Helicobacter pylori Infection Impairs the Mucin Production Rate andTurnover in the Murine Gastric Mucosa

Nazanin Navabi,a,b Malin E. V. Johansson,b Sukanya Raghavan,a,c Sara K. Lindéna,b

Mucosal Immunobiology and Vaccine Centre,a Department of Medical Biochemistry and Cell Biology,b and Department of Microbiology and immunology,c University ofGothenburg, Gothenburg, Sweden

To protect the surface of the stomach, the epithelial cells secrete a mucus layer, which is mainly comprised of the MUC5AC mu-cin. Further protection is provided by a thick glycocalyx on the apical surface of the epithelial cell, with the cell surface mucinMUC1 as a major component. Here, we investigate the production rate and turnover of newly synthesized mucin in mice andanalyze the effects of early colonization and chronic infection with H. pylori. Metabolic incorporation of an azido GalNAc ana-log (GalNAz) was used as a nonradioactive method to perform pulse experiments in the whole animal. First, the subcellularmovement of newly synthesized mucin and mucin turnover was determined in uninfected mice. Based on the time line for mucintransport and dissemination, 2, 6, and 12 h after GalNAz injection was selected to collect the stomachs from mice infected withH. pylori strain SS1 during early colonization (7 days) and chronic infection (90 days). The results demonstrated that the speedfrom the start of glycosylation to the final destination is faster for the membrane-bound mucin to reach the glycocalyx (2 h) thanfor the secretory mucins to become secreted into the mucus layer (5 h). Furthermore, infection with H. pylori reduces the rate ofmucin turnover and decreases the levels of Muc1. Since H. pylori colonizes this mucus niche, the decreased turnover rate indi-cates that H. pylori creates a more stable and favorable environment for itself by impairing the defense mechanism for clearingthe mucosal surface of pathogens by mucus flow.

Helicobacter pylori is a Gram-negative bacterium infecting halfof the world’s population. Despite the fact that most of the

individuals infected with this bacterium are asymptomatic, H. py-lori can cause gastric and duodenal ulcers and mucosally associ-ated lymphoid tissue lymphoma in a subset of infected individuals(1, 2).

In order to protect the stomach from invading pathogens, theepithelial cells of the mucosal surface constantly secrete a mucuslayer, which is mainly comprised of the secreted mucin MUC5AC.MUC5AC is produced by the surface epithelium and another se-creted mucin, MUC6, by the glands (3–6). The cell surface mucinMUC1 is a major component of the gastric glycocalyx, which pro-vides a protective barrier on the apical surface of epithelial cells (7)and may initiate a signaling pathway in response to invasion (8).Mucins are heavily glycosylated glycoproteins consisting of a pro-tein backbone with a large number of O-linked glycosaccharidesand a few N-glycan chains. Mucins are secreted via both constitu-tive and regulated pathways. The constitutive pathway causes acontinuous secretion of mucin to maintain the mucus layer wash-ing the mucosal surface, whereas the regulated pathway affords amassive discharge as a response to environmental and/or patho-physiological stimuli (9). Mucin release is accompanied by hydra-tion, resulting in approximately a thousand-fold expansion in vol-ume of the secretory granule contents (10).

H. pylori can bind to both MUC1 and MUC5AC via fucosy-lated and sialylated glycans in a pH-dependent manner (11,12). In the rhesus monkey model of H. pylori infection, mon-keys with mucins that bind to H. pylori more effectively have alower H. pylori density in their stomachs, indicating that mucinbinding to H. pylori aids in removing the bacteria from thegastric niche (13). Binding of H. pylori to MUC1 results inrelease of the extracellular domain of the mucin from the epi-thelial surface, thereby acting as a releasable decoy and pre-venting prolonged adherence (14). Mucin glycosylation

changes the ability of H. pylori to adhere to the mucins and isdependent on the time point of infection (13). During the acutephase of H. pylori infection in adult patients, there is an infil-tration of neutrophils, followed by all classes of inflammatorycells, predominantly lymphocytes, accompanied by transientgastric hypochlorhydia. However, in chronic infection, whilethere is also inflammatory cell infiltration (predominantlylymphocytic), this is accompanied by local gastric inflamma-tion (15). The time course of gastric mucin turnover and even-tual alteration of this process during acute and chronic phasesof infection with H. pylori is currently unknown. In this study,we used in vivo metabolic labeling to evaluate the productionrate and turnover of mucins in the stomach of noninfectedmice, and we compared it to mice with early colonization orchronic H. pylori infection. We also investigated the differencesin the levels of Muc1 and Muc5ac.

