Helicobacter pylori (H. pylori) is the strongest known risk for gastric cancer. The H. pylori cag type IV secretion system is an oncogenic locus that translocates peptidoglycan into host cells, where it is recognized by NOD1, an innate immune receptor. Beyond this, the role of NOD1 in H. pylori–induced cancer remains undefined. To address this knowledge gap, we infected two genetic models of Nod1 deficiency with the H. pylori cag+ strain PMSS1: C57BL/6 mice, which rarely develop cancer, and INS-GAS FVB/N mice, which commonly develop cancer. Infected C57BL/6Nod1–/− and INS-GASNod1−/− mice acutely developed more severe gastritis, and INS-GASNod1−/− mice developed gastric dysplasia more frequently compared with Nod1+/+ mice. Because Nod1 genotype status did not alter microbial phenotypes of in vivo–adapted H. pylori, we investigated host immunologic responses. H. pylori infection of Nod1−/− mice led to significantly increased gastric mucosal levels of Th1, Th17, and Th2 cytokines compared with Nod1 wild-type (WT) mice. To define the role of specific innate immune cells, we quantified cytokine secretion from H. pylori–infected primary gastric organoids generated from WT or Nod1−/− mice that were cocultured with or without WT or Nod1−/− macrophages. Infection increased cytokine production from gastric epithelial cells and macrophages and elevations were significantly increased with Nod1 deficiency. Furthermore, H. pylori infection altered the polarization status of Nod1−/− macrophages compared with Nod1+/+ macrophages. Collectively, these studies demonstrate that loss of Nod1 augments inflammatory and injury responses to H. pylori. Nod1 may exert its restrictive role by altering macrophage polarization, leading to immune evasion and microbial persistence.

Significance:

These findings suggest that manipulation of NOD1 may represent a novel strategy to prevent or treat pathologic outcomes induced by H. pylori infection.

Helicobacter pylori (H. pylori) is the most common bacterial infection worldwide, colonizing more than 4.4 billion people (1). Infection with this pathogen also represents the strongest known risk factor for gastric adenocarcinoma, the third leading cause of cancer-related death (2). However, only a percentage of colonized persons ever develop gastric neoplasia (3). One strain-specific virulence locus that augments cancer risk is the cag pathogenicity island (PAI), which encodes a type IV secretion system (T4SS) that translocates the oncoprotein CagA and microbial DNA into gastric epithelial cells (4–6). Following T4SS-mediated delivery, intracellular CagA undergoes tyrosine phosphorylation (5) and activates a eukaryotic phosphatase (SHP-2), leading to carcinogenic cellular responses.

In addition to CagA and DNA, the cag T4SS delivers peptidoglycan into host cells (7–11). Peptidoglycan is also delivered intracellularly via outer membrane vesicles (12). NOD1, which is expressed by most gastrointestinal epithelial cells, is an innate immune receptor and intracytoplasmic sensor of peptidoglycan components. Most gastrointestinal epithelial cells express NOD1 and activation of NOD1 by the muropeptide γ-D-glutamyl-meso-diaminopimelic acid (iE-DAP) leads to NF-κB–dependent cytokine production as well as induction of autophagy (13, 14). NOD1 is also expressed and activated within macrophages in vivo (15–17). NOD1 sensing of H. pylori peptidoglycan induces NF-κB activation and expression of type I IFN via IFN-regulatory-Factor 7, MIP-2, and β-defensin (7, 8, 13, 18). In humans, genetic variation in ATG16L1, which encodes a key effector of NOD1-dependent autophagy and inflammation, alters susceptibility to H. pylori infection (19).

However, NOD1 activation is tightly regulated by a negative autocrine feedback system, in which NOD1-dependent effectors such as AP-1 and TRAF3 concomitantly suppress the downstream effects of NOD1 activation (18, 20–22). We previously demonstrated that H. pylori–induced injury can be significantly attenuated in vitro and in outbred Mongolian gerbils by preactivation of NOD1 (22), yet the role of aberrant NOD1 activation by H. pylori in gastric carcinogenesis has not been fully investigated. Therefore, we utilized two mouse models of gastric injury and cancer on different genetic backgrounds to precisely define the role of Nod1 in inflammation and inflammation-related cancer that develops in response to H. pylori.

Bacteria

H. pylori strains PMSS1 (23) and 7.13 (24), both cag+ strains, were maintained on TSA blood agar plates (25). For in vitro and in vivo experiments, H. pylori was cultured in Brucella broth (Becton Dickinson) supplemented with 10% heat-inactivated new calf serum (Atlanta Biologicals) overnight at 37°C and 5% CO2.

Cells

AGS cells (ATCC CRL-1739) were purchased from ATCC, tested for Mycoplasma contamination on July 10, 2018, and determined to be Mycoplasma free. Cells passed for <10 passages were grown in RPMI1640 media (Gibco) supplemented with 10% heat-inactivated FBS (Atlanta Biologicals) at 37°C and 5% CO2. L-WRN fibroblasts (ATCC CRL-3276) were obtained from ATCC, tested for Mycoplasma contamination on July 10, 2018, and determined to be Mycoplasma free. L-WRN cells passed for <10 passages were grown in Advanced DMEM media (Gibco) supplemented with 10% FBS, 500 μg/mL G418 (Gibco), and 500 μg/mL hygromycin (Invitrogen) at 37°C and 5% CO2. Once cells became confluent, antibiotics were removed, and supernatants were collected. Mouse primary gastric epithelial cell monolayers were generated as reported previously (26). Briefly, gastric glands harvested from wild-type (WT) or Nod1−/− mice were embedded into Matrigel (Corning) and cultured in 50% L-WRN conditioned media at 37°C and 5% CO2. Once glands formed 3D gastroids, they were trypsinized and plated in collagen-coated plates or transwell filters (Corning) in 5% L-WRN conditioned media at 37°C and 5% CO2 to convert to 2D monolayers. Cell monolayers were then infected with H. pylori at a multiplicity of infection (MOI) of 30.

Bone marrow–derived macrophages were obtained from femurs of C57BL/6 WT and Nod1−/− mice. Briefly, marrows were treated with red blood cell lysis buffer (Becton Dickinson) and washed with PBS, and recovered white blood cells were plated in DMEM media (Gibco) supplemented with 10% FBS and 20 ng/mL of M-CSF (Peprotech) for 6 days at 37°C and 5% CO2 for differentiation.

