Scientific Abstract
Proposal No. IBD-0070R
Principal Investigator: William Stenson, M.D.
Applicant Organization: Washington University (St. Louis, Missouri, U.S.A.)
Project Title: Indoleamine 2,3-dioxygenase in inflammatory bowel disease
Period of Award: October 1, 2003 - September 30, 2005
Indoleamine 2,3-dioxygenase (IDO), an intracellular heme-containing enzyme, catalyzes the initial and rate limiting steps in tryptophan degradation via the kynurenine pathway. IDO is induced during inflammation by IFN-γ and other pro-inflammatory cytokines and acts to deplete the local microenvironment of the essential amino acid, tryptophan. The resulting low levels of extracellular tryptophan act as a signal to inhibit T-cell proliferation. Moreover, kynurenine and other metabolites of tryptophan produced through the kynurenine pathway also act to inhibit T-cell activation.
Munn et al. demonstrated an essential role for placental IDO in tolerance at the maternal-fetal interface in mice. Inhibition of IDO resulted in a T-cell mediated rejection of allogeneic fetuses, whereas syngeneic fetuses were unaffected by IDO inhibition. Tolerance at the maternal-fetal interface parallels that of the gastrointestinal tract, as the context in which the foreign antigens are presented dictates the tolerogenic response of the host. In the basal state, the gut is the site of the highest levels of IDO expression. Using Affymetrix gene chips, we demonstrated that IDO was expressed in ulcerative colitis at levels 4.4 times higher than in normal colon, making IDO one of the most highly overexpressed genes in ulcerative colitis. Although IDO is highly expressed in the GI tract and is overexpressed in inflammatory bowel disease, the functional role of IDO in the GI tract has not been explored.
To address the question of whether IDO plays a regulatory role in gastrointestinal inflammation, we have studied IDO in a Th-1 mediated model of colitis, 2,4,6-5 trinitrobenzene sulfonic acid (TNBS) colitis in SJL/J mice. IDO mRNA and protein are significantly induced over baseline in the setting of TNBS colitis. In the normal mouse colon, IDO is predominately expressed in lamina propria mononuclear cells (LPMNCs), whereas in the TNBS colitis model, there is significant expression in LPMNCs, epithelial cells and neurons. We also found that administration of 1-methyltryptophan, a specific inhibitor of IDO, led to significant augmentation in the inflammatory response to TNBS. These findings suggest that IDO plays an important role in the downregulation of Th-1 responses within the gastrointestinal tract and is likely to play a similar role in human IBD.
Our goal is to complete our work on the role of IDO in TNBS colitis and to extend this investigation to human IBD. The central hypothesis of this proposal is that in Th-1 mediated colitis, including Crohn’s disease, IDO expression is induced and that the resultant IDO activity blocks lymphocyte activation and downregulates the immune response.
Our first Specific Aim is to determine the effect of the baseline upregulation of IDO in the TNBS colitis model in the mouse. To determine if upregulation of IDO expression can block the development of Th-1 colitis, we will induce IDO expression in lamina propria mononuclear cells in SJL/J mice prior to the administration of TNBS. In the first set of experiments, we will produce a transgenic mouse in which IDO is expressed in macrophages and dendritic cells. We will do this by placing the IDO coding sequence behind the MHC Class II promoter. Our expectation is that in these mice IDO will be overexpressed in macrophages and dendritic cells in the lamina propria and elsewhere and that overexpression of IDO will diminish the inflammatory response when TNBS colitis is induced. In a second set of experiments, we will upregulate IDO by administration of CTLA-4-Ig fusion protein, an agent that has been demonstrated to induce IDO expression in dendritic cells. Our expectation is that administration CTLA-4-Ig fusion protein will induce IDO expression in lamina propria dendritic cells and that administration of TNBS to mice that have received CTLA-4-Ig fusion protein will result in less severe colitis than in control mice. We will also study IDO expression in epithelial cells isolated from colons of normal and TNBS treated mice.
Our second Specific Aim is to define the expression and regulation of IDO in human IBD. We will define the cellular distribution of IDO in human IBD using immunohistochemistry. We will also isolate lamina propria mononuclear cells from IBD surgical resections and normal human colon and assay IDO expression in unfractionated and FACS fractionated LPMNCs by Western blotting and realtime PCR. We will also assess IDO activity by measuring levels of tryptophan and kynurenine in supernatants from culture lamina propria mononuclear cells and in serum from normals and patients with inflammatory bowel disease.