Adoption of Exhaust Air Dust Testing in SPF Rodent Facilities.

Reliable detection of unwanted microbial agents is essential for meaningful health monitoring in laboratory animal facilities. Most rodents at our institution are housed in IVC rack systems to minimize aerogenic transmission of infectious agents between cages. The most commonly used rodent health monitoring systems expose live sentinel rodents to soiled bedding collected from other rodent cages on IVC racks and subsequently test these soiled-bedding sentinels for evidence of infection with excluded agents. However, infectious agents might go undetected when using this health surveillance method, due to inefficient organism shedding or transmission failure. In 2016, our institution switched the health monitoring methodology for the majority of our SPF rodent colonies to real-time PCR testing of environmental samples collected from the exhaust plenums of IVC racks. Here we describe our rationale for this conversion, describe some interesting health monitoring cases that arose soon after the conversion, and discuss a potential problem with the conversion-residual nucleic acids. We compared cost and implementation effort associated with 2 sampling methods, sticky swabs and in-line collection media. We also compared the ability of these 2 sampling methods to detect 2 prevalent microbes in our facilities, Helicobacter and murine norovirus. Our institution-wide switch to health monitoring by real-time PCR assay of exhaust air dust samples thus far has provided a sensitive, simple, and reliable approach for maintenance of SPF conditions in laboratory rodents and has dramatically reduced the use of live sentinel animals.

Effect of Chronic Vitamin D Deficiency on the Development and Severity of DSS-Induced Colon Cancer in Smad3-/- Mice.

Both human epidemiologic data and animal studies suggest that low serum vitamin D increases the risk of inflammatory bowel disease (IBD) and consequently IBD-associated colorectal cancer. We tested the hypothesis that vitamin D deficiency increases the risk for colitis-associated colon cancer (CAC) by using an established CAC mouse model, 129-Smad3tm1Par/J (Smad3-/-) mice, which have defective transforming growth factor ß-signaling and develop colitis and CAC after the administration of dextran sodium sulfate (DSS). After determining a dietary regimen that induced chronic vitamin D deficiency in Smad3-/- mice, we assessed the effects of vitamin D deficiency on CAC. Decreasing dietary vitamin D from 1 IU/g diet (control diet) to 0.2 IU /g diet did not decrease serum 25-hydroxyvitamin D (25(OH)D) levels in Smad3-/- mice. A diet devoid of vitamin D (< 0.02 IU/g diet [no added vitamin D]; vitamin D-null) significantly decreased serum 25(OH)D levels in mice after 2 wk (null compared with control diet: 8.4 mg/mL compared with 12.2 ng/mL) and further decreased serum levels to below the detection limit after 9 wk but did not affect weight gain, serum calcium levels, bone histology, or bone mineral density. In light of these results, Smad3-/- mice were fed a vitamin D-null diet and given DSS to induce colitis. Unexpectedly, DSS-treated Smad3-/- mice fed a vitamin D-null diet had improved survival, decreased colon tumor incidence (8% compared with 36%), and reduced the incidence and severity of colonic dysplasia compared with mice fed the control diet. These effects correlated with increased epithelial cell proliferation and repair early in the disease, perhaps reducing the likelihood of developing chronic colitis and progression to cancer. Our results indicate that vitamin D deficiency is beneficial in some cases of CAC, possibly by promoting intestinal healing.

Lack of Effect of Murine Norovirus Infection on the CD4+ CD45RBhigh T-cell Adoptive Transfer Mouse Model of Inflammatory Bowel Disease.

Murine norovirus (MNV) infection is highly prevalent in laboratory mice. Although MNV infection does not typically induce clinical disease in most laboratory mice, infection may nonetheless affect mouse models of disease by altering immune responses. We previously reported that MNV altered the bacterial-induced mouse model of inflammatory bowel disease (IBD) using Helicobacter-infected Mdr1a-/- mice. Therefore, we hypothesized that MNV infection would exacerbate another mouse model of IBD, the T-cell adoptive transfer (AT) model. In this model, Helicobacter infection is used to accelerate the progression of IBD induced by AT of naïve CD4+CD45RBhigh T cells into B6.129S7- Rag1tm1Mom/J (Rag1-/-) mice. We evaluated the effects of MNV infection in both Helicobacter-accelerated as well as Helicobacter-free AT models. In our studies, Helicobacter-infected Rag1-/- mice that received CD4+CD45RBhigh T cells through AT rapidly developed weight loss and typhlocolitis; MNV infection had no effect on disease severity or rate of progression. In the absence of Helicobacter infection, progression of IBD caused by AT of CD4+CD45RBhigh T cells was slower and typhlocolitis was less severe; this inflammation likewise was unaltered by MNV infection. These results indicate that MNV infection does not alter IBD progression and severity in the CD4+CD45RBhigh T-cell AT model in Rag1-/- mice.

