key: cord-342122-certy2v8
authors: Ruscitti, Piero; Di Benedetto, Paola; Berardicurti, Onorina; Panzera, Noemi; Grazia, Nicolò; Lizzi, Anna Rita; Cipriani, Paola; Shoenfeld, Yehuda; Giacomelli, Roberto
title: Pro-inflammatory properties of H-ferritin on human macrophages, ex vivo and in vitro observations
date: 2020-07-22
journal: Sci Rep
DOI: 10.1038/s41598-020-69031-w
sha: 
doc_id: 342122
cord_uid: certy2v8

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.

Liquid chromatography mass spectrometry identification of FeL in sera of patients with AOSD and MAS. In the same patients, who underwent BM biopsies, liquid chromatography mass spectrometry (LC-MS/MS) analysis has successfully identified both FeH and FeL along with many other proteins from the assessed bands. The samples were analysed using All Taxonomy in Uniprot and also an in-house curated database containing the FeH protein sequence only. Following analysis against All Taxonomy at 95% CI probability, a mixed population of proteins was identified from each assessed band, a filtering of the data to highlight the Macrophages genes expression after stimulation with ferritin, FeH, FeL. After stimulation of macrophages with ferritin, for 120 and 240 min, respectively, the mRNA expression of FeH, FeL, IL-10, IL-12, TGF-β, VEGF and NLRP3 remained unchanged, when compared with UT cells. The ferritin significantly increased the mRNA levels of IL-1β, IL-6 and TNF-α genes, when compared with UT cells [IL-1β mRNA levels in ferritin treated cells for 240 min 0.017 (0.0011-0.94) vs IL-1β mRNA levels in UT treated cells 0.00092 (8.547e − 5 to 0.0025), p = 0.02; IL-6 mRNA levels in ferritin treated cells for 240 min 0.011 (0.0026-0.022) vs IL-6 mRNA levels in UT treated cells 0.0018 (0.00021-0.0054), p = 0.02; TNF-α mRNA levels in ferritin treated cells for 120 min 0.0027 (1.07e − 5 to 0.0084) vs TNF-α mRNA levels in UT treated cells 0.00017 (1.470e − 6 to 0.00040, p = 0.02] (Fig. 2) . Furthermore, as reported in Fig. 3 , after stimulation with FeH, for 120 and 240 min, respectively, the mRNA expression of TGF-β and VEGF remained unchanged, when compared to UT cells. Concerning FeH and FeL genes, the levels of mRNA expression significantly decreased after 120 min of FeH stimulation, when compared with UT cells, but after 240 min the levels of mRNA expressions were returned comparable to those of UT cells [FeH mRNA levels, in FeH treated cells for 120 min 0.79 (0.60-1.51) vs FeH mRNA levels, in UT treated cells 2.31 (0.87-3.73), p = 0.02; FeL mRNA levels, in FeH treated cells for 120 min 0.14 (0.069-0.31) vs FeL mRNA levels, in UT treated cells 0.87 (0.30-1.53), p = 0.008]. The FeH stimulation, for both 120 and 240 min, induced a significant increase of mRNA levels of expressions of IL-1β, IL-6, IL-10, IL-12 and TNF-α, when compared with UT cells [IL-1β mRNA levels in FeH treated cells for 120 min 0.09 (0.049-0.10); IL-1β mRNA levels in 

After stimulation of macrophages with ferritin, for 120 and 240 min, the proteins expression of IL-1β, NLRP3 and IL-12 remained unchanged when compared to UT cells (Fig. 4) . Interestingly, when macrophages were stimulated with FeH for 240 min, the levels of proteins expression of IL-1β, NLRP3 and IL-12 were significantly increased when compared with UT cells (p = 0.049) (Fig. 5 ). Finally, we tested macrophages proteins expression of IL-6 and TNF-α, after stimulation with ferritin and FeH, for 120 and 240 min, but non-significant results were obtained (data not shown).

