The CRTH2 receptor in experimental ulcerative colitis

The etiology and pathogenesis of inflammatory bowel disease (IBD), such as Crohn`s disease and ulcerative colitis (UC) is poorly understood. Despite multiple therapeutic options, treatment for IBDs remains unsatisfactory, calling for new and more effective pharmacological targets. In the present project proposal, we will investigate the unknown role of a newly described prostaglandin D2 receptor, CRTH2 (chemoattractant receptor-homologous molecule expressed on T-Helper 2 cells) in the immunopathogenesis of UC. CRTH2 has already been shown to act as a key molecule in the chemoattraction of eosinophils and TH2 lymphocytes during allergic inflammation. The involvement of CRTH2 in the pathogenesis of UC will be tested in DSS (dextran sulfate sodium)-induced colitis, a well-established mouse model of UC. Preliminary studies indicate that the colonic submucosa is densely infiltrated by CRTH2-expressing leukocytes during DSS-induced colitis. In Aim 1 we will therefore focus on infiltrated CRTH2-expressing leukocytes in the colon submucosa and identify their nature by multiple-labeling immunofluorescence and flow cytometry. The extent of infiltration will be assessed by quantitative image analysis of colon sections. In Aim 2, we will switch our focus to the ligands of CRTH2, which include prostaglandin D2 (PGD2), its stable metabolites and 11-dehydro thromboxane B2 (TXB2), a stable metabolite of TXB2. Particularly, these stable metabolites have been shown to act as potent mediators of leukocyte attraction acting via CRTH2. We will measure plasma levels of PGD2 and metabolites by mass spectrometry (LC-MS) during the course of the disease and will identify their cellular source by immunohistochemical detection of their synthesizing enzymes. In Aim 3, the question of the cellular source of the CRTH2 ligands in the colon will be addressed in more detail. Here, we will focus on the enteric nervous system (ENS) and test the hypothesis that the ENS, which expands like a network throughout the entire gut, is a source of release for these CRTH2 ligands during inflammation. In Aim 4, we will pharmacologically inhibit the CRTH2 receptor and the synthesizing enzymes of CRTH2 ligands in in vivo experiments and investigate the effects of these treatments on colitis pathology and disease progress. Altogether, the present proposal intends to unravel the unknown role of the CRTH2 receptor in the pathogenesis of UC and explores the possibility whether CRTH2 could become a new and urgently needed pharmacological target in the treatment of ulcerative colitis. In addition, we will elucidate the little known function of the ENS as a source of chemoattractants for leukocytes during inflammation, which will help to clarify neuro-immune interactions in GI diseases and the completely unknown pathogenesis of enteric neuropathies.

In this project, the role of a new prostaglandin receptor, named CRTH2, was investigated in inflammatory bowel diseases (IBDs). Prostaglandins are lipid signaling molecules that play an important role in many inflammatory processes of the body. Each of the prostaglandins signal through different receptors whose intracellular response may cause common but also divergent mechanisms. For instance, prostaglandin D2 signals via two separate receptors, called DP and CRTH2. We hypothesized in our project that these two receptors would have different functions. In particular, we investigated whether the CRTH2 receptor was involved in driving intestinal inflammation and would function differently to DP, a receptor with anti- inflammatory properties. Indeed, we could show that the CRTH2 receptor plays a different role in intestinal inflammation than DP. Firstly, we investigated the presence of the receptors on human white blood cells and observed that the number of the receptors correlated with the disease severity of IBD patients in an opposing manner. Thus, in eosinophils, the amount of DP increased whereas CRTH2 diminished relative to the severity of disease. The situation was different in mucosal colonic biopsies taken from IBD patients. Here, the presence of CRTH2 correlated strongly with disease severity indicating that, in contrast to DP, CRTH2 plays a proinflammatory role in this disease. We could confirm the proinflammatory role of CRTH2 in experimental models of intestinal inflammation by applying specific pharmacological antagonists. Histochemical data showed us that the CRTH2 was present in white blood cells and also in the mucosal lining of the large intestine. Since different forms of IBD are hard to distinguish, we investigated serum and urine from IBD patients and compared them with samples from healthy matching individuals. In cooperative work, we measured a set of small molecule metabolites, such as amino acids, and could show that IBD patients could be distinguished from healthy individuals by a certain metabolite profile. IBDs are a massive social burden for both the disease-affected and the society, a new treatment is therefore urgently wanted. We believe that the results from our project have strong impact on the pharmacological and medical field. By investigating this receptor, we have created a new target for pharmacological intervention that could be quickly translated into clinical practice, as CRTH2 inhibitors are already clinically available. Finally, we have shown that IBDs can be distinguished by a specific profile of small molecules in blood and urine. This set of molecules could serve as biomarkers to control side effects in IBD therapy and allow better diagnoses and therapeutic decision-making.