Helicobacter pylori is responsible for most human stomach cancers. Gastric cancer also is overrepresented in populations consuming high-salt diets. Attempts to test the hypothesis that high salt promotes H. pylori carcinogenesis have been hindered by the lack of a wild-type mouse model. Based on pilot observations of unexpectedly early gastric adenocarcinoma in C57BL/6 × 129S6/SvEv (B6129) mice infected with Helicobacter felis, we conducted a study to characterize H. pylori infection in these mice and to determine whether high salt promotes tumorigenesis. Male and female mice were gavaged with H. pylori Sydney strain-1 or vehicle only and divided into four groups based on infection status and maintenance on a basal (0.25%) or high (7.5%) salt diet. In uninfected mice, the high-salt diet enhanced proliferation and marginally increased parietal cell mucous metaplasia with oxyntic atrophy. Lesions in H. pylori infected mice without regard to diet or gender were of equivalent severity and characterized by progressive gastritis, oxyntic atrophy, hyperplasia, intestinal metaplasia, and dysplasia. Infected mice on the high-salt diet exhibited a shift in antimicrobial humoral immunity from a Th1 to a Th2 pattern, accompanied by significantly higher colonization and a qualitative increase in infiltrating eosinophils. No mice developed anti-parietal cell antibodies suggestive of autoimmune gastritis. At 15 months of age infected mice in both dietary cohorts exhibited high-grade dysplasia consistent with gastric intraepithelial neoplasia. In summary, we report for the first time H. pylori–induced gastric intraepithelial neoplasia in a wild-type mouse model and show no additive effect of high-salt ingestion on tumor progression.

Helicobacter pylori, a WHO class I carcinogen, is responsible for most human gastric cancers (1, 2). Stomach tumors are the second leading cause of human cancer deaths (3). In addition to H. pylori infection, dietary factors, including deficiencies of vitamin C and E and high intake of salt and nitrates, have been linked epidemiologically to an increased risk of stomach cancer (4). An association between high-salt ingestion and gastric cancer has been suspected since the 1960s (5). Both inherently salty foods and those cured in salt for preservation have been implicated in this process (4). The European Cancer Prevention and INTERSALT Cooperative Research Group showed in a multinational study an association between urinary sodium concentration (a surrogate marker of salt ingestion) and gastric cancer risk (6). A prospective study in Japan reported a dose-dependent association between self-reported salt ingestion and risk of stomach cancer among middle-aged men but not women; however, the correlation weakened when stratified by geographic region (7). Based on epidemiologic evidence, a Joint WHO/FAO Expert Consultation concluded that high-salt intake “probably increase(s) the risk of stomach cancer” (8). Because of significant geographic overlap among H. pylori endemicity, high-salt diet, and gastric cancer as is found in the Far East, some investigators have postulated that high salt may act cooperatively with H. pylori to promote stomach cancer (9). However, direct proof for this hypothesis is lacking.

Mouse models have been used experimentally to test the potential relationship between high-salt intake and H. pylori infection (10, 11). However, to date, no such study has found evidence of an additive or synergistic promotional effect between gastric Helicobacter infection and high salt. For example, in a previous report from our group, C57BL/6 mice infected with H. pylori Sydney strain-1 (SS1) for 4 months, and maintained on a high-salt diet, exhibited higher gastric urease activity, serum gastrin, foveolar hyperplasia, and colonization levels than did those on a basal diet; however, no meaningful effect of salt on gastric inflammation or oxyntic atrophy scores was observed (11). A critical limitation of mouse models is that to date, there has been no identified wild-type (WT) strain susceptible to H. pylori–induced gastric carcinoma. Infection of inbred mice with H. pylori results in gastritis ranging in severity from mild in the BALB/c strain to moderately severe in the C57BL/6 (12). Because of the need for better mouse models of H. pylori carcinogenesis, we were intrigued by the observation of earlier-than-expected gastric carcinoma in a pilot study of Helicobacter felis infection in C57BL/6 × 129S6/SvEv (B6129) mice (8 months after inoculation versus 13 months in C57BL/6 mice).3

3

A.B. Rogers and J.G. Fox, unpublished data.

Based on those preliminary observations, we undertook the present study to address two questions: (a) Can H. pylori induce gastric neoplasia in WT B6129 mice? and (b) Will a high-salt diet promote tumorigenesis?

