Barry H. Rickman

Overexpression of Interleukin-1β Induces Gastric Inflammation and Cancer and Mobilizes Myeloid-Derived Suppressor Cells in Mice

Polymorphisms of interleukin-1beta (IL-1beta) are associated with an increased risk of solid malignancies. Here, we show that stomach-specific expression of human IL-1beta in transgenic mice leads to spontaneous gastric inflammation and cancer that correlate with early recruitment of myeloid-derived suppressor cells (MDSCs) to the stomach. IL-1beta activates MDSCs in vitro and in vivo through an IL-1RI/NF-kappaB pathway. IL-1beta transgenic mice deficient in T and B lymphocytes develop gastric dysplasia accompanied by a marked increase in MDSCs in the stomach. Antagonism of IL-1 receptor signaling inhibits the development of gastric preneoplasia and suppresses MDSC mobilization. These results demonstrate that pathologic elevation of a single proinflammatory cytokine may be sufficient to induce neoplasia and provide a direct link between IL-1beta, MDSCs, and carcinogenesis.

DNA damage induced by chronic inflammation contributes to colon carcinogenesis in mice

Chronic inflammation increases cancer risk. While it is clear that cell signaling elicited by inflammatory cytokines promotes tumor development, the impact of DNA damage production resulting from inflammation-associated reactive oxygen and nitrogen species (RONS) on tumor development has not been directly tested. RONS induce DNA damage that can be recognized by alkyladenine DNA glycosylase (Aag) to initiate base excision repair. Using a mouse model of episodic inflammatory bowel disease by repeated administration of dextran sulfate sodium in the drinking water, we show that Aag-mediated DNA repair prevents colonic epithelial damage and reduces the severity of dextran sulfate sodium-induced colon tumorigenesis. Importantly, DNA base lesions expected to be induced by RONS and recognized by Aag accumulated to higher levels in Aag-deficient animals following stimulation of colonic inflammation. Finally, as a test of the generality of this effect we show that Aag-deficient animals display more severe gastric lesions that are precursors of gastric cancer after chronic infection with Helicobacter pylori. These data demonstrate that the repair of DNA lesions formed by RONS during chronic inflammation is important for protection against colon carcinogenesis.

Helicobacter pylori Eradication Prevents Progression of Gastric Cancer in Hypergastrinemic INS-GAS Mice

Helicobacter pylori infection results in chronic gastritis, which may progress to gastric cancer. In this study, we investigated the efficacy of H. pylori eradication in preventing the progression of gastritis to gastric cancer in H. pylori-infected transgenic INS-GAS mice. H. pylori infection induced severe dysplasia and gastric cancer classified as high-grade and low-grade gastrointestinal intraepithelial neoplasia (GIN) in INS-GAS mice at 28 weeks postinfection (WPI). H. pylori eradication therapy using omeprazole, metronidazole, and clarithromycin was administered p.o. at 8, 12, or 22 WPI. Compared with untreated infected mice, H. pylori eradication at 8, 12, and 22 WPI significantly reduced the severity of dysplasia (P < 0.01). Moreover, H. pylori eradication at 8 WPI completely prevented the development of GIN (P < 0.001). Although not as effective as early antimicrobial treatment, prevention of progression to high-grade GIN was achieved by H. pylori eradication at 12 and 22 WPI (P < 0.05). Consistent with reduced gastric pathology, H. pylori eradication at all time points significantly down-regulated gastric Interferon-gamma, tumor necrosis factor-alpha, inducible nitric oxide synthase, and Reg 1 mRNA levels (P < 0.05) and reduced epithelial proliferation in the corpus (P < 0.01) compared with untreated infected mice. We concluded that H. pylori eradication prevented gastric cancer to the greatest extent when antibiotics are given at an early point of infection, but that eradication therapy given at a later time point delayed the development of severe dysplastic lesions.