Animals

All procedures were approved by the Animal Care Committee of Vanderbilt University (Nashville, TN). All mouse strains were bred and maintained in the same animal facility. C57BL/6 Nod1−/− mice were kindly provided by Dr. Dana Philpott from the University of Toronto (Toronto, Ontario, Canada). FVB/N INS-GAS Nod1−/− mice were generated by crossing FVB/N INS-GAS Nod1+/+ mice with C57BL/6 Nod1−/− mice for 12 generations. Nod1−/− and INS-GAS+/+ genotypes were confirmed by PCR and qPCR respectively. Mice were housed in the Animal Care Facility of Vanderbilt University Medical Center in standard plastic cages in a room with a 12-hour light/dark cycle at 21°C to 22°C and fed a standard rodent chow (5L0D; Purina). Access to food and water was free throughout all experiments. No special pretreatments (acid inhibition, antibiotics) were used prior to orogastric H. pylori inoculation or before sacrificing the animals. For C57BL/6 mice, both females and males were used; for FVB/N INS-GAS Nod1+/+ and Nod1−/− mice, only males were used in this study (Supplementary Table S1). Mice 6 to 8 weeks of age were challenged with 1 × 109H. pylori at two time points (days 0 and 2) as described previously (27) except for the 2-day time point, where animals were given a single challenge. All mice appeared healthy with no signs of distress noted throughout the infection period up until the time of sacrifice. Serum samples and gastric tissue were harvested. A single pathologist scored indices of inflammation, injury, and cancer as described previously (28). Specifically, the following variables were graded on a 0 to 3 scale (0, none; 1, mild; 2, moderate; 3, severe) in the gastric antrum and body: acute inflammation (polymorphonuclear cell infiltration) and chronic inflammation (mononuclear cell infiltration independent of lymphoid follicles); thus, a maximum inflammation score of 12 was possible for each animal. Dysplastic mucosa was graded as 0 (absent), 1 (focal), or 2 (extensive) and consisted of irregular, angulated, and occasionally cystically dilated glands with enlarged overlapping hyperchromatic nuclei. Carcinoma was defined as irregular, angulated, and cystically dilated glands with occasional cribriform architecture in the submucosa and muscularis propria, spreading laterally to the surface mucosal component (28). For quantitative H. pylori culture, serial dilutions of homogenized tissue were plated on selective antibiotic TSA blood agar plates (27).

Multiplex bead array

Gastric linear strips extending from the squamocolumnar junction through the proximal duodenum were lysed in 200 μL of IP lysis buffer (Thermo Fisher Scientific) containing protease and phosphatase inhibitors (Roche). Lysates were diluted 1:3 in assay buffer and mixed with magnetic beads according to the manufacturer's instructions (Millipore; ref. 27). Data were acquired and analyzed using the Millipore software platform.

RT2 profiler PCR array

RNA from primary gastric epithelial cell monolayers was isolated using the RNeasy Kit (Qiagen). Total RNA was treated with RNase free-DNase (Promega) overnight and then used for cDNA synthesis (Applied Biosystems). Real-time PCR master mix, plates, and running protocols were performed following the manufacturer's instructions for the mouse NF-κB gene target array (Qiagen). Data were analyzed using the online Data Analysis Center provided by Qiagen.

Real-time RT-PCR

Total RNA isolated from bone marrow–derived macrophages or AGS gastric epithelial cells (CRL-1739) was subjected to overnight treatment with RNase-free DNase, and then reverse transcribed to cDNA. qPCRs were performed to determine relative differences in expression levels of Nos2, TNFα, IL10, TGFβ, Light, and Ym1, in murine macrophages and CXCL8 and CXCL2 in AGS cells. Results were then normalized to corresponding levels of GAPDH. For NOD1 inhibition studies, AGS cells were transfected with a mix of shRNAs targeting NOD1 as described previously (22). Colonies were selected using puromycin (10 μg/mL) and tested for NOD1 expression by real-time RT-PCR and Western blot analysis.

CagA translocation

AGS cells cocultured with H. pylori were lysed in RIPA buffer containing phosphatase and protease inhibitors. Proteins were separated using 6% SDS-PAGE mini gels, transferred to PVDF membranes (Thermo Fisher Scientific), and membranes were blocked with 1% BSA (Sigma) overnight. Incubation with primary antibodies (mouse anti-PY99 (Santa Cruz Biotechnology), rabbit anti-CagA (Austral Biologicals), and mouse anti-GAPDH (Millipore) was performed for 1 hour followed by addition of respective HRP-conjugated secondary antibodies (anti–mouse-HRP or anti–rabbit-HRP; Promega). The reaction was developed using ECL (Thermo Fisher Scientific).

c-Jun immunofluorescence staining

Monolayers of primary gastric epithelial cells derived from C57BL/6WT, C57BL/6Nod1−/−, FVB/N INS-GASNod1+/+, and FVB/N INS-GASNod1−/− mice were infected for 1 hour with H. pylori strains 7.13 or PMSS1. After infection, monolayers were subjected to c-Jun immunofluorescence staining as previously described (26). Briefly, cells were fixed with 4% paraformaldehyde (Thermo Fisher Scientific) for 1 hour and then blocked with 5% goat serum (Sigma) in PBS for 1 hour. Samples were incubated with an anti–phospho-c-Jun antibody (1:500 dilution; Cell Signaling Technology) overnight at 4°C. Samples were then incubated with Alexa 488-anti-rabbit (1:1,000; Invitrogen), Alexa 546-Phalloidin (1:500; Invitrogen), and Hoechst 33342 (1:1,000; Invitrogen) for 1 hour at room temperature. Slides were mounted using ProLong Glass (Invitrogen), and images were acquired in an Olympus FV-1000 confocal microscopy.

Statistical analysis

The Mann–Whitney test was used for two-group comparisons, whereas one-way ANOVA with Newman–Keuls posttest was used for multiple group comparisons. Data were plotted and analyzed using Prism V. 5b (GraphPad software Inc). Statistical significance was set at a two-tailed P value of < 0.05.

Nod1 suppresses the early inflammatory response to H. pylori in C57BL/6 mice

We previously demonstrated that preactivation of NOD1 leads to attenuated cytokine production by H. pylori in vitro and reduced inflammation in an outbred model of infection (Mongolian gerbils; ref. 22). We also showed that expression of NOD1 per se and its target genes were significantly decreased in human gastric cancer specimens compared with samples with gastritis alone (22). Therefore, we sought to define mechanisms that regulate this potential anti-inflammatory response within the context of gastric carcinogenesis using genetic models of NOD1 deficiency.