Animals and study design. We carried out replicate experiments using C57BL/6 × 129S6/SvEv mice from different sources. The first study employed multigenerationally intercrossed C57BL/6 × 129S6/SvEv (B6;129S) mice (n = 52) maintained in our in-house colony. Mice in our colony are viral antibody-free for 11 murine viruses and negative for enteric Helicobacter spp., Salmonella spp., and Citrobacter rodentium, as well as endoparasites and ectoparasites. Because the relative strain contribution from the original C57BL/6 and 129S6/SvEv parents inherited by individual mice in this colony was unknown, we repeated the experiments using 66 male and 66 female B6129F1 mice purchased from Taconic Farms (Germantown, NY). For both experiments, animals were divided into four groups: (a) sham-inoculated mice maintained on a basal (0.25%) salt diet (Special Formulation Lab Diet, Purina Mills, Richmond, IN); (b) sham-inoculated mice fed a high-salt (7.5%) diet; (c) H. pylori–infected mice on the basal diet; or (d) H. pylori–infected mice on the high-salt diet. At 8 weeks of age, mice were gavaged with 108 colony-forming units (CFU) of H. pylori SS1 or broth only (0.2 mL) every other day for a total of three doses. Mice were housed at four to five per microisolator cage on hardwood shavings on a 12-hour day/night cycle with constant humidity and temperature control. At 6, 12, or 15 months of age, mice were necropsied following euthanasia by CO2 inhalation. From the combined experiments, 54 mice were evaluated at 6, 52 at 12, and 62 at 15 months of age. Twelve of the 180 original mice (86 females and 94 males) were excluded from analysis due to unrelated conditions. All mice were maintained in compliance with the USPHS Policy on Humane Care and Use of Laboratory Animals in a facility certified by the Association for the Assessment and Accreditation of Laboratory Animal Care. Protocols were approved by the Massachusetts Institute of Technology Committee on Animal Care.

Helicobacter pylori quantitative culture. For assessment of H. pylori colonization levels by quantitative culture, aseptically collected gastric corpus and antrum specimens from mice at 6 and 15 months were weighed, homogenized, plated onto selective medium, and incubated under microaerobic conditions at 37°C for 3 to 5 days as previously described (11). Helicobacter growth was confirmed morphologically by phase microscopy and Gram stain, and biochemically by urease, catalase, and oxidase reactions. Colonies were counted and reported as mean number of CFU per mg tissue. For statistical comparisons, normal distribution was confirmed by Kolmogorov-Smirnov test, and data were compared between groups by one-way ANOVA with Tukey after test. All statistical analyses were done with Prism 4 for Macintosh (GraphPad, San Diego, CA).

Serum antibodies. Sera collected from mice at 15 months were tested for antibodies specific to outer membrane antigens of H. pylori. H. pylori SS1 was cultured in Brucella broth containing 5% fetal bovine serum for 24 hours under microaerobic conditions. After three washes in PBS and examination for bacterial contaminants by Gram stain and phase microscopy, the pellet was resuspended in 4 mL of 1% N-octyl-β-glucopyranoside (Sigma, St. Louis, MO) for 30 minutes at room temperature. Insoluble material was removed by ultracentrifugation at 100,000 × g for 1 hour. After dialysis against PBS for 24 hours at 4°C, supernatant protein concentration was measured by the Lowry technique (Sigma). For serum IgG isotype measurement, 96-well plates were coated with 100 μL per well of 10 μg/mL H. pylori protein in carbonate buffer (pH 9.6) overnight at 4°C. After washing, H. pylori–specific serum antibodies were labeled with 1:2,000 biotinylated monoclonal antimouse antibody clones A85-1 and 5.7 for the specific detection of IgG1 and IgG2c, respectively (BD PharMingen, San Diego, CA). Extravidin peroxidase (Sigma) at 1:2,000 was followed by 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfunoic acid) diammonium salt (ABTS substrate, KPL, Gaithersburg, MD) for color development. Absorbance development at 405/592λ was recorded by an ELISA plate reader (Dynatech MR7000, Dynatech Laboratories, Inc., Chantilly, VA). Results were reported as mean absorbance from triplicate measurements at a sample dilution of 1:100. Total serum IgG1 and IgE concentrations were measured using commercial kits (Bethyl Laboratories, Montgomery, TX). Titers were compared for statistical purposes as described above.

Histopathology. At necropsy, the stomach and proximal duodenum were removed and incised along the greater curvature. Linear gastric strips from the lesser curvature extending from the squamocolumnar junction through proximal duodenum were fixed overnight in 10% neutral-buffered formalin, routinely processed and embedded, cut at 4 μm, and stained with H&E. A comparative pathologist (A.B.R.) blinded to treatment groups scored gastric lesions on an ascending scale from 0 to 4 using the criteria outlined in Table 1. Defining characteristics for dysplasia and gastric intraepithelial neoplasia (GIN) were adapted from consensus guidelines on murine models of intestinal cancer (13). Mean lesion scores were compared by Kruskal-Wallis nonparametric one-way ANOVA.

Table 1.