Validation studies for germ-free Smad3-/- mice as a bio-assay to test the causative role of fecal microbiomes in IBD.

While the association between microbiomes and inflammatory bowel disease (IBD) is well known, establishing causal relationships between the two is difficult in humans. Germ-free (GF) mice genetically susceptible to IBD can address this question. Smad3-/- mice with defective transforming growth factor ß signaling are a model of IBD and colon cancer. They develop IBD upon colonization with Helicobacter under specific pathogen-free conditions, suggesting a role of the microbiome in IBD in this model. Thus, we rederived Smad3-/- mice GF to determine the potential of using these mice for testing the causative role of microbiomes in IBD. We found that fecal microbiomes from mice with IBD cause more severe gut inflammation in GF Smad3-/- and wild type mice compared to microbiomes from healthy mice and that Helicobacter induces gut inflammation within the context of other microbiomes but not by itself. Unexpectedly, GF Smad3+/+ and Smad3+/- mice given IBD microbiomes develop IBD despite their lack of disease in SPF conditions upon Helicobacter infection. This was not unique to the background strain of our Smad3 model (129); both wild type C57BL/6 and 129 strains developed IBD upon fecal transfer. However, wild type Swiss Webster stock was not susceptible, indicating that the genetic background of recipient mice influences the severity of IBD following fecal transfer. Our data suggest that the microbiome is an independent risk factor contributing to IBD development, and careful characterization of new GF models is needed to understand potential sources of confounding factors influencing microbiome studies in these mice.

Obstructive Lymphangitis Precedes Colitis in Murine Norovirus-Infected Stat1-Deficient Mice.

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 Ifnαßγr-/- 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.

Murine norovirus inhibits B cell development in the bone marrow of STAT1-deficient mice.

Noroviruses are a leading cause of gastroenteritis in humans and it was recently revealed that noroviruses can infect B cells. We demonstrate that murine norovirus (MNV) infection can significantly impair B cell development in the bone marrow in a signal transducer and activator of transcription 1 (STAT1) dependent, but interferon signaling independent manner. We also show that MNV replication is more pronounced in the absence of STAT1 in ex vivo cultured B cells. Interestingly, using bone marrow transplantation studies, we found that impaired B cell development requires Stat1-/- hematopoietic cells and Stat1-/- stromal cells, and that the presence of wild-type hematopoietic or stromal cells was sufficient to restore normal development of Stat1-/- B cells. These results suggest that B cells normally restrain norovirus replication in a cell autonomous manner, and that wild-type STAT1 is required to protect B cell development during infection.

Effects of Murine Norovirus on Chlamydia pneumoniae-Accelerated Atherosclerosis in ApoE(-/-) Mice.

Chlamydia pneumoniae (Cpn), a common respiratory pathogen of humans, is associated with human cardiovascular disease and the acceleration of atherosclerosis in hyperlipidemic animal models. Our laboratory has demonstrated that murine norovirus (MNV), a prevalent infection of laboratory mice, can unpredictably alter atherosclerosis in hyperlipidemic Ldlr(-/-) and ApoE(-/-) mice. Given that MNV has a tropism for macrophages and may exacerbate atherogenesis, we investigated whether coinfection with MNV and Cpn might alter macrophage phenotypes in vitro and atherosclerosis in ApoE(-/-) mice. In the presence of oxidized low-density lipoprotein, coinfection of ApoE(-/-) bone marrow-derived macrophages (BMDM) with MNV and Cpn resulted in significant increases in gene expression of IL6, MCP1, iNOS, and TNFα compared with Cpn-monoinfected BMDM. On the basis of these findings, we hypothesized that concurrent MNV-Cpn infection might increase plaque lesion size in vivo. As expected, Cpn monoinfection of ApoE(-/-) mice increased mean plaque size by 62% compared with that in uninfected mice. However, MNV did not significantly alter plaque lesion size in MNV-Cpn-coinfected mice compared with Cpn-monoinfected mice. There were no differences in aortic cytokines locally at the site of plaque development or in peritoneal macrophages at 1 wk after infection in MNV-Cpn-coinfected mice compared with Cpn-monoinfected mice. MNV was not detected in the aortic tissue of MNV-infected mice at 1 or 8 wk after infection regardless of Cpn status. These data suggest that MNV infection does not appreciably alter plaque development in Cpn-accelerated atherosclerosis in ApoE(-/-) mice.

