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

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.


Supplementary materials
Additional material 1. The effects of FeL on gene expressions of macrophages. Lane 2). LC-MS/MS analysis to determine identification was requested.

Enzymatic Digestion
In-gel reduction, alkylation and digestion with trypsin were performed on the four gel bands prior to subsequent analysis by mass spectrometry. Cysteine residues were reduced with dithiothreitol and derivatised by treatment with iodoacetamide to form stable carbamidomethyl derivatives. Trypsin digestion was carried out overnight at room temperature after initial incubation at 37oC for 2 hours.

LC-MS/MS
Peptides were extracted from the gel pieces by a series of acetonitrile and aqueous washes. The extract was pooled with the initial supernatant and lyophilised. Each sample was then resuspended in 10µL of 50mM ammonium bicarbonate and analysed by LC/MS/MS. Chromatographic separations were performed using an EASY NanoLC system (ThermoFisherScientific, UK).
Peptides were resolved by reversed phase chromatography on a 75 µm C18 column using a three step linear gradient of acetonitrile in 0.1% formic acid.
The gradient was delivered to elute the peptides at a flow rate of 300 nL/min over 120 min. The eluate was ionised by electrospray ionisation using an Orbitrap Velos Pro (ThermoFisherScientific, UK) operating under Xcalibur v2.2.
The instrument was programmed to acquire in automated data-dependent switching mode, selecting precursor ions based on their intensity for sequencing by collision-induced fragmentation using a Top20 CID method. The MS/MS analyses were conducted using collision energy profiles that were chosen based on the mass-to-charge ratio (m/z) and the charge state of the peptide.

Database Searching
Raw mass spectrometry data were processed into peak list files using Proteome Discoverer (ThermoScientific; v2.1) ( Figure B).

Figure B:
Proteome discoverer nodal workflow for raw data processing Processed raw data was searched using the Mascot search algorithm (v2.6; www.matrixscience.com) against the Uniprot database with All Taxonomy. The data was also searched against an in-house curated database containing only the H-ferritin protein of interest (Table 1).  Scaffold allows statistical filtering of the data at the protein and peptide level.

LC/MS/MS analysis has successfully identified both H-ferritin
Mascot applies a 95% probability CI in the MOWSE scoring algorithm that is an identification threshold. This threshold is calculated as described on the Matrix Science website: "Given an absolute probability that a match is random, and knowing the size of the sequence database being searched, it becomes possible to provide an objective measure of the significance of a result. A commonly accepted threshold is that an event is significant if it would be expected to occur at random with a frequency of less than 5%." Any protein that is above this identity threshold is deemed significant.
Following searching against All Taxonomy at 95% CI probability, minimum of 1 peptide and 0% CI peptide probability, 50 proteins were detected in sample PR1_1. 65 proteins were detected in PR1_2, 57 proteins detected in PR1_3 and 80 proteins detected in PR1_4.
As the sample was a mixed population of proteins identified from each band, filtering of the data to highlight the protein of interest was performed. In the Scaffold software it is possible to filter the data using terms in the search box.
In the first instance, typing 'ferritin' in to the search box highlights the proteins from the individual samples. Figure D represents the protein identification and total peptide counts for the protein in each band.        J). This suggests that the protein being detected in the pathological sample is more likely to be the light-chain form after probability matching at 95% CI however, this should not be the true of the recombinant protein sample PR1_1 as it was specifically the H-ferritin protein form. Immunofluorescence staining of CD14 and CD68 in macrophages after 7 days of M-CSF stimulation. The picture shows that the percentage of CD14+ cells is