Matthew H. Myles

Analysis of Gene Expression in Ceca of Helicobacter hepaticus-Infected A/JCr Mice before and after Development of Typhlitis

ABSTRACT The inflammatory bowel diseases, Crohns disease and ulcerative colitis, are chronic inflammatory disorders of the gastrointestinal tract. The causes of these diseases remain unknown; however, prevailing theories suggest that chronic intestinal inflammation results from a dysregulated immune response to ubiquitous bacterial antigens. While a substantial body of data has been amassed describing the role of the adaptive immune system in perpetuating and sustaining inflammation, very little is known about the early signals, prior to the development of inflammation, that initiate and direct the abnormal immune response. To this end, we characterized the gene expression profile of A/JCr mice with Helicobacter hepaticus-induced typhlitis at month 1 of infection, prior to the onset of histologic disease, and month 3 of infection, after chronic inflammation is fully established. Analysis of the gene expression in ceca of H. hepaticus infected mice revealed 25 up-regulated and 3 down-regulated genes in the month-1 postinoculation group and 31 up-regulated and 2 down-regulated genes in the month-3 postinoculation group. Among these was a subset of immune-related genes, including interferon-inducible protein 10, monokine induced by gamma interferon, macrophage-induced protein 1 alpha, and serum amyloid A1. Semiquantitative real-time reverse transcriptase PCR confirmed the increased expression levels of these genes, as well as elevated expression of gamma interferon. To our knowledge, this is the first report profiling cecal gene expression in H. hepaticus-infected A/JCr mice. The findings of altered gene expression prior to the development of any features of pathology and the ensuing chronic disease course make this an attractive model for studying early host response to microbe-induced inflammatory bowel disease.

Characterization of cecal gene expression in a differentially susceptible mouse model of bacterial-induced inflammatory bowel disease

Background: A/JCr mice develop typhlitis in response to Helicobacter hepaticus infection, whereas C57BL/6 mice coexist with this bacterium in a “commensal” relationship and do not develop disease even during prolonged colonization. Methods: To determine mechanisms that control this balance between responsiveness and nonresponsiveness, the mucosal response of A/JCr and C57BL/6 mice to acute H. hepaticus colonization was evaluated using genome‐wide profiling. Transcription levels for a subset of gene discoveries were then evaluated longitudinally by semiquantitative real‐time reverse‐transcriptase polymerase chain reaction (RT‐PCR) to identify changes in gene expression that occur during progression from the acute to chronic phase of colonization. To determine whether chronic mucosal inflammation in A/JCr mice was mediated through a Th1 mechanism, as was inferred from the gene expression data, mice with typhlitis were treated with neutralizing antibody targeting IL‐12/23p40 or IFN‐gamma and the response to treatment was determined by cecal lesion severity and transcription of disease‐related genes. Results: A/JCr mice had a biphasic expression of proinflammatory genes that corresponded with the acute and chronic phases of disease. In contrast, C57BL/6 mice exhibited a less robust acute transcriptional response that waned by day 30 postinoculation. Sustained upregulation of proinflammatory signals and responsiveness to anti‐IL‐12/23p40 and anti‐IFN‐&ggr; antibody suggests that inflammation in A/JCr mice was mediated through a Th1 mechanism. Prolonged upregulation of SOCS3 during the acute response to colonization suggests that C57BL/6 mice maintain mucosal homeostasis, at least in part by attenuating responsiveness to cytokine signaling. Conclusions: Collectively, these findings provide a foundation for understanding the immunological mechanisms that confer resistance or susceptibility to H. hepaticus‐induced typhlitis. (Inflamm Bowel Dis 2007)

The Role of Estrogen Signaling in a Mouse Model of Inflammatory Bowel Disease: A Helicobacter Hepaticus Model

The pathogenesis of inflammatory bowel diseases (IBD), Crohns disease and ulcerative colitis, is due in part to interactions between the immune system, genetics, the environment, and endogenous microbiota. Gonadal sex hormones (GSH), such as estrogen, are thought to be involved in the development of IBD as variations in disease severity occur during pregnancy, menopause, or oral contraceptives use. In certain strains of mice, infection with Helicobacter hepaticus triggers IBD-like mucosal inflammation that is more severe in female mice than in males, suggesting a role for GSH in this model. To determine the role of estrogen signaling in microbiota-induced intestinal inflammation, estrogen receptor (ER) α and β knock-out (KO) mice, ER agonists, and adoptive transfers were utilized. We demonstrate that, when signaling is limited to ERβ on a non-CD4+ cell subset, disease is less severe and this correlates with decreased expression of pro-inflammatory mediators.

