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. Author manuscript; available in PMC: 2023 Aug 15.
Published in final edited form as: J Immunol. 2022 Jul 27;209(4):796–805. doi: 10.4049/jimmunol.2100795

Ornithine Decarboxylase in Gastric Epithelial Cells Promotes the Immunopathogenesis of Helicobacter pylori Infection

Yvonne L Latour *,†,2, Johanna C Sierra *,‡,2, Kara M McNamara *,§, Thaddeus M Smith *, Paula B Luis , Claus Schneider , Alberto G Delgado *, Daniel P Barry *, Margaret M Allaman *, M Wade Calcutt ‖, Kevin L Schey ‖, M Blanca Piazuelo *,‡, Alain P Gobert *,‡, Keith T Wilson *,†,‡,§,#
PMCID: PMC9378675  NIHMSID: NIHMS1817588  PMID: 35896340

Abstract

Colonization by Helicobacter pylori is associated with gastric diseases, ranging from superficial gastritis to more severe pathologies, including intestinal metaplasia and adenocarcinoma. The interplay of the host response and the pathogen affect the outcome of disease. One major component of the mucosal response to H. pylori is the activation of a strong, but inefficient immune response that fails to control the infection and frequently causes tissue damage. We have shown that polyamines can regulate H. pylori-induced inflammation. Chemical inhibition of ornithine decarboxylase (ODC), which generates the polyamine putrescine from L-ornithine, reduces gastritis in mice and adenocarcinoma incidence in gerbils infected with H. pylori. However, we have also demonstrated that Odc deletion in myeloid cells enhances M1 macrophage activation and gastritis. Here we used a genetic approach to assess the specific role of gastric epithelial ODC during H. pylori infection. Specific deletion of the gene encoding for ODC in gastric epithelial cells reduces gastritis, attenuates epithelial proliferation, alters the metabolome, and downregulates the expression of immune mediators induced by H. pylori. Inhibition of ODC activity or ODC knockdown in human gastric epithelial cells dampens H. pylori-induced NF-κB activation, CXCL8 mRNA expression, and IL-8 production. Chronic inflammation is a major risk factor for the progression to more severe pathologies associated with H. pylori infection, and we now show that epithelial ODC plays an important role in mediating this inflammatory response.

Introduction

Infection by Helicobacter pylori is the main risk factor for the development of gastric cancer (1), and the inflammatory response induced by this bacterium is considered necessary for the progression to gastric adenocarcinoma (2). Strategies proposed to reduce gastric cancer mortality include global antibiotic eradication and early detection through upper gastrointestinal endoscopy (3, 4). However, both interventions face challenges including the increased occurrence of antibiotic resistance in H. pylori and the cost-effectiveness of screening a significant portion of the population at risk in areas of high gastric cancer incidence (5, 6). Complementary strategies that limit the inflammatory response induced by H. pylori may improve disease outcome for infected individuals.

Polyamines are generated through a process that starts with the synthesis of putrescine from L-ornithine by the action of the rate-limiting enzyme ornithine decarboxylase (ODC)3 (7, 8). Then, putrescine is sequentially transformed to spermidine and spermine by spermidine synthase and spermine synthase, respectively (7). Polyamines are pleiotropic molecules that have major functions in embryogenesis, homeostasis, and aging (9–11). In addition, polyamines contribute to the regulation of the immune response during inflammation and infection, notably by altering histone modifications and chromatin structure and thus transcription of inducible effectors (12–14). Further, these molecular alterations can also affect DNA stability, and the global metabolism of polyamines has been also shown to support cell proliferation and oxidative damage (15, 16), thus favoring cell transformation and, potentially, carcinogenesis (8, 17). In this context, the ODC inhibitor difluoromethylornithine (DFMO) represents a promising potential treatment or chemopreventive for various cancers, including neuroblastoma, colorectal cancer or gastric adenocarcinoma in high-risk H. pylori-infected populations (16, 18–21).

