Microbes and Infection 13 (2011) 818e823www.elsevier.com/locate/micinf

Short communication

Evidence of involvement of the receptor for advanced glycationend-products (RAGE) in the adhesion of Helicobacter pylori

to gastric epithelial cells

Armando Rojas a,*, Ileana Gonzalez a, Boris Rodrıguez b, Jacqueline Romero a,Hector Figueroa a, Jorge Llanos a,c, Erik Morales a,d, Ramon Perez-Castro a

aBiomedical Research Labs, Medicine Faculty, Catholic University of Maule, 3605 San Miguel Ave., Talca, ChilebMicrobiology & Immunology Dept., National Center for Scientific Research, La Habana, Cuba

cCentre for Gastric Cancer, Regional Hospital of Talca, ChiledPathology Department, Regional Hospital of Talca, Chile

Received 9 December 2010; accepted 15 April 2011

Available online 12 May 2011

Abstract

The adherence of Helicobacter pylori to gastric epithelial cells is required for prolonged persistence in the stomach and for induction ofinjury. Here, we first reported a new role of the receptor for advanced glycation end-products (RAGE) on the adherence of H. pylori to gastricepithelial cells, assessed by different methods and binding to immobilized RAGE. RAGE-targeted knock-down in MKN74 cell line markedlyreduced not only the adhesion of H. pylori, but also the levels of IL-8 transcripts and protein released in response to infection. These data suggestthat RAGE may represent a new factor on the pathogenesis of H. pylori infection.� 2011 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Keywords: Helicobacter pylori; Receptor for advanced glycation end-products; Inflammation; Gastric epithelial cells; Gastric cancer; Hostepathogen interactions

1. Introduction

Helicobacter pylori is a gram-negative, spiral bacteriumthat colonizes gastric mucosa and plays an important role inthe pathogenesis of chronic gastritis, peptic ulcer, gastricadenocarcinoma and mucosa-associated lymphoid tissuelymphoma [1].

Although the vast majority of H. pylori in colonized hostsare free-living, w20% bind to gastric epithelial cells andadherence is required for prolonged persistence in the stomachand for induction of injury [2,3].

H. pylori has raised as a common infection in diabetics whodo not have metabolically controlled hyperglycemia [4,5].Furthermore, diabetic patients show a significantly lower

* Corresponding author. Tel.: þ56 71 203134; fax: þ56 71 413657.

E-mail address: arojasr@ucm.cl (A. Rojas).

1286-4579/$ - see front matter � 2011 Institut Pasteur. Published by Elsevier Ma

doi:10.1016/j.micinf.2011.04.005

eradication rate than non-diabetic subjects [6], suggesting thatdiabetics are H. pylori infection-prone individuals. Veryrecently, hyperglycemia has been shown to be a cofactor,increasing the risk of gastric cancer in patients infected byH. pylori [7]. The mechanisms for increased risk of gastriccancer in hyperglycemia are not fully understood.

Firstly described in 1992, the receptor for advanced glycationend-products (RAGE) has attracted increasing attention due toits diverse ligand repertoire and contribution to the inflamma-tory basis for diabetes, cancer, renal and heart failure, as well asneurodegenerative diseases [8,9]. Although expressed at verylow levels in normal tissues, chronic inflammatory conditionsincrease not only RAGE expression [10], but also the formationand release of some RAGE ligands [11,12].

Although advanced glycation end-products (AGEs) were thefirst reported activating ligands; many other, including damage-associated molecular patterns, have been identified [13].

sson SAS. All rights reserved.

819A. Rojas et al. / Microbes and Infection 13 (2011) 818e823

The structural analysis of ligandeRAGE interaction hasrevealed that the receptor recognized three-dimensional struc-tures, rather than specific amino acid sequences (i.e., primarystructure). Some authors have even postulated that RAGEmay interact with bacteria [14] and prions [15], throughmolecules sharing structural analogies to well-recognizedRAGE ligands [16].

RAGE is thought to function as a pattern-recognitionreceptor. Strikingly, Toll- like receptor 4, which belongs to thesame family, can serve as a receptor for H. pylori adhesion togastric epithelial cells [17].

Considering all these antecedents it was tempting to spec-ulate that RAGE expression may contribute to the adherenceof H. pylori to gastric epithelial cells.