MATERIALS AND METHODSAnimals. Six- to 8-week-old, specific-pathogen-free, female C57BL/6mice were purchased from Taconic (England) or Charles River (Ger-many). The mice were housed in individually ventilated cages at the Lab-oratory for Experimental Biomedicine (EBM) for the duration of thestudy. The animals had free access to water and food throughout theexperiment and were monitored daily. All experimental procedures were

Received 19 September 2012 Returned for modification 15 October 2012Accepted 18 December 2012

Published ahead of print 28 December 2012

Editor: B. A. McCormick

Address correspondence to Sara Lindén, sara.linden@gu.se.

Copyright © 2013, American Society for Microbiology. All Rights Reserved.

doi:10.1128/IAI.01000-12

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approved by the ethics committee for animal experiments (Gothenburg,Sweden).

Intraperitoneal GalNAz injection and tissue fixation. A total volumeof 0.5 ml per mouse of Click-iT reagent was prepared by dissolving 2.6 mgof the Click-iT reagent GalNAz (Invitrogen) in 50 �l dimethylsulfoxide(DMSO) and was diluted with 6 mg/ml bromodeoxyuridine (Sigma-Al-drich) in phosphate-buffered saline (PBS; 0.15 mol/liter NaCl, 5 mmol/liter sodium phosphate buffer, pH 7.4). The injections were given intra-peritoneally during the dark period. For the basal turnover study, a totalnumber of 24 mice (from Taconic, England) were used, and 2 mice weresacrificed every hour for 12 h. Although only two mice were harvestedeach hour, the location of newly synthesized mucin was consistent be-tween these two mice, and the incremental movement of newly synthe-sized mucin from one hour to the next was small. Furthermore, the resultsfrom the mice in the noninfected control group in the infection study(below) confirmed the consistency and reproducibility of the method. Forthe infection study, 18 mice (from Charles River, Germany) were sacri-ficed 2, 6, and 12 h after GalNAz injection. To keep the secreted mucuslayer as undisturbed as possible, the entire glandular part of the stomach(containing luminal material), along with a small piece of duodenum,were fixed in Carnoy’s methanol (60% dried methanol, 30% chloroform,10% glacial acetic acid).

Cultivation of H. pylori used for infection. The mouse-adapted H.pylori SS1 strain, stored at �70°C as aliquots in Luria-Bertani mediumcontaining 20% glycerol, was used as the stock culture. The bacteria weregrown for 3 days on Columbia iso-agar (Becton, Dickinson [BD]) platesand further cultured overnight in Brucella broth (BD) under microaero-philic conditions. Before infecting the mice, the optical density (OD) ofthe bacteria was adjusted to 1.5, corresponding to approximately 1 � 109

viable bacteria/ml.Infecting the mice. Mice were infected with 3 � 108 live H. pylori SS1

bacteria in 300 �l Brucella broth, administered orally using a feedingneedle under anesthesia, corresponding to approximately 100 times theminimal effective dose of colonization to ensure that all mice were colo-nized. For the mucin turnover study, a total of 18 mice were divided intothree groups: noninfected, early colonization with H. pylori (sacrificed 7days postinfection), and chronic H. pylori infection (sacrificed 90 dayspostinfection). As it was necessary to fix the entire stomach without open-ing it to keep the mucus layer intact, we determined the number of H.pylori organisms in the murine stomachs in a separate set of experiments.

Quantitative culture of H. pylori SS1 from the stomach. To evaluatebacterial colonization, one-half of each stomach was homogenized inBrucella broth using a tissue homogenizer (Ultra Turrax; IKA LaboratoryTechnologies, Staufen, Germany). Serial dilutions of the homogenateswere plated on BD modified Helicobacter agar (BD). After at least 7 days ofincubation at 37°C under microaerophilic conditions, visible colonieswith typical H. pylori morphology were counted, and the urease test wasperformed for any uncertain colonies. Plates with 10 to 100 colonies wereused for calculating the number of bacteria per stomach by multiplying bythe appropriate dilution factor.

