Background: The concept of ‘hyperferritinemic syndrome’ has recently been proposed, suggesting high levels of ferritin as pathogenic pro-inflammatory mediator [1] Ferritin is an intracellular iron storage protein, comprising 24 subunits, heavy (H) and light (L) based on molecular weight. The H-/L subunits ratio may be different in tissues, since the ferritin enriched in L subunits (L-ferritin) and the ferritin enriched in H subunits (H-ferritin) may be observed in different tissues, according to pathophysiologic status [1].

Objectives: We aimed to assess the presence of H- and L-ferritin as well as of CD68/H-ferritin and CD68/L-ferritin in bone marrow (BM) biopsies of adult macrophage activation syndrome (MAS) patients. In the same patients, we matched the findings of BM biopsies with sera to identify the main represented subunits of ferritin. Furthermore, we evaluated effects of ferritin, L-ferritin, and H-ferritin on human monocytes, assessing pro- and anti-inflammatory cytokines, and expression of NLRP3 inflammasome. Finally, we checked the ability of monocytes, which were treated with ferritin, to stimulate or not the proliferation of peripheral blood mononuclear cells (PBMCs).

Methods: Immunofluorescence analysis was performed to investigate the tissue presence of L- and H-ferritin in BM biopsies as well as of CD68/H-ferritin and CD68/L-ferritin. Liquid chromatography mass spectrometry (LC-MS/MS) based proteomics was performed to identify L- and H-ferritin in sera proteins. Human monocytes were cultured with M-CSF for 7 days and, after that, treated with ferritin, H-ferritin, and L-ferritin at 10nM, for 120 and 240 minutes. After stimulation, IL-1β, IL-6, IL-10, IL-12, IFN-γ, TGF-β, TNF, and VEGF were assessed by RT-PCR and, in case of positive finding, evaluated by western blot. NLRP3 inflammasome was also assessed. Finally, the proliferation of PBMCs when co-cultured with ferritin-treated monocytes was tested by a specific proliferation assay.

Results: Immunofluorescence showed an increased H-ferritin expression in the BMs of MAS patients, whereas L-ferritin did not. Conversely, LC-MS/MS identified that the L-ferritin was the dominant form, after stringent probability matching. In vitro , H-Ferritin induced a significant increased expression of IL-1β, IL-6, IL-12, and TNF after 240 minutes. Ferritin also induced a significant increased expression of IL-1β, IL-6, IL-12, and TNF after 240 minutes. Effects on pro-inflammatory cytokines were more marked with H-ferritin than ferritin. Conversely, no significant effects were retrieved analysing IFN-γ, IL-10, TGF-β, and VEGF after 240 minutes, after ferritin and H-ferritin stimulation. Furthermore, both ferritin and H-ferritin induced a direct effect on NLRP3 inflammasome. Finally, monocytes, which were treated with H-ferritin, stimulated the proliferation of co-cultured PBMCs.

Conclusion: In our work, results showed the presence of H-ferritin and CD68/H-ferritin cells in BM biopsies of MAS patients, by immunofluorescence. Conversely, LC-MS/MS identified L-ferritin in sera proteins of those patients. Furthermore, pro-inflammatory effects of ferritin and, particularly, of H-ferritin on human monocytes were observed in vitro , increasing pro-inflammatory cytokines and NLRP3 inflammosome. Finally, H-ferritin-treated monocytes stimulated the proliferation of co-cultured PBMCs.

REFERENCES:

[1]Rosario C, et al. BMC Med 2013; 11:185.

Disclosure of Interests: Piero Ruscitti Grant/research support from: Pfizer, Speakers bureau: BMS, MSD, Ely Lilly, SOBI, Paola Di Benedetto Grant/research support from: Paola Di Benedetto received grant from Dompè outside this work., Onorina Berardicurti: None declared, Noemi Panzera: None declared, Federica Sensini: None declared, Paola Cipriani Grant/research support from: Actelion, Pfizer, Speakers bureau: Actelion, Pfizer, Roberto Giacomelli Grant/research support from: Actelion, Pfizer, Speakers bureau: Abbvie, Roche, Actelion, BMS, MSD, Ely Lilly, SOBI, Pfizer