C57BL/6WT and C57BL/6Nod1−/− mice were challenged with the mouse-adapted H. pylori cag+ strain PMSS1 or broth alone, and stomachs were harvested and analyzed at 2 (acute), 20 (subacute), and 90 (chronic) days postchallenge. All mice challenged with H. pylori were successfully infected. H. pylori–infected C57BL/6Nod1−/− mice developed significantly more severe gastric inflammation 20 days postchallenge compared with their infected WT counterparts (P < 0.05; Fig. 1A and B). However, by 90 days postchallenge, both groups had similarly elevated inflammation scores (P = NS).

Figure 1.

Nod1 suppresses the early inflammatory response to H. pylori but does not alter microbial virulence phenotypes. A, Gastric mucosal inflammatory scores and colonization density for C57BL/6 WT (white bars) and Nod1−/− (gray bars) male (M) and female (F) mice infected with or without H. pylori strain PMSS1 for 2 days [WT uninfected (n = 6, 3M/3F) and infected (n = 9, 5M/4F); Nod1−/− uninfected (n = 9, 5M/4F) and infected (n = 9, 5M/4F)], 20 days [WT uninfected (n = 10, 5M/5F) and infected (n = 11, 5M/6F); Nod1−/− uninfected (n = 8, 7M/1F) and infected (n = 9, 4M/5F)], and 90 days [WT uninfected (n = 7, 4M/3F) and infected (n = 10, 5M/5F); Nod1−/− uninfected (n = 10, 5M/5F) and infected (n = 11, 5M/6F)]. Bars represent mean inflammation scores (left axis) or colony-forming units (CFU)/g tissue (right axis) ± SEM. *, P ≤ 0.05. B, Representative histologic images from uninfected or H. pylori–infected WT and Nod1−/− mice. C, Western blot analysis for CagA (phosphorylated, P-CagA; total, T-CagA) in AGS cells cocultured with output H. pylori strains isolated from C57BL/6 WT and Nod1−/− mice infected for 20 and 90 days. Bar graphs represent densitometric index of P-CagA over T-CagA, which reflects the intensity of CagA translocation. An H. pylori PMSS1 isogenic pgdA mutant strain (22) was included as a bacterial control to ensure that altering H. pylori peptidoglycan did not reduce CagA translocation.

Figure 1.

Nod1 suppresses the early inflammatory response to H. pylori but does not alter microbial virulence phenotypes. A, Gastric mucosal inflammatory scores and colonization density for C57BL/6 WT (white bars) and Nod1−/− (gray bars) male (M) and female (F) mice infected with or without H. pylori strain PMSS1 for 2 days [WT uninfected (n = 6, 3M/3F) and infected (n = 9, 5M/4F); Nod1−/− uninfected (n = 9, 5M/4F) and infected (n = 9, 5M/4F)], 20 days [WT uninfected (n = 10, 5M/5F) and infected (n = 11, 5M/6F); Nod1−/− uninfected (n = 8, 7M/1F) and infected (n = 9, 4M/5F)], and 90 days [WT uninfected (n = 7, 4M/3F) and infected (n = 10, 5M/5F); Nod1−/− uninfected (n = 10, 5M/5F) and infected (n = 11, 5M/6F)]. Bars represent mean inflammation scores (left axis) or colony-forming units (CFU)/g tissue (right axis) ± SEM. *, P ≤ 0.05. B, Representative histologic images from uninfected or H. pylori–infected WT and Nod1−/− mice. C, Western blot analysis for CagA (phosphorylated, P-CagA; total, T-CagA) in AGS cells cocultured with output H. pylori strains isolated from C57BL/6 WT and Nod1−/− mice infected for 20 and 90 days. Bar graphs represent densitometric index of P-CagA over T-CagA, which reflects the intensity of CagA translocation. An H. pylori PMSS1 isogenic pgdA mutant strain (22) was included as a bacterial control to ensure that altering H. pylori peptidoglycan did not reduce CagA translocation.

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To define mechanisms underpinning these temporal differences in inflammation, we initially investigated whether microbial factors contributed to these phenotypes. No significant differences in levels of H. pylori colonization were present between WT and Nod1−/− mice at any time point (Fig. 1A). We next determined whether selective pressure exerted by the genetic loss of Nod1 affected H. pylori virulence phenotypes in vivo. H. pylori strains were recovered from infected WT or Nod1−/− mice and assessed for the ability to translocate CagA in vitro as a measure of cag T4SS functionality. AGS gastric epithelial cells were infected with in vivo–adapted H. pylori strains and the ratio of phosphorylated CagA (intracellular) to total CagA was quantified by Western blot analysis. The vast majority of strains harvested at 20 days postinfection harbored a functional cag T4SS. However, as previously reported (25, 29), loss of cag T4SS function was present in many of the in vivo–adapted isolates harvested at 90 days postchallenge (Fig. 1C). Because activation of NOD1 is cag-dependent (7), this may partially explain the lack of difference in severity of inflammation between infected WT or Nod1−/− mice at the 90-day time point. Importantly, host Nod1 status had no effect on T4SS function at either time point (Fig. 1C). Thus, on a genetically defined background, loss of Nod1 resulted in more severe subacute inflammation and injury within the context of H. pylori infection and in conjunction with strains that harbored a functional cag T4SS; however, Nod1 deficiency did not alter H. pylori cag T4SS phenotypes.