Histological scoring of mouse stomach infected with H. pylori. He-matoxylin and eosin (H&E) staining was performed on fixed (Carnoy’smethanol) paraffin-embedded samples containing the full length of lon-gitudinally cut sections of the gastric specimen, starting at the forestom-ach and ending at the duodenum. A whole longitudinal section for eachmouse in all groups was examined for scoring of gastritis. Gastritis scoringfor corpus was performed from the limiting ridge of the stomach to thejunction between corpus and antrum, whereas scoring for antrum wasperformed on the segment from the junction between antrum and corpusto the junction between duodenum and antrum. The blinded gastritisscores were obtained based on the level of cellular infiltration, number ofabscesses, mucus metaplasia, and atrophy. The cellular infiltration (mi-gration of lymphocytes and neutrophils) was graded from 0 to 6, where 0is none; 1 is mild multifocal; 2 is mild widespread or moderate multifocal;3 is mild widespread and moderate multifocal, or severe multifocal; 4 is

moderate widespread; 5 is moderate widespread and severe multifocal;and 6 is severe widespread. In control uninfected mice, we could detect afew infiltrating immune cells, which were accepted as the baseline forscoring. The total number of abscesses was counted (including very smallborderline ones) in a full-length section in each animal and expressed as agrade from 0 to 3, where 0 is �8 abscesses, 1 is 9 to 15 abscesses, 2 is 16 to25 abscesses, and 3 is �25 abscesses. Mucus metaplasia and atrophy weregraded from 0 to 3, corresponding to absent, mild, moderate, and severe.

Fluorescent detection of GalNAz, Muc5ac, Muc1, and H. pylori infixed tissue. Carnoy’s fixed paraffin-embedded 5-�m sections weredewaxed, hydrated, and washed in PBS. Tissue sections were incubatedwith 40 �l of the reaction mix from the tetramethylrhodamine(TAMRA) glycoprotein detection kit (Invitrogen) and incubated atroom temperature for 2 h. After washing with PBS, samples wereblocked in 5% fetal bovine serum in PBS containing 0.05% Tween 20.Muc5ac was detected using the 45M1 antibody (1/1,000) (Sigma) andvisualized by an anti-mouse Alexa Fluor 488 conjugate (1/100) (Invit-rogen). Muc1 was detected using the CT2 antibody (1/200) (kind giftfrom Sandra Gendler, the Mayo Clinic, Scottsdale, AZ) followed byDonkey anti-Armenian hamster (1/200) (Jackson) and anti-donkeyAlexa Fluor 488 (1/100) or Alexa Fluor 647 (1/100) (Invitrogen). ForH. pylori detection, rabbit anti-H. pylori serum (1/1,000) (kindly pro-vided by Thomas Borén, Umeå University, Sweden) was visualizedusing anti-rabbit Alexa Fluor 405 (1/100) (Invitrogen). Fetal bovineserum (5%) in PBS was used for dilution of antibodies. Sections weremounted using Prolong antifade mounting media (Invitrogen). AZeiss LSM 510 META microscope was used for image acquisition andprocessing. Quantification of integrated fluorescence density was ob-tained using the Image J program, which is a public domain image-processing program (Wayne Rasband at the Research Services Branchof the National Institute of Mental Health, National Institutes ofHealth, Bethesda, MD). Briefly, the specimen of each mouse was stud-ied by outlining the surface and neck cells at the beginning of thecorpus and antrum. Care was taken to make sure it was always the samearea measured, as we detected some variations in mucin turnover de-pending on tissue localization. The threshold for each stain was ad-justed to the same level for all samples. The measurement was then setfor area, integrated density (the total intensity of fluorescence in thedefined area), and mean gray value.

