Thea Brabb

Smooth Muscle Cells Give Rise to Osteochondrogenic Precursors and Chondrocytes in Calcifying Arteries

Vascular calcification is a major risk factor for cardiovascular morbidity and mortality. To develop appropriate prevention and/or therapeutic strategies for vascular calcification, it is important to understand the origins of the cells that participate in this process. In this report, we used the SM22-Cre recombinase and Rosa26-LacZ alleles to genetically trace cells derived from smooth muscle. We found that smooth muscle cells (SMCs) gave rise to osteochondrogenic precursor- and chondrocyte-like cells in calcified blood vessels of matrix Gla protein deficient (MGP−/−) mice. This lineage reprogramming of SMCs occurred before calcium deposition and was associated with an early onset of Runx2/Cbfa1 expression and the downregulation of myocardin and Msx2. There was no change in the constitutive expression of Sox9 or bone morphogenetic protein 2. Osterix, Wnt3a, and Wnt7a mRNAs were not detected in either calcified MGP−/− or noncalcified wild-type (MGP+/+) vessels. Finally, mechanistic studies in vitro suggest that Erk signaling might be required for SMC transdifferentiation under calcifying conditions. These results provide strong support for the hypothesis that adult SMCs can transdifferentiate and that SMC transdifferentiation is an important process driving vascular calcification and the appearance of skeletal elements in calcified vascular lesions.

Differential tolerance is induced in T cells recognizing distinct epitopes of myelin basic protein.

Experimental allergic encephalomyelitis (EAE) is induced by T cell-mediated immunity to central nervous system antigens. In H-2u mice, EAE is mediated primarily by T cells specific for residues 1-11 of myelin basic protein (MBP). We demonstrate that differential tolerance to MBP1-11 versus epitopes in MBP121-150 is induced by expression of endogenous MBP, reflecting extreme differences in stability of peptide/MHC complexes. The diverse MBP121-150-specific TCR repertoire can be divided into three fine specificity groups. Two groups were identified in wild-type mice despite extensive tolerance, but the third group was not detected. Activated MBP121-150-specific T cells induce EAE in wild-type mice. Thus, encephalitogenic T cells that escape tolerance either recognize short-lived peptide/MHC complexes or express TCRs with unique specificities for stable complexes.

A Molecular Map of T Cell Development

Using a sensitive molecular marker for positive selection, the appearance of a particular functional TCR alpha chain sequence in cells from mice bearing a transgenic beta chain, we address several aspects of intrathymic T cell development. First, by examining specific TCR prior to and after maturation, we demonstrate how a restricted TCR repertoire is positively selected from a highly diverse immature TCR repertoire. Second, since this molecular marker is enriched in cells progressing toward the CD4 lineage and depleted in cells progressing toward the CD8 lineage, a map of the developmental pathway of alphabeta thymocytes can be inferred. Third, the first cells that show clear signs of positive intrathymic selection are identified.

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.

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.

Regulatory T Cells Maintain Long-Term Tolerance to Myelin Basic Protein by Inducing a Novel, Dynamic State of T Cell Tolerance

The pathogenesis of multiple sclerosis involves a breakdown in T cell tolerance to myelin proteins like myelin basic protein (MBP). Most MBP-specific T cells are eliminated by central tolerance in adult mice, however, the developmentally regulated expression of MBP allows MBP-specific thymocytes in young mice to escape negative selection. It is not known how these T cells that encounter MBP for the first time in the periphery are regulated. We show that naive MBP-specific T cells transferred into T cell-deficient mice induce severe autoimmunity. Regulatory T cells prevent disease, however, suppression of the newly transferred MBP-specific T cells is abrogated by activating APCs in vivo. Without APC activation, MBP-specific T cells persist in the periphery of protected mice but do not become anergic, raising the question of how long-term tolerance can be maintained if APCs presenting endogenous MBP become activated. Our results demonstrate that regulatory T cells induce naive MBP-specific T cells responding to nonactivated APCs to differentiate into a unique, tolerized state with the ability to produce IL-10 and TGF-β1 in response to activated, but not nonactivated, APCs presenting MBP. This tolerant response depends on continuous activity of regulatory T cells because, in their absence, these uniquely tolerized MBP-specific T cells can again induce autoimmunity.

