of June 19, 2018.This information is current as

InfectionHelicobacter pyloriof Th17 Responses during

InhibitionExpression on Gastric Mucosa and CagA-Dependent Downregulation of B7-H2

ReyesE.Yamaoka, David Y. Graham, Ellen J. Beswick and Victor

Taslima T. Lina, Irina V. Pinchuk, Jennifer House, Yoshio

http://www.jimmunol.org/content/191/7/3838doi: 10.4049/jimmunol.1300524August 2013;

2013; 191:3838-3846; Prepublished online 30J Immunol 

Referenceshttp://www.jimmunol.org/content/191/7/3838.full#ref-list-1

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The Journal of Immunology

CagA-Dependent Downregulation of B7-H2 Expression onGastric Mucosa and Inhibition of Th17 Responses duringHelicobacter pylori Infection

Taslima T. Lina,* Irina V. Pinchuk,*,† Jennifer House,‡ Yoshio Yamaoka,x

David Y. Graham,x Ellen J. Beswick,{,1 and Victor E. Reyes*,‡,1

Gastric epithelial cells (GECs) are the primary target for Helicobacter pylori infection and may act as APCs regulating local T cell

responses. We previously reported that H. pylori infection of GECs induces the expression of the T cell coinhibitory molecule

B7-H1 on GECs. This process contributes to the hyporesponsiveness of CD4+ effector T cells and accumulation of regulatory

T cells. In the present study, we investigated the impact of H. pylori cytotoxin-associated gene A (CagA) on the modulation of the

expression of the T cell costimulator B7-H2 by GECs. B7-H2 is involved in promoting Th17 type responses. H. pylori infection

downregulates B7-H2 expression by GECs in a CagA-dependent manner. IFN-g, which is increased in the H. pylori–infected

gastric mucosa, synergizes with H. pylori in downregulating B7-H2 expression by GECs. CagA-mediated modulation of B7-H2 on

GECs involves p70 S6 kinase phosphorylation. The CagA-dependent B7-H2 downregulation in GECs correlates with a decrease in

Th17 type responses in vitro and in vivo. Furthermore, CagA-dependent modulation of Th17 responses was inversely correlated

with the H. pylori colonization levels in vivo. Our data suggest that CagA contributes to the ability of H. pylori to evade Th17-

mediated clearance by modulating expression of B7-H2 and, thus, to the establishment of the H. pylori chronic infection. The

Journal of Immunology, 2013, 191: 3838–3846.

Helicobacter pylori is a Gram-negative, spiral-shapedbacterium that infects the gastric mucosa of .50% ofthe world’s population. H. pylori infection initially

occurs in childhood and becomes persistent. This chronic infec-tion leads to gastric inflammation (1) and is the major cause ofgastritis, gastric and duodenal ulcers, as well as gastric adeno-carcinoma (2–8).The cytotoxin-associated gene A (CagA) protein is a major

virulence factor of H. pylori. Patients infected with cagA+ strainshave higher levels of inflammatory responses and are at a higherrisk of developing peptic ulcer or gastric cancer (4, 9). CagA isencoded by the cagA gene within the cag pathogenicity island,a 40-kb chromosomal region that encodes for a type IV secretionsystem (T4SS).

Gastric epithelial cells (GECs) are the primary target forH. pylori infection. After H. pylori adheres to GECs, the CagA

protein is translocated into their cytosol via a T4SS (10). Once

inside GECs, CagA becomes phosphorylated and elicits multiple

cell responses, including disruption of epithelial tight junctions,

cytoskeleton rearrangement, changes in cellular adhesion proper-

ties and polarity, as well as secretion of proinflammatory media-

tors (11, 12). Despite the marked inflammatory response within

the H. pylori–infected gastric mucosa, the host immune response

is unable to clear H. pylori, resulting in persistent infection and

development of chronic gastric inflammation (13, 14). Studies by

us and others suggested that the imbalance in CD4+ T cells

responses to H. pylori is responsible for the host’s inability to

clear the infection (15–18). Th17 cells, whose hallmark cytokine

is IL-17A, are crucial in the clearance of extracellular bacteria

(19). IL-17A is primarily associated with gastric inflammation

during H. pylori infection and, when chronically present, may

contribute to the inflammation-associated carcinogenesis (19–21).

Alternatively, IL-17A–initiated recruitment of neutrophils is crit-

ical for the clearance of the bacteria (22). Although increased IL-

17A expression is observed during chronic gastric inflammation,

the levels produced are not sufficient to clear the infection. The

mechanisms responsible for the reduced Th17 responses during

the establishment of H. pylori persistence in the gastric mucosa

remain poorly understood. Dendritic cell–mediated skewing of

T cell balance toward suppressive regulatory T cells (Tregs) has

been suggested to be important in the downregulation of Th17 and

the establishment of the H. pylori persistence (23). However, it is

not known whether and how GECs, as primary targets for H. pylori

infection, contribute to regulation of Th17 cell responses during

the establishment of the chronic infection.During H. pylori infection GECs express MHC class II and may

act as local APCs (24, 25). In addition to the recognition of MHC-

bound peptides on APCs by TCR, the outcome of APC/T cell

*Department of Microbiology and Immunology, University of Texas Medical Branch,Galveston, TX 77555; †Department of Internal Medicine, University of Texas MedicalBranch, Galveston, TX 77555; ‡Department of Pediatrics, University of Texas MedicalBranch, Galveston, TX 77555; xDepartment of Medicine, Michael E. DeBakey Vet-erans Affairs Medical Center, Baylor College of Medicine, Houston, TX 77030; and{Department of Molecular Genetics and Microbiology, University of New MexicoHealth Sciences Center, Albuquerque, NM 87131

1E.J.B. and V.E.R. share senior authorship.