Deficient degradation of homotrimeric type I collagen,α1(I)3 glomerulopathy in oim mice

Col1a2-deficient (oim) mice synthesize homotrimeric type I collagen due to nonfunctional proα2(I) collagen chains. Our previous studies revealed a postnatal, progressive type I collagen glomerulopathy in this mouse model, but the mechanism of the sclerotic collagen accumulation within the renal mesangium remains unclear. The recent demonstration of the resistance of homotrimeric type I collagen to cleavage by matrix metalloproteinases (MMPs), led us to investigate the role of MMP-resistance in the glomerulosclerosis of Col1a2-deficient mice. We measured the pre- and post-translational expression of type I collagen and MMPs in glomeruli from heterozygous and homozygous animals. Both the heterotrimeric and homotrimeric isotypes of type I collagen were equally present in whole kidneys of heterozygous mice by immunohistochemistry and biochemical analysis, but the sclerotic glomerular collagen was at least 95-98% homotrimeric, suggesting homotrimeric type I collagen is the pathogenic isotype of type I collagen in glomerular disease. Although steady-state MMP and Col1a1 mRNA levels increased with the disease progression, we found these changes to be a secondary response to the deficient clearance of MMP-resistant homotrimers. Increased renal MMP expression was not sufficient to prevent homotrimeric type I collagen accumulation.

A Naturally Transmitted Epitheliotropic Polyomavirus Pathogenic in Immunodeficient Rats: Characterization, Transmission, and Preliminary Epidemiologic Studies.

We report the identification, pathogenesis, and transmission of a novel polyomavirus in severe combined immunodeficient F344 rats with null Prkdc and interleukin 2 receptor gamma genes. Infected rats experienced weight loss, decreased fecundity, and mortality. Large basophilic intranuclear inclusions were observed in epithelium of the respiratory tract, salivary and lacrimal glands, uterus, and prostate gland. Unbiased viral metagenomic sequencing of lesioned tissues identified a novel polyomavirus, provisionally named Rattus norvegicus polyomavirus 2 (RatPyV2), which clustered with Washington University (WU) polyomavirus in the Wuki clade of the Betapolyomavirus genus. In situ hybridization analyses and quantitative polymerase chain reaction (PCR) results demonstrated viral nucleic acids in epithelium of respiratory, glandular, and reproductive tissues. Polyomaviral disease was reproduced in Foxn1rnu nude rats cohoused with infected rats or experimentally inoculated with virus. After development of RatPyV2-specific diagnostic assays, a survey of immune-competent rats from North American research institutions revealed detection of RatPyV2 in 7 of 1,000 fecal samples by PCR and anti-RatPyV2 antibodies in 480 of 1,500 serum samples. These findings suggest widespread infection in laboratory rat populations, which may have profound implications for established models of respiratory injury. Additionally, RatPyV2 infection studies may provide an important system to investigate the pathogenesis of WU polyomavirus diseases of man.

In Vivo Tropisms and Kinetics of Rat Theilovirus Infection in Immunocompetent and Immunodeficient Rats

Rat theilovirus (RTV) is a cardiovirus related to Theilers murine encephalomyelitis virus. While RTV is a prevalent viral pathogen of rats used in biomedical research, the pathogenesis and characterization of RTV infections is not well understood. In the studies reported herein, we used immunohistochemistry to identify viral antigens in enterocytes of the small intestines of Sprague-Dawley (SD) rats. Fecal viral shedding in immunocompromised and immunocompetent rats following oral gavage with RTV1 was high for the first 2 weeks of infection with persistent shedding of high viral loads being observed in immunocompromised nude rats but not in immunocompetent rats. RTV was also detected in mesenteric lymph nodes and spleen of immunocompromised rats but not immunocompetent rats. In addition, the magnitude of serum antibody responses differed between immunocompetent rat strains with Brown Norway and SD rats having a significantly higher antibody response than CD or Fischer 344 rats. These data suggest that RTV1 has a tropism for the epithelial cells of the small intestine, immunocompetent rats have differing serum antibody responses to RTV infection, and sustained fecal shedding and extraintestinal dissemination of RTV1 occurs in rats deficient in T cell-dependent adaptive immunity. RTV infection in immunocompromised and immunocompetent rats has merit as a model for further studies of theilovirus pathogenesis following oral viral exposure.