Immunohistochemistry for Muc5ac. Rodent decloaker (BiocareMedical) was used for antigen retrieval at 80°C for 2 h. Sections were thentreated with 3% (vol/vol) hydrogen peroxide, washed, and blocked byrodent block M (Biocare Medical) for 30 min. The primary antibody(45M1) was diluted in antibody diluent (1:1,000; Dako) and incubated for1 h. The sections then were incubated with MM HRP-Polymer (BiocareMedical) for 20 min. Bound antibody was visualized with diaminobenzi-dine, and the sections were counterstained with Harris’ hematoxylin.

Statistical analysis. All statistical analyses were performed using one-way analysis of variance (ANOVA) with Dunnett’s post hoc test in theGraphPad Prism program (GraphPad Software).

RESULTSProduction rate and turnover of mucin in the noninfected mu-rine stomach. To obtain the basal production rate and turnover ofmucin in the murine stomach, the azido GalNAc analog GalNAzwas intraperitoneally injected, and samples were collected everyhour for 12 h. Previous experiments have demonstrated efficientincorporation of GalNAz into mucin O-glycans in cell culture andin the mouse intestine during biosynthesis (16, 17). Incorporationof GalNAz was much more prominent in the surface epitheliumthan in the glands, therefore we focused the analysis on the surfaceepithelium. The images from gastric specimens gathered in ourexperiment demonstrated metabolic incorporation of GalNAzinto newly synthesized glycoproteins from the first hour of injec-

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tion. At this time point, the newly synthesized mucins were local-ized at the supranuclear part of the surface epithelial cells(Fig. 1A), in the area where the Golgi apparatus previously hasbeen shown to be located. During the second and third hours, themucins moved through the cells toward the cell surface (Fig. 1B).Five hours after injection, some newly synthesized mucins weredetected on the cell surface, whereas the majority remained insidethe cell (Fig. 1C). After 6 h, a few cells had secreted their newlysynthesized mucins completely into the mucus layer, although themucins could still be detected inside the majority of the cells.During the time points 6 to 12 h postinjection, the proportion ofcells having released their newly synthesized mucins into the mu-cus layer increased with time (Fig. 1D, E, F, and G). However,some cells still had not released their newly synthesized mucinsafter 12 h.

Furthermore, our results demonstrate that after glycosylation,the membrane-bound mucin reached the glycocalyx (i.e., finaldestination) in less time than that required for the secretory mu-cins to become secreted into the mucus layer (Fig. 2A and B).Incorporation of GalNAz into the glycocalyx was first detected at 2h (Fig. 2A and B), whereas the first weak traces of GalNAz in thesecreted mucus layer appear after 5 h (Fig. 1C), and full colocal-ization of newly synthesized mucin with secreted Muc5ac was ob-vious at 10 h after GalNAz injection (Fig. 1E and G). In the fluo-rescent images, the colocalization of Muc5ac and newlysynthesized mucin inside the cells is not as obvious in all picturesas the colocalization in the mucus layer is. This can be explained bythe higher intensity of the fluorescence signal in the newly synthe-sized mucin, as well as a lower signal intensity from intracellularMuc5ac than that from Muc5ac in the mucus layer. Together, this

FIG 1 Confocal images of stomach sections from uninfected mice injected intraperitoneally with GalNAz. (A to F) The sections were collected every hour duringa 12-h time course. A TAMRA-conjugated reagent was used for detecting GalNAz incorporated into newly synthesized mucin (visualized in the red channel). The45M1 antibody was used to stain for Muc5ac (visualized in the green channel) and 4=,6-diamidino-2-phenylindole (DAPI) for nuclear DNA (visualized in theblue channel). Colocalization of Muc5ac and incorporated GalNAz (green and red) appears yellow in the images. (G) Schematic picture of the cellularlocalization and transfer of newly synthesized mucin. The rate of mucin transfer through the cells to the mucus layer varied slightly between cells, and in theschematic picture the average surface epithelial cell of noninfected mice is depicted.

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makes it more difficult to simultaneously visualize Muc5ac andGalNAz inside the cells (Fig. 2E). However, comparing the stain-ing in the red and green channels separately confirms colocaliza-tion of intracellular Muc5ac with newly synthesized mucin(Fig. 2C and D).