Received for publication February 28, 2013. Accepted for publication July 29, 2013.

This work was supported by National Institutes of Health Grants AI68712, DK090090-01,and CA127022; the American Gastroenterological Association Research ScholarAward; National Institutes of Health Grant 1U54RR02614; the University of TexasMedical Branch Clinical and Translational Sciences Award; a University of TexasMedical Branch Sealy Center for Vaccine Development predoctoral fellowship (toT.T.L.); American Cancer Society Grant RSG-10-159-01-LIB; National Institutesof Health Grant 8UL1TR000041; and by the University of New Mexico Clinicaland Translational Science Center.

Address correspondence and reprint requests to Dr. Victor E. Reyes, Department ofPediatrics, University of Texas Medical Branch, Children’s Hospital, 301 UniversityBoulevard, Galveston, TX 77555-0366. E-mail address: vreyes@utmb.edu

Abbreviations used in this article: CagA, cytotoxin-associated gene A; EpCAM,epithelial cell adhesion molecule; GEC, gastric epithelial cell; PMSS1, premouseSydney strain 1; ROR, retinoic acid–related orphan receptor; SS1, Sydney strain 1;Treg, regulatory T cell; T4SS, type IV secretion system; WT, wild-type.

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interactions depends on a second signal provided by engagementof the B7 costimulatory receptor (26). Our laboratory has previ-ously shown that GECs express the classical B7 costimulatorsB7-1 and B7-2, whose expression is increased during H. pyloriinfection (25). We also demonstrated that GECs express B7-H1,whose expression also increases during H. pylori infection andcontributes to the suppression of CD4+ effector T cell activity andupregulation of Tregs (18, 27).B7-H2 (ICOS ligand) is among the newer members of the B7

family of receptors and is known to have a costimulatory function onT cell activity upon binding to its receptor, ICOS (28). Recent studieshave implicated B7-H2/ICOS interaction in Th17 cell development,maintenance, and function (29–31). However, the role of B7-H2 inimmune responses to H. pylori is unknown. Thus, in this study weinvestigated the impact of H. pylori and its major virulence factorCagA on the modulation of B7-H2 as an important regulator ofTh17 cell responses. Our in vitro and in vivo studies showed thatH. pylori causes downregulation of B7-H2 on GECs and this effectdepends on the presence of H. pylori CagA via a process involvingp70 S6 kinase activation. CagA-dependent B7-H2 downregulationon GECs correlated with the decrease in Th17 responses and wasinversely correlated with the level of H. pylori colonization in vivo.Thus, this study points out a novel strategy used by H. pylori toimpair Th17 responses, and this impairment could contribute to theestablishment of persistent infection in the host.

Materials and MethodsAnimals

Female C57BL/6 mice were purchased from The Jackson Laboratory (BarHarbor, ME). Six- to 8-wk-old mice, which were tested negative for in-testinal Helicobacter spp., were used in the experiments. The University ofTexas Medical Branch Institutional Animal Care and Use Committee–approved protocol was followed.

Human tissue and cell lines

GECs were isolated from biopsy specimens as described previously (32).Briefly, biopsy specimens of the gastric antrum were obtained from con-senting patients undergoing gastroesophageal duodenoscopy for variousclinical indications in accordance with Institutional Review Board–approvedprotocol. Patients were considered infected when H. pylori was detected byboth rapid urease testing and histopathology, and for these studies they wereconfirmed to be infected by culture of H. pylori from biopsies. Biopsy tissuewas placed in calcium- and magnesium-free HBSS, supplemented with 5%FCS and penicillin plus streptomycin, and transported immediately to thelaboratory. The tissue was then placed in HBSS containing 0.1 mM EDTAand 0.1 mM DTT and agitated at 37˚C for 15–30 min to remove the mucus.The biopsy tissue was placed in dispase solution (2.4 U/ml; BoehringerMannheim, Mannheim, Germany) and agitated at 37˚C for 30 min witha change of fresh dispase solution after 15 min. The supernatant was col-lected and the cells were pelleted by centrifugation at 2003 g for 5 min. Thecells isolated were mostly (.90%) epithelial cells according to the mor-phology with May–Grunwald–Giemsa staining (Sigma-Aldrich, St. Louis,MO) and flow cytometry with immunofluorescence staining of anticytokeratinmAb. Human gastric carcinoma epithelial cells N87 and AGS as well asHs738.st/int human GECs were obtained from the American Type CultureCollection. HGC-27 was obtained from RIKEN (Japan) and was main-tained in RPMI 1640 with 10% FBS and 2 mM L-glutamine.

