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Ferritin is an iron-binding molecule, which comprises 24 subunits, heavy (FeH) and light (FeL) subunits, suggested to have a pathogenic role by the ‘hyperferritinemic syndrome’. In this work, we tested (1) FeH and FeL in bone marrow (BM) and sera in patients with macrophage activation syndrome (MAS); (2) pro-inflammatory effects of ferritin, FeL, and FeH on macrophages; (3) ability of FeH-stimulated macrophages to stimulate the proliferation of peripheral blood mononuclear cells (PBMCs); (4) production of mature IL-1β and IL-12p70 in extracellular compartments of FeH-stimulated macrophages. Immunofluorescence analysis and liquid chromatography mass spectrometry (LC–MS/MS) based proteomics were performed to identify FeL and FeH in BM and sera, respectively, in the same patients. Macrophages were stimulated with ferritin, FeH, and FeL to assess pro-inflammatory effects by RT-PCR and western blot. The proliferation of co-cultured PBMCs with FeH-stimulated macrophages was tested. Immunofluorescence showed an increased FeH expression in BMs, whereas LC–MS/MS identified that FeL was mainly represented in sera. FeH induced a significant increase of gene expressions of IL-1β, IL-6, IL-12, and TNF-α, more marked with FeH, which also stimulated NLRP3. FeH-stimulated macrophages enhanced the proliferation of PBMCs. The ELISA assays showed that mature form of IL-1β and IL-12p70 were increased, in extracellular compartments of FeH-stimulated macrophages. Our results showed FeH in BM biopsies of MAS patients, whereas, LC–MS/MS identified FeL in the sera. FeH showed pro-inflammatory effects on macrophages, stimulated NLRP3, and increased PBMCs proliferation.

Pro-inflammatory properties of H-ferritin on human macrophages, ex vivo and in vitro observations