showed that the levels of mature form of IL-1β, following stimulation with FeH for both 120 min and 240 min, were significantly increased when compared with UT cells [mature IL-1β levels in FeH treated cells for 120 min 13.73 pg/ml (13.49-13.96 ) vs mature IL-1β levels in UT cells 1.96 pg/ml (1.68-2.14); p = 0.048. Mature IL-1β levels in FeH treated cells for 240 min 24.49 pg/ml (22.83-26 .00) vs mature IL-1β levels in UT cells 1.96 pg/ml (1.68-2.14); p = 0.047] (Fig. 6A) . Similarly, IL-12p70 levels, following stimulation with FeH for both 120 min 

In this work, the results showed the presence of FeH in BM biopsies of AOSD patients complicated with MAS patients, whereas FeL was the predominant form in the sera of those. Furthermore, pro-inflammatory effects of FeH on human macrophages were observed in vitro, increasing pro-inflammatory cytokines and NLRP3. Finally, www.nature.com/scientificreports/ FeH-treated macrophages enhanced the proliferation of co-cultured PBMCs. Taking together all these results and considering that AOSD and MAS could be included in the so-called "hyperferritinaemic syndrome" 14 , our data could reinforce the hypothesis that higher levels of ferritin may not only be considered a consequence or an epiphenomenon of the inflammation, but it may actively play a role in pathogenic mechanisms of those diseases, thus enhancing the inflammatory burden. Our results showed that FeH was more represented than FeL in BM biopsies of patients with AOSD and MAS, as previously reported in affected tissues 18, 19, 23, 24 . Conversely, in sera of these patients, FeL was the predominant form, as reported by LC-MS/MS. Based on that discrepancy, we tested the inflammatory properties of ferritin, FeH, and FeL on human macrophages, and we observed that ferritin and, as particularly, FeH induced the expression of pro-inflammatory cytokines. Specifically, an increased gene expression of IL-1β, IL-6, IL-12, and TNF-α was observed. In this context, pro-inflammatory cytokines are largely overexpressed in patients with AOSD complicated with MAS [25] [26] [27] and may induce preferentially the expression of FeH, via FER2. The latter, after activation, stimulates the synthesis of FeH and the production of many pro-inflammatory cytokines, perpetuating a vicious pathogenic inflammatory circle [28] [29] [30] [31] [32] . In addition, the assessment of protein expressions showed the stimulation of macrophages with FeH induced a significant increase of IL-1β, and IL-12, when compared to UT cells, whereas ferritin and FeH did not. These discrepancies among ferritin, FeH, and FeL could be related to a different effect of these molecules on macrophages, as observed for gene expression. Furthermore, the production of mature form of IL-1β, in the extracellular compartment, was induced by FeH stimulation on macrophages. This finding could suggest a specific pathogenic link between FeH and IL-1β, which is a crucial mediator in AOSD and MAS, also because of clinical usefulness of IL-1 inhibition in those patients 33, 34 . In fact, multiple lines of evidence suggested the efficacy of IL-1 inhibition in the context of the hyperferritinaemic syndrome [35] [36] [37] . Furthermore, the lack of confirmation of protein expression of both IL-6 and TNF-α could reinforce this hypothesis. Moreover, the efficacy of IL-6 and TNF-α inhibition reported conflicting results 5, 38 . Additionally, paralleling with IL-1β, FeH induced a significant expression of NLRP3, a cytosolic innate immune signalling receptor, which is the main factor associated with the maturation and production of this cytokine 39 . Interestingly, our data could suggest a vicious cycle by FeH, as a further stimulator of NLRP3, since it could be an additional danger signal in triggering this factor. The activation of NLRP3 begins with the recognition of the danger or stressor, pathogen/damage-associated