Bacterial cultures and infection of GECs

H. pylori LC11 (cagA+) and RD26 (cagA+) strains were originally isolatedfrom duodenal ulcer and peptic ulcer patients, respectively (33, 34).H. pylori strain Sydney strain 1 (SS1) and premouse SS1 (PMSS1) (35)used to infect mice were gifts from Dr. J. Pappo (Astra Research Center)and Dr. Richard Peek (Vanderbilt University), respectively. These bacterialstrains were grown on tryptic soy agar plates supplemented with 5% sheep’sblood (Becton Dickinson, San Jose, CA) or on blood agar plates with2.5 mg/ml chloramphenicol (Technova, Hollister, CA) to maintain cagA2

strains at 37˚C under microaerophilic conditions. Bacteria were transferredafter 48 h into Brucella broth containing 10% FBS for overnight. Aftercentrifugation at 3000 rpm for 10 min, bacteria were resuspended in normal

saline. The concentration of bacteria was determined by measuring theOD530 using a spectrophotometer (DU-65; BD Biosciences) and compar-ing the value to a standard curve generated by quantifying viable organ-isms from aliquots of bacteria at varying concentrations that were alsoassessed by OD and colony formation. For specific multiplicity of infec-tion, the numbers of GECs were determined using trypan blue staining, andthe required number of bacteria was added after calculation. GECs weretreated with IFN-g (100 U/ml) for 48 h, then washed and incubated anadditional day in regular medium without IFN-g before infection. WhenIFN-g needed to be neutralized, anti–IFN-g-neutralizing Ab was added atan optimal concentration (10 mg/ml). As an isotype control, mouse IgG1 kAb was used in the same concentration. B7-H2 expression was measuredafter coculture with IFN-g or H. pylori–infected GECs in the presence ofeither anti–IFN-g-neutralizing Ab or isotype control Ab.

Construction of cagA isogenic mutant

For isogenic cagA mutants, portions of the genes were amplified by PCRand the amplified fragment was inserted into the pBluescript SK(+)(Stratagene, La Jolla, CA). After mutagenesis by insertion of a chloram-phenicol resistance gene cassette (a gift from Dr. D. E. Taylor, Universityof Alberta, Edmonton, AB, Canada) in the cagA gene, the obtained plas-mids (1–2 mg) were used for inactivation of chromosomal genes by naturaltransformation as previously described (36). Correct integration of the chlor-amphenicol resistance gene cassette into the H. pylori chromosome by doublecrossover recombination was confirmed by PCR amplification followed bySouthern blot hybridization.

Abs, recombinant proteins, and cell signaling inhibitors

PE-conjugated anti-human B7-H2 (clone M1H12), PE-conjugated anti-murine B7-H2 (clone HK5.3), allophycocyanin-conjugated anti-murineepithelial cell adhesion molecule (EpCAM; clone G8.8), and PE-conjugatedretinoic acid–related orphan receptor (ROR)gt (clone AFKJS-9) were pur-chased from eBioscience, as were the isotype controls. The viability dyeeFluor 780 (eBioscience, San Diego, CA) was included in the experiments tocontrol cell viability. Human rIFN-g (Roche) was used at 100 U/ml. Neu-tralizing Abs for IFN-g included the purified functional grade anti-humanIFN-g from eBioscience. The isotype control Ab used for IFN-g studies wasfunctional grade mouse IgG1 k from eBioscience. For cell signaling inhi-bition the following inhibitors were used: CAY10512 (10 mM; CaymanChemical, Ann Arbor, MI), AG-490 (100 ng/ml; Enzo Life Sciences,Farmingdale, NY), wortmannin (100 nM; Calbiochem, Billerica, MA),and rapamycin (100 ng/ml; Calbiochem, Darmstadt, Germany).

GEC/T cell coculture

For GEC/T cell coculture experiments, naive CD4+ T cells were isolatedfrom peripheral blood as previously described (17). Briefly, heparinizedvenous blood samples were collected from healthy volunteers negative forH. pylori (Institutional Review Board–approved protocol 06-122 at theUniversity of Texas Medical Branch). PBMCs were prepared from col-lected blood by density gradient centrifugation over Ficoll-Paque Plus.Naive CD4+ T cells were isolated from the peripheral blood by negativeselection using a MACS naive CD4+ T cell isolation kit II (MiltenyiBiotec, Bergisch Gladbach, Germany). GECs where exposed to H. pyloristrains for 8 h, and then supernatants from H. pylori–treated cultures werefiltered to remove bacteria, and GECs were washed twice with PBS toremove attached bacteria and replaced with filtered supernatants until 24 h.CD4+ T cells were preactivated for 1 h with anti-CD3/CD28 beads (Invi-trogen) according to the manufacturer’s instructions and added to each wellat 3:1 T cell/GEC ratio and then incubated at 37˚C with 5% CO2 for 2 d.

Western blot analysis

Western blot analysis was performed as previously described (37).

