Vitamin D deficiency has been associated with a wide range of diseases and multiple forms of cancer including breast, colon, and prostate cancers. Relatively recent work has demonstrated vitamin D to be critical in immune function and therefore important in inflammatory diseases such as inflammatory bowel disease (IBD). Because vitamin D deficiency or insufficiency is increasingly prevalent around the world, with an estimated 30%-50% of children and adults at risk for vitamin D deficiency worldwide, it could have a significant impact on IBD. Epidemiologic studies suggest that low serum vitamin D levels are a risk factor for IBD and colon cancer, and vitamin D supplementation is associated with decreased colitis disease activity and/or alleviated symptoms. Patients diagnosed with IBD have a higher incidence of colorectal cancer than the general population, which supports the notion that inflammation plays a key role in cancer development and underscores the importance of understanding how vitamin D influences inflammation and its cancer-promoting effects. In addition to human epidemiological data, studies utilizing mouse models of colitis have shown that vitamin D is beneficial in preventing or ameliorating inflammation and clinical disease. The precise role of vitamin D on colitis is unknown; however, vitamin D regulates immune cell trafficking and differentiation, gut barrier function and antimicrobial peptide synthesis, all of which may be protective from IBD and colon cancer. Here we focus on effects of vitamin D on inflammation and inflammation-associated colon cancer and discuss the potential use of vitamin D for protection and treatment of IBD and colon cancer.

Epidemiologic studies associate low serum vitamin D levels with an increased risk of colon cancer and inflammatory diseases such as inflammatory bowel disease (IBD). 129-Smad3tm1Par/J (Smad3−/−) mice are a model of bacterial-driven colitis and colon cancer when infected with Helicobacter bilis. Thus, we used this mouse model to determine whether increased dietary vitamin D would reduce inflammation and colon cancer. Smad3−/− mice were fed purified diet with either maintenance (1 IU vitamin D/g diet; maintenance) or increased concentrations of vitamin D (5 IU vitamin D/g diet; high vitamin D). One week after diet initiation, mice were inoculated with broth or H. bilis and were necropsied at several time points post-inoculation to assess inflammation, dysplasia, and neoplasia incidence. At 16 weeks post infection, 11% of mice fed high vitamin D diet had cancer compared to 41% of mice fed maintenance diet (p=0.0121). Evaluation at an early time point (1 week post-infection) showed that animals fed high vitamin D had decreased MAPK (p-p38 and p-JNK) activation in lamina propria leukocytes as well as decreased NFκB activation in colonic epithelial cells. Reduction in MAPK and NFκB activation correlated with decreased IBD scores (2.7 vs 15.5, p<0.0001) as well as decreased inflammatory cell infiltrates and reduced expression of proinflammatory cytokines in cecal tissue. These findings suggest that increased dietary vitamin D is beneficial in preventing inflammation-associated colon cancer through suppression of inflammatory responses during initiation of neoplasia or early stage carcinogenesis.

Patients with cystic fibrosis (CF) have altered fecal microbiomes compared to those of healthy controls. The magnitude of this dysbiosis correlates with measures of CF gastrointestinal (GI) disease, including GI inflammation and nutrient malabsorption. However, whether this dysbiosis is caused by mutations in the CFTR gene, the underlying defect in CF, or whether CF-associated dysbiosis augments GI disease was not clear. To test the relationships between CFTR dysfunction, microbes, and intestinal health, we established a germ-free (GF) CF mouse model and demonstrated that CFTR gene mutations are sufficient to alter the GI microbiome. Furthermore, flow cytometric analysis demonstrated that colonized CF mice have increased mesenteric lymph node and spleen TH17+ cells compared with non-CF mice, suggesting that CFTR defects alter adaptive immune responses. Our findings demonstrate that CFTR mutations modulate both the host adaptive immune response and the intestinal microbiome.