We have previously shown that mice with specific deletion in myeloid cells of the gene encoding for ODC, Odc1, hereafter termed Odc, exhibit increased gastric inflammation and reduced colonization in response to H. pylori (13); this is associated with increased polarization of macrophages toward an M1 phenotype (13). However, the specific contribution of ODC in gastric epithelial cells (GECs) to H. pylori pathogenesis remains unknown. To test this, we infected mice with specific deletion of the Odc gene in GECs and found that epithelial ODC supports H. pylori pathogenesis.

Materials and Methods

Model of H. pylori infection

C57BL/6 Odc1fl/fl mice were crossed with C57BL/6 Foxa3cre/+ mice containing a single copy of the Foxa3-cre1Khktransgene (13, 22). The resulting Odc1+/fl;Foxa3cre/+ mice were backcrossed once more with Odc1fl/fl mice to create Odc1fl/fl;Foxa3+/+ and Odc1fl/fl;Foxa3+/cre (OdcΔepi) mice. Mice were housed in a specific pathogen-free facility, with ventilated cage racks and on 12-hour light-dark cycle. Littermate Odcfl/fl and OdcΔepi male mice (6 to 12 weeks) were provided continuous water, fed ad libitum with 5L0D chow (LabDiet), and infected with 109 CFU of H. pylori PMSS1, a cagA+ strain with intact type IV secretion system function, as reported (23, 24). After 4 or 12 weeks, animals were sacrificed. Colonization was assessed by culture of serial dilutions of the gastric tissue lysates (23, 24). Histologic assessment of longitudinal sections of the gastric tissues stained by H&E was performed by a gastrointestinal pathologist (MBP) in a blinded manner using the modified Sydney System (13, 25). The antrum and corpus regions were each scored 0–3 for acute and chronic inflammation, and the scores for antrum and corpus were added together for a 0–12 scale. On the mice infected for 12 weeks, the extent of mucous metaplasia, the loss of parietal cells and the loss of chief cells were assessed on a 0–3 scale (absent, mild, moderate, marked) on H&E-stained sections containing the entire length of the stomach, using a slightly modified system based on Rogers et al (26). Mucous metaplasia is described as the presence of mucous-producing cells with foamy change, predominantly replacing parietal cells. GECs were isolated by dissociation and dispersion as reported (27).

These experiments were approved by the Vanderbilt University Medical Center Institutional Animal Care and Use Committee under protocols M/14/230, M1600091, and M1900067. Procedures followed institutional policies, AVMA Guidelines of Euthanasia, AAALAC guidelines, NIH regulations (Guide for the Care and Use of Laboratory Animals), and the United States Animal Welfare Act (1996).

Cells

AGS cells were obtained from ATCC and maintained in RPMI medium supplemented with 10% FBS and 10 mM HEPES. We also used AGS cells expressing a stable luciferase-based NF-κB reporter pGL4.32(luc2P/NF-κB-RE/Hygro; Promega) (29). Cells were treated with 5 mM DFMO for 7 days; DFMO was removed 24 h before the infection with H. pylori PMSS1 at a multiplicity of infection of 10. Putrescine (10 μM) was added 24 h before infection.

For transfections, AGS cells maintained in Opti-MEM I Reduced Serum Media (Invitrogen) were transfected with 100 nM ON-TARGETplus siRNAs (Dharmacon) directed against human ODC or LMNA (used as a control) using Lipofectamine 2000 (Invitrogen). After 16 h, cells were washed and maintained in fresh media for 36 h prior to infection.

NF-κB activation reporter assay

Lysates were prepared using Reporter Lysis Buffer (Promega), mixed with Luciferase Assay System (Promega), and luminescence was measured on a Synergy4 plate reader (BioTek).

mRNA analysis

Total RNA was isolated from longitudinal sections, encompassing both the antrum and corpus, of gastric tissues using the RNeasy Mini kit (QIAGEN).