Infection of mice, processing of stomach, and detection ofH. pylori in murine stomach

Experimental animal groups were inoculated by orogastric gavage with 108

colony-forming units (in 100 ml PBS) of H. pylori SS1 or PMSS1 strainsthree times during a week. The placebo group was inoculated with PBS.Four weeks later, animal serum was collected, mice were euthanized, andstomach was removed. Stomachs were dissected longitudinally in two tofour pieces and used for analysis of the H. pylori load, histopathology, RT-PCR, and flow cytometry analysis. For histopathology analysis one lon-gitudinal strip of stomach was placed in 10% normal buffered formalin for24 h at 4˚C, transferred into 70% ethanol solution, and stored at 4˚C untilneeded. Tissue was then embedded in paraffin and processed by H&E

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staining. Stomach tissue dissociation and enzymatic digestion was per-formed using a gentleMACS dissociator (Miltenyi Biotec, Auburn, CA)according to the manufacturer’s instructions. Briefly, DTT/EDTA treat-ment was performed similarly as described above for human tissue. Cellswere then washed twice with HBSS followed by treating with collagenase

I, II, and IV (100 U/ml). After the first round of dissociation, cells wereincubated with enzymatic mixture at 37˚C for 30 min. Tissue was thensubjected to the second round of dissociation and treated with DNAse (10mg/ml; Worthington Biochemical) at 37˚C for 15 min. Finally, tissue wasprocessed using the dissociator and washed twice with HBSS. The digested

FIGURE 1. Decreased expression of B7-H2 in H. pylori–infected GECs. (A) Gastric biopsy samples were collected from five H. pylori negative and five

positive patients. GECs were isolated and were analyzed for the B7-H2 mRNA expression by real-time RT-PCR. (B) Modulation of the B7-H2 surface

expression on GEC N87 by the infection with H. pylori 51B and LC11 strains (24 h after infection, H. pylori/GEC ratio of 10:1) as determined by flow

cytometry analysis. Upregulation of B7-H1 on N87 cells after treating with H. pylori 51B was shown as a positive control. (C) One of the representative

flow cytometry histograms obtained for GEC N87 infected with H. pylori RD26. (D) Dose-response analysis. N87 cells were infected with H. pylori RD26

strain at different ratio of H. pylori/GECs (10:1, 30:1, and 100:1) for 24 h and analyzed by flow cytometry. The data are expressed as mean fluorescence

intensity (MFI). Data represent the means 6 SD (n = 8). *p , 0.05, **p , 0.01.

FIGURE 2. H. pylori–mediated downregulation of B7-H2 on GEC involves CagA. (A) B7-H2 mRNA expression in N87 cells was analyzed using real-

time RT-PCR. RNA was isolated from uninfected, 2 h, and 4 h H. pylori cagA+ (WT) and cagA2 strain–infected GECs. mRNA level for B7-H2 was

normalized to 18S and compared with the level of B7-H2 mRNA of untreated N87 cells (n = 9). (B) Surface B7-H2 expression was analyzed by flow

cytometry on HS-738 and (C) N87 GEC lines after 24 h of infection with H. pylori cagA+ (WT) or cagA2 mutant. Data are expressed as percentage of

positive cells expressing B7-H2. (D) One representative histogram is shown for GEC N87 infected with H. pylori 51B WTor cagA2 mutant (n = 8). (E) B7-

H2 expression was analyzed by Western blot in N87 cells treated with H. pylori WT and cagA2 strain after 24 h infection. (F) Quantitative analysis of B7-

H2/b-actin ratio in GEC–infected sample is included (n = 3). *p , 0.05, ***p , 0.001.

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tissue was passed through a 40-mm cell strainer. Recovered cells werecounted and used for immunostaining followed by flow cytometry analysis.

Murine gastric tissue was homogenized and DNA was extracted usinga DNeasy blood and tissue kit (Qiagen, Valencia, CA) followed by a pu-rification of the DNA according to the manufacturer’s protocol. ExtractedDNA was used for the detection of H. pylori by real-time PCR usinga protocol originally described by Roussel et al. (38). A primer/probe set16SHP229BP for 16S gene was used for the quantification of the H. pyloribacterial load. To determine bacterial load, standard curves were generatedby PCR of serial dilutions of extracted H. pylori DNA. Quantification ofH. pylori in murine gastric mucosa and determination of absolute genomecopy number were calculated according to the method described by Rousselet al. (38). Murine GADPH gene amplification was used to control the equalloading of total DNA used in the PCR reaction.

Flow cytometry

Flow cytometry was performed as we described previously (17).

Real-time RT-PCR

Real-time RT-PCR was performed according to the a two-step real-timeRT-PCR protocol (Applied Biosystems, Foster City, CA). The appropri-ate Assays-on-Demand gene expression assay primers/probe mix (AppliedBiosystems) for human 18S and gene of interest (a 203 mix of unlabeledPCR primers and TaqMan MGB probe, FAM dye–labeled) and 2 ml cDNAwas added to the PCR reaction step. The reactions were carried out in 20ml final volume using a Bio-Rad Q5 real-time PCR machine according tothe following protocol: 2 min at 50˚C, 10 min at 95˚C (1 cycle) and 15 s at95˚C, and 1 min at 60˚C (40 cycles). Triplicate cycle threshold values wereanalyzed in Microsoft Excel using the comparative CT (DDCT) method asdescribed by the manufacturer (Applied Biosystems).