Loss of Nod1 alters cytokine production within H. pylori–infected gastric mucosa

Having shown that H. pylori increases inflammation in Nod1−/− mice despite similar cag T4SS function (Fig. 1), we next performed an unbiased survey to assess the role of Nod1-mediated immune effectors that may contribute to differences in inflammation within uninfected or infected gastric tissue. Infection with H. pylori induced an increase in the levels of numerous chemokines, as well as Th1, Th2, and Th17 cytokines in gastric mucosa harvested from both WT and Nod1-deficient mice when compared with uninfected controls. For ease of presentation, we have included cytokines and chemokines in Fig. 2 that (i) were statistically different in terms of levels of expression between Nod1+/+ and Nod1−/− mice and (ii) could be classified as either chemokines or Th1, Th2, or Th17 cytokines. The complete sets of data are contained in Supplementary Tables S2–S4. Expression levels of archetypal Th1-secreted cytokines such as IL12, Th1-associated proinflammatory chemokines such as IL1, the Th17 cytokines IL17 and IL23, and the Th2 cytokine IL9 were further increased in H. pylori–infected C57BL/6Nod1−/− compared with infected C57BL/6WT mice at each time point (2, 20, and 90 days postchallenge; Fig. 2A–C; Supplementary Tables S2–S4). In contrast to universal elevation of these cytokines and chemokines throughout the duration of infection, there were also more selected differences between H. pylori–infected WT versus Nod1-deficient mice that varied by time point (Fig. 2A–C; Supplementary Tables S2–S4). Specifically, at 20 days postchallenge when inflammation was significantly increased in Nod1-deficient mice (Fig. 2B), levels of G-CSF, IL7, IP-10, IFNα, IFNβ, and IL4 were exclusively increased in infected C57BL/6Nod1−/− compared with C57BL/6WT mice. In addition, the overall number of cytokines and chemokines that were elevated in infected WT mice increased from 2 to 90 days postchallenge (Fig. 2A–C). Thus, in addition to altering the level of inflammation in a time-dependent manner, loss of Nod1 also temporally modified the portfolio of chemokine and cytokine expression within H. pylori–infected gastric mucosa.

Figure 2.

Loss of Nod1 alters cytokine production in gastric mucosa of C57BL/6 mice following H. pylori infection. Expression of chemokines and cytokines in gastric mucosa of C57BL/6 WT and Nod1−/− mice infected with H. pylori strain PMSS1 for 2 (A), 20 (B), and 90 days (C). Bars, mean ± SEM. Light shaded bars, upregulated in Nod1−/− mice; dark shaded bars, upregulated in WT mice. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

Figure 2.

Loss of Nod1 alters cytokine production in gastric mucosa of C57BL/6 mice following H. pylori infection. Expression of chemokines and cytokines in gastric mucosa of C57BL/6 WT and Nod1−/− mice infected with H. pylori strain PMSS1 for 2 (A), 20 (B), and 90 days (C). Bars, mean ± SEM. Light shaded bars, upregulated in Nod1−/− mice; dark shaded bars, upregulated in WT mice. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

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H. pylori differentially alters cytokine production in primary gastric organoid systems in a Nod1-dependent manner

Our findings in gastric mucosa provided important insights into mechanisms through which Nod1 may restrain early proinflammatory responses to H. pylori. However, gastric tissue contains a myriad of cell types, including epithelial cells and macrophages among others. Gastroids are polarized, replenishable epithelial culture systems that can be readily generated from nontransformed gastric epithelium (30). We have previously developed and optimized gastroid models of H. pylori infection originating from murine tissue (31); therefore, we capitalized on this reductionist ex vivo model to begin to dissect the role of specific cell types in regulating phenotypes linked to Nod1 deficiency (Fig. 3). Polarized 2-dimensional gastroid monolayers were generated from uninfected C57BL/6WT and C57BL/6Nod1−/− mice, infected ex vivo with H. pylori strains PMSS1 or 7.13 for 6 or 24 hours, and then coculture total mRNA was subjected to real-time RT-PCR to quantify expression levels of NF-κB target genes. Compared with expression levels in gastroids generated from WT mice (Fig. 3A; Supplementary Fig. S1A), expression levels of numerous NF-κB target genes were significantly increased in infected Nod1-deficient gastroids at both 6 (Fig. 3B; Supplementary Fig. S1B) and 24 (Fig. 3C; Supplementary Fig. S1C) hours after H. pylori challenge (Fig. 3; Supplementary Fig. S1; Supplementary Tables S5–S9). Of note, the magnitude of these effects were amplified following infection with H. pylori strain PMSS1 versus strain 7.13, which likely reflects the genetic diversity inherent within this pathogen. Specific targets upregulated in Nod1-deficient gastroids infected by either H. pylori strain that were also upregulated acutely in vivo included Cxcl1 (e.g., KC). We therefore validated expression of KC at the protein level in gastric epithelial monolayers by demonstrating that KC protein was not only increased by H. pylori infection but that loss of Nod1 significantly augmented this effect (Fig. 3D). These data complement our previously published results (22), demonstrating that suppression of Nod1 in gastric epithelial cells augments H. pylori–induced NF-κB activation. However, based on prior data implicating MAP kinase activation as well as NF-κB activation in mediating downstream effects of Nod1 (32, 33), we also investigated the role of MAP kinase activation within the context of Nod1 in our gastroid system. As shown in Fig. 3E and F, we cocultured gastroids isolated from Nod1+/+ or Nod1−/− mice with H. pylori and examined c-Jun activation by immunofluorescence. In contrast to the pattern observed with NF-κB, Nod1 deficiency suppressed c-Jun activation in response to H. pylori infection (Fig. 3E and F). Thus, Nod1 can exert differing effects on signaling pathways following infection with H. pylori. Collectively, these results indicate that gastric epithelial cells likely represent an important source of cytokine production that is under Nod1-dependent control during H. pylori infection.

Figure 3.

H. pylori differentially alters cytokine production and MAP kinase activation in gastroids in a Nod1-dependent manner. RT2 profiler PCR array analysis for NF-κB gene targets in primary gastric epithelial cell organoids generated from C57BL/6 WT and Nod1−/− uninfected mice; control gastroids were uninfected ex vivo (A) or cocultured with H. pylori strain PMSS1 for 6 (B) or 24 (C) hours at MOI of 30. Black circles represent genes upregulated and gray circles represent genes downregulated in gastroids from Nod1−/− mice compared with WT mice. Solid line represents the linear regression of the WT samples, and dotted lines represent 95% confidence intervals. D, Protein levels of KC (Cxcl1) in supernatants of primary gastric epithelial cell organoids from WT or Nod1−/− mice cocultured with H. pylori PMSS1 for 24 hours at MOI of 30. Bars, mean ± SEM. **, P ≤ 0.01; and ***, P ≤ 0.001. E, Immunoflourescence for phospho–c-Jun nuclear translocation in primary gastric cell monolayers generated from C57BL/6 and INS-GAS Nod1+/+ and Nod1−/− uninfected mice, which were then infected with or without H. pylori strains PMSS1 or 7.13 (MOI 30) for 1 hour. Green, phospho–c-Jun; blue, DAPI; red, phalloidin; magnification, ×60.F, Quantification of nuclear c-Jun is shown adjacent to representative images. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.

Figure 3.

