Characterization of Innate immune receptors

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DESCRIPTION (provided by applicant): Celiac disease (Cd) is a T cell mediated disease of the small intestine. It is triggered by dietary gluten proteins from cereals and affects 1 in 130 US citizens. While Cd can lead to severe malabsorption, most patients present with minor or atypical (nondiarrheal) symptoms. Cd is associated with secondary autoimmunity, such as type 1 diabetes or autoimmune thyroiditis, and longterm Cd that has not been treated with a strict gluten free diet can lead to (gastrointestinal) malignancy. Gluten peptides that have been deamidated by the Cd autoantigen tissue transglutaminase bind strongly to HLA-DQ2 or -DQ8, the essential genetic predisposition for Cd. This results in intestinal Th1 T cell activation and mucosal destruction (adaptive immunity). Apart from triggering adaptive immunity, recent data suggest that gluten (gliadin) can also stimulate innate immunity, i.e., the immediate and relatively nonspecific immune response to common foreign antigens, such as bacterial polysaccharide or viral RNA. The variable contribution of innate immunity to gliadin could explain why only 2- 5% of those individuals that carry HLA-DQ2 or -DQ8 finally develop Cd. The responsible gliadin peptide(s) and the receptors mediating innate immunity to gluten remain to be identified. Our preliminary results show that a peptic-tryptic digest of gliadin which contains roughly 1000 different gliadin peptides triggers a marked innate immune response in human dendritic but also intestinal epithelial cells, as assessed by release of the inflammatory mediators interleukin 8 and MCP-1. Based on these data we plan to: 1. Identify the gliadin peptide(s) that drive innate immunity after their chromatographic separation by using dendritic (intestinal epithelial) as indicators and MALDI-TOF mass spectrometry. 2. Isolate and characterize the innate immune receptor(s) on these cells that trigger(s) the innate immune response to gliadin by use of affinity chromatography of labeled cell membranes on the identified immobilized stimulatory gliadin peptide(s) and MALDI-TOF mass spectrometry. Identity and function of the receptor(s) will further be confirmed by use of function blocking antibodies, siRNA and signaling pathway inhibitors. We anticipate that identification of the receptor(s) responsible for the innate immune response to gliadin will lead to a better understanding of the pathogenesis of Cd, and possibly to novel nondietary therapies to treat this common intestinal disorder. PUBLIC HEALTH RELEVANCE Celiac disease (Cd) is a common immune disease of the small intestine that affects 1 in 130 US citizens. It is triggered by ingestion of the storage proteins of wheat (glutenins and especially gliadins) and related cereals. Cd that is not treated by strict dietary gluten exclusion can lead to severe malabsorption and malignancy, and is associated with secondary autoimmunity, such as type 1 diabetes. While the adaptive (T cell mediated) immune response to gluten peptides that leads to destruction of the resorptive intestinal mucosa is well understood, there is a yet ill defined innate immune response (the immediate and relatively nonspecific reaction to foreign antigens, such as bacterial polysaccharide or viral RNA) to gliadins. The responsible gliadin peptide(s) and the receptors mediating innate immunity to gluten remain to be identified. We plan to identify the gliadin peptide(s) that drive innate immunity in human dendritic and intestinal epithelial cells and characterize their responsive innate immune receptor(s) by use of proteomic techniques, affinity chromatography and functional studies. We anticipate that identification of the receptor(s) responsible for the innate immune response to gliadin will lead to a better understanding of the pathogenesis of Cd, and possibly to novel nondietary therapies to treat this common intestinal disorder.