molecular patterns (PAMPs/DAMPs), by the sensor pattern recognition receptors (PRRs) 40 . Once activated, NLRP3 nucleates the assembly of an inflammasome, by interacting with an adaptor apoptosis speck-like protein (ASC), recruits and activates procaspase-1 to generate active caspase-1 and then converts the cytokine precursors pro-IL-1β into mature and biologically active IL-1β 41, 42 . After that, a series www.nature.com/scientificreports/ of inflammatory mechanisms and pyroptotic cell death are triggered 43 . Taking together all these observations and considering its involvement in AOSD 44, 45 , the direct stimulation of NLRP3 by FeH could provide further insights to the pathogenesis of these diseases, linking the typical hyperferritinemia with the production of a crucial pathogenic mediator. Additionally, FeH induced a significant expression of intracellular IL-12 as well as promoted its release in the extracellular compartment. It has been reported that IL-12 is a pro-inflammatory cytokine produced by dendritic cells, macrophages and B cells in response to microbial pathogens 46 . On this basis, we could speculate that IL-12, increased in our experimental conditions, could play a pro-inflammatory role. Interestingly, it has been shown that after over-expression of IL-12, the phenotype of M2 macrophages could be re-directed to that of M1-like macrophages 47 . Although it is presently known that functional polarization of macrophages is an over-simplified description of macrophage heterogeneity and plasticity, two classical different phenotypes of macrophages were described, considered the end-stage phenotypes of a continuum of functional states, classically activated (or inflammatory) macrophages (M1) and the other alternatively activated (or wound-healing) macrophages (M2) 48, 49 . In this context, a differential cytokine production is a key feature of polarized macrophages 49 . The M1 phenotype is typically IL-12 high and IL-10 low , whereas M2 macrophages are typically IL-10 high and IL-12 low50 . Furthermore, the stimulation of macrophages with FeH, enhanced the proliferation of co-cultured PBMCs. Taking together these results and previous observations [51] [52] [53] [54] , it could be possible to hypothesize that the stimulation with FeH could orientate the macrophages toward an M1 phenotype, suggesting the need of further studies to entirely clarify these issues. In addition, in inflammatory infiltrate of AOSD and MAS, a specific subset of macrophages was reported, displaying a specific CD68/H-ferritin phenotype expressing IL-12, which cannot be observed in normal tissues 19, 23 . Finally, considering that the protein expressions did not confirmed the gene expressions observed following stimulation with ferritin on pro-inflammatory mediators, it could be possible to attribute the pro-inflammatory effects to FeH subunits of the ferritin. In this study, the effects FeL on pro-inflammatory cytokines were also tested, but non-significant results were obtained. In fact, it has been recently reported that a compensatory increase of FeL, after deletion of FeH, could reduce the cytokine levels, the multi-organ dysfunction and the mortality in a murine model of sepsis 55 . In fact, an inhibitory action of FeL was shown on NF-kB activation, a key signalling pathway which is implicated in the pathogenesis of sepsis but also of other inflammatory diseases 55 . On the contrary, a stimulatory effect of FeH on NF-kB was described acting as a pro-inflammatory cytokine on hepatic stellate cells 56 . Additionally, it has been reported that FeH could modulate macrophage response to immune stimuli 57 . Taking together all these findings, it is possible to suggest that the pro-inflammatory effects of ferritin could be mainly attributed to FeH than FeL, suggesting also a possible therapeutic target to be investigated in future specific designed studies.

