BioProject

Abstract Human type 1 diabetes mellitus (T1DM) arises through autoimmune destruction of pancreatic β cells and is modeled in many respects by the lymphopenic and spontaneously diabetic BioBreeding (BB) DRlyp/lyp rat. Previously, pre-onset expression profiling of whole DRlyp/lyp pancreatic lymph nodes (PLN) revealed innate immune activity, specifically that of mast cells and eosinophils. Furthermore, we observed that pancreatic islets of DRlyp/lyp rats as well as those of diabetes-inducible BB DR+/+ rats potentially recruit innate cells through eotaxin expression. Here we determine that lifelong eotaxin expression begins before 40 days of life and localized specifically to β cells. In this report, we find PLN mast cells more abundant in DRlyp/lyp compared to related BB DR+/+ rats (2.1 +/-0.9% versus 0.9+/-0.4% of total cells, p<0.0001). DRlyp/lyp PLN mast cell gene expression profiling revealed an activated population and included significant overrepresentation of transcripts for mast cell protease 1, cationic trypsinogen, carboxypeptidase A, IL-5, and phospholipase Cγ. In the DR+/+ rat, which develops T1DM upon depletion of TREG cells, mast cells displayed gene expression consistent with the negative regulation of degranulation, including significant overrepresentation of transcripts encoding tyrosine phosphatase SHP-1, lipid phosphatase SHIP, and E3 ubiquitin ligase c-Cbl. To recapitulate negative mast cell regulation observed in the DR+/+, we treated DRlyp/lyp rats with the mast cell “stabilizer” cromolyn which significantly (p<0.05) delayed T1DM onset. These findings are consistent with a growing body of evidence in humans and animal models where a role for mast cells in the initiation and progression of autoimmune disease is emerging. Keywords: Expression profile between disease and control Overall design: Rat mast cells were isolated from freshly harvested, female d65 DR+/+ and DRlyp/lyp PLN (n=8 female animals per group) and total RNA was extracted using TRIzol Reagent (Invitrogen Life Technologies, Carlsbad CA). The GeneChip® Rat Genome 230 2.0 array was selected for this study and possesses more than 31,000 probes sets, representing more than 17,734 unique UniGenes. Purified RNA (~50ng) was amplified using the Two-Cycle cDNA Synthesis Kit (Affymetrix, 900432) and cRNA was synthesized, labeled, fragmented, and hybridized to the Rat Genome 230 2.0 array in accordance to standard Affymetrix protocols (Affymetrix, Santa Clara, CA). Each RNA pool was analyzed in duplicate from independent RNA amplifications (4 arrays total). After hybridization, arrays were washed, stained with PE-conjugated streptavidin (Molecular Probes, Eugene, OR), and scanned