Bio-Plex

The levels of total and phosphorylated cell signaling proteins in N87 cellsinfected with H. pylori strains—IL-17A from T cell/GEC coculture andIL-17, IL-21, IL-22, IL-23, and IL-6 from murine serum collected fromH. pylori–infected mice—were measured using a Luminex array (Millipore,Billerica, MA) according the manufacturer’s instructions. Samples wereanalyzed using Bio-Plex Manager software (Bio-Rad).

Statistical analysis

The results were expressed as the means 6 SD of data obtained from atleast three independent experiments done with triplicate sets in each ex-periment, unless otherwise indicated. Differences between means wereevaluated by ANOVA using a Student t test for multiple comparisons. Thep values , 0.05 were considered statistically significant.

ResultsH. pylori downregulates B7-H2 expression on GECs

To assess B7-H2 expression during H. pylori infection, mRNAlevels were examined in GECs isolated from gastric mucosa biopsysamples from H. pylori–infected and uninfected individuals usingreal-time RT-PCR. B7-H2 mRNA expression was significantly de-creased in GECs from H. pylori+ subjects when compared withGECs from uninfected controls (Fig. 1A). To determine whetherH. pylori directly induced a reduction of B7-H2 by GECs, a humanGEC line (N87) was infected with H. pylori 51B, LC-11, or RD26strains. A significant decrease in surface B7-H2 expression on GECswas observed at 24 h after infection with all H. pylori strains (Fig.1B, 1C). Fig 1B shows differential regulation of B7-H1 and B7-H2by N87 cells infected with H. pylori 51B. This effect was dose-dependent (Fig. 1D). Similar results were observed when other hu-man GEC lines (e.g., AGS, HGC-27, and HS-738 cells) were infected(not shown). Thus, our results indicate that H. pylori infectiondownregulates B7-H2 expression on human GECs in a dose-dependent manner.

Downregulation of B7-H2 expression depends on the presenceof H. pylori CagA

Because CagA is an importantH. pylori virulence factor capable ofeliciting multiple host cell responses (39), we sought to determine

whether downregulation of B7-H2 is influenced by H. pylori CagA.Infection of GECs with H. pylori 51B wild-type (WT) strain (Fig.2A) led to as much as 50% decrease of B7-H2 mRNA as comparedwith uninfected controls at the time points examined (2, 4 h). Incontrast, the H. pylori cagA2 mutant had a very limited effect onB7-H2 expression compared with the controls. These data wereconfirmed at the protein level. In contrast to the cagA+ H. pyloristrain, infection with the cagA2 mutant had a minor effect on thereduction of B7-H2 surface expression by GECs (Fig. 2B–D).Western blot analysis of N87 cells treated under the same conditionsprovided an independent approach to validate decreased B7-H2protein levels in cells infected with H. pylori WT as comparedwith cells infected with cagA2 strain (Fig. 2E, 2F). These resultssuggested that the major H. pylori virulence factor CagA is in-volved in the B7-H2 downregulation on GECs.

IFN-g and H. pylori have synergistic effects on B7-H2downregulation

IFN-g is produced within the H. pylori–infected gastric mucosa(40), and previously we showed a synergistic effect of IFN-g andH. pylori on B7-H1 upregulation on GECs (18). Thus, we ex-amined whether IFN-g could modulate H. pylori–mediated B7-H2downregulation on GECs. N87 cells treated with either IFN-g orH. pylori alone showed significant decreases in B7-H2 expression.However, treatment with both IFN-g and H. pylori resulted incomplete abrogation of B7-H2 expression. B7-H2 expression de-creased after culturing GECs with IFN-g, and decreased expres-sion was more prominent when GECs were pretreated with IFN-gprior to infection with H. pylori (Fig. 3). Blocking the IFN-g withneutralizing anti–IFN-g mAb prevented the decrease in the levelsof B7-H2 expression. Our results clearly showed a synergisticeffect of IFN-g and H. pylori in reduced B7-H2 expression byGECs. Interestingly, the synergism of IFN-g and H. pylori in de-creasing B7-H2 expression was less obvious when cells wereinfected with a cagA2 strain (Fig. 3). This result supports a keyrole of CagA in B7-H2 downregulation during H. pylori infection.

FIGURE 3. IFN-g synergizes H. pylori–mediated B7-H2 downregula-

tion on gastric epithelium. N87 cells were treated with IFN-g (100 U/ml)

for 48 h. Cells treated with IFN-g were washed and cultured with medium

for 24 h before they were either infected with H. pylori 51B WT and

cagA2 strain in the presence or absence of IFN-g–neutralizing Ab. B7-H2

expression on N87 GECs was measured by flow cytometry after 24 h.

Graph represents the mean 6 SD (n = 8). *p , 0.05.