H. pylori localization and inflammation in the murine stom-ach differs between early colonization and chronic infection.Early H. pylori colonization (7 days postinfection) was associatedwith low gastritis scores, mild to moderate immune cell infiltra-tion, and few abscesses. In contrast, during chronic infection (90days postinfection), all mice showed moderate widespread andmore severe multifocal infiltration, with a higher number of ab-

scesses along with mild mucus metaplasia and atrophy (Fig. 3Aand B). Immunofluorescence staining for H. pylori demonstratedthe presence of this bacterium in the gastric lumen both during theearly colonization and chronic infection of all mice and in cryptsof the antrum of some mice during acute infection (Fig. 2F). Therewas no statistical difference in the number of H. pylori bacteriapresent in the mouse stomachs during early colonization com-pared to that during chronic infection (Fig. 3C).

Changes in mucin production during early colonization andchronic infection with H. pylori. Based on the time line for mucintransport and dissemination obtained from the time course studyof uninfected mice, we selected the time points 2, 6, and 12 h after

FIG 2 GalNAz is incorporated into the glycocalyx 2 h after GalNAz injection. (A) Close-up confocal image of the surface mucus cells of a section from anuninfected mouse; red arrows denote GalNAz in the glycocalyx, which is visualized as red. White arrows point at the black holes where the nucleus is present(DAPI was not used to show the nucleus here, as the broad spectra from DAPI poses a risk for spillover of signal into the red channel). (B) Schematic picture ofnewly synthesized mucin in the glycocalyx and supranuclear area of gastric mucus-producing epithelium in noninfected mice. At this time point there was a cleargap between the GalNAz in the lower cytoplasmic area and the GalNAz in the glycocalyx. (C) Close-up confocal image of Muc5ac staining in a gastric section froman uninfected mouse, visualized as green. (D) Close-up confocal image of newly synthesized mucin in the same gastric section as that shown in panel C, visualizedin red. (E) Immunostaining of Muc5ac (brown) in the antrum of a noninfected mouse. The line indicates secreted Muc5ac, and arrows denote intracellularMuc5ac. (F) Confocal images of H. pylori in an antral crypt from a mouse 7 days postinfection. H. pylori is visualized as white, Muc5ac as green. Arrows denoteH. pylori.

FIG 3 Gastritis scores and CFU counts from mouse stomachs infected with H. pylori. Gastric scores of antrum (A) and corpus (B) of mice during earlycolonization and chronic H. pylori infection (***, P � 0.001 compared to control by ANOVA with Dunnett’s post hoc test). (C) H. pylori CFU counts fromstomachs of infected mice (no significant difference; two-tailed t test).

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GalNAz injection to compare the rates and turnover of gastricmucin production in noninfected mice to those of mice duringearly colonization or chronic H. pylori infection. In the antrum,the newly produced mucins displayed a slower movement fromsynthesis to secretion at the cell surface during infection. Thiseffect was most pronounced during early colonization (Fig. 4 and5); i.e., a high level of GalNAz was detected in the supranuclearregion of the surface epithelial cells in noninfected and chronicallyinfected mice at 2 h after GalNAz injection. In contrast, theamount of incorporated GalNAz was very low at this time point inmice during early colonization (Fig. 4A, D, and G). At the 6-h timepoint, the newly synthesized mucin was present inside the cells innoninfected mice (Fig. 4B) and already shed from the mucus layer

at the 12-h time point (Fig. 4C). In contrast, mice with both earlycolonization and chronic infection had a lower intensity of thestained, incorporated GalNAz at 6 h postinjection (Fig. 4E and H),and plenty of newly synthesized mucin was still present inside thecells after 12 h, although some had been released (Fig. 4F and I).Thus, at all three time points the newly synthesized mucin hadprogressed further toward the mucus layer in uninfected micethan that in mice in early colonization. In addition, infection withH. pylori resulted in a reduction of the total amount of Muc1(Fig. 6A). Similarly, there also seemed to be a trend toward adecrease in the total amount of Muc5ac, although this was notsignificant(Fig. 6C).