For RNA sequencing, RNA quality control, cDNA libraries, and Next Generation Sequencing (PE150) were performed using TapeStation System (Agilent), NEBNext Ultra II Directional RNA Library Prep kit (New England BioLabs), and Illumina NovaSeq6000 with NovaSeq 6000 SP Reagent Kit (Illumina), respectively. Adapter sequences were removed and read quality was checked using fastp (30). Transcripts were quantified and mapped to the indexed mouse genome (M23, GRCm38) using Salmon (31). Transcript-level quantification was then summarized to the gene level, annotated, and prepared for differential gene expression analysis using the R package tximeta (32). The R/Bioconductor package DESeq2 was then used to identify differentially expressed genes in each condition using a Benjamini-Hochberg test adjusted for false discovery rate (33). Pathway analysis was performed using the Database for Annotation, Visualization, and Integrated Discovery (DAVID). RNA-Seq data can be accessed on the Gene Expression Omnibus repository (https://www.ncbi.nlm.nih.gov/geo/) using the accession number GSE181917 (token for Reviewers: mbapkmcofrehdwn).

Expression of Odc, Cxcl1, Ccl5, Ccl3, Tnf, Ifng, and Il17a was also assessed by real-time PCR as previously described (13, 27).

IL-8 expression

Total RNA was isolated using the RNeasy Mini kit (QIAGEN) from AGS cells 3 h post-infection and expression of CXCL8, the gene which encodes for human IL-8, was assessed by real-time PCR. IL-8 protein levels were determined in AGS cell culture supernatants using the Human IL-8 DuoSet ELISA kit per the manufacturer’s instructions (R&D Systems) 6 h post infection. IL-8 concentrations were normalized to the protein concentration of lysed cells from the same well.

Western blot

Protein isolation, electrophoresis separation, transfer to nitrocellulose, and hybridization with rabbit polyclonal anti-ODC antibody (Abcam 97395, 1:1000) or a mouse monoclonal anti-β-actin antibody (Sigma A1978, 1:10000) was performed as described (28).

Immunofluorescence

Immunofluorescence staining for ODC was performed as previously described (13) using a rabbit polyclonal anti-ODC (Lisa Shantz (Penn State College of Medicine) and David Feith (University of Virginia), 1:2000) and goat anti-rabbit IgG, Alexa Fluor 488-labeled (Thermo Fisher A11008).

Immunohistochemistry and image analysis

Sections of paraffin-embedded tissues were deparaffinized and antigen retrieval was performed (26). Tissues were incubated with prediluted rabbit polyclonal anti-Ki-67 (Biocare PRM325AA), prediluted rabbit monoclonal anti-myeloperoxidase (MPO, Biocare PP023AA), or rabbit polyclonal anti-CD3 (Abcam ab5690, 1:150), and processed as described (28). Ki-67 slides were imaged and analyzed using a Cytation C10 Confocal Imaging Reader and Gen 5+ software (Agilent BioTek). The average number of MPO and CD68 positive cells per 5 high powered fields (HPF) were quantified by a gastrointestinal pathologist (MBP) in a blinded manner.

Polyamine quantification

The polyamines putrescine, spermidine, and spermine were quantified by LC-MS as previously described (34).

Metabolomic analysis

Gastric tissues from uninfected and infected mice from each genotype were homogenized and processed as previously described (35). We used XCMS (https://xcmsonline.scripps.edu) to generate chromatographic alignment, peak picking, and statistical comparisons. Metabolomics data has been deposited to MetaboLights (https://www.ebi.ac.uk/metabolights/) with accession number MTBLS3235.

Statistics

Prism 9.3 software (GraphPad Inc.) was used for statistical analysis and all the results are expressed as mean ± SEM. Data that were not normally distributed according to the D’Agostino & Pearson normality test were square root transformed, and distribution was re-assessed. Student’s t test or ANOVA with the Tukey test were used to determine significant differences between two or multiple groups, respectively. Fisher’s exact test was used to determine significant differences between the proportions of two groups.

Results

Reduced gastric inflammation in OdcΔepi mice infected with H. pylori

We first found that OdcΔepi mice showed a significant reduction in Odc mRNA levels (Fig. 1A) and ODC protein expression (Fig. 1B and 1C) in isolated GECs when compared to littermate Odcfl/fl mice. Polyamine levels in GECs were quantified, and we found a significant reduction in the concentrations of putrescine, spermidine, and spermine in OdcΔepi mice compared to Odcfl/fl animals (Fig. 1D).

FIGURE 1.

FIGURE 1.