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B7-H2 downregulation involve activation of mTOR/p70 S6kinase

Previous studies showed that H. pylori CagA protein can activateNF-kB, MAPK, STAT3, and PI3K pathways (41–44). To under-stand the underlying mechanisms regulating B7-H2 decreasedexpression during H. pylori infection, we first globally analyzedpathways activated in H. pylori–infected GECs using a Luminexcell signaling array. Our data demonstrated that, in addition to NF-kB and STAT3 pathways, H. pylori infection of GECs also leadsto the activation of mTOR/p70 S6 kinase within the first 5 min ofinfection (Fig. 4A, 4B). In contrast, no significant phosphorylationof p70 S6 kinase was observed in GECs infected with cagA2

strain (Fig. 4C, 4D). Thus, we examined the role of these path-ways in H. pylori–mediated downregulation of B7-H2 by usingspecific inhibitors. We observed that downregulation of B7-H2 by

the cagA+ H. pylori strain was blocked in the presence of rapa-mycin, a p70 S6 kinase/mTOR–specific inhibitor (Fig. 4E). In con-trast, inhibition of PI3K, STAT3, and NF-kB pathways withpharmacological inhibitors did not affect H. pylori–mediateddownregulation of B7-H2 expression (Fig. 4F). These results sug-gest that p70 S6 kinase is a key signaling pathway in H. pylori–mediated downregulation of B7-H2 on GECs.

CagA+ H. pylori infection reduces induction of Th17 by humangastric epithelium

Because B7-H2 has been implicated in Th17 cell differentiation,we examined whether the CagA-dependent B7-H2 downregulationimpaired the capacity of GECs to induce Th17 cell differentiationfrom naive CD4+ T cells. Our results showed that there is a smallinduction of RORg expressing CD4+ T cells when naive CD4+

FIGURE 4. Activation of mTOR/p70 S6 kinase involved in the H. pylori–mediated downregulation of B7-H2 expression. N87 cells were incubated with

H. pylori 51B WT and cagA2 strain. Cells were lysed after 5, 15, 30, 45, and 60 min. Cell lysates from GECs exposed to H. pylori 51B WT and cagA2

strain were analyzed for (A, C) phosphorylated and (B, D) total p70 S6 kinase using Luminex bead arrays, respectively. (E) GECs were treated with

rapamycin (100 ng/ml), an inhibitor of mTOR/p70 S6 kinase pathway, or (F) with wortmannin (100 nM), AG-490 (100 ng/ml), and CAY10512 (10 mM)

inhibitors of PI3K, STAT3, and NF-kB pathways, respectively, for 1 h and then were infected with H. pylori. B7-H2 expression was analyzed using flow

cytometry 24 h later. Data are expressed as a percentage of positive cells. Results represents as the means 6 SD (n = 8). *p , 0.05, **p , 0.01.

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T cells were cocultured with N87 cells infected with H. pylori WT,but this induction was significantly increased when cells wereinfected with a cagA2 strain (Fig. 5A). The presence of Th17 wasfurther confirmed by measuring IL-17A in coculture supernatantsby a Luminex array (Fig. 5B). Analysis of mRNA levels inparallel cultures confirmed these findings by showing increasedRORg and IL-17A mRNA levels in CD4+ T cells (Fig. 5C, 5D).These data demonstrate the critical role of CagA in maintaininglow-level Th17 responses, and this may contribute to H. pyloriimmune evasion.

Downregulation of B7-H2 on GECs during murine H. pyloriinfection depends on CagA and correlates with the decrease ofTh17 responses and increase in H. pylori colonization

To understand the relevance of our observations in vitro to theH. pylori–associated immunopathophysiology, we used a mousemodel of H. pylori infection. Mice infected with H. pylori PMSS1,which contains a functional T4SS, had downregulated B7-H2 ex-pression by gastric epithelial (EpCAM+) cells. Interestingly, infec-tion of mice with H. pylori SS1 strain in which the T4SS is defectiveand cannot deliver CagA in GECs (45) resulted in upregulation ofB7-H2 (Fig. 6A, 6B). No significant difference was observed ingastric mucosal inflammation in mice infected with either strain atweek 4. However, the H. pylori load was drastically different be-tween the mice infected with SS1 and PMSS1 strains. Mice infectedwith PMSS1 strain had .100-fold higher bacterial burden whencompared with those infected with SS1 strain (Fig. 6C). Further-more, analysis of serum cytokine profile demonstrated that in con-trast to the mice infected with SS1 strain, which had significantserum levels of IL-17 and IL-21 (p , 0.05), mice infected withPMSS1 strain failed to upregulate proinflammatory Th17 cytokines(Fig. 7). Serum levels of IL-6, IL-22, and IL-23 were also increasedsignificantly inH. pylori SS1–infected mice (Fig. 7). Taken together,our in vivo data correlate with our in vitro findings and suggest thatCagA-mediated downregulation of B7-H2 might be involved in theprevention of the Th17-mediated clearance of the H. pylori duringonset of the infection.

