylate several downstream kinases, one of which is MSK1, which is constitutively localized in the nucleus, where it regulates transcription by phosphorylating among others, histone H3, p65/RelA, and CBP/p300 (39), leading to nucleosomal modification and increased accessibility of the transcription ma- FIGURE 6. HP0175 stimulates the PI3K/Ras and PI3K/Rac1 signaling chinery to promoters. HP0175 activated MSK1 in a time-depen- pathways leading to the activation of ERK and p38 MAPKs. A, THP-1 dent manner (data not shown). MSK1 activity could be partially cells were left untreated or pretreated with anti-TLR4-neutralizing Ab (10 inhibited by pretreatment with either U0126 or SB203580 (Fig. ␮ Ϫ g/ml) or isotype IgG for 1 h, followed by incubation without ( ) or with 7A). A combination of U0126 and SB203580 completely blocked (ϩ) HP0175. Cells were lysed and immunoprecipitated with anti-p85 Ab, MSK1 activation to the extent observed using H89, suggesting that and the immunoprecipitate was probed with anti-phosphotyrosine Ab. The blot was reprobed with anti-p85 Ab. B and C, Cells were left untreated or ERK and p38 MAPK act synergistically to activate MSK1. Ex- treated with wortmannin, followed by HP0175 treatment, as described pression of MSK1 was effectively inhibited by siRNA directed Downloaded from above. Cell lysates were incubated with 5 ␮g of GST-Raf RBD (B)or against MSK1 (Fig. 7B). Under these conditions, HP0175-induced GST-PBD (C) immobilized on glutathione-Sepharose. Bound Ras (B)or IL-6 release was inhibited (Fig. 7C), lending further support to the Rac1 (C) was detected by Western blotting using anti-Ras (B) or anti-Rac1 view that MSK1 plays a central role in HP0175-induced IL-6 re- (C) Ab. Blots were reprobed with anti-actin Ab to ensure equal loading in lease from THP-1. all lanes. THP-1 cells were transfected with different dn constructs (D and E), as indicated in the figure, followed by treatment without (Ϫ) or with HP0175-induced phosphorylation of p65 and histone H3

ϩ http://www.jimmunol.org/ ( ) HP0175. Cell lysates were immunoblotted with phospho-ERK1/2 Ab depends on MSK1 (D) or with phospho-p38 MAPK Ab (E). Blots were reprobed with anti- ERK1/2 (D) or with anti-p38 MAPK (E) Ab to ensure equal loading in all Recruitment of NF-␬B to its target depends on the integration of lanes. Blots shown are representative of results obtained in three separate signals coming from stimulus-dependent activation of multiple sets of experiments. signaling pathways, leading to covalent modifications of NF-␬B that regulate its transcriptional activity. Protein kinase A and MSK1 phosphorylate p65 on Ser276 (40), enabling the p50-p65 HP0175-induced IL-6 gene expression. U0126 or SB203580 complex to interact with the transcriptional coactivators p300 and could partially inhibit luciferase gene expression, whereas a CBP (41). HP0175-induced p65 phosphorylation was inhibited in combination of both inhibitors completely inhibited luciferase cells transfected with dn MSK1 (Fig. 7D). At the same time, trans- by guest on June 26, 2015 gene expression (Fig. 5C), suggesting that ERK and p38 MAPK fection of cells with a p65 construct bearing the S276A mutation activation are critical in sustaining HP0175-driven IL-6 pro- inhibited HP0175-stimulated IL-6 release (Fig. 2E), suggesting a moter activation. role of p65 phosphorylation on S276 in activating IL-6 production. Covalent modification of histones by acetylation, phosphoryla- PI3K/Ras and PI3K/Rac signaling is involved in tion, and methylation regulates transcription of a subset of genes in HP0175-induced IL-6 release a context-specific manner (32). MSK1 phosphorylates histone on 10 10 HP0175-triggered IL-6 release in PBMCs and THP-1 was inhib- Ser (42). HP0175-induced H3 Ser phosphorylation was inhib- ited by wortmannin, an inhibitor of PI3K (Fig. 4), and HP0175 was itable by dn MSK1 (Fig. 7E), affirming the role of MSK1 in H3 10 found to stimulate PI3K activity, as evidenced by the phosphory- Ser phosphorylation. lation of the p85 subunit of PI3K (Fig. 6A). This was abrogated when cells were pretreated with neutralizing anti-TLR4 Ab (Fig. Association of phosphohistone H3 and p65 with the IL-6 6A), suggesting TLR4-dependent activation of PI3K. Wortmannin promoter in HP0175-stimulated cells inhibited HP0175-triggered Ras (Fig. 6B) and Rac1 activation To delineate the relationship of phosphorylation of H3 and p65 to (Fig. 6C). Our results therefore suggested that HP0175 activates IL-6 gene transcription, ChIP assays were performed. Immunopre- PI3K/Ras and PI3K/Rac1 signaling. HP0175-stimulated ERK ac- cipitation with an Ab specific for p-Ser10 H3 after stimulation of tivation was inhibited by dn Ras (Fig. 6D), whereas p38 MAPK THP-1 cells with HP0175 resulted in reproducible enrichment of activation was partially inhibited by dn Rac1 (Fig. 6E). Taken IL-6-specific gene sequence in stimulated vs unstimulated samples together, our results suggested that HP0175 signaling induced by PCR (Fig. 7F), affirming H3 phosphorylation at Ser10 on the TLR4/PI3K/Ras-dependent ERK activation and TLR4/PI3K/ nucleosomes distributed along the IL-6 promoter. Anti-phospho- Rac1-dependent p38 MAPK activation. Ser10 H3 immunoprecipitates from cells transfected with dn MSK1 contained greatly reduced amounts of promoter sequences (Fig. HP0175-induced p38 MAPK activation depends on TAK1 and 7F), confirming that MSK1 plays a critical role in nucleosomal on PI3K/Rac1 signaling modification at the IL-6 promoter. When Ab was omitted from the The MAPK kinase kinase TAK1 is known to phosphorylate nu- immunoprecipitation reaction, no promoter sequence was retrieved clear factor-inducing kinase, MAPK kinase 3/6, and MAPK kinase (data not shown), confirming the specificity of the reaction. p65- 4 to regulate the NF-␬B and the MAPK signaling pathways (37, transfected cells when stimulated with HP0175 showed enrich- 38). Kinase-dead TAK1 (K63W) inhibited HP0175-induced IL-6 ment of IL-6-specific sequence in ChIP assays with p65-specific release (Fig. 2E). TAK1 K36W inhibited p38 MAPK activation Ab (Fig. 7G). Reduced amounts of promoter sequence were im- partially (Fig. 6E), suggesting that TAK1 signaling lies upstream munoprecipitated in cells transfected with p65(S276A), affirming 7956 H. pylori INDUCES TLR4-DEPENDENT IL-6 FROM MACROPHAGES

FIGURE 7. Role of MSK-1 in HP0175-stimulated IL-6 production in THP-1 cells. A, Cells were left untreated or pretreated with inhibitors (at concentrations described under Fig. 1) for 30 min, followed by incubation without or with HP0175 for 30 min. Cell lysates were immunoprecipitated with anti-MSK1 Ab, and the immunoprecipitate was used to measure the phosphorylation of CREB-tide, as described in Materials and Methods. B, THP-1 cells were transfected with Lit28i polylinker control shortcut siRNA (scrambled) or MSK1 kinase siRNA mix using TransPass R2 siRNA transfection reagent. Downloaded from Cells were lysed and immunoblotted with anti-MSK1 Ab. C, THP-1 cells were transfected, as described for B. Cells were then left untreated or treated with HP0175 for 24 h, and release of IL-6 was measured by ELISA. D and E, Cells were transfected with either empty vector or dn constructs, followed by treatment without (Ϫ) or with (ϩ) HP0175 for 60 min. Cells were lysed and immunoprecipitated with the anti-p65 (D) or anti-H3 (D) Abs, followed by Western blotting using anti-phosphoserine Ab (D) or anti-phospho-Ser10 H3 Ab (E). Blots were reprobed with appropriate Abs. F and G, ChIP assays. For ChIP analyses, cells were transfected with either empty vector or different constructs, as indicated before treatment with HP0175 for 60 min. Treated cells were fixed with formaldehyde and immunoprecipitated with anti-phospho-Ser10 H3 Ab (F) or with anti-p65 Ab (G). Immunoprecipitates were analyzed by semiquantitative PCR with IL-6 promoter-specific primers. Gels in F and G are representative of results obtained in three separate experiments. PCR using http://www.jimmunol.org/ the same primers was also performed with 1% of input (bottom panel) to ensure loading. that phosphorylation of p65 on Ser276 promotes its recruitment to recombinant protein preparations with polymyxin-agarose. HP0175 the IL-6 promoter. was also purified from the