LAUSR

Introduction and Objective: Type 1 diabetes (T1D) is characterized by β-cell loss and insulin insufficiency. Receptor interacting protein kinase 1 (RIPK1) regulates survival and death signaling in non-islet cell types via its kinase and scaffolding functions. We observed that RIPK1 is expressed in mouse and human β-cells in situ and that Ripk1 is upregulated in aged NOD islets and β-cells from T1D donors. We hypothesized that RIPK1 kinase activity regulates cytokine-induced β-cell cytotoxicity. Methods: We treated control and Ripk1 gene-edited (Ripk1Δ) NIT-1 β-cells as well as control and RIPK1 kinase dead (RIPK1-D138N) mouse islets with TNFα+IFNγ for up to 72 hrs, then assessed gene expression, kinase signaling, caspase activity, and cell death. We also tested a RIPK1 kinase inhibitor (SZM679) and activator (BV6). Results: RNAseq revealed that TNFα- (Nfkb1, Jnk1, Casp3, Casp7) and IFNγ- (Sting1, cGas, Jak1, Stat2) related genes are differentially expressed in Ripk1Δ versus control NIT-1 β-cells. In islets, Ripk1, Jnk1, Jak1, Sting1, and Casp3 were upregulated with TNFα+IFNγ treatment, and these effects were blocked in RIPK1-D138N islets. Kinome profiling showed that TNFα- (IKBKB, ASK1/3, JNK1/2/3, MLKL) and IFNγ- (PKR, MTOR) related kinases are differentially activated in Ripk1Δ versus control NIT-1 β-cells. TNFα+IFNγ treatment increased RIPK1 phosphorylation at Ser161, caspase 3/7 activity, and cell death in NIT-1 β-cells, and these effects were blocked with SZM679 treatment or in Ripk1Δ cells. Similarly, TNFα+IFNγ treatment increased RIPK1 phosphorylation, caspase activity, and cell death in mouse islets, effects that were amplified with BV6 treatment and blocked in RIPK1-D138N islets. Conclusion: Our data indicate that RIPK1 kinase function promotes cytokine-induced β-cell cytotoxicity via actions on gene expression, kinase signaling, caspase activity, and cell death. Future studies are needed to determine whether therapies targeting RIPK1 kinase activity could protect β-cells in T1D. C.J. Contreras: None. N. Mukherjee: None. K.A. Colglazier: Employee; Eli Lilly and Company. E. Mather: None. R.C. Branco: None. E.P. Cai: None. A.T. Templin: None. National Institutes of Health (P30 DK097512 to Indiana University Center for Diabetes and Metabolic Diseases, P30 DK017047 to University of Washington Diabetes Research Center, T32 DK064466 to Indiana University Diabetes and Obesity Research Training Program); Ralph W. and Grace M. Showalter Research Trust (08065700002B); U.S. Department of Veterans Affairs (IK2 BX004659, I01 BX001060)