Helicobacter hepaticus Infection Promotes Colon Tumorigenesis in the BALB/c-Rag2 / Apc Min/ Mouse

ABSTRACT Adenomatous polyposis coli (APC) mutations are linked to human and mouse colorectal cancers. The Apc multiple intestinal neoplasia (Min) mouse mutation causes adenomas to develop throughout the small and large intestines. The BALB-Min (C.B6-ApcMin/+) congenic strain was generated by backcrossing into BALB/c the ApcMin allele from C57BL/6J-ApcMin/+ mice. BALB-Min mice have a low tumor multiplicity (27.4 small intestine tumors/mouse) and a relatively long life span (>1 year) that makes them amenable to long-term studies. To investigate the interplay of the adaptive immune system and intestinal tumorigenesis, the immunodeficient compound mutant strain BALB-RagMin (C.Cg-Rag2−/−ApcMin/+) was generated. BALB-RagMin mice had a significant increase in tumors in the small, but not large, intestine relative to their BALB-Min counterparts (43.0 versus 24.0 tumors/mouse, respectively). The results suggest that the adaptive immune system plays a role in either the elimination or the equilibrium phase of cancer immunoediting in the small intestine in this model. We investigated the effect of the enterohepatic bacterial pathogen Helicobacter hepaticus on liver and intestine tumorigenesis in BALB-RagMin mice. H. hepaticus-infected BALB-RagMin mice developed moderate hepatitis, moderate typhlitis, and mild colitis. There were no differences in small intestine and cecal tumor multiplicity, regionality, or size relative to that in uninfected mice. However, H. hepaticus-infected BALB-RagMin mice had a significant increase in colon tumor incidence relative to uninfected BALB-RagMin mice (23.5% versus 1.7%, respectively). The data suggest that H. hepaticus, which is present in many research colonies, promotes colon tumorigenesis in the BALB-RagMin mouse and that it has the potential to confound colon tumorigenesis studies.

Decreased expression of colonic Slc26a3 and carbonic anhydrase iv as a cause of fatal infectious diarrhea in mice.

ABSTRACT Citrobacter rodentium causes epithelial hyperplasia and colitis and is used as a model for enteropathogenic and enterohemorrhagic Escherichia coli infections. Little or no mortality develops in most inbred strains of mice, but C3H and FVB/N mice exhibit fatal outcomes of infection. Here we test the hypothesis that decreased intestinal transport activity during C. rodentium infection results in fatality in C3H/HeOu and FVB/N mice. Susceptible strains were compared to resistant C57BL/6 mice and to inbred strains SWR and SJL of Swiss origin, which have not been previously characterized for outcomes of C. rodentium infection. Mortality in susceptible strains C3H/HeOu and FVB/N was associated with significant fluid loss in feces, a remarkable downregulation of Slc26a3 and carbonic anhydrase IV (CAIV) message and protein expression, retention of chloride in stool, and hypochloremia, suggesting defects in intestinal chloride absorption. SWR, SJL, and C57BL/6 mice were resistant and survived the infection. Fluid therapy fully prevented mortality in C3H/HeOu and FVB/N mice without affecting clinical disease. Common pathogenic mechanisms, such as decreased levels of expression of Slc26a3 and CAIV, affect intestinal ion transport in C. rodentium-infected FVB and C3H mice, resulting in profound electrolyte loss, dehydration, and mortality. Intestinal chloride absorption pathways are likely a potential target for the treatment of infectious diarrhea.

Gastric Helicobacter species as a cause of feline gastric lymphoma: A viable hypothesis