HEK293 cell line. Both preparations were found to induce IL-6 release from THP-1, ruling out the Discussion possibility of contaminating bacterial products being responsible H. pylori infection leads to chronic inflammation and a Th1- for the observed effect. This view was further strengthened by our skewed immune response with production of proinflammatory cy- observation that inactivation of the HP0175 gene led to Ͼ70% by guest on June 26, 2015 tokines, one of which is IL-6. The role of TLRs in the pathophys- reduction in IL-6-inducing ability of H. pylori, and that significant iology of Helicobacter infection is a matter of debate. Most of the reduction of IL-6-inducing ability occurred when HP0175 was im- reports have focused on epithelial cells. Some groups have argued munodepleted from aqueous extracts of H. pylori. This suggested that the response of epithelial cells to H. pylori is TLR2, but not that HP0175 is one of the major IL-6-inducing factors. ELISA-like TLR4 dependent (43, 44). Others have reported that TLR4 expres- binding assays demonstrated that HP0175 (purified either from sion is enhanced in the gastric epithelium following H. pylori in- E. coli or from HEK293) could interact directly with TLR4, ruling fection (19) and that enhanced TLR4 expression stimulates NF- out the requirement of accessory molecules or copurified bacterial ␬B-driven IL-8 promoter activity (45). It is likely that TLRs 2, 4, products in this interaction. In vitro analysis has shown that and 5 all have a role in the context of H. pylori infection. It is HP0175 is secreted by H. pylori rather than being released by pertinent to point out that our studies have focused not on gastric nonspecific lysis (22). Assuming that HP0175 release also occurs epithelial cells, but on cells of the monocytic lineage. Consid- in vivo, it is likely to be a factor of potential importance in the ering that mononuclear cell infiltration in the lamina propria pathophysiology of H. pylori infection, because HP0175 would be and increased expression of inflammatory cytokines character- likely to reach mucosal macrophages after the disruption of the ize H. pylori-induced chronic infection (33), it is of obvious epithelial cell junction during infection. importance to elucidate the effectors of H. pylori that drive the HP0175-triggered TLR4-dependent signaling pathways acti- macrophage response to gain insight into how inflammation is vated the MAPKs ERK and p38 MAPK in a PI3K/Ras, Rac1- induced by H. pylori. The present study is a step toward this dependent manner. This in turn activated MSK1, a nuclear kinase end. TLR4-mediated signals play a major role in eliciting in- central to HP0175-driven NF-␬B activation (Fig. 8). NF-␬B acti- flammatory cytokines from macrophages. TLR4-interacting vation was necessary for driving HP0175-induced IL-6 gene ex- molecules are likely to deliver signals of consequence in dis- pression. Considering that MSK1 has been reported to phosphor- ease progression and outcome. ylate histone H3 as well as the p65 subunit of NF-␬B, we Based on our earlier observations that HP0175 interacts with conjectured that MSK1 was most likely influencing HP0175-in- TLR4 (25), we investigated the probable involvement of TLR sig- duced chromatin modifications at the IL-6 promoter, thereby reg- naling pathways in HP0175-induced IL-6 production in THP-1 ulating IL-6 gene expression. HP1075-stimulated histone H3 phos- cells. rHP0175 expressed in E. coli elicited IL-6 release from phorylation on Ser10 and p65 phosphorylation were dependent on THP-1 and from PBMCs. In view of the fact that several reports in MSK1, and ChIP assays showed that association of p65 and phos- the past on the effects of recombinant proteins were later found to pho-Ser10 H3 with the IL-6 promoter was dependent on MSK1. be due to contaminating bacterial products, care was taken to rule MSK1 therefore plays a central role in TLR4-dependent IL-6 in- out this possibility. Contaminating LPS was removed by treating duction elicited by HP0175 in macrophages. The Journal of Immunology 7957

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