2. Materials and methods

2.1. Cell lines

We used two gastric adenocarcinoma cell lines, widely usedin H. pylori adhesion studies. The MKN45 cell line was kindlysupplied by Dr. Alejandro Corvalan (Pontifical CatholicUniversity of Chile). MKN74 was purchased from the RikenCell Bank (Tsukuba, Japan). Both cell lines were routinelymaintained in RPMI-1640 supplemented with 10% FCS, 100units/ml penicillin, and 100 mg/ml streptomycin, underconditions of 5% CO2 in air at 37 �C. RAGE is differentiallyexpressed in these cell lines (MKN45, RAGE-negative);(MKN74, RAGE-positive) as previously reported [18], andconfirmed in our lab by western blotting and RT-PCR. In alladherence assays, 24 h before infection antibiotics and fetalcalf serum were removed from cell cultures.

Cell viability was checked out in every experimentalapproach by using a Vybrant� MTT Cell Proliferation (Invi-trogen) to exclude any effect on cell viability produced byantibody incubations or transfection procedures.

2.2. H. pylori strains and culture conditions

H. pylori clinical isolates 225 (Cag Aþ, s1m1, babA2) and236 (Cag A�, s2m2, babA2), recovered from gastric biopsyspecimens, were used in this study. Bacterial stock preservedat �80 �C of both strains were grown on Columbia agar baseplates with 7% horse blood for 3 days at 37 �C undermicroaerophilic conditions using an anaerobic chamber.

To determine whether or not RAGE may contribute to theadhesion of H. pylori to gastric epithelial cells, three differentapproaches were used to quantify bacterial adherence:

2.3. Adherence assays

2.3.1. CFU countingBacteria were recovered from plates with phosphate-buffered

saline (PBS) and washed by centrifugation at 5000 rpm/min for5 min at 4 �C, suspended in PBS to 1 OD600 (approximately109CFU/ml), and inoculated(100ml) into 24-well plate confluentcell monolayers to achieve a multiplicity of infection (MOI) of

100. As control, 100 ml of PBS was added to each host cell lines.Infectionwas carried out at 37 �C in 5%CO2 for 6 h. Afterwards,plates werewashed 5 timeswith PBS and cells lysedwith 1ml of0.1% saponin in PBS [19]. The suspensions were serial dilutedand total cell-associated bacteria were estimated by counting thenumber of CFU on H pylori selective plates. Data are expressedCFU/106 cells.

2.3.2. ELISA assayThe adhesion of H. pylori was analyzed by a cellular

ELISA essentially as previously described [20]. Briefly, gastricepithelial cell lines were cultured in 96 well plates and allowedto form confluent monolayers. Infection was carried out at100:1 bacteria to cell ratio and incubated for 90 min at 37 �C.Cells were extensively washed and fixed at 4 �C with 8%paraformaldehyde for 60 min. Endogenous peroxidase wasinactivated by the addition of 1% hydrogen peroxide inmethanol. After washing, a rabbit anti-H pylori polyclonalantibody was added overnight at 4 �C, followed by addition ofHRP-labeled goat anti-rabbit IgG antibody for 30 min at roomtemperature. Tetramethylbenzidine was added and the reactionstopped with 1 M HCL. Finally, the plates were read ina microplate reader at 450 nm. OD values/106 cells were usedas the index of the number H. pylori adhering to cells.

2.3.3. Urease activity assayBriefly, gastric epithelial cell lines were cultured in 48 well

plates until confluent. H. pylori strains were added in 100:1bacteria to cell ratio and after 6 h, plates were washed toremove non-adherent bacteria. Afterwards, saline solutioncontaining 2% (w/v) urea and 0.03% (w/v) phenol red wasadded and bacterial urease activity was assayed by measuringthe optical density at 540 nm as described [21]. Absorbancevalues//106 were used as an index of H pylori adhered to cells.

In all adherence assays, a goat anti-RAGE polyclonalantibody (10 mg/ml), used as internal control to block RAGE,was added to cell cultures 4 h before infection. An irrelevantpolyclonal antibody was used as negative control.