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ODC expression and polyamine levels in naïve mice with specific deletion of Odc in GECs. GECs were isolated from the stomach of Odcfl/fl and OdcΔepi mice. Levels of Odc mRNA (A), ODC protein (B), and polyamines (D) were analyzed by real time PCR, Western blot, and LC-MS, respectively; densitometric analysis of the immunoblot is depicted in (C); n = 2–3 mice per genotype. Each symbol is a different mouse. In all panels, *P<0.05 versus GECs from Odcfl/fl mice.

Next, Odc fl/fl and OdcΔepi mice were infected with H. pylori strain PMSS1 for four weeks. We evaluated ODC expression in the gastric mucosa by immunofluorescence. H. pylori-infected Odcfl/fl mice exhibited increased expression of ODC throughout the epithelium compared to uninfected mice, which was substantially reduced in the infected OdcΔepi mice (Figure 2A). This confirmed successful knockdown of ODC in the epithelium. Inflammation levels were evaluated in H&E-stained sections (Fig. 2B). Gastric tissues from Odcfl/fl-infected mice exhibited increased epithelial hyperplasia and significant recruitment of immune cells compared to uninfected animals (Fig. 2B). There was an obvious attenuation of inflammation in infected OdcΔepi mice (Fig. 2B). Accordingly, when we scored acute and chronic inflammation in the antrum and corpus of infected mice, we found significantly less gastritis in OdcΔepi mice than in Odcfl/fl animals (Fig. 2C). However, colonization density was similar between Odc fl/fl and OdcΔepi mice (Fig. 2D).

FIGURE 2.

FIGURE 2.

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Effect of Odc deletion in GECs on H. pylori pathogenesis. Littermate Odcfl/fl and OdcΔepi mice were infected with H. pylori PMSS1 for 4 weeks. (A) Representative immunofluorescence images of ODC in gastric tissues of uninfected and infected mice. n = 5 mice per group. Note strong staining of the antral-corpus transition zone in the Odcfl/fl mice. (B) H&E staining from the gastric tissues of uninfected and infected mice. (C) Gastric inflammation scores derived from H&E-stained tissues, n = 4–6 uninfected mice and n = 19 infected mice per genotype, data pooled from two independent experiments. (D) Colonization density was assessed by plating serial dilution of stomach lysates from C. In A and B, scale bars are 100 μm. *P<0.05 and **P<0.01.

H. pylori-induced disease manifests as chronic active inflammation in infected individuals, therefore, we infected Odcfl/fl and OdcΔepi mice for 12 weeks to assess the impact of epithelial ODC on chronic gastritis. Similar to our 4-week model, Odc fl/fl mice exhibited increased inflammation and epithelial hyperplasia compared to uninfected mice, which was attenuated in the OdcΔepi mice (Fig. 3A). These findings are reflected in the scored gastric inflammation (Figure 3B). Additionally, we found no difference in the level of H. pylori colonization (Fig. 3C). Loss of parietal cells and loss of chief cells were very mild and predominantly seen in the transitional mucosa of the junction of the antrum and corpus, however, infected OdcΔepi mice exhibited significantly less parietal cell loss than infected Odcfl/fl mice (Fig. 3D). Mucous metaplasia, only observed in one Odcfl/fl animal, was mild and located in the proximal corpus (Fig. 3E). There are no observed differences in the epithelium of OdcΔepi mice compared to Odcfl/fl mice at baseline.

FIGURE 3.

FIGURE 3.

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Effect of Odc deletion in GECs on chronic H. pylori pathogenesis. Littermate Odcfl/fl and OdcΔepi mice were infected with H. pylori PMSS1 for 12 weeks. (A) H&E staining from the gastric tissues of uninfected and infected mice. (B) Gastric inflammation scores derived from H&E-stained tissues, each symbol is a different mouse, n = 4 uninfected mice and n = 5–10 infected mice per genotype. (C) Colonization density was assessed by plating serial dilution of stomach lysates in B. (D) Percent of cases from B exhibiting no loss or loss of parietal cells and chief cells. (E) Percent of cases from B exhibiting mucous metaplasia. In A, scale bars are 100 μm. *P<0.05, **P<0.01, and ****P<0.0001.

