Immuno-spin trapping to detect immune-derived free radicals in type 1 diabetes (TECH2P.903)

Abstract

Abstract In autoimmune Type 1 diabetes (T1D), islet-infiltrating leukocytes synthesize noxious free radicals to collectively mediate b-cell lysis and enhance diabetogenic T cell responses as a pro-inflammatory third signal. We recently demonstrated a crucial role for NADPH oxidase (NOX)-dependent superoxide in T1D, as NOX-deficient Non-obese diabetic (NOD.Ncf1m1J) mice were highly resistant to autoimmune diabetes due to an enhanced alternatively-activated M2 macrophage phenotype, diminished pro-inflammatory cytokine and Type I interferon synthesis upon Toll-like receptor stimulation, dampened Th1 cytokine responses, and blunted CD8 T cell cytotoxicity. To further define the synergy between free radicals and T1D, we employed a 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) immuno-spin trapping technique to identify the generation of free radicals upon T cell activation and infection with Coxsackie B4 (CB4) virus, a putative diabetogenic enterovirus and hypothesized that NOD.Ncf1m1J immune cells would exhibit severe reductions in DMPO adducts. In the absence of superoxide, NOD.Ncf1m1J macrophages and CD4 T cells exhibited a significant decrease in DMPO adducts upon CB4 infection and autoantigen stimulation, respectively, in contrast to wild type cells. Future studies will utilize this technique to visualize the interface between free radicals and immune cells during spontaneous T1D progression and determine if immuno-spin trapping can be used as a biomarker during spontaneous T1D progression.