H. pylori differentially alters cytokine production and MAP kinase activation in gastroids in a Nod1-dependent manner. RT2 profiler PCR array analysis for NF-κB gene targets in primary gastric epithelial cell organoids generated from C57BL/6 WT and Nod1−/− uninfected mice; control gastroids were uninfected ex vivo (A) or cocultured with H. pylori strain PMSS1 for 6 (B) or 24 (C) hours at MOI of 30. Black circles represent genes upregulated and gray circles represent genes downregulated in gastroids from Nod1−/− mice compared with WT mice. Solid line represents the linear regression of the WT samples, and dotted lines represent 95% confidence intervals. D, Protein levels of KC (Cxcl1) in supernatants of primary gastric epithelial cell organoids from WT or Nod1−/− mice cocultured with H. pylori PMSS1 for 24 hours at MOI of 30. Bars, mean ± SEM. **, P ≤ 0.01; and ***, P ≤ 0.001. E, Immunoflourescence for phospho–c-Jun nuclear translocation in primary gastric cell monolayers generated from C57BL/6 and INS-GAS Nod1+/+ and Nod1−/− uninfected mice, which were then infected with or without H. pylori strains PMSS1 or 7.13 (MOI 30) for 1 hour. Green, phospho–c-Jun; blue, DAPI; red, phalloidin; magnification, ×60.F, Quantification of nuclear c-Jun is shown adjacent to representative images. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.

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NOD1 regulates cytokine production in multiple innate immune cells following H. pylori infection

Gastritis is an initiating event for the development of most gastric adenocarcinomas, and macrophages are required for gastritis to develop in response to H. pylori (34). Therefore, we enriched our gastroid monolayer system by adding macrophages derived from WT or Nod1-deficient mice to epithelial cells derived from the same mice.

Gastroid epithelial monolayers from uninfected WT or Nod1-deficient mice were seeded in the upper chamber of a transwell system and macrophages from the same mice were placed in the lower chamber in different combinations. H. pylori strain PMSS1 was then added to epithelial monolayers for 24 hours and cytokine production was quantified in supernatants. Loss of Nod1 in H. pylori–infected epithelial cells cocultured with WT macrophages resulted in significantly enhanced levels of chemokines and cytokines, including KC, MIP-2, IL6, MCP-1, and IL1α (Fig. 4A) when compared with infected WT epithelial cells cocultured with WT macrophages. However, loss of Nod1 in both epithelial cells and macrophages resulted in a markedly enhanced cytokine response to H. pylori (Fig. 4B). These results indicate that loss of Nod1 in multiple innate immune cells likely contributes to enhanced inflammation observed in H. pylori–infected Nod1-deficient mice.

Figure 4.

Nod1 regulates cytokine production in multiple innate immune cells following H. pylori infection. Cytokine and chemokine expression as determined by multiplex bead array from bone marrow (BM)–derived macrophages harvested from C57BL/6 WT or Nod1−/− mice placed in the lower chamber of a transwell system in coculture, with primary gastric epithelial cell (GEC) monolayers isolated from the same mice placed in the upper chamber and then infected with H. pylori strain (upper chamber) for 24 hours at MOI of 30. A, Comparison of H. pylori–infected cocultures of WT bone marrow macrophages and WT gastric epithelial cells with cocultures of WT bone marrow macrophages and Nod1−/− gastric epithelial cells. B, Comparison of H. pylori–infected cocultures of Nod1−/− bone marrow macrophages and WT gastric epithelial cells with cocultures of Nod1−/− bone marrow macrophages and Nod1−/− gastric epithelial cells. Dotted lines represent 95% confidence interval. Black circles represent upregulated and gray circle represents downregulated chemokines or cytokines. C, Quantification of macrophage differentiation marker expression by real-time RT-PCR using RNA isolated from bone marrow–derived macrophages (bottom chamber) cocultured in transwell systems with corresponding gastric epithelial cells (upper chamber), uninfected or infected with H. pylori (upper chamber) for 24 hours at MOI of 30. Primer sequences are shown in Supplementary Table S14. Bars, mean ± SEM. Light bars, cells derived from C57BL/6 WT mice; dark bars, cells derived from C57BL/6 Nod1−/− mice. *, P ≤ 0.05.

Figure 4.

Nod1 regulates cytokine production in multiple innate immune cells following H. pylori infection. Cytokine and chemokine expression as determined by multiplex bead array from bone marrow (BM)–derived macrophages harvested from C57BL/6 WT or Nod1−/− mice placed in the lower chamber of a transwell system in coculture, with primary gastric epithelial cell (GEC) monolayers isolated from the same mice placed in the upper chamber and then infected with H. pylori strain (upper chamber) for 24 hours at MOI of 30. A, Comparison of H. pylori–infected cocultures of WT bone marrow macrophages and WT gastric epithelial cells with cocultures of WT bone marrow macrophages and Nod1−/− gastric epithelial cells. B, Comparison of H. pylori–infected cocultures of Nod1−/− bone marrow macrophages and WT gastric epithelial cells with cocultures of Nod1−/− bone marrow macrophages and Nod1−/− gastric epithelial cells. Dotted lines represent 95% confidence interval. Black circles represent upregulated and gray circle represents downregulated chemokines or cytokines. C, Quantification of macrophage differentiation marker expression by real-time RT-PCR using RNA isolated from bone marrow–derived macrophages (bottom chamber) cocultured in transwell systems with corresponding gastric epithelial cells (upper chamber), uninfected or infected with H. pylori (upper chamber) for 24 hours at MOI of 30. Primer sequences are shown in Supplementary Table S14. Bars, mean ± SEM. Light bars, cells derived from C57BL/6 WT mice; dark bars, cells derived from C57BL/6 Nod1−/− mice. *, P ≤ 0.05.

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We also capitalized on this system to assess cytokine expression in macrophages per se. RNA was isolated from macrophages subjected to uninfected and infected transwell cocultures and expression of prototype M1 (classically activated), M2 (alternatively activated), and Mreg (regulatory) macrophage genes was determined by real-time RT-PCR. Uninfected WT macrophages harbored a predominant M2 phenotype, which shifted dramatically to an M1 phenotype after H. pylori infection. In contrast, uninfected Nod1-deficient macrophages displayed a more profound M2 phenotype than uninfected WT macrophages; following H. pylori infection, the profile of Nod1-deficient macrophages shifted to a hybrid M1/M2 phenotype. There were no significant differences in Mreg profiles between macrophages from WT or Nod1-deficient mice (Fig. 4C). Thus, loss of Nod1 alters cytokine profiles in both epithelial cells and macrophages during coincubation experiments in response to H. pylori.