Reduced gastric hyperplasia and immune infiltration in OdcΔepi mice infected with H. pylori

Since polyamines are important for cell survival and inhibition of ODC activity can lead to cytostasis, we evaluated cell proliferation in gastric tissues from Odcfl/fl and OdcΔepi mice using immunohistochemistry for Ki-67. There was increased Ki-67 immunostaining of GECs in Odcfl/fl mice infected with H. pylori compared to uninfected controls, and the number of proliferating cells was significantly reduced in H. pylori-infected OdcΔepi mice (Fig. 4A). These results were confirmed by the quantification of the positive nuclei (Fig. 4B).

FIGURE 4.

FIGURE 4.

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Effect of Odc deletion in GECs on cellular proliferation and immune cell infiltration. (A) Representative immunohistochemistry images of Ki-67 and (B) quantification of positive nuclei in gastric tissues infected or not with H. pylori for 4-weeks; n = 5–6 uninfected mice and n = 13–15 infected mice per genotype, data pooled from three independent experiments. (C) Representative images of gastric tissues immunostained for MPO and (D) quantification of the number of MPO+ cells per high power field (H.P.F); n = 3–5 uninfected mice and n = 5–6 infected mice per genotype, data pooled from three independent experiments. (E) Representative images of gastric tissues immunostained for CD3 and (F) quantification of the number of CD3+ T cells per H.F.P.; n = 3–4 uninfected mice and n = 5–6 infected mice per genotype, data pooled from three independent experiments. In A, C, and E, scale bars are 100 μm. *P<0.05, **P<0.01, and ***P<0.001.

To further investigate the alteration in cellular composition and level of inflammation overserved in the gastric mucosa of OdcΔepi mice, we assessed the population of MPO-expressing cells (macrophages and neutrophils) and T cells (CD3) by immunohistochemistry. Consistent with the decrease in scored gastric inflammation, infected OdcΔepi mice also exhibit a significant decrease in the infiltration of MPO+ (Fig. 4C and 4D) and CD3+ (Fig. 4E and 4F) cells found in the tissues of infected Odcfl/fl mice.

These data indicate that ODC in GECs controls polyamine levels and supports inflammation and GEC proliferation during H. pylori infection.

Metabolic pathways affected by Odc deletion during H. pylori infection

To gain further insights into the functional role of epithelial ODC, we assessed the effect of Odc deletion in GECs on the gastric metabolomic signatures of the stomach. We found 282 and 326 metabolites significantly altered (fold change ≥1.5 and P<0.05) by epithelial Odc deletion in the gastric tissues of uninfected and H. pylori-infected mice, respectively.

Pathway analysis revealed that the putrescine degradation pathway was reduced in OdcΔepi gastric tissues at baseline, as expected. We also found that glutathione redox reactions and glycolysis, two pathways potentially related to the propensity for proinflammatory responses (36, 37), were also affected in uninfected OdcΔepi animals (Fig. 5A; Supplemental Table 1). When we compared Odcfl/fl and OdcΔepi mice that were infected with H. pylori, we also observed a significant downregulation of different polyamine-associated pathways, such as putrescine degradation as well as spermine and spermidine degradation; in addition, the lipoxin biosynthesis pathway, which is known for their anti-inflammatory effects (38, 39), were upregulated in OdcΔepi mice (Fig. 5B).

FIGURE 5.

FIGURE 5.

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Metabolomic changes in gastric tissues affected by Odc deletion. Mice were infected or not with H. pylori PMSS1. After 4 weeks, the metabolome of the gastric tissues was determined by ultra-high performance liquid chromatography; n = 4 uninfected mice and n = 8 infected mice per genotype. Bubble plot of metabolomic analysis comparing uninfected Odcfl/fl and OdcΔepi mice (A) and H. pylori-infected Odcfl/fl and OdcΔepi animals (B) were generated using the XCMS website. The full list of pathways is shown in Supplemental Table 1. Putrescine, spermidine and spermine were quantified by LC-MS in strips of whole stomach tissues of Odcfl/fl and OdcΔepi mice, infected or not with H. pylori (C); n = 7–8 uninfected mice and n = 12–13 infected mice per genotype. **P<0.01 and ***P<0.001.