2.4. Direct adhesion to immobilized RAGE

H. pylori suspensions (100 ml) were added to RAGE/Fcchimera (R&D Systems) and to human IgG Fc fragment ascontrol) immobilized at 10 mg/ml on a goat anti-human IgG (Fcspecific) antibody coated plate (1 mg/well) and incubated at37 �C in 5% CO2 for 90 min. After incubation, non-adherentbacteria were removed by serial washes with PBS. Afterwards,the wells were allowed to react with a rabbit anti-H. pyloriantiserum (Dako; 1:5000 dilution) in PBS containing 1%BSAat37 �C in 5% CO2 for 1 h, followed by peroxidase-conjugatedgoat anti-rabbit IgG antibody (Santa Cruz Biotechnology;1:1000 dilution) 37 �C for 1 h, in 5% CO2. After washing withPBS containing 0.05% Tween-20, the remaining enzymeactivity was visualized by the addition of tetramethylbenzidine,and the reaction stopped with 1MHCL. Finally, the plates wereread at 450 nm. Specific adhesion of H. pylori to immobilizedRAGE was determined from the difference in absorbance with

820 A. Rojas et al. / Microbes and Infection 13 (2011) 818e823

respect to the RAGE chimera-immobilized wells and IgG-Fcfragment coated wells. Additional internal controls included theuse of a non-specific polyclonal antibody and wells withoutbacterial suspension. In inhibition experiments, wells contain-ing the immobilized RAGE-Fc chimera were preincubated for2 h with an excess of a RAGE ligand (AGE-BSA/100 mg/ml)before adding bacterial suspension.

2.5. DNA transfection and RNA interference

The full-length RAGE cloned into pcDNA3.1 was kindlysupplied fromDr. E. Rieber (Institute of Immunology, TechnicalUniversity of Dresden) [22] and used for transfection of the celllineMKN45 by using the CalPhosMammalian Transfection Kit(Clontech). Stably transfected expressing clones were selectedby geneticin (0.5 mg/ml; Invitrogen). Single clones wereanalyzed for RAGE expression by Western blotting.

RAGE short hairpin RNA (shRNA) and control shRNA(Santa Cruz Biotechnology) were transfected into MKN74cells according to manufacturer’s instructions. Stable RAGE-targeted knock-down were obtained by puromicyn selectionand cloning. RAGE expression was checked by immunoblot-ting. RAGE-targeted knock-down and control cells were theninfected at a MOI of 100, and incubated at different times at37 �C in 5% CO2 and assayed for bacterial adhesion and IL-8transcript and released protein levels.

2.6. Semiquantitative RT-PCR

TotalRNAwas isolated byusing anRNeasymini kit (Qiagen).Reverse transcription-PCR (RT-PCR) was performed by usingthe Onestep RT-PCR (Qiagen) kit for the detection of RAGE andIL-8 transcript levels and glyceraldehide-3-phosphate dehydro-genase (GAPDH) as internal control. The specific primersused were as follows: RAGE, 50 -gatccccgtcccaccttctcctgtagc-30(sense) and 50-acgctcctcctcttcctcctggttttctg-30(anti-sense);GAPDH, 50-gtcggagtcaacggattt-30(sense) and 50-actccacgacg-tactcagc-30(anti-sense); IL-8, 50-atgacttccaagctggccgtg-30(sense)and 50 -ttatgaattctcagccctcttcaaaaacttctc-30(anti-sense).

The quantitative nature of the RT-PCR was validated by thelinearity of the determination curve at various concentrationsof RNA.

2.7. IL-8 ELISA

The enzyme-linked immunosorbent assay (ELISA)-systemfrom R&D was used to analyze IL-8 secretion of H. pylori-infected epithelial cells. Following infection, the culturemedium was centrifuged (10 min, 10,000 g, 4 �C) and the IL-8content was determined according to the manufacturer’sinstructions.

2.8. Western blotting

Protein extracts (40 mg) were then separated by 10% SDSpolyacrylamide gel electrophoresis (SDS-PAGE). The sepa-rated proteins were electrotransferred onto a PVDF

membrane. After non-specific blocking, the membrane wasincubated overnight with a primary mouse monoclonal anti-body for RAGE (1: 400 Santa Cruz Biotech.), followed byHRP-labeled goat anti-mouse IgG antibody (1:2000) for 1.5 hat room temperature, and then visualized by an enhancedchemiluminescence detection kit. The immunoreactive bandswere analyzed with a Geliance photo-imaging system (PerkinElmer).

2.9. Statistical analysis

Results are expressed as means � S.E.M. from data derivedfrom at least three independent experiments. Statistical anal-ysis was performed using one-way ANOVA followed bya parametric Dunnett’s test. A P value <0,05 was taken assignificant.

3. Results

The putative contribution of RAGE on the adhesion ofH. pylori to gastric epithelial cells was firstly evidenced whenthe treatment with a blocking anti-RAGE antibody priorinfection, diminished by 30% the adhesion of H. pylori to thegastric epithelial cell line MKN74, which constitutivelyexpresses RAGE, as assessed by CFU counting. The same pre-treatment with the blocking antibody did not produce anydetectable effects on the adherence of H. pylori to the RAGE-negative cell line MKN45 [17] (Fig. 1A).

