Scientific Abstract
Proposal No. IBD-0098
Principal Investigator: James Anderson, M.D., Ph.D.
Applicant Organization: University of North Carolina at Chapel Hill (U.S.A.)
Project Title: The role of intestinal flora in transcriptional and functional control of the tight junction barrier
Period of Award: January 1, 2004 – February 28, 2005
Inflammatory bowel diseases are thought to result from an abnormal immune response to normal intestinal flora with a primary or secondary contribution from a defective epithelial barrier. Significant effort has been applied to understanding the role of the immune response as the primary defect while relatively less has focused on the barrier. Recently, progress has been made in characterizing the proteins that create and regulate a critical component of the epithelial barrier, the tight junction.
In the proposed studies, we will answer three major questions. First, does normal commensal intestinal flora regulate the tight junction and does this occur through controlling the expression profile of tight junction proteins? Second, can a single probiotic species (Lactobacillus plantarum) by itself induce changes in the barrier? Third, we will study the colitis-prone IL-10 knockout mice to document barrier changes that correlate with the progression of colitis and ask whether L. plantarum is able to prevent these changes. We will compare mice (129) that are germ-free (GF), normally-colonized specific pathogen free (SPF) and mono-associated (L. plantarum 299v) comparing mid-jejunum, ileum, cecum and colon for apparent permeability for non-charged solutes: [3H]-mannitol and graded sizes of fluorescent dextrans (reflecting the size of pro-inflammatory bacterial products), histology and freeze-fracture electron microscopy, mRNA transcript and protein profiles for the claudins and other proteins that control the barrier and immunofluorescence microscopy for tight junction proteins.
We predict that germ-free gut will show a leaky phenotype and that commensals and probiota will induce a tighter phenotype coinciding with a different claudin profile. Induction of this profile in IL-10 deficient mice will correlate with a tighter barrier and protection from colitis. These studies will provide highly novel information about regulation of the intestinal tight junction barrier by bacteria and changes occurring during colitis. With the background provided by these studies, our long-term goal will be to directly test a causal connection between the barrier and inflammation by creating a primary defect in the barrier of mice through genetic modification of tight junction proteins. These studies will contribute to the rationale for use of probiotic bacteria in the treatment of IBD and refocus research on how to enhance the intestinal barrier to prevent IBD.