Lack of Effect of Murine Norovirus Infection on a Mouse Model of Bacteria-Induced Colon Cancer

Murine norovirus (MNV) is endemic in mouse research facilities in the United States and Europe, with a prevalence as high as 58% to 64%. Because of MNV's orofecal route of infection, clinically silent persistent infections in some mouse strains, and proclivity for macrophage and dendritic cells, its presence in mouse colonies has potential to alter phenotypes in experimental mouse models, particularly those involving inflammation and immunologic responses. Although MNV is subclinical, not causing overt disease in immunocompetent mice, we found that MNV infection can accelerate bacteria-induced inflammatory bowel disease (IBD) progression in Mdr1a^(-/-) mice. The studies presented here examined whether MNV infection also affects the phenotype of a bacterially driven mouse model of inflammation-associated colon cancer in genetically susceptible Smad3^(-/-) mice. In vitro culture of bone-marrow—derived macrophages (BMDM) was used to determine whether MNV4 influenced macrophage cytokine production. For in vivo studies, Smad3-/- mice were infected with MNV4 one week prior to infection with Helicobacter. Mice were monitored for 17 to 32 wk for development of IBD and colon cancer, and tissues were analyzed histopathologically. Although in vitro infection of BMDM with MNV4 led to increased inflammatory cytokine production, infection with MNV4 in vivo did not result in any statistically significant differences in survival, IBD scores, tumor incidence, or tumor phenotype in Smad3^(-/-) mice. In addition, MNV infection alone did not result in IBD or colon cancer. Therefore MNV infection alone or in conjunction with Helicobacter does not alter the development or progression of IBD or colon cancer in Smad3^(-/-) mice

Murine Norovirus: An Intercurrent Variable in a Mouse Model of Bacteria-Induced Inflammatory Bowel Disease

Murine norovirus (MNV) has recently been recognized as a widely prevalent viral pathogen in mouse colonies and causes disease and mortality in mice with impaired innate immunity. We tested the hypothesis that MNV infection would alter disease course and immune responses in mice with inflammatory bowel disease (IBD). FVB.129P2-Abcb1a^(tm1Bor) N7 (Mdr1a−/−) mice develop spontaneous IBD that is accelerated by infection with Helicobacter bilis. As compared with controls, Mdr1a−/− mice coinfected with MNV4 and H. bilis showed greater weight loss and IBD scores indicative of severe colitis, demonstrating that MNV4 can modulate the progression of IBD. Compared with controls, mice inoculated with MNV4 alone had altered levels of serum biomarkers, and flow cytometric analysis of immune cells from MNV4-infected mice showed changes in both dendritic cell (CD11c+) and other nonT cell (CD4− CD8−) populations. Dendritic cells isolated from MNV4-infected mice induced higher IFNγ production by polyclonal T cells in vitro at 2 d after infection but not at later time points, indicating that MNV4 infection enhances antigen presentation by dendritic cells early after acute infection. These findings indicate that acute infection with MNV4 is immunomodulatory and alters disease progression in a mouse model of IBD

Characterization of Dextran Sodium Sulfate-Induced Inflammation and Colonic Tumorigenesis in <i>Smad3</i>^−/− Mice with Dysregulated TGFβ

<div><p>There are few mouse models that adequately mimic large bowel cancer in humans or the gastrointestinal inflammation which frequently precedes it. Dextran sodium sulphate (DSS)-induces colitis in many animal models and has been used in combination with the carcinogen azoxymethane (AOM) to induce cancer in mice. <i>Smad3</i>^−/− mice are deficient in the transforming growth factor beta (TGFβ) signaling molecule, <i>SMAD3</i>, resulting in dysregulation of the cellular pathway most commonly affected in human colorectal cancer, and develop inflammation-associated colon cancer. Previous studies have shown a requirement for a bacterial trigger for the colitis and colon cancer phenotype in <i>Smad3^−/−</i> mice. Studies presented here in <i>Smad3^−/−</i> mice detail disease induction with DSS, without the use of AOM, and show a) <i>Smad3</i>^−/− mice develop a spectrum of lesions ranging from acute and chronic colitis, crypt herniation, repair, dysplasia, adenomatous polyps, disseminated peritoneal adenomucinosis, adenocarcinoma, mucinous adenocarcinoma (MAC) and squamous metaplasia; b) the colon lesions have variable galactin-3 (Mac2) staining c) increased DSS concentration and duration of exposure leads to increased severity of colonic lesions; d) heterozygosity of <i>SMAD3</i> does not confer increased susceptibility to DSS-induced disease and e) disease is partially controlled by the presence of T and B cells as <i>Smad3</i>^−/−<i>Rag2</i>^−/− double knock out (DKO) mice develop a more severe disease phenotype. DSS-induced disease in <i>Smad3</i>^−/− mice may be a useful animal model to study not only inflammation-driven MAC but other human diseases such as colitis cystica profunda (CCP) and pseudomyxomatous peritonei (PMP).</p></div

Helicobacter infection is required for inflammation and colon cancer in Smad3-deficient mice