Last, we confirmed by a targeted approach that putrescine concentration was increased in the whole gastric tissues of infected Odcfl/fl mice compared to control animals (Fig. 5C). There was a significant reduction of putrescine to basal levels in H. pylori infected OdcΔepi mice (Fig. 5C). However, there was no effect of epithelial-specific Odc deletion on the level of spermidine or spermine in the gastric tissues of infected mice (Fig. 5C; Supplemental Table 1).

Reduced expression of immune response-associated genes in OdcΔepi mice infected with H. pylori

To better understand the effect of epithelial ODC on the transcriptomic changes of gastric tissues during H. pylori infection, we performed RNA sequencing from Odcfl/fl and OdcΔepi mice. We identified 26983 sequences in the analysis that comprised 19084 known genes and 7899 unknown sequences (Supplemental Table 2). The differential expression analysis included genes that were altered 2-fold or more between the two groups with an adjusted P-value of less than 0.05. We found that 148 genes, essentially coding for immune effectors (such as chemokines Cxcl5 and Cxcl9, or the defense response-related genes Mbl1, Ido1, and Nox1) were significantly upregulated in infected Odcfl/fl mice versus uninfected controls (Fig. 6A; Supplemental Table 2). In addition, 33 genes, involved in fatty acid metabolism (Ugt3a1, Apoa5) or cellular respiration (Cyp4a14, Cyp1a2), were downregulated with infection (Fig. 6A; Supplemental Table 2). In contrast, only 12 and 7 genes were upregulated and downregulated, respectively, in the infected OdcΔepi mice compared with the uninfected animals (Fig. 6A; Supplemental Table 2). Thus, when we compared H. pylori infected OdcΔepi mice to infected Odcfl/fl mice (Fig. 6A; Supplemental Table 2), we found 106 genes downregulated, comprising mainly immune effectors (Il1b, Cxcl5, Cxcl9, Ido1, and Nox1). According to this result, we selectively analyzed the expression of multiple genes involved in the immune response and we generated the heatmap depicted in Fig. 6B. Overall, cytokines (Il17a, Il1b, Il12b, Il2, and Ifng), chemokines (Cxcl3, Cxcl19, Cxcl10, Ccl5, and Ccl20) and immune cell-associated genes (Ccr4, Cxcr6, Cd74, Cd8a, Myd88, Cd4, and Cd86) exhibited decreased expression in OdcΔepi compared to Odcfl/fl mice during infection. Importantly, we identified only 29 genes and 2 unidentified genes upregulated and downregulated, respectively, when comparing uninfected OdcΔepi and Odcfl/fl mice without infection (Fig. 6B; Supplemental Table 2), demonstrating that the main transcriptomic changes orchestrated by epithelial ODC occur under pathophysiological conditions and not at the basal level.

FIGURE 6.

FIGURE 6.

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Determination of the effect of Odc deletion in GECs on the gastric transcriptome. Odcfl/fl and OdcΔepi mice were infected or not with H. pylori. After 4 weeks, gastric RNA was analyzed by RNA-Seq; n = 4 mice per group. (A) Volcano plots for paired comparisons; the full list of differentially expressed genes is shown in Supplemental Table 2. (B) Heat map representing the level of genes encoding for chemokines, cytokines, and immune effectors.

Analysis of the differentially expressed genes between Odcfl/fl and OdcΔepi mice infected with H. pylori using the Database for Annotation, Visualization and Integrated Discovery (DAVID) software evidenced that pathways related to innate or specific immune responses (Fig. 7A) were significantly downregulated in the infected OdcΔepi mice, confirming that ODC in GECs supports gastric inflammation. Only one pathway related to the immune response, “Response to cytokine,” was significantly upregulated in the infected OdcΔepi mice (Fig. 7B).

FIGURE 7.

FIGURE 7.

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Pathway analysis performed from the transcriptomic analysis, showing the pathways significantly downregulated (A) and significantly upregulated (B) in the gastric tissues of OdcΔepi mice infected with H. pylori compared to infected Odcfl/fl animals.