FIG 4 Confocal images of antrum from uninfected and H. pylori-infected mice. Uninfected mice (A to C), mice during early infection (D to F), and chronic H.pylori infection (G to I) 2, 6, and 12 h, respectively, after intraperitoneal GalNAz injection. Newly synthesized mucin (incorporated GalNAz) is visualized as red,Muc5ac as green, and Muc1 as blue. Colocalization of green and blue results in gray, whereas colocalization of green and red results in yellow.

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We also studied the changes in corpus and duodenum of thesemice. The results in the noninfected mice demonstrated that thesynthesis and turnover of newly synthesized mucins are slower inthe corpus than in the antrum (compare Fig. 7A to C to 4A to C).In the corpus, the rate of production and transfer of newly synthe-sized mucin from the perinuclear region toward the mucus layerwas similar in noninfected mice and in mice during early coloni-zation with H. pylori (Fig. 7A to F). This is in contrast to theantrum, where the mucin production rate and turnover were vis-ibly decreased by early H. pylori colonization compared to that innoninfected mice (see representative images in Fig. 4 and the sche-matic picture in Fig. 5).

Similar to the antrum, the total amount of Muc1 was reducedduring both early colonization and chronic infection with H. py-lori in corpus (Fig. 6B), whereas Muc5ac changes were less obviousbetween uninfected and infected groups (Fig. 6D).

We did not detect any changes in mucin production in themouse duodenum during different time points of infection withH. pylori (data not shown).

DISCUSSION

In the present study, we followed in real time the process of mucinproduction in the healthy murine stomach, as well as in miceinfected with H. pylori during early colonization and chronic in-fection, using metabolic labeling in a pulse experiment. In linewith previous studies of the distal colon (18), our results from thestomach demonstrated a continuous production and secretion ofmucins. GalNAz was already incorporated into mucins 1 h afterinjection and then moved through the cell to its final destinationin the glycocalyx or mucus layer. We found that the rate of pro-duction of newly synthesized mucin was faster in the antrum ofnoninfected mice than in the corpus. Furthermore, the speed fromthe start of glycosylation for the membrane-bound mucin to reachthe glycocalyx was faster than that for the secretory mucins to getsecreted into the mucus layer. During H. pylori infection, mucinproduction, production rate, and turnover were hampered.

The results of our histological scoring showed that H. pyloriproduced only a mild inflammation in mice, although the severityof gastritis during chronic infection was slightly higher than thatduring early colonization with H. pylori. This finding was compa-rable those of to previous studies on the effect of this bacterium onthe murine stomach (19). Despite the mild gastritis caused by thisbacterium, we detected a reduction in the rate of mucin produc-tion and turnover and a decrease in the level of Muc1 but nosignificant changes in the Muc5ac level. Together, these resultsshow that there are no major changes in the total amount of mucinin the gastric tissue and the mucus layer, as the amount of Muc5acis vastly higher than the amount of Muc1. However, the produc-tion and secretion rate of the mucin is decreased, and the rate atwhich the secreted mucus washes harmful agents away from theepithelial surface is decreased. H. pylori-infected rhesus monkeysand children (aged 3 to 18 years) secreting mucins with weaker H.pylori binding capacity develop infections with higher H. pyloridensity and more severe gastritis (13, 20). Patients with primarySjogren’s syndrome are known to produce fewer mucins and arealso reported to present a more severe H. pylori-associated pathol-ogy (21). These results indicate the ability of secreted mucins tobind to and transport away H. pylori as a mechanism for protect-ing the gastric epithelium. Thus, the impaired speed of the wash-ing of the mucosal surface caused by the slower mucin secretionrate is likely to provide H. pylori with a more stable environment.The presence of H. pylori inside the crypts of mice during acuteinfection coincides with the time when we see the most severeslowdown of mucin turnover. Thus, slowing down the mucus flowmay enable close association of H. pylori with the epithelial cellsand aid in the colonization process. Later in the infection, themucus flow appears to be less hampered, and then we detect H.pylori mainly in the lumen.

The reduced production of Muc1 by the host is also likely to bebeneficial to the bacterium, as mice lacking Muc1 are more sus-ceptible to infection by H. pylori (22). H. pylori binding to gastric

FIG 5 Schematic picture of cellular localization and transfer of newly synthesized mucin during infection. Mucin transfer is depicted on an average surfaceepithelial cell of noninfected mice and compared to those of mice with early and chronic infection in antrum and corpus.

