Scientific Abstract

Proposal No. IBD-0017
Principal Investigator:  Lawrence J. Saubermann, M.D.
Applicant Organization:  Boston Medical Center (Massachusetts, U.S.A.)
Project Title:  Immune modulation effects of PPARgamma in inflammatory bowel disease
Period of Award:  August 1, 2002 - July 31, 2004

Chronic inflammatory bowel diseases (IBD), including Crohn’s disease and ulcerative colitis, are felt to represent an abnormal host immune response to luminal bacteria in the genetically susceptible individual.  Current therapies are directed at reducing the inflammation and restoring a normal immunologic homeostasis.  Recently, peroxisome proliferator-activated receptor-gamma (PPARγ), a nuclear transcription factor that is highly expressed in colonic tissues, has been shown to have important anti-inflammatory effects following activation.  These effects include inhibition of relevant cytokines and chemoattractants.  In association with these effects, our preliminary in vitro investigations using stimulated human intestinal epithelial cells has indicated that PPARγ activation significantly reduces T helper 1 (Th1)-associated chemokines, CXCL10 (IP-10) and CXCL9 (Mig).  Furthermore, PPARγ activation also appears to reduce expression of the corresponding chemokine receptor, CXCR3 on human-derived monocytes.  This information combined with prior animal investigations into PPARγ's effects on intestinal inflammation, indicate an important and potentially novel use of PPARγ activation in reducing Th1-associated inflammation, such as occurs in Crohn’s disease. Therefore, we plan to formally investigate the in vitro and ex vivo potential of PPARγ activation to inhibit chemotaxis in the setting of intestinal inflammation.

We plan to examine the effects of PPARγ activation, and activation of its heterodimeric binding partner, RXRα, on human intestinal epithelial cell chemokines, human intestinal lamina propria T lymphocytes, and the functional chemotactic migration of the T lymphocytes from subjects with inflammatory bowel disease as compared to normal controls.  In the first series of experiments, Th1 and Th2 inflammatory stimuli will be administered separately to HT-29 and Caco2 epithelial cells, in the presence and absence of PPARg/RXRa activation.  Chemokine expression analysis will be accomplished using RT-PCR to evaluate cellular messenger RNA levels and ELISA measurements of secreted proteins. Northern blot or RNA protection assays will be used to confirm RT-PCR results if needed. The second set of experiments will examine the effects of PPARγ/RXRα activation on chemokine receptor expression and the ability to recruit Th1 or Th2 lymphocytes to sites of inflammation.  This will be accomplished through the isolation of intestinal and peripheral blood T lymphocytes from individuals with and without IBD.  Comparison of chemokine receptor expression will be done through the use of RT-PCR and flow cytometric analysis.  The third goal of this investigation is aimed at ex vivo testing of the functional effects of PPARγ/RXRα activation on the chemotactic potential of intestinal and peripheral blood T cells from individuals with IBD in comparison to normal controls.  Standard chemotactic assays will be performed using the supernatants from the stimulated epithelial cells as chemoattractants and compared to control chemokines.

By these investigations on human intestinal cell lines and lymphocytes, the potential effects of PPARγ/RXRα activation on modulating the immunologic response to chronic intestinal inflammation will be further understood.  The promising preliminary indications that activation of this nuclear transcription factor complex could reduce a pro-inflammatory Th1 response has direct relevance to inflammation associated with Crohn’s disease.  The information from these studies will aid in directing further clinical studies in IBD using these widely available activators of these nuclear transcription factors.