Murine gastric histopathology scoring paradigm

CriterionScore
1234
Inflammation Patchy infiltration of mixed leukocytes in mucosa and/or submucosa. Add 0.5 for significant granulocytes. Multifocal-to-coalescing leukocyte infiltration not extending below submucosa Marked increase in leukocytes with lymphoid follicles ± extension into tunica muscularis Effacing transmural inflammation 
     
Epithelial defects Rare dilated glands and/or attenuated epithelium Frequent dilated glands, some large, surface epithelial “tattering” Surface erosions and gland atrophy Full thickness mucosal ulceration, gland atrophy and fibrosis 
     
Hyalinosis Red refractile droplets and crystals: Ym1/Ym2 (34) Slightly increased surface epithelial red glassy (hyalin) intracytoplasmic droplets in cardia Cytoplasmic hyalin droplets and/or crystals in cardia and proximal corpus Coalescing hyalin droplets and crystals extending to distal corpus Diffuse hyalin droplets and large extracellular crystals affecting entire corpus 
     
Mucous metaplasia* Rare small foci in corpus Moderately large foci affecting <1/3 of parietal cells Large foci affecting 1/3-2/3 of parietal cells Foamy change affecting >2/3 of parietal cells 
     
Oxyntic gland atrophy ∼50% chief cell loss, ∼25% parietal cell loss 100% chief cell loss, ∼50% parietal cell loss ∼75% parietal cell loss, no chief cells >75% parietal cell loss, no chief cells 
     
Foveolar hyperplasia ∼1.5× normal isthmus length ∼2× normal isthmus length ∼3× normal isthmus length ≥4× normal isthmus length 
     
Intestinal metaplasia Rare small foci, usually near cardia Moderately large foci affecting cardia and <1/3 of corpus Large foci affecting 1/3-2/3 of corpus Metaplasia affecting >2/3 of corpus 
     
Dysplasia Focal, irregularly shaped gastric glands (analogous to colonic aberrent crypt foci) including elongated, slit, trident, and back-to-back forms. Increased gland distortion, pleomorphism, branching, stratification; indefinite dysplasia or atypical hyperplasia. Add 0.5 for marked branching or herniation. Severe loss of gland organization and columnar orientation, marked cell atypia, visible mitoses; gastric intrapeithelial neoplasia (GIN). Add 0.5 for carcinoma in situ or invasion without frank malignancy. Unequivocal invasive (adeno)carcinoma extending into submucosa, ± vascular/lymphatic invasion 
CriterionScore
1234
Inflammation Patchy infiltration of mixed leukocytes in mucosa and/or submucosa. Add 0.5 for significant granulocytes. Multifocal-to-coalescing leukocyte infiltration not extending below submucosa Marked increase in leukocytes with lymphoid follicles ± extension into tunica muscularis Effacing transmural inflammation 
     
Epithelial defects Rare dilated glands and/or attenuated epithelium Frequent dilated glands, some large, surface epithelial “tattering” Surface erosions and gland atrophy Full thickness mucosal ulceration, gland atrophy and fibrosis 
     
Hyalinosis Red refractile droplets and crystals: Ym1/Ym2 (34) Slightly increased surface epithelial red glassy (hyalin) intracytoplasmic droplets in cardia Cytoplasmic hyalin droplets and/or crystals in cardia and proximal corpus Coalescing hyalin droplets and crystals extending to distal corpus Diffuse hyalin droplets and large extracellular crystals affecting entire corpus 
     
Mucous metaplasia* Rare small foci in corpus Moderately large foci affecting <1/3 of parietal cells Large foci affecting 1/3-2/3 of parietal cells Foamy change affecting >2/3 of parietal cells 
     
Oxyntic gland atrophy ∼50% chief cell loss, ∼25% parietal cell loss 100% chief cell loss, ∼50% parietal cell loss ∼75% parietal cell loss, no chief cells >75% parietal cell loss, no chief cells 
     
Foveolar hyperplasia ∼1.5× normal isthmus length ∼2× normal isthmus length ∼3× normal isthmus length ≥4× normal isthmus length 
     
Intestinal metaplasia Rare small foci, usually near cardia Moderately large foci affecting cardia and <1/3 of corpus Large foci affecting 1/3-2/3 of corpus Metaplasia affecting >2/3 of corpus 
     
Dysplasia Focal, irregularly shaped gastric glands (analogous to colonic aberrent crypt foci) including elongated, slit, trident, and back-to-back forms. Increased gland distortion, pleomorphism, branching, stratification; indefinite dysplasia or atypical hyperplasia. Add 0.5 for marked branching or herniation. Severe loss of gland organization and columnar orientation, marked cell atypia, visible mitoses; gastric intrapeithelial neoplasia (GIN). Add 0.5 for carcinoma in situ or invasion without frank malignancy. Unequivocal invasive (adeno)carcinoma extending into submucosa, ± vascular/lymphatic invasion 
*

Foamy change (resembling Brunner's glands) predominantly affecting parietal cells, with PAS+ and Alcian blue+ mucins.

Foveolar cell columnar heightening, globoid cells with PAS/Alcian blue+ mucins (often mixed), ± brush border, or goblet cells.

