Audrey Seamons

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.

Competition between two MHC binding registers in a single peptide processed from myelin basic protein influences tolerance and susceptibility to autoimmunity.

Experimental allergic encephalomyelitis (EAE) is an animal model for multiple sclerosis induced by stimulating myelin basic protein (MBP)-specific T cells. The MBP-specific repertoire in B10.PL mice is shaped by tolerance mechanisms that eliminate MBP121–150–specific T cells. In contrast, MBPAc1–11–specific T cells escape tolerance and constitute the encephalitogenic repertoire. To determine if this differential tolerance is caused by differences in the abundance of MBP epitopes generated by processing, MBP peptides were eluted from I-Au complexes and analyzed by mass spectrometry. Peptides were identified from both the NH2-terminal and MBP121–150 regions. Unexpectedly, MBPAc1–18 and Ac1–17, which contain the MBPAc1–11 epitope, were much more abundant than MBP121–150 peptides. The results demonstrate that competition between two I-Au binding registers, a low affinity register defined by MBPAc1–11 and a high affinity register defined by MBP5–16, prevents most of the NH2-terminal naturally processed peptides from binding in the MBPAc1–11 register. The small fraction of MBPAc1–18 bound in the MBPAc1–11 register is not sufficient to induce tolerance but provides a ligand for MBPAc1–11–specific T cells during disease. These results provide a basis for both the lack of tolerance to MBPAc1–11 and the ability of this epitope to become a target during autoimmunity.

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.

Immune tolerance to myelin proteins

Multiple sclerosis (MS) is a demyelinating disorder of the central nervous system. It is believed to be an autoimmune disease arising from a breakdown of immune tolerance in T cells specific for myelin antigens. The heterogeneity in clinical signs and pathology observed in MS patients suggests a complex pathogenesis in which the specificity of the pathogenic T cells and the tolerance mechanisms that are compromised vary among individual patients. In this review, we summarize some of the features of the diverse immune pathology observed in MS and the animal models used to study this disease. We then describe the current state of knowledge regarding the expression of the major myelin protein antigens believed to be targeted in MS and the mechanisms of immune tolerance that operate on T cells that recognize these antigens.

Bacterial Infection of Smad3/Rag2 Double-Null Mice with Transforming Growth Factor-β Dysregulation as a Model for Studying Inflammation-Associated Colon Cancer

Alterations in genes encoding transforming growth factor-beta-signaling components contribute to colon cancer in humans. Similarly, mice deficient in the transforming growth factor-beta signaling molecule, Smad3, develop colon cancer, but only after a bacterial trigger occurs, resulting in chronic inflammation. To determine whether Smad3-null lymphocytes contribute to increased cancer susceptibility, we crossed Smad3-null mice with mice deficient in both B and T lymphocytes (Rag2(-/-) mice). Helicobacter-infected Smad3/Rag2-double knockout (DKO) mice had more diffuse inflammation and increased incidence of adenocarcinoma compared with Helicobacter-infected Smad3(-/-) or Rag2(-/-) mice alone. Adoptive transfer of WT CD4(+)CD25(+) T-regulatory cells provided significant protection of Smad3/Rag2-DKO from bacterial-induced typhlocolitis, dysplasia, and tumor development, whereas Smad3(-/-) T-regulatory cells provided no protection. Immunohistochemistry, real-time reverse transcriptase-polymerase chain reaction, and Western blot analyses of colonic tissues from Smad3/Rag2-DKO mice 1 week after Helicobacter infection revealed an influx of macrophages, enhanced nuclear factor-kappaB activation, increased Bcl(XL)/Bcl-2 expression, increased c-Myc expression, accentuated epithelial cell proliferation, and up-regulated IFN-gamma, IL-1alpha, TNF-alpha, IL-1beta, and IL-6 transcription levels. These results suggest that the loss of Smad3 increases susceptibility to colon cancer by at least two mechanisms: deficient T-regulatory cell function, which leads to excessive inflammation after a bacterial trigger; and increased expression of proinflammatory cytokines, enhanced nuclear factor-kappaB activation, and increased expression of both pro-oncogenic and anti-apoptotic proteins that result in increased cell proliferation/survival of epithelial cells in colonic tissues.

