Glycoxidated ferritin induces the release of microparticles positive for Toll-like receptors derived from peripheral blood CD14+ cells

Abstract

Both increased serum ferritin levels and Toll-like receptor (TLR) activation show independent association with the inflammatory processes. During inflammation, cell activation and apoptosis are accompanied by the release of membranederived microparticles (MPs), which are considered to be mediators of intercellular communication as they induce specific responses in target cells. The aim of this study was to determine whether glycated and glycoxidated ferritin induce in vitro release TLR microparticles from CD14+ peripheral blood mononuclear cells. Peripheral blood mononuclear cells were stimulated with glycated, glycoxidated and native ferritin. The release of microparticles from CD14+ cells, the presence of TLR2+ and TLR4+ on the microparticles surface and the presence of interleukins-6 and -8 (IL-6 and IL-8) inside the microparticles after stimulation were determined by flow cytometry. The role of nuclear factor κB (NF-κB) was evaluated by pretreatment of the cells with the Bay 11-7085 inhibitor. Glycated and glycoxidated ferritin induced the release of microparticles from CD14+ cells, the majority of which expressed TLR2+ and TLR4+ on their surface and contained IL-6 and IL-8. These effects were dependent on NF-κB activation. Our findings show that glycated and glycoxidated ferritin might be involved in the release of microparticles and stimulation of inflammatory responses.

Glycated ferritin increases the in vitro expression of TLR2 and TLR4 in peripheral blood CD14+ cells obtained from patients with prediabetes

“…Our recent studies have shown that glycated ferritin serves as a ligand for TLR and that it promotes its in vitro expression in PBMC-derived CD14+ cells from normoglycemic subjects [19][20]. Results from the present study support the finding that glycated ferritin promotes the increased expression of TLR2 and TLR4 in CD14+ PBMC cells from subjects with prediabetes to a significantly greater extent than in cells from normoglycemic subjects.…”

“…Activation of NF-κB is the canonical downstream event of the TLR-ligand interaction [8]. In a previous report, we showed that glycated ferritin induces the expression of TLR2, TLR4 and cytokines in PBMC CD14+ cells through differentiation of the myeloid primary response 88 (MyD88)/NF-κB pathway [20]. Although we did not study the independent signaling of MyD88, studies with glycated proteins have shown that NF-κB is activated through the MyD88-dependent pathway [18,37,38].…”

“…To obtain glycated ferritin, 1 mg of native ferritin was dissolved in 1 mL of 20 mmol/L phosphate buffered saline (PBS), pH 7.4, and was then incubated in a solution of 500 mL of 20 mmol/L PBS, pH 7.4 with 0.4 M D-(+)-glucose, containing 1 mmol/L EDTA, 0.1 mg/mL chloramphenicol and 0.02% NaN 3, pH 7.4 at 37°C for 7 days under a nitrogen atmosphere. Glycated ferritin was incubated with 1.3 g/500 mL NaBH 4 for 1 h at 4°C and dialyzed against 20 mmol/L PBS, pH 7.4 containing 0.1 mmol/L EDTA for 24 h to remove free glucose [19][20]. Finally, the concentration of glycated ferritin was estimated with the Bradford technique [29] and the protein modification was evaluated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE) [30].…”

“…Recently, we have been able to show that ferritin undergoes in vitro glycation and promotes both the expression of TLR2 and TLR4 as well as the activation of NF-κB in PBMC-derived CD14+ cells in normoglycemic subjects [19][20]. In the present study, we compared the expression of TLR2, TLR4, IL-6 and IL-8 in PBMC-derived CD14+ cells from subjects with prediabetes and with a high level of serum ferritin to that found in cells from normoglycemic subjects both at baseline and after stimulation of the cells with glycated ferritin.…”

Vascular Cell Adhesion Molecule 1 and Adiponectin Receptor 1 in Plasma-Derived Extracellular Vesicles of Type 2 Diabetes Patients

“…The flow cytometry analysis (FACS) of the vesicles was executed using calibrated fluorescent latex beads of 1.0 and 0.1 μm diameter to select the capture area of the EVs. 14 20,000 Annexin V+ events were evaluated in the 0.1 -1.0 μm area per assay using a FACSCalibur™ cytometer from Becton Dickinson (San Diego, CA) and the Summit Software Informer ® program (Figure 1).…”