Special stains, immunohistochemistry, and proliferation index. Selected tissues from infected and control animals were characterized with special stains and immunohistochemistry. Acidic (intestinal type) mucins were shown with pH 2.5 Alcian blue followed by periodic acid-Schiff (PAS) to stain remaining neutral (gastric type) mucins. Apoptosis was shown by caspase-3 immunohistochemistry using a rabbit polyclonal antibody specific for the cleaved (activated) isoform (Cell Signaling Technologies, Beverly, MA). Cell proliferation labeling indices (LI) were determined by Ki-67 immunohistochemistry using the ARK kit (DAKO, Carpinteria, CA) as described previously (10). From representative male and female mice in each of the four groups, labeled nuclei from 10 well-oriented proximal corpus glands per animal were counted in a blinded fashion. Statistics were done as described for colonization and serology.

High salt increased Helicobacter pylori colonization and altered antibody responses.H. pylori–infected B6129 mice on the high-salt diet maintained significantly higher gastric bacterial burdens than did animals on the basal diet, in agreement with our previous observations in C57BL/6 mice (11). Mean H. pylori colonization in mice on the basal diet was 9,474 ± 4,798 CFU/mg stomach, whereas bacterial load in mice on the high-salt diet was 70,579 ± 15,863 CFU/mg (P < 0.0001). Moreover, colonization dropped significantly between 6 and 15 months in mice on the basal diet but increased in mice on the high-salt diet (Fig. 1). In mice on the basal diet, the decline in bacterial burden was especially marked in the corpus. In both dietary cohorts, H. pylori colonization of the corpus was roughly equivalent to that of the antrum at 6 months, but antral levels were significantly increased relative to the corpus at 15 months (Fig. 1). There were no consistent differences in colonization between genders (data not shown). Stomach tissue from sham-inoculated mice was negative for H. pylori in culture.

Figure 1.

H. pylori colonization of the gastric corpus and antrum as determined by quantitative culture in mice on a basal versus high-salt diet at 6 and 15 months of age. Mice on the high-salt diet had significantly greater colonization at both time points (P < 0.05). Note the statistically significant decrease in bacterial burden in mice on the basal diet between 6 and 15 months versus a slight increase over the same timeframe in mice on the high-salt diet. In both dietary cohorts, bacterial levels were equivalent between the corpus and antrum at 6 months but slightly higher in the antrum at 15 months.

Figure 1.

H. pylori colonization of the gastric corpus and antrum as determined by quantitative culture in mice on a basal versus high-salt diet at 6 and 15 months of age. Mice on the high-salt diet had significantly greater colonization at both time points (P < 0.05). Note the statistically significant decrease in bacterial burden in mice on the basal diet between 6 and 15 months versus a slight increase over the same timeframe in mice on the high-salt diet. In both dietary cohorts, bacterial levels were equivalent between the corpus and antrum at 6 months but slightly higher in the antrum at 15 months.

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We did serology on mice 15 months of age for H. pylori–specific IgG2c (Th1) and IgG1 (Th2) antibodies (14). Only infected mice (groups 3 and 4) developed significant antibody titers against H. pylori (Fig. 2). There were no significant gender differences within groups, although infected females on the high-salt diet exhibited slightly higher titers than males on the same diet (Fig. 2). However, diet had a major effect on anti-H. pylori IgG2c/IgG1 ratio. Mean IgG2c antibody titer was nearly 50% higher in infected mice on the standard basal versus high-salt diet (absorbance, 1.5 versus 1.1, respectively; P = 0.002). Conversely, mean IgG1 titer was significantly higher in mice on the high-salt diet (absorbance, 0.5 versus 0.2; P = 0.003). The net result was a dramatic increase in Th2/Th1 (IgG1/IgG2c) anti-H. pylori antibody ratio in mice on the high-salt diet (P < 0.0001). We found no significant differences between groups for total (as opposed to H. pylori specific) serum IgG1 and IgE concentrations nor did we detect serum anti-parietal cell antibodies by immunohistochemistry (data not shown). Thus, we found no systemic or parietal cell-specific serologic evidence of autoimmunity or hypersensitivity induction attributable to H. pylori infection or high-salt gastric injury, either alone or in combination.

Figure 2.

Circulating anti-H. pylori IgG titers in male and female 15-month-old mice as determined by ELISA. A, note increased levels of the Th1-associated antibody IgG2c in H. pylori–infected mice on the basal diet (group 3) compared with the high-salt diet (group 4). B, Th2-associated antibody IgG1 is significantly higher in infected mice on the high-salt diet. C, anti-H. pylori Th1/Th2-type antibody ratio is significantly higher in infected mice on the basal diet, consistent with a Th2 shift in mice on the high-salt diet. Anti-H. pylori titers in uninfected mice (groups 1 and 2) were below the lower threshold of significance (dashed horizontal line).

Figure 2.