Serum biomarkers in a mouse model of bacterial-induced inflammatory bowel disease

Background: The diagnosis and classification of inflammatory bowel disease (IBD) require both clinical and histopathologic data. Serum biomarkers would be of considerable benefit to noninvasively monitor the progression of disease, assess effectiveness of therapies, and assist in understanding disease pathogenesis. Currently, there are limited noninvasive biomarkers for monitoring disease progression in animal IBD models, which are used extensively to develop new therapies and to understand IBD pathogenesis. Methods: Serum biomarkers of early and late IBD were identified using multianalyte profiling in mdr1a−/− mice with IBD triggered by infection with Helicobacter bilis. The correlation of changes in these biomarkers with histopathology scores and clinical signs in the presence and in the absence of antibiotic treatment was determined. Results: Serum levels of interleukin (IL)–11, IL‐17, 10‐kDa interferon‐&ggr;‐inducible protein (IP‐10), lymphotactin, monocyte chemoattractant protein (MCP)–1, and vascular cell adhesion molecule (VCAM)–1 were elevated early in IBD. In late, more severe IBD, serum levels of IL‐11, IP‐10, haptoglobin, matrix metalloproteinase–9, macrophage inflammatory protein (MIP)–1&ggr;, fibrinogen, immunoglobulin A, MIP‐3 beta (&bgr;), VCAM‐1, apolipoprotein (Apo) A1, and IL‐18 were elevated. All late serum biomarkers except Apo A1 correlated with histopathology scores. Antibiotic treatment improved clinical signs of IBD and decreased mean serum values of many of the biomarkers. For all biomarkers, the individual pathology scores correlated significantly with individual serum analyte levels after treatment. Conclusions: Serum analyte measurement is a useful, noninvasive method for monitoring disease in a mouse model of bacterial‐induced IBD.

Endogenous Myelin Basic Protein Is Presented in the Periphery by Both Dendritic Cells and Resting B Cells with Different Functional Consequences

Multiple sclerosis is an inflammatory disease believed to be triggered by erroneous activation of self-reactive T cells specific for myelin proteins such as myelin basic protein (MBP). Inflammation is limited to the CNS, suggesting that the myelin-specific T cells encounter their Ags only after they cross the blood-brain barrier. However, our previous studies in mice showed that MBP epitopes are constitutively presented in lymphoid tissues. Here we identified which APCs in lymph nodes present endogenous MBP epitopes and determined the functional consequences of this presentation for both naive and activated MBP-specific T cells. Both CD8α+ and CD8α− dendritic cells were potent stimulators of proliferation for both naive and previously activated/memory MBP-specific T cells. Surprisingly, resting B cells also presented endogenous MBP that was acquired using a BCR-independent mechanism. Interaction with resting B cells triggered proliferation of both naive and activated MBP-specific T cells. Activated/memory MBP-specific T cells proliferating in response to resting B cells presenting endogenous MBP did not produce cytokines and became more refractory to subsequent stimulation. Interestingly, cytokine production by activated/memory T cells was triggered by resting B cells if the number of MBP epitopes presented was increased by adding exogenous MBP peptide. These results suggest that activated MBP-specific T cells may become less pathogenic in vivo following encounter with resting B cells presenting steady-state levels of endogenous MBP but can expand and remain pathogenic if the amount of MBP presented by B cells is increased, which could occur during chronic demyelinating disease.

Characterization of dextran sodium sulfate-induced inflammation and colonic tumorigenesis in Smad3-/- mice with dysregulated TGFβ

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 (TGFβ) 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).

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

BackgroundInterleukin-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.MethodsWe 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.ResultsTreatment 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.ConclusionsResults 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.

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.