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epithelial cells is inhibited by MUC1, irrespective of whether ornot they bind to this mucin. Strains that bind to MUC1 are re-moved from the epithelial cell surface as a result of the proteinacting as a releasable decoy (13). Strains that would otherwise bindto alternative cell surface ligands are inhibited by steric hindrancecaused by MUC1.

Our results showed no significant changes in the amount ofMuc5ac between infected and noninfected mice. Similarly, mostimmunohistochemical studies on human biopsy specimens fromH. pylori-infected individuals detect no difference in MUC5ACexpression (23). One study, however, demonstrated a reduction ofMUC5AC-producing cells in H. pylori-infected individuals (24).

A previous study using the gastric cancer cell line KATO-IIIinfected with H. pylori in vitro found that mucin synthesis is de-creased upon infection, supporting our in vivo results. However,in their study they found no change in mucin secretion (25). Thisdifference in results relating to the secreted mucins compared toours can be explained by many experimental factors. The in vitroexperiments were performed with at least a 1,000-fold higher bac-terial/mammalian cell ratio than our in vivo experiments. Further-more, the in vitro experiment was performed under conditionsthat are incompatible with H. pylori survival. However, the factorthat most likely has the most importance with regard to measure-

ment of secreted mucins is that the in vitro experiment was shortterm (22 h). The detected decrease in mucin synthesis may haveresulted in a decrease in mucin secretion at later time points, sim-ilar to our in vivo experiments. Byrd et al. also found a transientdecrease in MUC1 that was recovered by the 22-h time point.However, the bacteria in their study were shown to be nonviable.Therefore, the reduction in MUC1 may depend on the viability ofH. pylori. We have reported in a previous study that MUC1 is shedand coats H. pylori, which leads to a decrease in MUC1 levels at asimilar time point using viable bacteria (14).

In patients with chronic gastritis, depletion of the MUC1 ex-tracellular subunit, but not the transmembrane subunit, has beenreported (26), which is compatible with MUC1 acting as a releas-able decoy (14). Thus, the current study is the first to show down-regulation of the cytoplasmic domain of Muc1 in vivo. It is inter-esting that the in vivo model of H. pylori inhibition of mucinsynthesis (used in this study) has far greater sensitivity than the invitro model (24). The effects on mucin production and secretionachieved in vitro are similar to (or smaller than) the ones we detectin vivo, although the in vitro model uses at least a 1,000-fold higherconcentration of H. pylori. As the effect on mucin synthesis in vitrois dependent on the concentration of H. pylori (25), factors such asviability of the bacteria and/or the host immune system are likely

FIG 6 Quantification of Muc1 and Muc5ac during early and chronic H. pylori infection. Comparison of the integrated density of fluorescence as a measure ofMuc1 in antrum (A) and corpus (B), as well as Muc5ac in antrum (C) and corpus (D). Data were compared to the control (n � 6) by ANOVA with Dunnett’spost hoc test (**, P � 0.01; NS, not significant).

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to be an important part of H. pylori-dependent mucin inhibitionin vivo.

In conclusion, H. pylori colonization in the mucus niche of themurine stomach in vivo leads to decreased mucin production andsecretion rate and decreased levels of Muc1 in the mucosa. Thisindicates disruption in the mucus defense mechanism of clearingthe mucosal surface from pathogens by impairing the mucus flow.

ACKNOWLEDGMENTS

The work was supported by the Swedish Research Council (Vetenskapsrå-det K2008-58X-20693-01-4 and 521-2011-2370), the Swedish ResearchCouncil Formas (221-2011-1036), the Swedish Cancer Foundation (Can-cerfonden), Mucosal Immunobiology and Vaccine Center (Gothenburg,

Sweden), the Jeansson Foundation, the Åke Wiberg Foundation, theGoljes Memorial Foundation, and the Magnus Bergvall Foundation.

We thank the Center of Cellular Imaging core facility of the Gothen-burg University for their assistance with confocal microscopy studies.

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