Circulating anti-H. pylori IgG titers in male and female 15-month-old mice as determined by ELISA. A, note increased levels of the Th1-associated antibody IgG2c in H. pylori–infected mice on the basal diet (group 3) compared with the high-salt diet (group 4). B, Th2-associated antibody IgG1 is significantly higher in infected mice on the high-salt diet. C, anti-H. pylori Th1/Th2-type antibody ratio is significantly higher in infected mice on the basal diet, consistent with a Th2 shift in mice on the high-salt diet. Anti-H. pylori titers in uninfected mice (groups 1 and 2) were below the lower threshold of significance (dashed horizontal line).

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Helicobacter pylori infection was the predominant disease determinant. Histopathologic scores stratified by group were essentially identical between B6129F1 and B6;129S mice (data not shown); therefore, data from both experiments were combined. The generic strain designation assigned to the combined groups of mice was B6129. In sham-inoculated B6129 mice on the high-salt diet (group 2), there was a very slight increase in inflammation, oxyntic atrophy, mucous metaplasia, and hyperplasia scores consistent with mild atrophic gastritis at 6 and 12 months compared with uninfected mice on the basal diet (group 1; Fig. 3). However, because these mild changes occurred sporadically in both groups, there was no statistically significant difference between them. For all lesion criteria except hyalinosis and mucous metaplasia (data not shown), statistically significant increases (P < 0.05) were evident in H. pylori–infected mice (groups 3 and 4) at 15 months compared with uninfected mice (Fig. 3). Inflammation, epithelial defects, and oxyntic atrophy scores reached significantly higher levels by 6 months, and hyperplasia, intestinal metaplasia, and dysplasia were significantly increased by 12 months (Fig. 3). Interestingly, except for mucous metaplasia, mean lesion grades were slightly higher for mice on the basal diet (group 3) than for mice on the high-salt diet (group 4; Fig. 3). These differences were not statistically significant.

Figure 3.

Gastric histopathology scores for all mice at 6, 12, and 15 months based on the criteria outlined in Table 1. Compared with uninfected mice on the basal diet (group 1) or high-salt diet (group 2), H. pylori–infected mice on either the basal (group 3) or high-salt (group 4) diet had significantly higher mean scores for inflammation, epithelial defects, and oxyntic atrophy by 6 months, and for hyperplasia, intestinal metaplasia, and dysplasia by 12 months that persisted to the end of the study. There were no statistically significant differences between groups for hyalinosis or mucous metaplasia (data not shown).

Figure 3.

Gastric histopathology scores for all mice at 6, 12, and 15 months based on the criteria outlined in Table 1. Compared with uninfected mice on the basal diet (group 1) or high-salt diet (group 2), H. pylori–infected mice on either the basal (group 3) or high-salt (group 4) diet had significantly higher mean scores for inflammation, epithelial defects, and oxyntic atrophy by 6 months, and for hyperplasia, intestinal metaplasia, and dysplasia by 12 months that persisted to the end of the study. There were no statistically significant differences between groups for hyalinosis or mucous metaplasia (data not shown).

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Helicobacter pylori–infected B6129 mice developed gastric intraepithelial neoplasia. Compared with histologically normal mice (Fig. 4A-B), mice with mucous metaplasia secreted mixed acidic intestinal-type and neutral gastric-type mucins (Fig. 4C-D). Infected mice on both diets developed chronic active gastritis, oxyntic atrophy, hyperplasia, intestinal metaplasia, and dysplasia, recapitulating Correa's multistage model of H. pylori carcinogenesis (15). By 12 months of age, mice infected with H. pylori exhibited severe gasritis and epithelial changes, including glandular ectasia and mineralization, surface irregularities, marked hyperplasia, and early dysplasia with glandular distortion and pleomorphism (Fig. 4E-F). Although not quantified, there was a subjective increase in the proportion of infiltrating eosinophils in mice on the high-salt diet. In some mice, there was complete oxyntic atrophy with surface epithelial proliferation, inflammation, and fibrosis (Fig. 4G). Intestinal metaplasia (incomplete or type II) was manifested by columnar elongation of foveolar epithelium interspersed with rare goblet cells (Fig. 4H; ref. 16). By 15 months, B6129 mice in both dietary cohorts developed H. pylori–associated high-grade dysplasia consistent with GIN (13). Dysplastic changes were characterized by marked surface epithelial hyperplasia, absence of oxyntic cells, disorganization and branching of glands, loss of columnar glandular orientation, cell stratification, pleomorphism and atypia, and nuclear changes, including anisokaryosis, loss of basal polarity, vesicular (euchromatic) chromatin pattern, and increased mitotic figures with occasional bizarre forms (Fig. 4I-L). In some mice, there was glandular herniation into the muscularis mucosae (Fig. 4J). However, invasion of neoplastic glands into the submucosa or lymphatics was not observed. Globoid dysplasia characterized by disorderly stratification of cells expanded by large cytoplasmic mucus vacuoles with nuclear margination (somewhat resembling signet ring cells) was evident multifocally (Fig. 4M; ref. 16). Intestinal metaplasia was confirmed by the demonstration of acidic and mixed mucins within apical cytoplasmic droplets, and on the surface of atypical cells by pH 2.5 Alcian blue/PAS stain (Fig. 4N).