These alterations were further confirmed when we quantified selected chemokine and cytokine mRNA expression levels in the tissues of Odcfl/fl and OdcΔepi mice. Cxcl1, Ccl5, Ccl3, Tnf, Ifng, and Il17a mRNA expression was significantly induced after H. pylori infection in Odcfl/fl mice and significantly reduced in infected OdcΔepi mice (Fig. 8).

FIGURE 8.

FIGURE 8.

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Targeted mRNA analysis. The expression of genes encoding for different chemokines and cytokines in the gastric tissues of Odcfl/fl and OdcΔepi mice, infected or not with H. pylori, was analyzed by RT-real-time PCR. n = 7–8 uninfected mice and n = 12–13 infected mice per genotype. *P<0.05, **P<0.01, and ***P<0.001.

ODC inhibition diminishes the innate response of human GECs to H. pylori

We then assessed the direct effect of ODC on the innate activation of GECs. We found a marked and significant reduction in the levels of putrescine and spermidine, but not spermine, when uninfected and H. pylori infected AGS cells were treated with the ODC inhibitor DFMO as previously observed in cell culture (13, 40) (Fig. 9A). In parallel, the activation of NF-κB (Fig. 9B), the expression of the classic proinflammatory GEC H. pylori-response gene CXCL8 (Fig. 9C), and the subsequent protein expression of IL-8 (Fig. 9D) induced by H. pylori were inhibited by DFMO. These molecular events were restored when DFMO-treated cells were supplemented with the ODC product putrescine (Fig. 9B–D). Using siRNA directed against ODC (Fig. 9E), we confirmed the reduction of CXCL8 mRNA and IL-8 protein expression by DFMO was the result of ODC inhibition. Silencing of ODC in AGS cells led to a significant reduction in H. pylori-stimulated CXCL8 mRNA expression (Fig. 9F) and IL-8 protein production (Fig. 9G) as was seen with DFMO treatment.

FIGURE 9.

FIGURE 9.

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Effect of ODC inhibition or knockdown on H. pylori-induced innate response in GECs. (A-D) AGS cells were treated with DFMO and/or putrescine, and then infected or not with H. pylori PMSS1. (A) Polyamine levels measured by mass spectrometry. NF-κB activity (B) and CXCL8 mRNA expression (C) were assessed after infection for 3 h. (D) IL-8 protein concentration was assessed after 6 h. (E-G) AGS cells were transfected with siRNA directed against ODC or LMNA control, and then infected or not with H. pylori PMSS1. ODC (E) and CXCL8 (F) mRNA expression was assessed after infection for 3 h. (G) IL-8 protein concentration was assessed after 6 h. **P<0.01 and ***P<0.001; n = 3–6.

Discussion

Polyamines are ubiquitous molecules, but decades of investigation have shown that their roles in homeostasis and pathophysiology are disease-, time- and cell-specific. We have previously demonstrated that H. pylori induced ODC expression in myeloid cells attenuates the antimicrobial/proinflammatory response of innate immune cells, thus supporting bacterial persistence and pathogenesis (13). Here, we questioned the role of ODC in GECs since these cells are the first in contact with the bacterium, produce chemokines that attract immune cells to the gastric mucosa, and are prone to malignant transformation. Thus, we found that genetic deletion of Odc in GECs reduces polyamine levels and protects mice from H. pylori-induced inflammation and proliferation. We also confirmed by metabolomics and transcriptomics substantive effects of epithelial ODC on inflammatory pathways. Together, these data indicate that the expression of ODC in the gastric epithelium supports H. pylori pathogenesis and further demonstrates that polyamines can have multiple and varying effects according to the type of cells in which they are generated.