DiscussionDuring H. pylori infection the host mounts an immune response,but this response is insufficient to clear the infection, leading tothe establishment of a persistent infection and development ofchronic inflammation. Infiltration of CD4+ T cells into the gastricmucosa is among the major factors contributing to the ongoinginflammation. At the same time, these cells are required for theimmunization-induced protective responses (46–48). Recent datasuggested that Th17 type responses are required for the clearanceof the bacteria (49). The exact mechanisms implicated in theH. pylori–mediated escape of host immunity to prevent clearanceof H. pylori remain far from understood. In this study, we dem-onstrated that H. pylori infection of GECs leads to a decrease ofB7-H2, which is a positive costimulatory ligand, and that thisprocess might be important in H. pylori–mediated escape of Th17-mediated bacterial clearance.Costimulatory interactions between B7 family ligands and their

receptors play important roles in the growth, development, anddifferentiation of T cells. Recent data demonstrated that inter-actions between B7-H2 on APCs with its putative receptor ICOSon T and B cells regulate adaptive immune responses (30, 50).Stimulation of ICOS was demonstrated to be critical for the de-velopment of human IL-17–producing CD4+ T cells (31). Fur-thermore, Bauquet et al. (29) demonstrated that ICOS was criticalfor maintaining effector memory Th17 cells. B7-H22/2 knockoutmice also were noted to have lower Th17 responses to chlamydialinfection than do WT mice (51). The blockade of B7-H2/ICOSsignaling inhibited Th1 and Th17 cells responses in chronic in-flammatory conditions such as rheumatoid arthritis (30). Takentogether, these findings implicate B7-H2/ICOS signaling as animportant mediator in the activation of Th17 cells in inflamma-tion. Because expression of ICOS ligand, B7-H2, was previouslyreported on intestinal epithelial cells (52), and owing to its im-portance in activation of Th17 responses, we measured the ex-pression of B7-H2 in human biopsy samples and found thatepithelial cells in gastric biopsies isolated from H. pylori–infectedpatients had decreased levels of B7-H2 expression compared with

FIGURE 5. H. pylori–mediated Th17 devel-

opment from activated naive T cells in coculture

with N87. N87 cells were treated with either

media, with H. pylori WT, or with H. pylori

cagA2 strain (H. pylori/GEC ratio of 10:1) for

8 h. After treatment, the GECs were washed

and cocultured with preactivated human CD4+

T cells (3:1 T cell/GEC ratio) for 2 d and were

analyzed for (A) RORg expression in T cells by

flow cytometry, (B) IL-17A production in su-

pernatant by Luminex array, (C) RORg mRNA

levels, and (D) IL-17A mRNA levels in T cells

by real-time PCR. mRNA was normalized to

18S and compared with untreated cells (n = 6).

*p , 0.05, **p , 0.01.

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uninfected biopsy samples. B7-H2 expression on colonic andairway epithelium was previously noted (52). However, little isknown about the role of B7-H2 costimulation in the responsesassociated with bacterial immunopathogenesis and clearance. Inthe present study, we demonstrated for the first time, to our knowl-edge, that H. pylori significantly downregulated B7-H2 expression ingastric mucosa, particularly on GECs.Because CagA has been shown to play an important role in

H. pylori–mediated pathogenesis and immune evasion mechanisms(53), we sought to investigate its role in the observed B7-H2 down-

regulation. In this study using a CagA isogenic mutant and theircorresponding parental strains, we showed that CagA plays acrucial role in downregulating B7-H2 expression on GECs.Compared with the untreated cells, H. pylori cagA2 strains alwaysshowed some downregulation of B7-H2, suggesting that othercomponents of H. pylori might also have an influence in down-regulating B7-H2 expression, but they are less effective than theWT CagA+ strains. Our in vivo data using C57BL/6 mice alsoshowed that H. pylori mediated transfer of CagA via a T4SSsignificantly downregulates B7-H2 expression in the GECs in the

FIGURE 6. H. pylori–mediated downregulation of B7-H2 on GECs in vivo involves CagA function and is inversely correlated with bacterial clearance.

(A) C57BL/6 mice were challenged with H. pylori strain PMSS1, which express functional T4SS and can deliver CagA, or with H. pylori SS1, which does

not. Gastric mononuclear cells were isolated 4 wk after H. pylori challenge using enzymatic digestion, and expression of B7-H2 and epithelial cell marker

EpCAM was analyzed by flow cytometry. (B) Level of the B7-H2–expressing epithelial cells (EpCam+) in the gastric mucosa from the cells was measured

by flow cytometry. (C) Infection rate was determined by quantification of H. pylori genome copy per half of stomach based on the analysis of H. pylori 16S

gene amplification by real-time PCR. Each datum point represents a single mouse tested in quadruplicate. Average bars of infection rate were calculated

from five mice per group and are demonstrated as means 6 SD. *p , 0.05.

FIGURE 7. In vivo infection with H. pylori expressing functional T4SS and that can deliver CagA fails to upregulate Th17 type responses. C57BL/6

mice were challenged with H. pylori strain PMSS1 or with H. pylori SS1. Blood was collected 4 wk after H. pylori challenge, and cytokine profile was

analyzed using a Luminex bead array. Data are represented as means 6 SD (n = 12). *p , 0.05.