Figure 4.

Histopathology of gastric disease induced by H. pylori infection and/or high-salt diet in B6129 mice. Uninfected mouse stomach showing normal microscopic architecture on H&E stain (A) and a thin surface lining of gastric-type neutral mucins (red) by AB/PAS (B). Tissue from uninfected mouse on high-salt diet exhibiting functional loss of parietal cell mass due to mucous metaplasia characterized by foamy change of parietal cell cytoplasm on H&E stain (C) and intestinal-type acidic mucins (blue) and increased gastric-type neutral mucins (red) in the oxyntic mucosa, along with coexpression of mixed mucins by surface epithelial cells (azure) as shown by Alcian blue/PAS stain (D). Tissues from 12-month H. pylori–infected mice on basal (E) or high-salt (F) diet exhibiting marked inflammation and mucosal hyperplasia with glandular ectasia, mineralization, and early dysplasia. G, complete oxyntic atrophy with compensatory epithelial hyperplasia and focal fibrosis. H, intestinal metaplasia characterized by epithelial cell columnar elongation and rare goblet cells (arrow). Tissues from 15-month mice on basal (I) or high-salt (J) diet showing high-grade dysplasia consistent with GIN; note herniation of dysplastic glands into muscularis mucosae (J, arrow). K, glandular dysplasia characterized by loss of columnar orientation, elongation, branching and infolding, irregular shapes and sizes, and cell stratification. L, dysplastic glands with many mitotic figures (arrows). M, globoid dysplasia characterized disorganized stratification of cells with large cytoplasmic mucus vacuoles and nuclear margination. N, mucous droplets in dysplastic glands containing either acidic (blue) or mixed mucins (azure), consistent with incomplete (type II) intestinal metaplasia. Dysplastic and hyperplastic mucosal epithelium showing intercurrently high apoptosis and proliferation with caspase-3+ apoptotic cells in glandular lumens (arrows, O) and numerous mitotically active Ki-67+ epithelial lining cells (arrows, P). Stains: H&E (A, C, E-L), pH 2.5 Alcian blue/PAS (B, D, N), caspase-3 immunohistochemistry (O), Ki-67 immunohistochemistry (P). Barm 160 μm (A-D, G, O, P), 400 μm (E, F, I, J), 40 μm (H, K, L), 80 μm (M and N).

Figure 4.

Histopathology of gastric disease induced by H. pylori infection and/or high-salt diet in B6129 mice. Uninfected mouse stomach showing normal microscopic architecture on H&E stain (A) and a thin surface lining of gastric-type neutral mucins (red) by AB/PAS (B). Tissue from uninfected mouse on high-salt diet exhibiting functional loss of parietal cell mass due to mucous metaplasia characterized by foamy change of parietal cell cytoplasm on H&E stain (C) and intestinal-type acidic mucins (blue) and increased gastric-type neutral mucins (red) in the oxyntic mucosa, along with coexpression of mixed mucins by surface epithelial cells (azure) as shown by Alcian blue/PAS stain (D). Tissues from 12-month H. pylori–infected mice on basal (E) or high-salt (F) diet exhibiting marked inflammation and mucosal hyperplasia with glandular ectasia, mineralization, and early dysplasia. G, complete oxyntic atrophy with compensatory epithelial hyperplasia and focal fibrosis. H, intestinal metaplasia characterized by epithelial cell columnar elongation and rare goblet cells (arrow). Tissues from 15-month mice on basal (I) or high-salt (J) diet showing high-grade dysplasia consistent with GIN; note herniation of dysplastic glands into muscularis mucosae (J, arrow). K, glandular dysplasia characterized by loss of columnar orientation, elongation, branching and infolding, irregular shapes and sizes, and cell stratification. L, dysplastic glands with many mitotic figures (arrows). M, globoid dysplasia characterized disorganized stratification of cells with large cytoplasmic mucus vacuoles and nuclear margination. N, mucous droplets in dysplastic glands containing either acidic (blue) or mixed mucins (azure), consistent with incomplete (type II) intestinal metaplasia. Dysplastic and hyperplastic mucosal epithelium showing intercurrently high apoptosis and proliferation with caspase-3+ apoptotic cells in glandular lumens (arrows, O) and numerous mitotically active Ki-67+ epithelial lining cells (arrows, P). Stains: H&E (A, C, E-L), pH 2.5 Alcian blue/PAS (B, D, N), caspase-3 immunohistochemistry (O), Ki-67 immunohistochemistry (P). Barm 160 μm (A-D, G, O, P), 400 μm (E, F, I, J), 40 μm (H, K, L), 80 μm (M and N).