Gastric Helicobacter spp. are associated with chronic inflammation and neoplastic transformation in humans as well as domestic and laboratory species. The present study examined the association of Helicobacter heilmannii (Hhe) infection in pet cats with feline gastric mucosa associated lymphoid tissue (MALT) lymphoma. Tissues were collected via gastric biopsy or at necropsy from 47 pet cats with clinical signs of gastrointestinal disease, including vomiting and inappetance, and classified as gastritis (14/47), lymphoma (31/37), or normal (2/47). Tissues positive for argyrophilic organisms with Warthin-Starry stain (29/47) were assessed by fluorescent in situ hybridization (FISH) for the presence of Hhe strains 1-4 as well as with a fifth probe that detected Helicobacter salomonis, Helicobacter bizzozeronii, or Helicobacter felis. A significant association of positive Warthin-Starry status with Hhe infection was found in cases of sick cats (22/29; p<0.05 by Chi-square; chi(2)=7.034). Interestingly, a significant association between Hhe status and a diagnosis of lymphoblastic or lymphocytic lymphoma was observed as well in a subset of 24 Warthin-Starry positive lymphoma cases: of lymphoblastic lymphoma cases, 13/17 were positive for Hhe (p<0.05; chi(2)=4.854). Hhe strains 2 and 4 were most commonly found (18/29 and 17/29, respectively) among sick cats, although a higher than expected number of cats was also positive for Hhe1, which initial reports have described as rare in cats and common in humans. The association found between a positive Hhe status with the presence of feline gastric lymphoma, especially lymphoblastic lymphoma, argues for the need to conduct prospective studies to better identify the frequency and strain distribution of Hhe infection in both healthy and clinically ill cats, particularly those cats with gastric lymphoma.

Cytolethal Distending Toxin Promotes Helicobacter cinaedi-Associated Typhlocolitis in Interleukin-10-Deficient Mice

ABSTRACT Helicobacter cinaedi colonizes a wide host range, including rodents, and may be an emerging zoonotic agent. Colonization parameters, pathology, serology, and inflammatory responses to wild-type H. cinaedi (WTHc) were evaluated in B6.129P2-IL-10tm1Cgn (IL-10−/−) mice for 36 weeks postinfection (WPI) and in C57BL/6 (B6) mice for 12 WPI. Because cytolethal distending toxin (CDT) may be a virulence factor, IL-10−/− mice were also infected with the cdtBHc and cdtB-NHc isogenic mutants and evaluated for 12 WPI. Consistent with other murine enterohepatic helicobacters, WTHc did not cause typhlocolitis in B6 mice, but mild to severe lesions developed at the cecocolic junction in IL-10−/− mice, despite similar colonization levels of WTHc in the cecum and colon of both B6 and IL-10−/− mice. WTHc and cdtB mutants also colonized IL-10−/− mice to a similar extent, but infection with either cdtB mutant resulted in attenuated typhlocolitis and hyperplasia compared to infection with WTHc (P < 0.03), and only WTHc infection caused dysplasia and intramucosal carcinoma. WTHc and cdtBHc mutant infection of IL-10−/− mice elevated mRNA expression of tumor necrosis factor alpha, inducible nitric oxide synthase, and gamma interferon in the cecum, as well as elevated Th1-associated serum immunoglobulin G2ab compared to infection of B6 mice (P < 0.05). Although no hepatitis was noted, liver samples were PCR positive at various time points for WTHc or the cdtBHc mutant in approximately 33% of IL-10−/− mice and in 10 to 20% of WTHc-infected B6 mice. These results indicate that WTHc can be used to model inflammatory bowel disease in IL-10−/− mice and that CDT contributes to the virulence of H. cinaedi.

Gastrin Is an Essential Cofactor for Helicobacter-Associated Gastric Corpus Carcinogenesis in C57BL/6 Mice

We have previously described a synergistic interaction between hypergastrinemia and Helicobacter felis infection on gastric corpus carcinogenesis in FVB/N mice housed under specific-pathogen-free conditions. However, gastrin-deficient (GAS-KO) mice on a mixed C57BL/6/129Sv genetic background maintained in conventional housing were reported to develop spontaneous gastric antral tumors. Therefore, we investigated the role of gastrin in Helicobacter-associated gastric carcinogenesis in H. felis-infected mice on a uniform C57BL/6 background housed in specific-pathogen-free conditions. Hypergastrinemic transgenic (INS-GAS) mice, GAS-KO mice, and C57BL/6 wild-type mice were infected with H. felis for either 12 or 18 months. At 12 months postinfection, INS-GAS mice had mild corpus dysplasia, while B6 wild-type mice had either severe gastritis or metaplasia, and GAS-KO mice had only mild to moderate gastritis. At 18 months postinfection, both INS-GAS and B6 wild-type mice had both severe atrophic gastritis and corpus dysplasia, while GAS-KO mice had severe gastritis with mild gastric atrophy, but no corpus dysplasia. In contrast, both GAS-KO and B6 wild-type mice had mild to moderate antral dysplasia, while INS-GAS mice did not. H. felis antral colonization remained stable over time among the three groups of mice. These results point to a distinct effect of gastrin on carcinogenesis of both the gastric corpus and antrum, suggesting that gastrin is an essential cofactor for gastric corpus carcinogenesis in C57BL/6 mice.