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murine gastric mucosa. These in vitro and in vivo data reveal anovel mechanism whereby H. pylori uses one of its importantvirulence factors, CagA, to create a favorable environment for itspersistence via suppression of the positive costimulators requiredfor an efficient effector T cell response.Because cytokines play an important role in regulating immune

function, and IFN-g has been detected in H. pylori–infected gas-tric tissues in both humans and mice (54), we also investigatedwhether IFN-g has any role in B7-H2 expression. Our resultsshowed that IFN-g synergizes with the effect of H. pylori cagA+

strains in downregulating B7-H2 expression by GECs. Severalstudies showed induction of B7-2, B7-H1, and B7-DC in differentclassical APCs by IFN-g (55, 56). Stanciu et al. (57) showed asynergistic effect of respiratory syncytial virus and IFN-g in theupregulation of B7-H1 and B7-DC in respiratory epithelial cells.Additionally, their study showed that treatment of respiratory syn-cytial virus infected cells with IFN-g causes downregulation ofB7-H2 and B7-H3 expression. Previous findings from our groupshowed that IFN-g and H. pylori synergize in B7-H1 upregulation(18). Thus, the synergistic effect of IFN-g and H. pylori in B7-H2downregulation could result from an IFN-g–mediated increase inexpression by GEC receptors that are used by H. pylori (32).CagA interacts with several intracellular components of signal

transduction and activates NF-kB, MAPK, STAT3, and PI3K/Aktpathways (41–44). Previous reports have highlighted the fact thatH. pylori activates STAT3 to modulate host immune responses(43). Although our results showed activation of these pathwaysby CagA-expressing H. pylori strains, our data imply that CagA-mediated activation of the NF-kB, STAT3, and PI3K pathways isnot required for the H. pylori–mediated downregulation of B7-H2on GECs. Furthermore, to our knowledge, our data demonstratefor the first time that CagA contributes to the H. pylori–mediatedactivation of the mTOR/p70 S6 kinase pathway. Serine/threonineprotein kinase mTOR acts in a signaling pathway downstream fromPI3K/Akt and regulates the activation of the p70 S6 kinase, whichis required for translational regulation of ribosomal proteins (58).Using specific cell signaling inhibitors we showed that H. pyloriuses CagA to manipulate B7-H2 expression by activating p70 S6kinase pathway to prevent GECs from providing positive costim-ulation needed for protective Th17 cells.Previously our group showed that H. pylori uses its CagA and

VacA proteins to induce TGF-b production from GECs, whichcauses inhibition of CD4+ effector T cell proliferation and in-duction of Tregs (17). In this study, using in vitro GEC/T cellcocultures we showed that H. pylori uses CagA cytotoxin todownregulate Th17 cell type responses. A significant downregu-lation of Th17 cell transcription factor RORgt and IL-17A wasobserved when GECs were exposed to the H. pylori strainsexpressing CagA, but not in presence of the cagA2 mutant. Ourin vivo data with the H. pylori mouse model support this in vitroobservation. We have further shown that H. pylori CagA-mediatedB7-H2 downregulation correlates with a decrease in Th17 typeresponses detected in murine serum and an increase in H. pyloricolonization of the gastric mucosa. Although our data showedincreased H. pylori bacterial load and decreased Th17 type re-sponse in PMSS1-infected mice compared with those infectedwith the SS1 strain, we did not observe severe inflammatory changesin any of the H. pylori–infected mice. This might be becauseH. pylori infection in the mouse model results mostly in lympho-cytic gastritis, which does not progress to severe inflammation. Anoptimum induction of chronic gastritis can be achieved using Heli-cobacter felis as a model (59). Th17 cells have been suggested tohave dual roles in both infection control on the one hand and pre-neoplastic changes on the other hand. Several studies showed that

protective immunity against H. pylori infection requires a strongTh17 response (60). Our study showed reduced Th17 cell cyto-kines and increased bacterial load in the PMSS1-infected micecorrelate with results from another recent study (49). Horvathet al. (49) showed that mice lacking IL-23 when infected withH. pylori showed reduced IL-17 production and increased bacte-rial load in their stomachs. Another study suggested that ICOS-induced signaling is essential for IL-21–mediated regulation ofIL-23R expression in differentiated Th17 cells and for IL-23–driven expansion of Th17 cells (29). Our study also supports theimportance of B7-H2/ICOS signaling in Th17 cell developmentbecause the downregulation of B7-H2 expression in the gastricmucosa of PMSS1 H. pylori–infected mice correlates with de-creased Th17 type responses.Our data suggest a novel CagA-dependent mechanism, which

involves downregulation of B7-H2 on GECs, a primary target forH. pylori infection, used by the bacteria to avoid a Th17 cell–mediated clearance. Thus, our in vitro and in vivo studies suggestthat H. pylori use the T4SS to downregulate Th17 cell responses,which are critical for clearing the pathogen. Therefore, the H.pylori CagA delivery system may be an important target in vaccinedevelopment for achieving acceptable levels of immune protectionand for designing a therapeutic strategy to treat patients infectedwith this prevalent and deadly human pathogen.

AcknowledgmentsWe thank technical assistant Yu Lin for excellent assistance in performing

the animal work.

DisclosuresThe authors have no financial conflicts of interest.

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