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.

TCR signaling regulates thymic organization: lessons from TCR-transgenic mice

Abstract Recently described changes in thymic architecture in a variety of T-cell receptor (TCR)-transgenic mouse models have implications for our understanding of the processes underlying stromal-cell organisation, Here, Joan Goverman and colleagues discuss the new findings.

Inhibition of Retinoic Acid Biosynthesis by the Bisdichloroacetyldiamine WIN 18,446 Markedly Suppresses Spermatogenesis and Alters Retinoid Metabolism in Mice

Background: WIN 18,446, one of the class of compounds known as bisdichloroacetyldiamines, inhibits spermatogenesis. Results: WIN 18,446 strongly and irreversibly inhibits the retinal dehydrogenase ALDH1A2 in vitro, alters vitamin A status, and blocks spermatogenesis in mice. Conclusion: Inhibition of spermatogenesis by WIN 18,446 cannot be reversed by oral retinoic acid supplementation. Significance: WIN 18,446 is a useful tool to study retinoid metabolism and spermatogenesis in vivo. Knowledge of the regulation of testicular retinoic acid synthesis is crucial for understanding its role in spermatogenesis. Bisdichloroacetyldiamines strongly inhibit spermatogenesis. We reported previously that one of these compounds, WIN 18,446, potently inhibited spermatogenesis in rabbits by inhibiting retinoic acid synthesis. To understand how WIN 18,446 inhibits retinoic acid synthesis, we characterized its effects on human retinal dehydrogenase ALDH1A2 in vitro as well as its effects on retinoid metabolism in vivo using mice. WIN 18,446 strongly and irreversibly inhibited ALDH1A2 in vitro. In vivo, WIN 18,446 treatment completely abolished spermatogenesis after 4 weeks of treatment and modestly reduced adiposity in mice fed a chow diet. Effects of WIN 18,446 on retinoid concentrations were tissue-dependent. Although lung and liver retinyl ester concentrations were lower in WIN 18,446-treated animals, adipose retinyl ester levels were increased following the treatment. Interestingly, animals treated with WIN 18,446 had significantly higher circulating retinol concentrations compared with control mice. The effect on spermatogenesis by WIN 18,446 was not prevented by simultaneous treatment with retinoic acid, whereas effects on other tissues were partially or completely reversed. Cessation of WIN 18,446 treatment for 4 weeks reversed most retinoid-related phenotypes except for inhibition of spermatogenesis. Our data suggest that WIN 18,446 may be a useful model of systemic acquired retinoic acid deficiency. Given the effects observed in our study, inhibition of retinoic acid biosynthesis may have relevance for the treatment of obesity and in the development of novel male contraceptives.

Crosspresentation by nonhematopoietic and direct presentation by hematopoietic cells induce central tolerance to myelin basic protein

Central tolerance plays a critical role in eliminating self-reactive T cells specific for peripheral antigens. Here we show that central tolerance of MHC class I-restricted T cells specific for classic myelin basic protein (MBP), a component of the myelin sheath, is mediated by both bone marrow (BM)-derived and nonBM-derived cells. Unexpectedly, BM-derived cells induce tolerance directly by using classic MBP that they synthesize, whereas nonBM-derived cells mediate tolerance by crosspresenting classic MBP acquired from an exogenous source. Thus, tolerance to tissue-specific antigens can involve multiple cell types and mechanisms in the thymus, which may account for the limited spectrum of autoimmune syndromes observed when expression of tissue-specific antigens is impaired only in thymic epithelial cells.