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

Implications of myelin basic protein processing and presentation on T cell activation and tolerance

Thesis (Ph. D.)--University of Washington, 2004Experimental autoimmune encephalomyelitis (EAE) is an animal model for multiple sclerosis which can be induced by immunization with myelin antigens such as myelin basic protein (MBP). Immune tolerance influences the repertoire of MBP-specific T cells in B10.PL mice. MBPAc1--11-specific T cells escape tolerance while MBP121--150-specific T cells undergo tolerance in vivo. This differential tolerance induction may reflect unstable binding of MBPAc1--11 to I-Au compared to stable binding of peptides within MBP121--150.To determine which MBP epitopes are naturally processed from whole MBP, peptides eluted from I-Au isolated from MBP-pulsed splenocytes were analyzed by mass spectrometry. Two nested sets of peptides were found, N-terminal MBP peptides and peptides containing MBP125--135. N-terminal peptides MBPAc1--18 and MBPAc1--17 were the most abundant peptides processed from whole MBP. Our investigations indicate that MBPAc1--18 can bind in the register that presents MBPAc1--11, however, most MBPAc1--18 binds in a more stable register, MBP5--16, allowing MBPAc1--11-specific T cells to escape tolerance. Additionally, endogenously derived MBP peptides are constitutively presented by B cells and dendritic cells (DCs) which are able to stimulate activated MBP121--150-specific T cells. However, only DCs are able to stimulate naive MBP121--150-specific T cells, implicating DCs as the antigen presenting cell involved in the maintenance of peripheral tolerance to MBP.To investigate why two different MBPAc1--11-specific T cell receptor transgenic mouse lines exhibit differences in spontaneous EAE incidence, T cell responses to MBP were compared between the two lines. While differences in T cell proliferative responses were not detected, T cells isolated from the Tg line with lower spontaneous EAE incidence did not produce IFN-gamma and lower percentages of T cells produced cytokines compared to the line with higher incidence of spontaneous EAE.We also analyzed the structural basis for cross-reactivity exhibited by T cells recognizing two distinct epitopes within MBP121--150. Mutational analysis of the CDRI and 3 regions of one cross-reactive T cell receptor indicated that the same amino acids within CDR1 and 3 are used to recognize both epitopes

Protective Effects of ALDH1A Enzyme Inhibition on Helicobacter-Induced Colitis in Smad3−/− Mice are Associated with Altered α4ß7 Integrin Expression on Activated T Cells

Many inflammatory bowel disease (IBD) patients require surgical intervention due to limited pharmacological treatment options. Antibodies targeting α4ß7, a gut-homing integrin, are one of the most promising IBD treatments. As retinoic acid (RA) regulates expression of gut-homing proteins including α4ß7 integrin, we tested if ALDH1A enzymes in the RA synthesis pathway could be targeted for IBD treatment using a potent inhibitor, WIN 18,446. Age- and sex-matched Smad3−/− mice were fed a diet with and without WIN 18,446 for 3 weeks before triggering inflammation with Helicobacter bilis infection. Colitis was evaluated by histopathology one week following the IBD trigger, and T cell subsets were evaluated before and after the IBD trigger. WIN 18,446 treatment significantly reduced IBD severity in Smad3−/− mice and reduced expression of α4ß7 integrin on multiple activated CD4+ T cell subsets. This change was associated with increased ratios of induced regulatory T cells to Th17 cells during the inflammatory response in the draining lymph nodes. These studies indicate that RA reduction via ALDH1A enzyme inhibition is a potential new target for IBD treatment. Further studies are needed to examine its effects on other types of immune cells, to evaluate the efficacy window for this target, and to determine its efficacy in other animal models of IBD

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

ALDH1A Inhibition Suppresses Colitis and Alters α4β7 Integrin Expression on Activated T Cells in <i>Mdr1a</i>^−/− Mice

There are limited pharmacological treatment options for inflammatory bowel disease (IBD), and some of these options are expensive and administered by injection or infusion. Thus, new cheaper and easier (oral) treatment options are needed. ALDH1A enzymes produce retinoic acid that can affect intestinal diseases such as IBD by regulating immune cells in the gut. We previously demonstrated that an orally deliverable ALDH1A inhibitor, WIN 18,466, can suppress colitis in an acute mouse model of IBD. Here, we tested the efficacy of ALDH1A inhibition in a chronic mouse model of IBD. Mdr1a−/− mice were treated with a diet containing WIN 18,446 starting 1 week prior to inducing colitis by H. bilis inoculation. Treatment was continued until the study end point and colitis was monitored based on clinical symptoms and confirmed by histological analysis. Immune cell phenotypes in colon-draining lymph nodes (cMLN) were analyzed. WIN 18,446 treatment reduced clinical symptoms and improved histopathologic colitis scores. This was associated with decreased expression of the gut homing integrin, α4β7, on T cells in cMLN; increased expression of CD103, a protein associated with tissue-resident memory T cells; and changes in dendritic cells, plasmacytoid dendritic cells and B cells in inhibitor-treated mice. ALDH1A inhibition broadly influences immune cells during colitis and is a potential new target for IBD treatment. Future studies will be needed to determine the efficacy of ALDH1A inhibition on active colitis and to evaluate its relative efficacy in comparison to approved drugs

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

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

Histopathology scores of DSS-treated <i>Smad3^−/−</i> and <i>Smad3/Rag-DKO</i> mice treated with different doses of DSS.

<p><i>Smad3^+/−</i>, <i>Smad3^−/−</i> and <i>Smad3/Rag-DKO</i> (DKO) mice were treated with either a single DSS cycle or 9 cycles of DSS. Experimental endpoint was 17 weeks. A) IBD score, B) Invasion score, C) Summed dysplasia score and (D) Distribution score (as described in materials and methods) are shown for individuals in each treatment group. Negative control groups were all statistically different (significance not shown in figure) from their respective DSS-treated group except for untreated water vs. 1.5% DSS <i>Smad3^−/−</i> in (B). <i>*P</i>≤<i>0.05, **P</i>≤<i>0.01.</i></p