RAGE-positive MKN74 cells were pretreated, prior infec-tion, with RAGE ligands (HMGB1 and AGE-BSA, 6 h, at100 ng/ml and 10 mg/ml, respectively). The incrementsobserved in adhesion, ranging from 65 to 70% (Fig. 1B), wereparalleled by increased levels of both RAGE transcripts andprotein levels (see insert). As expected, no effects wereobserved when RAGE-negative MKN45 cells were pretreatedwith both RAGE ligands.

Next, we tested the RAGE contribution to H. pylori adhe-sion by ELISA. The results depicted in Fig. 1C, were quitesimilar to those obtained by classical CFU counting, therebysupporting our assumption that RAGE contributes to theadhesion of H. pylori to gastric epithelial cells. In thisexperimental approach, treatment with a blocking anti-RAGEpolyclonal antibody diminished the adhesion of H. pylori tothe RAGE-positive MKN74 cell line by 35%. When this cellline was pretreated by two RAGE ligands (HMGB1 andAGE-BSA), as described in the CFU counting method,adherence increments ranged between 63 and 79%, respec-tively (Fig. 1D). As already observed, no effects were detectedwhen RAGE-negative MKN45 cells were pretreated with bothRAGE ligands.

A third set of experiments was then performed, andadherence was assessed by determining urease activity. Oncemore the results obtained, as depicted in Fig. 1E and F, areconsistent with those obtained by the previous microbiologicaland immunochemical approaches, thus clearly suggesting thatRAGE contributes to the adhesion of H. pylori to gastricepithelial cells.

Fig. 1. Adhesion of H. pylori strains 225 and 236 to RAGE-negative (MKN-45) and RAGE-positive (MKN-74) gastric epithelial cell lines. Adherence was

determined by CFU counting (panels A and B), ELISA (C and D) and urease activity (E and F). Experiments were performed in the presence and absence of anti-

RAGE polyclonal antibody (10 mg/ml, 4 h before infection). The effect of preincubation with RAGE ligands was also evaluated on the adhesion of H. pylori

clinical isolates to both cell lines. Increments in adhesion were paralleled by increased levels of both RAGE transcript and protein (see insert).*P < 0.05;

**P < 0,01.

821A. Rojas et al. / Microbes and Infection 13 (2011) 818e823

Because RAGE may oligomerize with other molecules andthus contribute to adherence, we intend to clarify whether ornot RAGE may interact directly with H. pylori, based onspecific adhesion to immobilized RAGE, assessed by ELISA.The results obtained confirm our previous observations thatRAGE contributes to adhesion, as evidenced by the directbinding of H. pylori to immobilized RAGE in a dose-depen-dent manner. This binding was markedly interfered (60%) byan excess of RAGE ligand (AGE-BSA, 100 mg/ml) added 2 hbefore bacterial suspension (Fig. 2A).

We further tested whether or not RAGE-targeted knock-down in MKN74 cell line may have any consequence onH. pylori adhesion. As depicted in Fig. 2B, transfection ofRAGE-shRNA reduced RAGE protein levels by 85% (seeinsert). Consistent with this result, we observed that targetedknock-down of RAGE in MKN74 cell line significantlyreduced the adhesion of H. pylori, assessed by ELISA, whencompared to both untransfected and shRNA control cells.Similar results were also obtained when adherence wasassessed by the urease method (data not shown). Additionally,MKN45 cell line was transfected with a full-length RAGEconstruct, and expression of RAGE on this cell line was par-alleled with an increase in H. pylori adhesion, assessed byELISA, as shown in Fig. 2C. Interestingly, H. pylori infectionproduced an increase in RAGE expression, in a time-

dependent fashion, which was not observed in RAGE-targetedknock-down cells, as depicted in Fig. 2D.

Because RAGE is thought to function as a pattern-recog-nition receptor, we finally tested whether RAGE-mediatedbinding may also contribute to trigger a proinflammatoryresponse, as assessed by the detection of both IL-8 transcriptsand protein levels.

Strikingly, reduced IL-8 transcript and released proteinlevels were detected in RAGE-targeted knock-down cells ina time-dependent fashion (Fig. 3A and B).

