COX2 is a genetic susceptibility locus with respect to Crohn's disease (CD), but the nature of the link between COX2 and CD is unclear. Mice deficient in COX2 develop Crohn's disease-like inflammation when challenged with a cholate-containing diet. This project will investigate the mechanism behind the pathology in this model in order to gain insight into the etiology of CD. Pilot data implicates COX2 in both intestinal epithelial barrier integrity and the resolution of inflammation in this model, such that COX2 deficiency appears to inhibit epithelial tissue repair while also prolonging the inflammatory response. It is thus hypothesized that in the absence of COX2, a positive feedback loop develops between altered barrier integrity and prolonged inflammation, with each reinforcing the other.
Aim 1 seeks to characterize the direct effects of cholate and COX2 on barrier integrity in this model. Time course experiments, in vitro studies, and bacterial ablation will all be employed to isolate the direct effects of cholate on barrier integrity from the confounding effects of inflammation. Barrier integrity will be partially characterized through the use of in vivo translocation assays together with measurements of trans-epithelial electrical resistance within a Ussing chamber.
Aim 2 further investigates this hypothesis by studying the effect of COX2 on intestinal stem cell (ISC) proliferation and differentiation within a primary enteroid culture system. Preliminary data indicates that COX2 inhibition within crypts limits crypt expansion, while both exogenous PGE2 and macrophage conditioned media enhance expansion. The role of crypt-specific COX2 will be further investigated using crypt and stem cell- specific COX2 knock-outs. In parallel, the effect of COX2 in macrophages on ISC proliferation and differentiation will be more fully investigated using the enteroid culture system together with bot hypothesis- driven and discovery-driven LC-MS/MS techniques. Finally, Aim 3 will investigate this hypothesis by studying the effect of COX2 including macrophage COX2 on the resolution of inflammation in this model. LC-MS/MS methods for measuring a full panel of pro- inflammatory and inflammation resolving lipid signals will be developed. This LCMS panel will help determine the effect of COX2 on the kinetic balance of pro-inflammatory and inflammation resolving signals within the inflammatory microenvironment of the model. This panel, together with novel lipidomic and proteomic discovery, will also inform the investigation into the effect of macrophage COX2 on ISCs in Aim 2. Given the high morbidity associated with CD, this work will hopefully uncover targets for the amelioration or suppression of this chronic inflammatory disorder.
Crohn's disease (CD) is a chronic and relapsing inflammatory disorder of the GI tract, whose cause is not completely understood. Cyclooxygenase 2 (COX2), an important enzyme involved in lipid signaling and inflammation, has been linked to Crohn's disease in genetic studies; but the nature of the link between COX2 and CD is not well understood. The goal of this proposal is to use a novel mouse model of CD to understand the manner in which both COX2 and its signaling products produce CD-like symptoms in the presence of specific environmental triggers.
| Meriwether, David; Sulaiman, Dawoud; Wagner, Alan et al. (2016) Transintestinal transport of the anti-inflammatory drug 4F and the modulation of transintestinal cholesterol efflux. J Lipid Res 57:1175-93 |