Inhibition of Helicobacter hepaticus-Induced Colitis by IL-10 Requires the p50/p105 Subunit of NF-κB

Defects within the innate immune system sensitize NF-κB-deficient (p50−/−; p65+/−) mice to Helicobacter hepaticus (Hh)-induced colitis. Because IL-10 plays a central role in the inhibition of Hh-induced colitis, we hypothesized that the ability of IL-10 to inhibit the innate inflammatory response to Hh may be compromised in NF-κB-deficient mice. To test this hypothesis, we evaluated the ability of an IL-10-Ig fusion protein with IL-10-like properties to inhibit Hh-induced colitis in RAG-2−/− (RAG) and p50−/−; p65+/−; RAG-2−/− (3X/RAG) mice. As expected, IL-10-Ig efficiently inhibited the development of colitis in RAG mice. In contrast, the ability of IL-10-Ig to inhibit colitis was compromised in 3X/RAG mice. The defect in response to IL-10-Ig appeared to be primarily the result of the absence of the p50/p105 subunit, because the ability of IL-10-Ig to inhibit colitis was also compromised in p50−/−; RAG-2−/− (p50/RAG) mice. Radiation chimeras demonstrated that the presence of p50/p105 within hemopoietic cells of the innate immune system was necessary for efficient inhibition of colitis by IL-10-Ig. Consistent with a defect in the suppressive effects of IL-10 in the absence of p50/p105, we found that the ability of IL-10 to control LPS-induced expression of IL-12 p40 was significantly compromised in macrophages lacking p50/p105. These results suggest that the absence of the p50/p105 subunit of NF-κB within hemopoietic cells of the innate immune system interferes with the ability of IL-10 to suppress inflammatory gene expression and Hh-induced colitis.

Combination of sulindac and antimicrobial eradication of Helicobacter pylori prevents progression of gastric cancer in hypergastrinemic INS-GAS mice.

Helicobacter pylori infection causes severe dysplasia manifested as gastrointestinal intraepithelial neoplasia (GIN) after 28 weeks post-H. pylori infection (WPI) in cancer-prone, hypergastrinemic male INS-GAS mice. We examined the efficacy of the nonsteroidal anti-inflammatory drug sulindac (400 ppm in drinking water) alone, the CCK2/gastrin receptor antagonist YM022 (45 mg/kg/wk) alone, and sulindac or YM022 combined with H. pylori eradication therapy to prevent H. pylori-associated gastric cancer in male INS-GAS mice. Treatments started at 22 WPI, and mice were euthanized at 28 WPI. In uninfected mice, all treatments significantly delayed development of spontaneous GIN (P < 0.05). In H. pylori-infected mice, sulindac alone or YM022 alone had no protective effect on H. pylori-associated GIN. Importantly, sulindac exacerbated the severity of H. pylori-associated gastritis despite decreased gastric prostaglandin E(2) levels. However, sulindac combined with H. pylori antimicrobial eradication reduced the incidence of GIN (P < 0.05), whereas YM022 combined with antimicrobial eradication did not reduce GIN. In infected mice, sulindac or YM022 treatment did not alter gastric expression of the proinflammatory cytokines Ifn-gamma and Tnf-alpha and mucosal cell proliferation. Sulindac or YM022 combined with antimicrobial eradication down-regulated mRNA levels of Ifn-gamma and Tnf-alpha and mucosal cell proliferation (P < 0.05). We conclude that sulindac enhances H. pylori gastritis and may promote inflammation-mediated gastric carcinogenesis. The combination of sulindac and antimicrobial H. pylori eradication was beneficial for reducing proinflammatory cytokine mRNA in the stomach and preventing progression from severe dysplasia to gastric cancer in H. pylori-infected INS-GAS mice.