4. Discussion

In the present study we showed, for the first time, thatRAGE contributes to the adhesion of H. pylori to gastricepithelial cells. Adherence of H. pylori is dependent on theexpression of bacterial adhesins and cognate host counter-receptors [2]. The H. pylori genome encodes outer membraneproteins (OMP) in an unusually high percentage (4%) [23],which may function as adhesins and contribute to pathogen-esis. Some of these OMP are known to be involved in bindingto gastric epithelial cells, but the corresponding receptors inhost cells are yet unknown.

The finding that H. pylori may interact directly with RAGEis particularly interesting because this pathogen also use the

Fig. 2. Binding of H. pylori to immobilized RAGE, the effects of RAGE-targeted knock-down and RAGE full-length transfection on H. pyloi binding to gastric

epithelial cells and the effect of H. pylori infection on RAGE expression. H. pylori suspensions (100 ml, 106 and 105 CFU) bind to immobilized RAGE in a dose-

dependent manner. This binding was markedly reduced by preincubation (2 h) with an excess of AGE-BSA (100 mg/ml) before adding 106 CFU bacterial

suspension. Internal controls included adhesion of 106 CFU bacterial suspension to wells without immobilized RAGE-Fc chimera and wells with immobilized

human IgG Fc fragment (Panel A). Both RAGE-targeted knock-down on a RAGE-expressing cell line (Panel B) and RAGE full-length transfection on a non-

RAGE-expressing cell line (Panel C) have marked effects on H. pylori adhesion to gastric epithelial cells Infection in both cases were carried out at a MOI of 100,

and adhesion assessed by ELISA. Infection of H. pylori to RAGE-expressing cells (solid bars) also increased RAGE expression which is not observed when

infection was carried out on RAGE-targeted knock-down cells (open bars) (Panel D). *P < 0.05; **P < 0,01.

822 A. Rojas et al. / Microbes and Infection 13 (2011) 818e823

pattern-recognition receptor TLR-4, as a receptor for bindingto gastric epithelial cell [17]. Recently some authors have evenpostulated that RAGE might function as a receptor forMycobacterium tuberculosis [24].

It is known that cellular stress, such as H. pylori infection,may result in the release of RAGE ligands such as 100/cal-granulins and HMGB1 [25]. The activation of RAGE initiatesa complex signaling cascade including nuclear factor kappa B(NF-kB) and mitogen-activated protein kinase (MAPK) path-ways. Additionally, RAGE-mediated cellular stimulationpromotes increased expression of the receptor itself. Thispositive feedback loop, suggests that RAGE functions as aninflammation-perpetuating receptor.

In this context, the finding that H. pylori infection increaseRAGE expression is quite significant considering that

Fig. 3. Effect of RAGE-targeted knock-down on IL-8 production triggered by infect

infection were markedly reduced in RAGE-targeted knock-down (solid bars) cells

increased RAGE expression is strictly associated with ampli-fication of the inflammatory cascade. This notion is particu-larly interesting because several evidences, derived from bothepidemiological studies and basic research, have shown thatorgan-specific carcinogenesis is linked to the development ofa chronic local inflammatory milieu [25,26], as reported forH. pylori-induced gastric inflammation and the occurrence ofgastric cancer.

Currently, it is known that H. pylori infection is associatedwith increased production of interleukin-8 (IL-8) by gastricepithelium, as an inflammatory mediator. The results pre-sented herein demonstrated that RAGE not only contribute tothe adhesion of H. pylori, but also to the inflammatoryresponse of gastric epithelial cells triggered by bacteriabinding.

ion. Both IL-8 transcripts (Panel A) and released protein (Panel B) triggered by

when compared to control cells (open bars).*P < 0.05; **P < 0,01.

823A. Rojas et al. / Microbes and Infection 13 (2011) 818e823

In spite of the exact nature of the interaction betweenH. pylori and RAGE remains to be elucidated, these resultsmay represent new insights into the pathogenesis of H. pyloriinfection.

Because of RAGE expression is markedly increased in dia-betes, these results may explain, at least in part, not only whyH. pylori has raised as a common infection with significantcolonization rates in diabetics, but also the contribution ofhyperglycemia as a cofactor increasing the risk of gastric cancerin patients infected by H. pylori. However, the significance ofthese findings is not only restricted to diabetics, considering thatH. pylori adhesion to gastric epithelium is a crucial event topromote the induction and release of proinflammatorymediators,which in turn, increases RAGE expression and thus resulting inthe perpetuation of inflammation at gastric epithelium.

Acknowledgments

This work was supported by FONDECYT grant 1090340.

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