Regulation of the CX3CR1/CX3CL1 signalling axis by diabetes-induced methylglyoxal accumulation promotes monocyte activation

Purpose: Diabetes mellitus is a major risk factor for cardiovascular disease, which remains the leading cause of death. Diabetic patients have significantly accelerated atherosclerosis, making them more susceptible to serious consequences such as a myocardial infarction. Monocytes are known to be essential for the development and progression of an atherosclerotic plaque. However, the mechanism by which the diabetic milieu influences atherosclerosis development is still not understood and is the subject of the present work.

Methods: Patients with type 2 diabetes mellitus (T2DM) and healthy individuals were recruited to isolate primary human monoyctes by negative immunological magnetic isolation. For mouse studies, leptin-receptor deficient mice and wild-type controls were used. Mice monocytes were isolated from the bone marrow. FACS and Western Blot were used to analyse protein expression. CX3CL1/fractalkine, was used to analyse CX3CR1-dependent chemotaxis and adhesion of monocytes. Monocyte migration behaviour was investigated using transwell migration assays and the results were documented and analysed by Invitrogen Cell Counter.

Results: In primary human monocytes isolated from T2DM patients, we observed a significant upregulation of CX3CR1 – an important adhesion molecule for monocyte attraction towards atherosclerotic plaque – compared to healthy inviduals. These findings were confirmed in monocytes isolated from diabetic mice. Interestingly, we identified methylglyoxal (MG), a by-product of  glycolysis responsible for numerous protein modifications, as the main component of the diabetic milieu responsible for CX3CR1 upregulation. Primary human monocytes incubated with pathologically relevant concentrations of MG for 24 hours  revealed significantly increased surface expression of CX3CR1 compared to unstimulated controls.
To test the functional relevance, fluorescence-stained monocytes with and without MG stimulation were allowed to bind to immobilised  CX3CL1, and the CX3CL1-bound monocytes were measured. After exposure a significantly increased adhesion of MG-stimulated monocytes was observed.
Excitingly, methyglyoxal-stimulated monocytes and monocytes isolated from diabetic patients also exhibited a pro-migratory phenotype, as  confirmed by increased chemokinesis in a transwell migration assay. These findings were further validated by a chemotaxis experiment investigating migration towards CX3CL1. Stimulation with fractalkine resulted in enhanced monocyte migration. This effect was further augmented by prior incubation with MG through augmented CX3CR1 expression. This pro-migratory phenotype could be traced at the level of  signalling pathways, where an increased phosphorylation of Akt, a serine/threonine kinase important for migration control, could be detected by methylglyoxal incubation. Interestingly, this effect was further enhanced by CX3CL1 ligation.

Conclusions: The present study reveals a novel mechanism of diabetes-induced monocyte activation. MG was identified as the key metabolite of the diabetic milieu leading to the upregulation of CX3CR1. This induces a pro-migratory and pro-adhesive phenotype of monocytes, which is critical  for monocyte adhesion and transmigration and may contribute to enhanced monocyte recruitment into atherosclerotic plaque regions. The results thus provide a rationale for detoxification of methylglyoxal accumulation in diabetes as a potential therapeutic approach to attenuate monocyte activation.