H. pylori induces sustained acute inflammation, characterized by polymorphonuclear cell infiltration of the gastric mucosa in most infected individuals. This response is characterized by the production of multiple pro-inflammatory chemokines and cytokines. High levels of these pro-inflammatory mediators are associated with elevated risk of severe disease (41, 42). Global assessment of the gastric transcriptome from Odcfl/fl and OdcΔepi mice revealed a significant downregulation in the expression of chemokines, cytokines, and other immune-associated genes in infected OdcΔepi mice. We confirmed by RT-PCR that not only the genes encoding for chemokines expressed in epithelial cells, but also those encoding for effectors related to the innate immune system (Tnf, Il1b, Cxcl1) and to T cell activation (Ifng, Il17a, Il2) are regulated by epithelial-specific Odc deletion. Further, we also found that ODC expression in cultured GECs favors NF-κB activation and chemokine synthesis. In this context, we postulate that ODC in GECs induces and/or sustains chemokine production, resulting in recruitment of leukocytes in the infected gastric mucosa and thus to the development of a robust innate and adaptive immune response. Because we previously reported that putrescine in macrophages dampens the M1 response (13), a future goal is further elucidating the cellular/molecular mechanism by which ODC supports chemokine synthesis in GECs.

Early studies showed that polyamine levels increase during cell cycle progression and their depletion inhibits G1- to S-phase transition (15, 43). Here we used Ki-67 as a marker of cell proliferation in gastric tissues, and we observed increased proliferation in infected Odcfl/fl animals, which is consistent with previous studies in H. pylori-infected humans and mice (44, 45). It has been shown that H. pylori can interact directly with progenitor cells deep in the glands and accelerates their proliferation leading to hyperplastic changes (46). We showed that GEC proliferation was significantly reduced in infected OdcΔepi versus Odcfl/fl mice, suggesting that polyamines are important mediators of epithelial proliferation in the context of H. pylori infection and that polyamine depletion may be a logical strategy to prevent uncontrolled proliferation of GECs with carcinogenic potential.

Based on data demonstrating that the ODC inhibitor DFMO reduced gastritis and carcinogenesis in H. pylori-infected gerbils (16, 21), our group is conducting a clinical trial of this drug in patients with precancerous gastric lesions in Latin America (ClinicalTrials.gov Identifier: NCT02794428). Further, we have shown that DFMO induces direct oxidative DNA damage to H. pylori and specific mutations in the cagY gene, which results in reduction in the functionality of the Type 4 secretion system and thus less CagA translocation in GECs (47). This was observed in vitro and in H. pylori-infected gerbils treated with DFMO (47). Because we now show an overall protective effect of gastric epithelial Odc deletion in H. pylori-induced inflammation and a reduction of GEC proliferation, we suggest that a key protective effect of DFMO treatment in H. pylori infection is due to the inhibition of epithelial ODC. Moreover, we also show that DFMO treatment or ODC knockdown attenuates H. pylori-stimulated expression of CXCL8 and the IL-8 protein for which it encodes, in human GECs. Therefore, our present work further supports the use of DFMO in H. pylori-infected patients to limit the development of more advanced gastric pathology.

Supplementary Material

1
2

Key Points:

  • Epithelial ODC contributes to H. pylori-induced pathogenesis.

  • Genetic deletion of ODC in epithelial cells reduces gastric inflammation.

  • DFMO limits inflammation through inhibition of epithelial ODC.

Acknowledgments

This work was funded by NIH grants R21AI142042, R01CA190612, P01CA116087, P01CA028842, and R01DK128200 (KTW); Veterans Affairs Merit Review grants I01BX001453 and I01CX002171 (KTW); Department of Defense grants W81XWH-18-1-0301 and W81XWH-21-1-0617 (KTW); the Thomas F Frist Sr. Endowment (KTW); and the Vanderbilt Center for Mucosal Inflammation and Cancer (KTW). YLL was supported by T32AI138932, and KMM was supported by T32CA009592. Metabolomic analysis were supported in part by Core Scholarships from the Vanderbilt University Medical Center Digestive Disease Research Center funded by NIH grant P30DK058404 and the Vanderbilt Ingram Cancer Center support grant P30CA068485.

Abbreviations used in this article:

DFMO

difluoromethylornithine

GEC

gastric epithelial cell

ODC

ornithine decarboxylase

References

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Supplementary Materials

1
NIHMS1817588-supplement-1.xlsx (11.8KB, xlsx)
2
NIHMS1817588-supplement-2.xlsx (4.2MB, xlsx)

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