Abstract 2137: Ligands Of The Receptor For Advanced Glycation End Products Induce Trained Immunity In Mouse Bone Marrow-Derived Macrophages

Background: The bone marrow undergoes a process of "training," and glucose-related mechanisms play a role in the accelerated development of atherosclerosis in diabetic mouse models. In diabetes, the accumulation of advanced glycation end-products (AGEs), resulting from the nonenzymatic glycation of proteins, is observed. We hypothesized that the receptor for AGEs (RAGE) ligand carboxymethyllysine (CML) induces trained immunity in macrophages, contributing to the pathogenesis of diabetic complications. Methods: Bone marrow-derived macrophages (BMDMs) from Wild Type (WT) C57BL/6J mice were exposed to CML-AGE for 24 hrs, followed by a 48-hr washout period without CML-AGE. Subsequently, BMDMs were stimulated with LPS or CML-AGE vs. vehicle for 24 hrs. The roles of RAGE and Diaphanous 1 (DIAPH1) were explored using Agerfl/fl Lysm Cre+/+, control Lysm Cre+/+, Diaph1fl/fl Lysm Cre+/- and control Lysm Cre+/- mice. Results: In CML-AGE-trained BMDMs from WT mice, significant upregulation of pro-inflammatory IL1b, IL6, and TNFα, and downregulation of anti-inflammatory IL10, was observed, but not in Ager-deleted BMDMs. Seahorse Analyzer studies revealed an increased reliance on glycolysis in CML-AGE-trained Lysm Cre+/+ vs. Agerfl/fl Lysm Cre+/+ and Lysm Cre+/- vs. Diaph1fl/fl Lysm Cre+/- macrophages In vivo, CML-AGE induced trained immunity in BMDMs and splenic monocytes isolated from Ager- and -Diaph1-expressing mice but not those devoid of Ager or Diaph1. Western blotting indicated that CML-AGE training induced upregulation of H3K27ac (histone acetylation marks) and H3K4me3 (histone methylation marks). The non-selective methylation inhibitor MTA blocked the development of CML-AGE-induced trained immunity, with reduced Il6 and Tnfa expression. CML-AGE stimulation or training in control but not Ager- or Diaph1-devoid BMDMs led to a decrease in Mito-ER distance; cristae number, volume density, and area; while increasing the interaction of DIAPH1 with Mitofusin 2 (MFN2), which was recently identified as a mediator of mitochondrial dysfunction. Conclusions: CML-AGE induces trained immunity through modulation of cellular metabolism, epigenetic marks, and mitochondrial dysfunction, ultimately activating proinflammatory gene expression