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Helicobacter pylori infection simultaneously up-regulated epithelial proliferation and apoptosis. In dysplastic regions, high rates of intercurrent apoptosis and proliferation were shown by immunohistochemistry. Lumens of ectatic glands contained many caspase-3+ epithelial cells, whereas epithelial lining cells of the same glands exhibited a high rate of proliferation as shown by nuclear Ki-67 immunoreactivity (Fig. 4O-P). We rarely detected caspase-3+ cells within the gastric mucosa, probably because of rapid phagocytic removal by neighboring viable cells. To quantitatively compare proliferation differences, we determined the Ki-67 LI for representative mice at 12 months from each of the four groups. In uninfected mice, the high-salt diet resulted in a moderate but statistically significant (P = 0.003) increase in proliferation (Fig. 5). As expected, H. pylori infection significantly increased Ki-67 LI in mice on either diet compared with uninfected controls (Fig. 5; P < 0.001). In contrast to observations in uninfected mice, however, H. pylori–infected mice on the high-salt diet had a lower proliferation index than that of mice on the basal diet (P < 0.001). Taken together, these data are in agreement with our previous study in INS-GAS mice indicating that up-regulated proliferation and not down-regulated apoptosis is the primary mechanism of foveolar hyperplasia in H. pylori–infected mice (10).

Figure 5.

Epithelial cell Ki-67 LI as a measure of proliferation in mice at 12 months. High-salt diet alone increased cell proliferation by a modest but statistically significant level in uninfected mice. H. pylori infection markedly increased proliferation, but in contrast to uninfected mice, this increase was significantly greater in mice on the basal diet than on the high-salt diet. Representative photomicrographs of Ki-67 immunohistochemistry from each group (top of each matching column).

Figure 5.

Epithelial cell Ki-67 LI as a measure of proliferation in mice at 12 months. High-salt diet alone increased cell proliferation by a modest but statistically significant level in uninfected mice. H. pylori infection markedly increased proliferation, but in contrast to uninfected mice, this increase was significantly greater in mice on the basal diet than on the high-salt diet. Representative photomicrographs of Ki-67 immunohistochemistry from each group (top of each matching column).

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H. pylori infection is now recognized as the single greatest risk factor for human gastric cancer (17). High-salt ingestion has been proposed to increase the risk of stomach carcinoma as well (7). However, in contrast to H. pylori infection, which increases the risk of stomach cancer up to 10×, epidemiologic associations between high-salt and gastric tumors are less consistent and rarely raise the adjusted odds ratio above 2 (18). Thus, although H. pylori has been designated a class I carcinogen (1), a Joint WHO/FAO Expert Consultation could only conclude that high-salt ingestion “probably increase(s) the risk of stomach cancer” (8). Indeed, a prospective study in the United States found no association between self-reported seasoning of food with salt and subsequent risk of stomach cancer, whereas food salting was significantly associated with high blood pressure (19). Thus, the hypothesis that high-salt ingestion promotes gastric cancer remains controversial.

In the present study, B6129 mice infected with H. pylori developed chronic active gastritis and, by 15 months of age, high-grade dysplasia consistent with GIN (13). Neither we nor others have observed this high degree of gastric dysplasia by 15 months in gastritis-susceptible inbred C57BL/6 mice following infection with H. pylori (20). B6129 mice fed a high-salt diet showed a shift in anti-H. pylori humoral immunity from a Th1 to a Th2 phenotype. This humoral immunity shift is not specific to B6129 mice. Our group observed a similar increase in Th2-associated IgG1 relative to Th1-associated IgG2a in H. pylori–infected INS-GAS mice on a FVB strain background (10). However, the salt-associated shift in humoral immunity to a Th2 pattern seemed to be H. pylori specific. We found no increase in total IgG1 or IgE consistent with hypersensitivity nor did we detect anti-parietal cell antibodies suggestive of autoimmune gastritis. Further studies will be needed to determine whether antibody responses reflect cell-mediated immunity within the gastric mucosa. For example, in contrast to previous work from our group showing that a Th2-invoking helminth infection may reduce the risk of Helicobacter-induced gastric cancer (21), we found no protective effect of the high-salt diet on dysplastic progression. Direct chemical injury to a gastric mucosa already impaired by Helicobacter-induced gastritis may offset any beneficial effect from the salt-induced humoral immunity shift. Moreover, innate immunity plays a critical role in H. pylori infection. For example, the cyclooxygenase-2/prostaglandin E synthase pathway was shown to be a critical mediator of proinflammatory and tumorigenic phenotypes in the murine gastric mucosa, both in transgenic mice and in Helicobacter infection models (22).