Accumulating evidence suggests that intestinal microbial organisms may play an important role in triggering and sustaining inflammation in individuals afflicted with inflammatory bowel disease (IBD). Moreover, individuals with IBD are at increased risk for developing colorectal cancer, suggesting that chronic inflammation may initiate genetic or epigenetic changes associated with cancer development. We tested the hypothesis that bacteria may contribute to the development of colon cancer by synergizing with defective transforming growth factor-β (TGF-β) signaling, a pathway commonly mutated in human colon cancer. Although others have reported that mice deficient in the TGF-β signaling molecule SMAD3 develop colon cancer, we found that SMAD3-deficient mice maintained free of the Gram-negative enterohepatic bacteria Helicobacter spp. for up to 9 months do not develop colon cancer. Furthermore, infection of SMAD3-/- mice with Helicobacter triggers colon cancer in 50% to 66% of the animals. Using real-time PCR, we found that Helicobacter organisms concentrate in the cecum, the preferred site of tumor development. Mucinous adenocarcinomas develop 5 to 30 weeks after infection and are preceded by an early inflammatory phase, consisting of increased proliferation of epithelial cells; increased numbers of cyclooxygenase-2-positive cells, CD4+ T cells, macrophages; and increased MHC class II expression. Colonic tissue revealed increased transcripts for the oncogene c-myc and the proinflammatory cytokines interleukin-1α (IL-1α), IL-1β, IL-6, IFN-γ, and tumor necrosis factor-α, some of which have been implicated in colon cancer. These results suggest that bacteria may be important in triggering colorectal cancer, notably in the context of gene mutations in the TGF-β signaling pathway, one of the most commonly affected cellular pathways in colorectal cancer in humans

Squamous metaplasia and dysplasia in distal colons of DSS-treated <i>Smad3^−/−</i> mice.

<p><i>Smad3^+/−</i>, <i>Smad3^−/−</i> and WT mice were treated with either 1.5% DSS for one or 9 cycles. Experimental endpoint was 17 weeks. (A) Squamous cell metaplasia was scored as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079182#pone-0079182-t003" target="_blank">Table 3</a>. Pairwise comparisons between DSS-treated groups were via Mann-Whitney test. B) The same region of the distal colon as in (A) was scored for squamous cell dysplasia. No high grade (grade 3 or 4) dysplasia was detected. Incidence of dysplasia was compared via Fisher exact test. <i>*P</i>≤<i>0.05, **P</i>≤<i>0.01, ***P</i>≤<i>0.001, ****P</i>≤<i>0.0001</i>.</p

Dysplasia and invasive neoplasia scoring schema.

a<p>dysplasia score is assigned 0–4 for each section of large bowel, summed dysplasia are the sum of the scores in the individual segments (cecum, proximal colon, mid colon, distal colon).</p>b<p>number of invasive tumors is multiplied by size weight in each segment of the large bowel (cecum, proximal colon, mid colon, distal colon). Invasive tumor score is the sum of these values across the bowel).</p>c<p>diagnostic criteria for hyperplasia, adenoma, carcinoma and to distinguish invasion vs. pseudoinvasion (herniation) after Boivin et al 2003.</p

Patterns of galectin-3 expression.

<p><i>Smad3^−/−</i>3% DSS (A–C). Sections of formalin-fixed paraffin-embedded colon from a mouse with regions of normal proximal colon (A) mucosal adenomatous hyperplasia (B) and mucinous adenocarcinoma (C and D) immunohistologically stained for galectin-3. (A) In normal proximal colonic mucosa, galectin-3 expression is restricted to well-differentiated apical colonocytes with strong nuclear and lesser cytoplasmic signal. (B) Within adenomas there is loss of signal in the proliferative cells (arrows, mitotic figures) and staining cytoplasmic signal with indistinct to absent nuclear signal. (C) Within invasive crypts deep within the tunica muscularis (see A) there is loss of signal with gradual increase in signal as cells migrate into the peritoneal cavity and presumably differentiate to a mucinous phenotype. (D) <i>Smad3/Rag-DKO</i>, 1.5% DSS Strong cytoplasmic signal is present in the neoplastic epithelium lining mucinous peritoneal cysts and in free floating cells within the mucin pools (<b>P</b>).</p

Mucinous neoplasia and high grade dysplasia induced by DSS.

<p>(A) <i>Smad3/Rag-DKO</i> DSS cycles. A cecal-colic multicystic mass (arrow) is present adjacent to the pancreas (asterisk). (B) <i>Smad3/Rag-DKO</i> 1.5%DSS. Multiple serosal masses are indicated on the mid to distal colon. (C) <i>Smad^−/−</i> DSS cycles. A large multicystic mesenteric mass. (D) <i>Smad^−/−</i> DSS cycles. Expansile mass in (A). Pancreas (asterisk) and mesenteric lymph node (upper right). Box region presented in (G). (E) Herniated proliferative mucosa (<b>H</b>) with compression the tunica muscularis (<b>TM</b>) with preservation of the submucosa (arrowhead). At left are invasive angular glands, dissecting mucin in the TM with focal penetration of the serosa (<b>S</b>) and intraperitoneal mucus (asterisk). (F) <i>Smad^−/−</i> DSS cycles. Mesenteric implant of mucinous cysts (asterisk) with mucin-producing epithelial lined glands (arrowheads). Note abscess (<b>A</b>) and mesenteric lymph node (<b>ML</b>). (G) Boxed region in (D). Note dissecting lakes of mucin and isolated epithelium and epithelial rafts within the cysts. (H) Higher magnification of asterisked region in (F). (I) <i>Smad3^+/−</i>3%DSS. Within mesenteric cysts are free large round cells (arrowhead) and clumps of basophilic cells (arrow) and rare signet rings (inset, Smad3−/− DSS cycles) (J) Foci of high grade dysplasia with in a hyperplastic polyp.</p

Grading of DSS IBD.

*<p>These are scored for each section of large bowel (cecum, proximal colon, mid colon and distal colon) and summed for total IBD score.</p>**<p>Two extent scores are included in the IBD score. Extent 1 =  % of intestine affected in any manner; Extent 2 = % percent of intestine affected by the most severe score.</p