Loss of Nod1 accelerates gastric carcinogenesis in a mouse model of stomach cancer

Our studies described above using a mouse model of H. pylori–induced gastritis demonstrated that C57BL/6Nod1−/− mice develop more severe inflammation in response to H. pylori infection than C57BL/6WT mice (Fig. 1A and B). Because specific genetic backgrounds can influence disease outcome in different mouse models (35), we next determined whether the increased inflammatory phenotype induced by loss of Nod1 in C57BL/6 mice could be recapitulated in H. pylori–infected mice on a different genetic background that are also susceptible to gastric cancer.

INS-GASNod1+/+ and INS-GASNod1−/− mice on a FVB/N background were challenged with H. pylori strain PMSS1 or broth alone, and stomachs were harvested and analyzed 2, 20, 40, and 90 days postchallenge. The 40-day time point was added due to accelerated inflammation and damage previously observed in H. pylori–infected INS-GAS mice at this time point (36). All mice challenged with H. pylori were successfully infected. H. pylori–infected INS-GASNod1−/− mice developed significantly more severe acute and chronic inflammation compared with their infected WT Nod1 counterparts at 20 and 40 days postchallenge (Fig. 5A and B). Of interest, loss of Nod1 per se in uninfected INS-GAS mice led to increased levels of inflammation and injury at 40 and 90 days postchallenge compared with uninfected INS-GAS mice with a WT Nod1 genotype (Fig. 5A and B). Thus, on two genetically distinct backgrounds, loss of Nod1 resulted in a time-dependent pattern of more severe inflammation and injury within the context of H. pylori infection.

Figure 5.

Loss of Nod1 accelerates gastric carcinogenesis in mice. A, Gastric mucosal inflammatory scores and colonization density for male INS-GAS Nod1+/+ mice and INS-GAS Nod1−/− mice infected with or without H. pylori strain PMSS1 for 2 days [Nod1+/+ uninfected (n = 5) and infected (n = 5); Nod1−/− uninfected (n = 5) and infected (n = 5)], 20 days [Nod1+/+ uninfected (n = 10) and infected (n = 10); Nod1−/− uninfected (n = 10) and infected (n = 10)], 40 days [Nod1+/+ uninfected (n = 5) and infected (n = 6); Nod1−/− uninfected (n = 5) and infected (n = 6)], and 90 days [Nod1+/+ uninfected (n = 5) and infected (n = 8); Nod1−/− uninfected (n = 5) and infected (n = 13)]. Bars represent mean inflammation scores (left axis) or CFU/g tissue (right axis) ± SEM. *, P ≤ 0.05; **, P ≤ 0.01. B, Representative histologic images from uninfected or H. pylori–infected INS-GAS Nod1+/+ mice and INS-GAS Nod1−/− mice. Higher magnification insets of dysplastic foci are shown below other histologic images. C, Frequency of gastric dysplasia in INS-GAS Nod1+/+ and INS-GAS Nod1−/ mice, uninfected or infected with H. pylori strain PMSS1 for 40 or 90 days. White bars, no dysplasia; gray bars, dysplasia. ***, P ≤ 0.001; ****, P ≤ 0.0001.

Figure 5.

Loss of Nod1 accelerates gastric carcinogenesis in mice. A, Gastric mucosal inflammatory scores and colonization density for male INS-GAS Nod1+/+ mice and INS-GAS Nod1−/− mice infected with or without H. pylori strain PMSS1 for 2 days [Nod1+/+ uninfected (n = 5) and infected (n = 5); Nod1−/− uninfected (n = 5) and infected (n = 5)], 20 days [Nod1+/+ uninfected (n = 10) and infected (n = 10); Nod1−/− uninfected (n = 10) and infected (n = 10)], 40 days [Nod1+/+ uninfected (n = 5) and infected (n = 6); Nod1−/− uninfected (n = 5) and infected (n = 6)], and 90 days [Nod1+/+ uninfected (n = 5) and infected (n = 8); Nod1−/− uninfected (n = 5) and infected (n = 13)]. Bars represent mean inflammation scores (left axis) or CFU/g tissue (right axis) ± SEM. *, P ≤ 0.05; **, P ≤ 0.01. B, Representative histologic images from uninfected or H. pylori–infected INS-GAS Nod1+/+ mice and INS-GAS Nod1−/− mice. Higher magnification insets of dysplastic foci are shown below other histologic images. C, Frequency of gastric dysplasia in INS-GAS Nod1+/+ and INS-GAS Nod1−/ mice, uninfected or infected with H. pylori strain PMSS1 for 40 or 90 days. White bars, no dysplasia; gray bars, dysplasia. ***, P ≤ 0.001; ****, P ≤ 0.0001.

Close modal

Unlike C57BL/6 mice, which do not develop cancer prior to 15 months after H. pylori infection (37), INS-GAS mice rapidly develop premalignant lesions as early as 6 to 12 weeks following H. pylori challenge (36). At 40 days postchallenge, none of the uninfected or H. pylori–infected INS-GASNod1+/+ developed gastric dysplasia. However, loss of Nod1 led to the development of dysplasia in 50% of uninfected mice, and the frequency of dysplasia rose significantly in conjunction with H. pylori infection (Fig. 5C). At 90 days postchallenge, these effects were magnified. Infection of INS-GASNod1+/+ with H. pylori significantly increased the prevalence of dysplasia compared with uninfected WT controls, which did not demonstrate any evidence of premalignant lesions. In uninfected INS-GASNod1−/− mice, the prevalence of gastric dysplasia was significantly higher than in uninfected INS-GASNod1+/+ mice; however, infection with H. pylori similarly augmented this effect as 95% of infected INS-GASNod1−/− mice developed gastric dysplasia by this time point.

To assess the levels of microbial colonization in each group, we quantified colony-forming units (CFU) from each infected animal. There were no differences in colonization between WT and Nod1−/− INS-GAS mice at any time point (Fig. 5A). Collectively, these data indicate that the increased injury phenotype that develops in H. pylori–infected Nod1-deficient mice is not an artifact of a single host genetic background.