Taking together all these data, a contributory role of ferritin as a pathogenic mediator rather than being a product of inflammation could be suggested, but, additionally, ferritin is proposed to be a biomarker for the disease in early diagnosing and in monitoring the clinical response to therapies 58 . In fact, hyperferritinemia, may identify a more aggressive subset of diseases, and, as observed in cAPS and MAS, its reduction, after treatment, is associated with a lower mortality [59] [60] [61] . In addition, recent evidence from coronavirus disease 2019 (COVID-19), identified hyperferritinemia and IL-6 as predictors of poor prognosis, suggesting that the mortality of these patients is related to a hyper-inflammatory process [62] [63] [64] . This finding could thus hypothesize the inclusion of COVID-19, at least for a more severe subset of patients, in the "hyperferritinaemic syndrome", since sharing pathogenic mechanisms, clinical features, and possibly therapeutic targets.

In spite of suggesting possible pro-inflammatory properties of ferritin and particularly of FeH, our work is affected by some limitations, such as the relative low number of assessed patients, which could limit the external validity. However, it must be pointed out that AOSD and MAS are very rare diseases and it is very challenging to get matched BM biopsies and peripheral blood samples. An additional challenge is the severity of the diseases rapidly evolving into a life-threatening clinical picture, complicating even more the collection of biologic samples from affected patients. Taking together these observations, further confirmatory and mechanicistic studies are needed to fully elucidate these issues.

In conclusion, pro-inflammatory effects of FeH on human macrophages could be suggested, since it increased the expression of the pro-inflammatory cytokines and NLRP3, and enhanced the proliferation of co-cultured PBMCs. Considering these results and previous observations [51] [52] [53] , it could be possible to hypothesize that the stimulation with FeH could orientate the macrophages toward an M1 phenotype, suggesting the need of further studies to entirely clarify this issue. Taking together all these results and considering that AOSD and MAS could be included in the so-called "hyperferritinaemic syndrome", our data could reinforce the hypothesis that higher levels of ferritin may not only be considered a consequence of the inflammation, but it may actively play a role in the pathogenic mechanisms of those diseases enhancing the inflammatory burden. In addition, given that these pro-inflammatory effects could be mainly attributed to FeH, it could be also possible to speculate a possible new therapeutic target to be tested to improve the management of these patients.

Patients. Four patients with AOSD complicated with MAS were assessed at the time of diagnosis, collecting BM biopsies and sera, which were analysed at the same time. All these patients were admitted to the Rheumatology Clinic of L' Aquila University, Italy, and fulfilled the diagnostic criteria proposed by for AOSD and MAS in rheumatic diseases [20] [21] [22] . In this study, we also evaluated 2 BM biopsies derived from BM-donors, used as HCs. The local Ethics Committee approved the study (ASL1 Avezzano-Sulmona-L'Aquila, L' Aquila, Italy, protocol number 0122353/17) that was performed according to Good Clinical Practice guidelines and Declaration of Helsinki. Each patient provided informed consent for purposes of the study.

Scientific RepoRtS | (2020) 10:12232 | https://doi.org/10.1038/s41598-020-69031-w www.nature.com/scientificreports/ Histological analysis of biopsies. Four BM biopsies were evaluated, which derived from patients with AOSD complicated by MAS. The immunofluorescence analysis was performed on paraffin sections (thickness 3 µm) and antigen retrieval was carried out using target retrieval solution (DAKO, USA). Samples were stained with anti-FeH, anti-FeL antibodies (Santa Cruz Biotechnology, USA), as reported previously 18, 19 . The immunoreaction was revealed by using a secondary antibody (Alexa fluor, Sigma-Aldrich, USA). Cell nuclei were visualized using 4′,6-diamidino-2-phenylindole. The fluorescence was assessed by using an Olympus BX53 fluorescence microscope.

Enzymatic digestion. Four sera of patients with AOSD were mixed with 50 mM sodium acetate (Sigma Aldrich, Germany), Ph4.8, heated at 70 °C for 10 min and centrifugated at 15000 g for 30 min at 4 °C. Maintaining the solution at PH 5.2 (4 °C), ammonium sulfate (Sigma Aldrich, Germany) (50% of saturation) was added and the final solution was centrifuged at 15000g for 30 min at 4 °C. The final pellet was re-suspended in PBS (PH 7.0). Proteins contained in the final solution and recombinant FeH (Abcam, UK) were separated by 15% SDS-PAGE gel. The band with a molecular weight 18-25 kDa, with the same weight of recombinant FeH were collected. In-gel reduction, alkylation and digestion with trypsin were performed on the four gel bands before to a subsequent analysis by a mass spectrometry, as previously reported 65 .