Protective links between vitamin D, inflammatory bowel disease and colon cancer.

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.

Murine Norovirus Infection Variably Alters Atherosclerosis in Mice Lacking Apolipoprotein E.

Macrophages play a key role in the development of atherosclerosis. Murine noroviruses (MNV) are highly prevalent in research mouse colonies and infect macrophages and dendritic cells. Our laboratory found that MNV4 infection in mice lacking the LDL receptor alters the development of atherosclerosis, potentially confounding research outcomes. Therefore, we investigated whether MNV4 likewise altered atherosclerosis in ApoE(-/-) mice. In the presence of oxidized LDL, MNV4 infection of ApoE(-/-) bone marrow-derived macrophages increased the gene expression of the inflammatory markers inducible nitric oxide synthase, monocyte chemoattractant protein 1, and IL6. In addition, proteins involved in cholesterol transport were altered in MNV4-infected ApoE -/- bone marrow-derived macrophages and consisted of increased CD36 and decreased ATP-binding cassette transporter A1. MNV4 infection of ApoE(-/-) mice at 12 wk of age (during the development of atherosclerosis) had a variable effect on atherosclerotic lesion size. In one study, MNV4 significantly increased atherosclerotic plaque area whereas in a second study, no effect was observed. Compared with controls, MNV4-infected mice had higher circulating Ly6C-positive monocytes, and viral RNA was detected in the aortas of some mice, suggesting potential mechanisms by which MNV4 alters disease progression. Plaque size did not differ when ApoE -/- mice were infected at 4 wk of age (early during disease development) or in ApoE -/- mice maintained on a high-fat, high-cholesterol diet. Therefore, these data show that MNV4 has the potential to exert a variable and unpredictable effect on atherosclerosis in ApoE(-/-) mice. We therefore propose that performing experiments in MNV-free mouse colonies is warranted.

Isolation of segmented filamentous bacteria from complex gut microbiota.

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.

Potential for using a hermetically-sealed, positive-pressured isocage system for studies involving germ-free mice outside a flexible-film isolator.

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.

Effects of murine norovirus on atherosclerosis in ldlr(-/-) mice depends on the timing of infection.

We previously reported that murine norovirus (MNV), a virus prevalent in United States research institutions, increased atherosclerotic lesion size in Ldlr(-/-) mice when the mice were infected 8 wk after feeding an atherogenic diet. To determine whether the timing of MNV infection relative to atherosclerosis development altered the disease phenotype and to examine potential mechanisms by which MNV influences the disease process, we fed Ldlr(-/-) mice an atherogenic diet for 16 wk. Three days after initiating the atherogenic diet, half of the mice received MNV4 and the other half vehicle only (clarified cell-culture lysate; controls). Both groups of mice developed large aortic sinus lesions (control compared with MNV4: 133 ± 8 × 10³ µm² compared with 140 ± 7 × 10³ µm²) that were not significantly different in size. Because the timing of MNV infection relative to atherosclerosis development and hypercholesterolemia differed between our previous and the current studies, we examined whether hypercholesterolemia altered MNV4-induced changes in bone-marrow-derived macrophages. MNV4 infection increased the potential of macrophages to take up and store cholesterol by increasing CD36 expression while suppressing the ABCA1 transporter. Thus, the effects of MNV4 infection on atherosclerotic lesion size appear to be dependent on the timing of the infection: MNV4 infection promotes only established lesions. This effect may be due to MNV4's ability to increase cholesterol uptake and decrease efflux by regulating CD36 and ABCA1 protein expression.

Infection with murine norovirus 4 does not alter Helicobacter-induced inflammatory bowel disease in Il10(-/-) mice.