Nod1 deficiency increases gastric mucosal cytokine production in H. pylori–infected INS-GAS mice

We next sought to elucidate the mechanisms through which loss of Nod1 resulted in increased inflammation and premalignant lesions among H. pylori–infected INS-GAS mice by quantifying Nod1-mediated immune effectors within uninfected or infected gastric tissue. Similar to our results in C57BL/6 mice (Fig. 2A–C; Supplementary Tables S2–S4), infection with H. pylori induced an increase in the levels of chemokines, as well as Th1, Th2, and Th17 cytokines in both WT and Nod1-deficient mice when compared with uninfected controls and these changes were augmented within the context of Nod1 deficiency (Fig. 6A–D; Supplementary Tables S10–S13). For ease of presentation, we have included cytokines and chemokines in Fig. 6 that (i) were statistically different in terms of levels of expression between Nod1+/+ and Nod1−/− mice and (ii) could be classified as either chemokines or Th1, Th2, or Th17 cytokines. The complete sets of data are contained in Supplementary Tables S10–S13. Because inflammation was more severe in Nod1 deficient INS-GAS mice at 20 and 40 days postinfection (Fig. 5A and B), we focused on selected differences between H. pylori–infected WT versus Nod1-deficient mice at these time points (Fig. 6B and C). Specifically, at 20 days postchallenge, levels of IL12p40 and TNFα were exclusively increased in infected INS-GASNod1−/− compared with INS-GASWT mice and at 20 and 40 days postinfection, levels of IL6 were exclusively increased in the same pattern. There was also a shift toward more chemokines and cytokines being increased in INS-GASNod1+/+ mice compared with their Nod1-deficient counterparts as the duration of infection progressed (Figs. 6A–D). Collectively, these results and results from C57BL/6 mice demonstrate that loss of Nod1 alters the severity of mucosal damage as well as the portfolio of chemokine and cytokine expression within H. pylori–infected gastric mucosa in two different murine models of gastric injury. However, the effects of Nod1 deficiency wane over time, suggesting that Nod1 exerts its effects predominantly on innate immune responses, which is consistent with our epithelial:macrophage gastroid coculture results.

Figure 6.

Nod1 deficiency increases gastric mucosal chemokine/cytokine production in H. pylori–infected INS-GAS mice and human gastric epithelial cells. Expression of chemokines and cytokines in gastric mucosa of INS-GAS Nod1+/+ and INS-GAS Nod1−/− mice infected with H. pylori strain PMSS1 for 2 (A), 20 (B), 40 (C), and 90 (D) days. Bars, mean ± SEM. Light shaded bars, upregulated in INS-GAS Nod1−/− mice; dark shaded bars, upregulated in INS-GAS Nod1+/+ mice. *, P ≤ 0.05; **, P ≤ 0.01. E, AGS cells stably transfected with either nontargeting shRNA or shRNA specific for NOD1 were cocultured with strains 7.13 or PMSS1 at MOI 30 for 6 and 24 hours. Expression levels of CXCL8 (left axis) and CXCL2 (right axis) were determined by real-time RT-PCR. Primer sequences are shown in Supplementary Table S14. Data represent mean ± SEM from at least two experiments. *, P ≤ 0.05; **, P ≤ 0.01.

Figure 6.

Nod1 deficiency increases gastric mucosal chemokine/cytokine production in H. pylori–infected INS-GAS mice and human gastric epithelial cells. Expression of chemokines and cytokines in gastric mucosa of INS-GAS Nod1+/+ and INS-GAS Nod1−/− mice infected with H. pylori strain PMSS1 for 2 (A), 20 (B), 40 (C), and 90 (D) days. Bars, mean ± SEM. Light shaded bars, upregulated in INS-GAS Nod1−/− mice; dark shaded bars, upregulated in INS-GAS Nod1+/+ mice. *, P ≤ 0.05; **, P ≤ 0.01. E, AGS cells stably transfected with either nontargeting shRNA or shRNA specific for NOD1 were cocultured with strains 7.13 or PMSS1 at MOI 30 for 6 and 24 hours. Expression levels of CXCL8 (left axis) and CXCL2 (right axis) were determined by real-time RT-PCR. Primer sequences are shown in Supplementary Table S14. Data represent mean ± SEM from at least two experiments. *, P ≤ 0.05; **, P ≤ 0.01.

Close modal

Finally, because H. pylori is a human gastric pathogen, we ascertained the effects of infection on Nod1-dependent signaling in human gastric epithelial cells. For these studies, stably transfected human AGS gastric epithelial cells harboring either NOD1-targeting or control shRNA were used as described previously (22). Inhibition of NOD1 prior to infection with the H. pylori cag+ strains PMSS1 or 7.13 increased production of CXCL8 and CXCL2, confirming our data in mouse gastroids that suppression of Nod1 can augment inflammatory responses to this pathogen (Fig. 6E).

The innate immune system is exquisitely poised to detect and respond to bacteria residing at mucosal surfaces (38, 39). However, chronic mucosal pathogens have developed multiple strategies to subvert this facet of the immune response (40). H. pylori has coevolved with its cognate human host for over 100,000 years and is uniquely adapted to survive for decades within the harsh environment of the stomach (41). This has necessitated the development of mechanisms to induce inflammation as well as strategies to evade detection and downregulate the host immune response.

H. pylori harbors multiple pathogen-associated mucosal patterns that interact differently with innate immune effectors than the respective counterparts in other acute mucosal pathogens. For example, H. pylori FlaA is a noninflammatory molecule in terms of its ability to activate TLR5 (42). H. pylori LPS contains an anergic lipid A core that induces an attenuated TLR4-mediated response (43, 44). The cag T4SS delivers peptidoglycan into host cells, where it is recognized by NOD1 (7–11), and although NF-κB activation is a prototypical response to NOD1 activation, we and others have shown that preactivation of NOD1 suppresses subsequent H. pylori–induced NF-κB signaling via activation of a negative feedback loop, and that deacetylation of peptidoglycan allows H. pylori to evade host clearance (22, 45–47). More recent work has also indicated that NOD1 may suppress gastric inflammation in response to H. pylori. Tran and colleagues. used a mouse model of gastritis to demonstrate that H. pylori promotes the activation of IL33, a mediator of Th2 immune responses, via Nod1 signaling (48). Importantly, H. pylori cag+ strains specifically activated Nod1 in mouse gastric epithelial cells, leading to enhanced levels of IL33 in gastric mucosa and splenocytes, which was linked with reduced IFNγ responses (48). Our current results are consistent with these data, as loss of Nod1 in two independent models of H. pylori–induced inflammation and injury augmented damage within the gastric niche. However, there are additional pathways that can regulate NF-κB activation following H. pylori infection that are independent of NOD1. Gall and colleagues. demonstrated that NF-κB activation can be induced by TNF receptor–associated factor (TRAF)-interacting protein with forkhead-associated domain (TIFA), and that this occurs independently from NOD1-mediated NF-κB activation (49). Furthermore, NF-κB activation following H. pylori infection occurs in a temporally regulated manner, with TIFA induction occurring early, which is subsequently followed by NOD1-dependent NF-κB activation (49). Selective activation of these additional pathways under different experimental conditions may account for differences in our current results when compared with previous results published by Viala and colleagues (7). Therefore, future studies should focus on assessing both NOD1 and TIFA innate immune signaling pathways in primary organoid systems as well as animal models of infection, utilizing both WT H. pylori cag+ strains and mutant strains with defective cag secretion systems, to precisely elucidate the combinatorial contributions of these constituents to H. pylori pathogenesis.