The most frequently studied animal models of H. pylori infection are the Mongolian gerbil and the mouse (2325). Gerbils infected with H. pylori develop an antral gastritis/duodenitis that mimics the ulcerogenic form of the human disease (26). Unlike mice but similar to humans, the bacterial cag pathogenicity island seems to be a critical disease determinant in gerbils (27). The gerbil model should prove highly useful towards addressing questions specifically related to cagA and other H. pylori virulence determinants in vivo. Our group showed previously in gerbils that high dietary salt alone provoked oxyntic atrophy and foveolar hyperplasia (28). Moreover, others have shown a synergistic promoting effect of H. pylori and high salt on N-methyl-N-nitrosurea (MNU)–initiated gastric cancer in the gerbil model (29). However, chemical initiation was required to induce cancer, as no tumors developed in gerbils untreated with MNU regardless of H. pylori infection status or diet. Thus, to date, the gerbil model has failed to show a contribution of high salt in the promotion of gastric tumorigenesis in the absence of chemical initiation. Importantly, pathogenic gastric Helicobacter spp. in both gerbils and mice reproduce Correa's histologic progression of inflammation, oxyntic atrophy, epithelial hyperplasia, intestinal metaplasia, and dysplasia/cancer (30), attesting to their appropriateness as models of human disease.

In contrast to the antral-predominant disease seen in the Mongolian gerbil, stomach lesions in mice infected with gastric Helicobacter spp. are usually most severe in the cardia and corpus (25). Corpus gastritis and parietal cell mucous metaplasia contribute to the sometimes severe atrophic gastritis observed in murine models (11). C57BL/6 mice infected with H. pylori SS1 for 4 months and maintained on a high-salt diet exhibited higher gastric urease activity, serum gastrin, bacterial colonization, and foveolar proliferation levels than did those on a basal diet; however, no meaningful effect of salt on gastric inflammation or oxyntic atrophy scores was observed (11). High salt likewise increased foveolar proliferation in uninfected but not infected mice in the present study. Thus, induction of proliferation in the absence of dysplasia does not seem to increase the risk of carcinogenesis in this model. In agreement with this concept, H. pylori infection was shown to induce DNA damage in Big Blue mice (containing a chromogen-inducible lambda phage transgene for in vitro mutation quantitation), but no increase in mutation frequency was evident in animals fed a high-salt diet (31).

To our knowledge, this is the first report of H. pylori–induced gastric intraepithelial neoplasia in any wild-type mouse model. In agreement with a previous study using INS-GAS mice on a FVB background (10), we observed no promotional effect of high salt on H. pylori tumorigenesis in B6129 mice. Wild-type and genetically engineered mice on a 129S background are used widely in inflammatory bowel disease studies due to their high susceptibility to bacterial-induced colitis and cancer (25). Additional work will be required to determine whether inbred 129S mice are susceptible to H. pylori–induced gastric tumorigenesis, or whether the combined contribution of C57BL/6 and 129S6/SvEv parental strains was necessary to induce GIN in the present study. Whereas H. felis infection results in stomach cancer in C57BL/6 and other susceptible strains of mice (32), to date, the only published murine model of gastric carcinogenesis due to H. pylori is the INS-GAS mouse (10, 33). INS-GAS mice are transfected with a humanized gastrin transgene linked to a rat insulin promoter and develop constitutive hypergastrinemia and gastric cancer within 7 months of H. pylori infection. However, previous work from our group showed that H. pylori–infected INS-GAS mice fed a high-salt diet exhibited no increase in tumor progression over those fed the basal diet. Indeed, in agreement with our present study, gastritis and dysplasia scores in INS-GAS mice on the high-salt diet were slightly lower than those for mice on the basal diet, although like the present study, those differences were not statistically significant (10). As is the case with humans, male INS-GAS mice infected with H. pylori were more susceptible to tumorigenic progression than females (10), in contrast to the present study in B6129 mice showing no difference between genders. Further studies will be required to determine the host factors governing gender-dimorphic disease expression in mouse models.

In summary, B6129 mice infected with H. pylori SS1 developed chronic active gastritis, epithelial defects, oxyntic atrophy, intestinal metaplasia, and foveolar hyperplasia, and by 15 months, high-grade dysplasia consistent with GIN (13). High-salt ingestion induced slightly increased gastric epithelial proliferation in uninfected mice. In infected mice, high salt resulted in an H. pylori–specific shift in humoral immunity from a Th1 to a Th2 phenotype, accompanied by increased gastric bacterial colonization. Importantly, we report for the first time gastric intraepithelial neoplasia in WT mice infected with H. pylori and show that high-salt ingestion in this model neither promotes nor prevents tumorigenesis. The B6129 mouse model provides a new opportunity to study H. pylori carcinogenesis in vivo.

Grant support: NIH grants AI37750, RR07036 and ES02109 (J.G. Fox).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Vivian Ng and Kristen Clapp for technical assistance, Kathleen Cormier and the DCM Histology Lab for tissue preparations, Nataliya Sundina for serology, and Shilu Xu for quantitative culture.

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