Infection of laboratory mice with murine noroviruses (MNV) is widely prevalent. MNV alters various mouse models of disease, including the Helicobacter bilis-induced mouse model of inflammatory bowel disease (IBD) in Mdr1a(--) mice. To further characterize the effect of MNV on IBD, we used mice deficient in the immunoregulatory cytokine IL10 (Il10(-/-) mice). In vitro infection of Il10(-/-) bone marrow-derived macrophages (BMDM) with MNV4 cocultured with H. bilis antigens increased the gene expression of the proinflammatory cytokines IL1ß, IL6, and TNFα as compared with that of BMDM cultured with H. bilis antigens only. Therefore, to test the hypothesis that MNV4 infection increases inflammation and alters disease phenotype in H. bilis-infected Il10(-/-) mice, we compared the amount and extent of inflammation in Il10(-/-) mice coinfected with H. bilis and MNV4 with those of mice singly infected with H. bilis. IBD scores, incidence of IBD, or frequency of severe IBD did not differ between mice coinfected with H. bilis and MNV4 and those singly infected with H. bilis. Mice infected with MNV4 only had no appreciable IBD, comparable to uninfected mice. Our findings suggest that, unlike in Mdr1a(-/-) mice, the presence of MNV4 in Il10(-/-) mouse colonies is unlikely to affect the IBD phenotype in a Helicobacter-induced model. However, because MNV4 altered cytokine expression in vitro, our results highlight the importance of determining the potential influence of MNV on mouse models of inflammatory disease, given that MNV has a tropism for macrophages and dendritic cells and that infection is widely prevalent.

Increased dietary vitamin D suppresses MAPK signaling, colitis, 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-Smad3(tm1Par)/J (Smad3(-/-)) mice are a model of bacteria-driven colitis and colon cancer when infected with Helicobacter bilis (H. 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 postinoculation to assess inflammation, dysplasia, and neoplasia incidence. At 16 weeks postinfection, 11% of mice fed high vitamin D diet had cancer compared with 41% of mice fed maintenance diet (P = 0.0121). Evaluation at an early time point (1 week postinfection) 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.

Characterization of dextran sodium sulfate-induced inflammation and colonic tumorigenesis in Smad3(-/-) mice with dysregulated TGFß.

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. Smad3(-/-) mice are deficient in the transforming growth factor beta (TGFß) signaling molecule, SMAD3, 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 Smad3(-/-) mice. Studies presented here in Smad3(-/-) mice detail disease induction with DSS, without the use of AOM, and show a) Smad3(-/-) 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 SMAD3 does not confer increased susceptibility to DSS-induced disease and e) disease is partially controlled by the presence of T and B cells as Smad3(-/-) Rag2(-/-) double knock out (DKO) mice develop a more severe disease phenotype. DSS-induced disease in Smad3(-/-) 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).

High-fat diet-induced obesity exacerbates inflammatory bowel disease in genetically susceptible Mdr1a-/- male mice.

Obesity is a chronic inflammatory disease and a risk factor for disorders such as heart disease, diabetes, and cancer. A high-fat diet (HFD), a risk factor for obesity, has also been associated with inflammatory bowel disease (IBD). A proinflammatory state characterized by systemic and local increases in cytokine and chemokine levels are noted in both obesity and IBD, but it is unclear whether obesity is a risk factor for IBD. To examine any association between obesity and IBD, we chose FVB.129P2- Abcb1a(tm1Bor)N7 (Mdr1a(-/-)) mice, because this strain develops IBD spontaneously with age without a chemical or bacterial "trigger." In addition, its background strain, FVB, has been used for diet-induced obesity studies. Mdr1a(-/-) mice and wild-type (WT) mice were fed a HFD (∼60% calories from fat) or a low-fat diet (LFD; ∼11% calories from fat) for 12 wk. Obesity phenotypes examined included body weight measurements, glucose metabolism changes, and adiposity at termination of the study. IBD was determined by clinical signs, necropsy, and histopathology. We found that compared with those fed the LFD, both the Mdr1a(-/-) and WT mice fed the HFD had greater weight gains and elevated plasma leptin concentrations (P < 0.0001). When all mice were analyzed, weight gain was also associated with inflammation in mesenteric fat (R(2) = 0.5; P < 0.0001) and mesenteric lymph nodes (R(2) = 0.4; P < 0.0001). In contrast, the HFD was not associated with IBD in WT mice, whereas it exacerbated spontaneous IBD in Mdr1a(-/-) mice (P = 0.012; Fisher's exact test). Although a HFD and obesity were not associated with IBD in WT mice, our studies suggest that they are likely risk factors for IBD in a genetically susceptible host, such as Mdr1a(-/-) mice.

