Abstract Body: Background In diabetes, the accumulation of advanced glycation end-products (AGEs), resulting from the nonenzymatic glycation of proteins, is amplified. We hypothesized that the receptor for AGEs (RAGE) ligand carboxymethyllysine (CML)-AGE induces trained immunity in macrophages, contributing to diabetic complications.
Methods Bone marrow-derived macrophages (BMDMs) from Wild Type (WT) C57BL/6J mice were exposed to CML-AGE (25 µg/ml) for 24 hours (h), followed by 48h washout without CML-AGE. Subsequently, CML-AGE treated BMDMs were re-stimulated with LPS (100 ng/ml) or CML-AGE (25 µg/ml) vs vehicle for 24h and subjected to molecular analysis.
Results In CML-AGE trained BMDMs from WT mice, significant upregulation of pro-inflammatory IL1b, IL6, and TNFα mRNA, along with downregulation of anti-inflammatory IL10, was observed in response to LPS or to CML-AGE, but not in Ager, Diaph1, or Clf1 deleted BMDMs. Seahorse Analyzer studies revealed a metabolic shift with increased reliance on glycolysis in CML-AGE-trained LysmHomo vs Agerfl/fl LysmHomo and Diaph1fl/fl LysmHet and Coffl/fl LysmHet macrophages.
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 effectively blocked the development of CML-AGE induced trained immunity. CML-AGE stimulation or training in control but not Ager or Diaph1 or CFl1 devoid BMDMs led to a decrease in Mito-ER distance, while increasing the interaction of DIAPH1 with Mitofusin 2, recently identified as a mediator of mitochondrial dysfunction. Disruption of actin dynamics with latrunculin B inhibited CML-AGE induced training in BMDMs as did myeloid deletion of Cfl1. Additionally, acidic extracellular pH conditions support the induction of trained immunity by CML-AGE in BMDMs. Buffering the extracellular pH prevented these cytokine changes, indicating the role of the microenvironment in modulating the training response. Histone analysis revealed a significant increase in euchromatin distribution in CML-AGE trained BMDMs vs control groups.
Conclusions CML-AGE induces trained immunity in macrophages via Ager, Diaph1, and CFL1 and underscore the interplay between mitochondrial dynamics and the extracellular environment.