In this study, Nod1−/− genotypes did not alter the microbial phenotype of H. pylori output derivatives in terms of T4SS function. However, Nod1-deficient mice developed more severe inflammation compared with WT mice when colonized with H. pylori strains harboring a functional cag T4SS. Using a multiplex cytokine array to examine distinct host immune effectors that may contribute to this increase in inflammation, we observed that, as expected, H. pylori infection broadly increased levels of Th1, Th2, and Th17 cytokines, and these changes were augmented in the presence of Nod1 deficiency. However, differences stratified on the basis of Nod1 genotype waned over time. To dissect this further, we utilized an innovative reductionist system in which the effects of H. pylori on WT or Nod1−/− epithelial cells alone or epithelial cells cocultured with macrophages of varying Nod1 genotype could be ascertained. We determined that loss of Nod1 in both epithelial cells and macrophages augmented the production of proinflammatory cytokine production in response to H. pylori infection, further supporting the premise that NOD1 primarily alters innate immune responses to this pathogen. To more fully define the respective contributions of each of these cellular constituents, however, would require eliminating the macrophage and the gastric epithelial cell component of Nod1-dependent signaling, both individually and in combination. This could be done by crossing Nod1-floxed mice and Foxa3-Cre mice (which directs expression of Cre recombinase to gastric epithelium; ref. 50) in conjunction with selective depletion of macrophages using agents such as clodronate liposomes and our current results have provided an important framework for these future studies.

In addition to differences in cytokine levels between Nod1+/+ and Nod1−/− mice, we also found differences in the levels of certain cytokines when we compared C57BL/6 with INS-GAS FVB/N mice. These results likely reflect differences in the genetic backgrounds of the mice under study as well as the presence of hypergastrinemia, which is inherent to INS-GAS mice bearing the human gastrin transgene (51). Gastrin exerts growth factor–like effects on gastric epithelial cells, which may induce the production of certain chemokines and cytokines (51). Furthermore, INS-GAS mice are on a FVB/N genetic background that has previously been shown to augment the risk for carcinogenesis when compared with C57BL/6 mice. Specifically, this may be due to a polymorphism within the Ptch1 gene, which encodes an inhibitory receptor for ligands of the Hedgehog gene family (52). Within the context of our results, it was notable that, among others, expression levels of IL9 were downregulated in INS-GAS Nod1−/− mice when compared with C57BL/6 Nod1−/− mice. IL9 is a cytokine produced by Th9 cells, a subpopulation of CD4+ T cells, and several studies have demonstrated that IL9 suppresses tumor growth (53, 54). Thus, decreased levels of IL9 in H. pylori–infected INS-GAS Nod1−/− mice may contribute to the enhanced carcinogenic phenotype seen in these mice following infection with this pathogen.

Alterations in the composition of functional macrophage phenotypes within a specific inflamed niche can significantly alter the risk for carcinogenesis (55). M1 (classically activated) macrophages clear pathogens via intracellular microbicidal activity and by secreting inflammatory mediators that promote a Th1-type response, while simultaneously dampening Th2-type responses (55). M2 (alternatively activated) macrophages promote wound healing by secreting components of the extracellular matrix and anti-inflammatory effectors that promote Th2-directed responses while dampening Th1 responses (5). Mregs (regulatory macrophages) are also anti-inflammatory, but fail to deposit extracellular matrix (55). Our results utilizing epithelial gastroid:macrophage cocultures with or without H. pylori revealed that NOD1 likely plays a role in macrophage polarization as Nod1 WT macrophages cocultured with epithelial cells developed a profound M1 phenotype following exposure to H. pylori compared with a mixed M1/M2 phenotype exhibited by infected Nod1−/− macrophages. Such a hybrid phenotype may render Nod1-deficient macrophages ineffective in dampening respective Th1- or Th2-directed responses, thereby leading to a more severe pattern of global inflammation; our current results provide an important framework for defining such mechanisms in future work. Thus, the capacity of NOD1 to regulate macrophage phenotypes may also contribute to the ability of H. pylori to evade host immune clearance.

In conclusion, this study demonstrates that loss of NOD1 augments inflammatory responses to H. pylori within the context of gastric carcinogenesis. NOD1 may exert its restrictive role by altering macrophage polarization, thereby leading to immune evasion and microbial persistence. These studies lay the foundation for further exploration into the role of NOD1–H. pylori interactions in human hosts and suggest that manipulation of NOD1 may represent a novel strategy to prevent or treat pathologic outcomes induced by H. pylori infection.

R.M. Peek Jr is the editor of Gastroenterology at AGA. No potential conflicts of interest were disclosed by the other authors.

Conception and design: G. Suarez, R.M. Peek Jr

Development of methodology: G. Suarez, R.M. Peek Jr

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): G. Suarez, J. Romero-Gallo, M.B. Piazuelo, J.C. Sierra, A.G. Delgado, M.K. Washington

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): G. Suarez, K.T. Wilson, R.M. Peek Jr

Writing, review, and/or revision of the manuscript: G. Suarez, M.K. Washington, S.C. Shah, K.T. Wilson, R.M. Peek Jr

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): G. Suarez, J. Romero-Gallo, R.M. Peek Jr

Study supervision: G. Suarez, R.M. Peek Jr

This work was supported by NIH R01-DK58587, R01-CA77955, P01-CA116087, P30-DK058404, and P01-CA028842.

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.

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