Lineage targeted MHC-II transgenic mice demonstrate the role of dendritic cells in bacterial-driven colitis.

BACKGROUND: Inflammatory bowel disease (IBD) pathogenesis involves an inadequately controlled immune reaction to intestinal microbiota, and CD4(+) T cells, dependent on MHC class II (MHC-II) processing and presentation by antigen-presenting cells (APC), play important roles. The role of professional APC (macrophages and dendritic cells [DCs]) and nonprofessional APC (intestinal epithelial cells [IECs]) in microbial-driven intestinal inflammation remains controversial. METHODS: We generated transgenic animals on an MHC-II(-/-) genetic background in which MHC-II is expressed on 1) DC via the CD11c promoter (CD11cTg) or 2) IEC via the fatty acid binding protein (liver) promoter (EpithTg). These mice were crossed with Rag2(-/-) mice to eliminate T and B cells (CD11cTg/Rag2(-/-) and EpithTg/Rag2(-/-)). Helicobacter bilis (Hb) infection and adoptive transfer (AT) of naïve CD4 T cells were used to trigger IBD. RESULTS: CD11cTg/Rag2(-/-) mice infected with Hb+AT developed severe colitis within 3 weeks post-AT, similar to disease in positive control Rag2(-/-) mice infected with Hb+AT. CD11cTg/Rag2(-/-) mice given AT alone or Hb alone had significantly less severe colitis. In contrast, EpithTg/Rag2(-/-) mice infected with Hb+AT developed mild colitis by 3 weeks and even after 16 weeks post-AT had only mild lesions. CONCLUSIONS: MHC-II expression restricted to DCs is sufficient to induce severe colitis in the presence of T cells and a microorganism such as Hb within 3 weeks of AT. Expression of MHC-II solely on IEC in the presence of a microbial trigger and T cells was insufficient to trigger severe colitis.

Interleukin-7 receptor blockade suppresses adaptive and innate inflammatory responses in experimental colitis.

BACKGROUND: Interleukin-7 (IL-7) acts primarily on T cells to promote their differentiation, survival, and homeostasis. Under disease conditions, IL-7 mediates inflammation through several mechanisms and cell types. In humans, IL-7 and its receptor (IL-7R) are increased in diseases characterized by inflammation such as atherosclerosis, rheumatoid arthritis, psoriasis, multiple sclerosis, and inflammatory bowel disease. In mice, overexpression of IL-7 results in chronic colitis, and T-cell adoptive transfer studies suggest that memory T cells expressing high amounts of IL-7R drive colitis and are maintained and expanded with IL-7. The studies presented here were undertaken to better understand the contribution of IL-7R in inflammatory bowel disease in which colitis was induced with a bacterial trigger rather than with adoptive transfer. METHODS: We examined the contribution of IL-7R on inflammation and disease development in two models of experimental colitis: Helicobacter bilis (Hb)-induced colitis in immune-sufficient Mdr1a-/- mice and in T- and B-cell-deficient Rag2-/- mice. We used pharmacological blockade of IL-7R to understand the mechanisms involved in IL-7R-mediated inflammatory bowel disease by analyzing immune cell profiles, circulating and colon proteins, and colon gene expression. RESULTS: Treatment of mice with an anti-IL-7R antibody was effective in reducing colitis in Hb-infected Mdr1a-/- mice by reducing T-cell numbers as well as T-cell function. Down regulation of the innate immune response was also detected in Hb-infected Mdr1a-/- mice treated with an anti-IL-7R antibody. In Rag2-/- mice where colitis was triggered by Hb-infection, treatment with an anti-IL-7R antibody controlled innate inflammatory responses by reducing macrophage and dendritic cell numbers and their activity. CONCLUSIONS: Results from our studies showed that inhibition of IL-7R successfully ameliorated inflammation and disease development in Hb-infected mice by controlling the expansion of multiple leukocyte populations, as well as the activity of these immune cells. Our findings demonstrate an important function of IL-7R-driven immunity in experimental colitis and indicate that the therapeutic efficacy of IL-7R blockade involves affecting both adaptive and innate immunity.

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.