1215 Lipoxygenase regulates Macrophage Migration during Islet Inflammation

12/15 -Lipoxygenase regulates Macrophage Migration during Islet Inflammation Abhishek Kulkarni 1, 2, 3, Annie Pineros 2, Sara Ibrahim 2, 3, Marimar Hernadez-Perez 2, Sarah Tersey 1, Raghavendra Mirmira 1 and Ryan M. Anderson 1. Affiliations: 1. Department of Medicine, The University of Chicago, IL 60637 2. Center for Diabetes and Metabolic Disease, Indiana University School of Medicine, Indianapolis, IN 46202 3. Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN 46202

Background Pancreatic islet inflammation is a shared characteristic of type 1 and type 2 diabetes pathogenesis Macrophage inflammation infiltration exacerbates pancreatic 12/15 -Lipoxygenase (12/15 -LOX), an enzyme involved in polyunsaturated fatty acid metabolism, has also been implicated in islet inflammation Two complementary model systems were used for this study: zebrafish and mice Figure : Sources of pancreatic islet inflammation Hypothesis: 12/15 -LOX promotes macrophage migration to the islets

β-cell injury assay Methods Transwell Migration Assay Receptor expression quantification Peritoneal Macrophages (WT/12/15 -LOX KO) Upper chamber Porous membrane Bottom chamber Metronidazole (X) X X 4 hours X X X 12/15 -LOX KO Isolate peritoneal macrophages Conditioned islet media X Wild Type Migrated Macrophages X Wash, fix and stain Quantify macrophages in controls and 12/15 -LOX inhibited zebrafish NTR-β cells α-cells Macrophages Quantify macrophages Quantify expression of chemokine receptors by flow cytometry

A Control Insulin Macrophage C Control Insulin Macrophage 4 ng 12 -LOX Mo B Results E F G H Insulin Macrophage 10μM ML 355 D Insulin Macrophage Figure A-H: Inhibition of 12/15 -LOX prevents migration of macrophages in the islet in response to injured β-cells due to downregulation of CXCR 3

Conclusion and Future Direction • Inhibition of 12/15 -LOX prevents macrophage infiltration in the pancreatic islets via downregulation of CXCR 3 expression • The effect of 12/15 -LOX on macrophage migration as well as the mechanism is conserved across species • Zebrafish prove to be excellent models for studying pancreatic islet inflammation in vivo due to their optical transparency and ease of genetic manipulation • Next, the molecular mechanism of the regulation of CXCR 3 expression by 12/15 -LOX needs to be determined • In vivo murine studies as well as in vitro migration assays with human monocytes in the presence of 12/15 LOX inhibitors would further consolidate the effects of 12/15 -LOX on the cell migration in complex model systems

Proposed Model/ Summary ML 355 CXCR 3 β-cell stress Macrophage β-cells 12/15 -LOX CXCR 3 HYPERGLYCEMIA