PROJECT SUMMARY Type 1 diabetes (T1D) is an autoimmune disease characterized by the destruction of pancreatic b-cells, however, the mechanisms leading to b-cells destruction are not completely understood. We identified a critical role for proinflammatory lipids produced by macrophages (MΦ) in T1D onset. Such lipids were generated by activation of the Ca2+-independent phospholipase A2β (iPLA2b). As a member of the PLA2 family, iPLA2b hydrolyzes membrane phospholipids at the sn-2 position to release a fatty acid, such as arachidonic acid (AA), which can be metabolized to bioactive oxidized lipids (eicosanoids). Several of these lipids are profoundly proinflammatory and we find that they are dramatically elevated during the pre-diabetic phase, suggesting their critical contribution to T1D onset. The MΦ are among the first to infiltrate islets and they can be induced to a proinflammatory (M1) or anti-inflammatory (M2) phenotype. In T1D, MΦM1 predominate. We find that inhibition or genetic global reduction of iPLA2b decreases proinflammatory iPLA2b-derived lipids (piDLs) production by MΦ in the pre-diabetic phase, disfavors MΦM1, and reduces insulitis and T1D incidence in the non-obese spontaneous diabetes-prone (NOD) mice. Importantly, non-diabetic children at high risk for developing T1D, exhibit a similar plasma lipid signature. These findings raise the possibility that by reducing MΦ-iPLA2b, piDLs production and induction of MΦM1 can be mitigated and that this will be beneficial in preventing or delaying T1D onset. Among the piDLs are DHETEs, generated downstream of iPLA2b via CYP450/soluble epoxide hydrolase (sEH). Inflammation relief has been reported with sEH inhibition, offering a select iDL-generating target to counter T1D. We hypothesize that reducing MΦ-piDLs will disfavor MΦM1 and counter T1D development. Recent advances in the CRISPR-Cas9 field have facilitated cell-specific and select genetic modification of genes. Our findings present a novel, and not yet considered scenario, where reducing iPLA2b in MΦ using CRISPR-Cas9 can be developed into an immunotherapy to counter T1D in humans. We propose to address this under Aim 1. Assess the impact of MΦ-iDLs on T1D development. We will determine the impact of MΦ-iDLs on T1D onset and progression using NOD mice with conditional modification of iPLA2b in MΦ. Aim 2. Establish genetically-modified MF as potential immunotherapy to counter T1D development. Develop CRISPR- Cas9 protocols to generate and assess functionality of MΦ derived from NOD bone marrow monocytes with reduced iPLA2b or sEH1 expression and determine their impact on T1D incidence. Significance. Our proposal addresses two novel concepts: (1) piDLs produced by MΦ are important contributors to b-cell death in T1D and (2) manipulating lipid-generating enzymes in MΦ (a readily accessible pool) can counter T1D. We feel that exploiting these will allow generation of novel immunotherapeutic avenues to prevent or delay T1D diagnosis in ...