Kristiaan Wouters^1, Xiaodi Zhang¹, Jean L Scheijen¹, Philippe Vangrieken¹, Jaycey Kelly¹, Thom E Kusters¹, Julia IP van Heck², Rinke Stienstra^{2, 3} , Casper G Schalkwijk¹.

¹ Dept. Internal Medicine, CARIM School for Cardiovascular Diseases, MUMC+, Maastricht, The Netherlands ² Dept. Internal Medicine, Radboud University Medical Centre, Nijmegen, The Netherlands. ³ Division of Human Nutrition and Health, Wageningen University, Wageningen, Netherlands. kristiaan.wouters@maastrichtuniversity.nl

Background: Immune cell activation leads to increased glycolysis. During glycolysis, the reactive dicarbonyl methylglyoxal (MGO) is formed, which may activate immune cells. We investigated whether MGO affects immune cell recruitment, inflammation, and trained immunity.

Methods: C57BL/6J mice received a MGO injection (25 µg or 1 mg), and were euthanized within 24h, or received 50 mM MGO in drinking water for 12 weeks. Bone marrow was isolated and cultured for 7 days into bone marrow derived macrophages (BMDMs), and stimulated with LPS. Inflammation was assessed with flow cytometry, qPCR and nitric oxide (NO) assays. Human monocytes were trained with β-glucan with or without aminoguanidine or MGO. After 6 days, cells were restimulated with LPS, and assessed with ELISA. MGO was quantified with LC-MS/MS.

Results: Neither short-term MGO spikes nor long-term exposure to MGO affected immune cell recruitment, plasma cytokines or hepatic inflammation. A single high-dose MGO injection enhanced LPS-induced NO production (from 7.0±1.2 to 10.3±0.84 µM; p<0.05 ) and inflammatory gene expression (fold changes for IL1b: 4668 ± 252 to 13062 ± 4943, TNF: 20 ± 5.8 to 51.7 ± 9.1, iNOS: 1140 ± 118 to 8612 ± 1598, for all p<0.05) in BMDMs from these mice, suggesting trained immunity induction. In primary human monocytes, β-glucan-induced innate training enhanced MGO formation (313.1 ± 71.8 to 549.1 ± 16.9 nmol/gram protein). Moreover, β-glucan-induced trained immunity response for TNF (2.4± 0.8 to 1.0±0.3 ng/mL, p<0.05) was blunted by the MGO scavenger aminoguanidine, while it was enhanced by adding additional MGO (0.8±0.06 to 1.2± 0.02ng/mL, p<0.05).

Conclusion: MGO is involved in trained immunity, but not in direct immune cell activation or recruitment.