IFN-y plays an important role in the generation and perpetuation of mucosal inflammation in many animal models of inflammatory bowel disease (IBD) and is central to the induction and perpetuation of Crohn's disease (CD). Cytokines indicative of Th1 polarization (IL-12, IL-18, TL1A, IFN-y) are elevated in inflamed murine and human mucosa. CD patients treated with anti-IFN-y MAb showed marked clinical improvement (decreased CDAI) and reduction in CRP. We have discovered IFNG regulatory region haplotypes positively and negatively associated with inflammatory bowel disease (IBD), further stressing the importance of studying altered IFN-y regulation in CD pathogenesis. Global suppression of IFN-y as treatment for IBD may have serious drawbacks, because Th1 cytokines are essential for protection against several classes of pathogens, and are central to maintaining the appropriate balance of immune responses. Our overall goal, therefore, is to determine whether IFN-y expression might be selectively suppressed in the mucosa by identifying mucosa-specific molecular mechanisms involved in regulation of IFNG expression as potential targets of intervention. We established the existence of mucosa-specific IFNG promoter elements by identifying two regions with enhancer activity that was mucosa-specific. Such elements might be targeted in several ways to achieve selective attenuation of mucosal IFN-y production. We have demonstrated that this strategy can work, in principle, because transfection of discrete IFNG promoter elements into both peripheral and lamina propria T cells attenuates IFN-y protein production. Furthermore, two epigenetic molecular mechanisms, histone acetylation and IFNG methylation, have recently been reported to play critical roles in the regulation of IFNG transcription. We have shown that mucosal T cells exhibit altered histone acetylation and methylation of the IFNG promoter, compared to peripheral T cells. These data provide the foundation of our continuation proposal, based on the hypothesis that there are mucosa-specific molecular mechanisms, both cis-regulatory and epigenetic, which specifically control mucosal Tcell IFN-yproduction and which may present opportunities for regionally directed attenuation of IFN-y expression. Our studies are designed to identify IFNG promoter sequence targets and to develop molecular reagents that attenuate mucosal, without eliminating systemic, IFN-y expression. Our hypothesis will be addressed by studies described in the following Specific Aims: 1) Use fine promoter analysis of two defined IFNG regions to characterize mucosa specific cis-regulation. 2) Define mucosa specific epigenetic mechanisms of Tcell IFNG regulation. 3) Design competitive oligonucleotide silencing and RNA interference modalities targeted to regulatory regions and methylation sites identified from Aims 1 and 2, to attenuate in vitro mucosal IFNG transcription. Understanding regulation of mucosal cytokine production in IBD could identify opportunities for therapeutic development and potentially prevention for these diseases which affect as many as 1 million Americans.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37DK043211-21
Application #
8249360
Study Section
Gastrointestinal Mucosal Pathobiology Study Section (GMPB)
Program Officer
Hamilton, Frank A
Project Start
1991-09-20
Project End
2016-02-29
Budget Start
2012-03-01
Budget End
2013-02-28
Support Year
21
Fiscal Year
2012
Total Cost
$380,727
Indirect Cost
$152,747
Name
Cedars-Sinai Medical Center
Department
Type
DUNS #
075307785
City
Los Angeles
State
CA
Country
United States
Zip Code
90048
Gonsky, Rivkah; Deem, Richard L; Landers, Carol J et al. (2014) IFNG rs1861494 polymorphism is associated with IBD disease severity and functional changes in both IFNG methylation and protein secretion. Inflamm Bowel Dis 20:1794-801
Gonsky, Rivkah; Deem, Richard L; Targan, Stephan R (2013) Multiple activating and repressive cis-promoter regions regulate TNFSF15 expression in human primary mononuclear cells. Cytokine 63:36-42
Gonsky, Rivkah; Deem, Richard L; Landers, Carol J et al. (2011) Distinct IFNG methylation in a subset of ulcerative colitis patients based on reactivity to microbial antigens. Inflamm Bowel Dis 17:171-8
Gonsky, Rivkah; Deem, Richard L; Targan, Stephan R (2009) Distinct Methylation of IFNG in the Gut. J Interferon Cytokine Res 29:407-14