Germ-free mice are used to examine questions about the role of the gut microbiota in development of diseases. Generally these animals are maintained in semi-rigid or flexible-film isolators to ensure their continued sterility or, if colonized with specific microbiota, to ensure that no new species are introduced. Here, we describe the use of a caging system in which individual cages are hermetically sealed and have their own filtered positive airflow. This isopositive caging system requires less space and reduces animal housing costs. By using strict sterile techniques, we kept mice germ-free in this caging system for 12 weeks. We also used this caging system and approach to conduct studies evaluating a) the stability of the microbiome in germ-free mice receiving a fecal transplant and b) the stability of dietary-induced microbiota changes in fecal-transplanted mice. As has been shown in fecal transfer studies in isolators, we found that the transferred microbiota stabilizes as early as 2 weeks post transfer although recipient microbiota did not completely recapitulate those of the donors. Interestingly, we also noted some sex effects in these studies indicating that the sex of recipients or donors may play a role in colonization of microbiota. However, a larger study will be needed to determine what role, if any, sex plays in colonization of microbiota. Based on our studies, an isopositive caging system may be utilized to test multiple donor samples for their effects on phenotypes of mice in both normal and disease states even with limited available space for housing.

Segmented filamentous bacteria (SFB) modulate the ontogeny of the immune system, and their presence can significantly affect mouse models of disease. Until recently, the inability to successfully culture SFB has made controlled studies on the mechanisms by which these bacteria exert their influence problematic. Here, we report a new method for selecting SFB from complex microbial mixtures, providing researchers a simple and cost-effective means to prepare pure infective inocula for prospective studies and also to compare individual SFB isolates.

Murine noroviruses (MNV) are highly prevalent in laboratory mice, can cause persistent infections, and have been shown to infect macrophages, dendritic cells, and B cells. To address the potential impact of MNV infection on research outcomes, numerous studies have been conducted with various mouse models of human disease and have generated mixed results ranging from no impact to significant disease. Many of these studies included histologic evaluations after MNV infection, and similarly these results have been variable as to whether MNV induces lesions despite the fact that localization of MNV by viral culture and molecular techniques have demonstrated systemic distribution regardless of mouse immune status. The aim of this review is to summarize the histologic findings that have been reported with MNV infection in several mouse models. The studies demonstrate that experimental infection of MNV in wild-type mice results in minimal to no histologic changes. In contrast, immunodeficient mice consistently have detectable MNV-induced lesions that are typically inflammatory and, in the most severe cases, accompanied by necrosis. In these, the liver is commonly affected with more variable lesions reported in the lung, gastrointestinal tract, mesenteric lymph nodes, brain, and spleen. In specific disease models including atherosclerosis, MNV infection had a variable impact that was dependent upon the mouse model, viral strain, timing of infection, or other experimental variables. It is important to recognize the reported MNV lesions to help discern the possible influence of MNV infection on data generated in mouse models.

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 (MNV) is an RNA virus that can prove lethal in mice with impaired innate immunity. We found that MNV-4 infection of Stat1 mice was not lethal, but produced a 100% penetrant, previously undescribed lymphatic phenotype characterized by chronic-active lymphangitis with hepatitis, splenitis, and chronic cecal and colonic inflammation. Lesion pathogenesis progressed from early ileal enteritis and regional dilated lymphatics to lymphangitis, granulomatous changes in the liver and spleen, and, ultimately, typhlocolitis. Lesion development was neither affected by antibiotics nor reproduced by infection with another enteric RNA virus, rotavirus. MNV-4 infection in Stat1 mice decreased expression of vascular endothelial growth factor (Vegf) receptor 3, Vegf-c, and Vegf-d and increased interferon (Ifn)-γ, tumor necrosis factor-α, and inducible nitric oxide synthase. However, anti-IFN-γ and anti-tumor necrosis factor-α antibody treatment did not attenuate the histologic lesions. Studies in Ifnαβγr mice suggested that canonical signaling via interferon receptors did not cause MNV-4-induced disease. Infected Stat1 mice had increased STAT3 phosphorylation and expressed many STAT3-regulated genes, consistent with our findings of increased myeloid cell subsets and serum granulocyte colony-stimulating factor, which are also associated with increased STAT3 activity. In conclusion, in Stat1 mice, MNV-4 induces lymphatic lesions similar to those seen in Crohn disease as well as hepatitis, splenitis, and typhlocolitis. MNV-4-infected Stat1 mice may be a useful model to study mechanistic associations between viral infections, lymphatic dysfunction, and intestinal inflammation in a genetically susceptible host.