LC-MS/MS. Peptides were extracted from the gel pieces, the peptides were resolved by reversed phase chromatography and the evaluate was ionised by electrospray ionisation using an Orbitrap Velos Pro (Thermo Fisher Scientific, UK) operating under Xcalibur v2.2, as previously reported 65 . The report of LC-MS/MS analysis is reported in Additional material 2.

Database searching. Raw mass spectrometry data were processed into peak list files using Proteome Dis- Monocytes isolation and differentiation. Peripheral blood monocytes were obtained from healthy donors by direct isolation using whole blood collected in 1 mM ethylenediaminetetracetic acid (EDTA) (Sigma-Aldrich, USA) and mixed with 50 µl/ml RosetteSep human monocytes enrichment cocktail (Stemcell, USA), according to the manufacturer's protocol. The derived enriched human monocytes were plated 2 × 10 5 /cm 2 in Roswell Park Memorial Institute (RPMI) 1,640 Medium (EuroClone, Europe), supplemented with 10% foetal bovine serum (FBS; Gibco, USA), 2 mmol/l l-glutamine (EuroClone, Europe) and 100 U penicillin, 1,000 U streptomycin (Biochrom AG, Germany). Additionally, these cells were cultured for 7 days with 50 ng/ml macrophage colony-stimulating factor (M-CSF) (PromoKine, Germany), changing the medium every 2 days. Cells were incubated at 37 °C in a humidified atmosphere consisting of 5% CO 2 . Purity of cells was assessed by immunofluorescence staining. Briefly, the cells were fixed with 4% paraformaldehyde (EMS, PA), incubated 20 min with protein block (DAKO, USA) and successively with anti-CD14-antibody (Invitrogen, USA) and anti-CD68 antibody (Santa Cruz Biotechnology, USA). The visualization of the anti-CD14-antibody was performed using an Alexa Fluor 488-conjugated (Invitrogen, USA) and the visualization of the anti-CD68-antibody was performed using an Alexa Fluor 555-conjugated (Invitrogen, USA). After counterstained with 4′,6-diamidino-2-phenylindole (DAPI), images were obtained using an Olympus BX53 fluorescence microscope. The number of CD14+ and CD68+ cells was counted to assess their purity. At day 0, before M-CSF stimulation CD14+ cells were 77.2% and CD68+ cells 22.8%, out of the total percentage of cells, respectively. After 7 days of stimulation with M-CSF, CD14+ cells were 15.4% and CD68+ cells 84.6%, out of the total percentage of cells, respectively (Additional Material 3).

To establish the optimal concentration of ferritin, FeH and FeL, in our system, a dose/response curve was performed, evaluating the IL-1β mRNA expression. Each experiment was performed in triplicate (data not show). The derived macrophages were cultured in 10% FBS medium, considering the untreated (UT) condition, and supplemented with selected dose of 10 nM ferritin (Mybiosource, USA), 10 nM FeH (Abcam, UK) and 10 nM FeL (Abcam, UK), for 120 and 240 min. 

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The authors thank Mrs. Federica Sensini for her technical assistance.

All authors made substantial contributions to the conception or design of the work, the acquisition and interpretation of data. All authors contributed to the critical review and revision of the manuscript and approved the final version. All the authors agreed to be accountable for all aspects of the work. P.R.: study conception and design, data interpretation, literature search, figures creation, writing, paper revision and acceptance; P.D.B.: study conception and design, data interpretation, literature search, figures creation, writing, paper revision and acceptance; O.B.: data collection, data interpretation, literature search, paper revision and acceptance; N.P.: performed experiments, data interpretation, paper revision and acceptance; N.G.: performed experiments, data interpretation, paper revision and acceptance; A.R.L.: performed experiments, data interpretation, paper revision and acceptance; P.C.: data collection, data interpretation, literature search, paper revision and acceptance; Y.S.: data interpretation, data interpretation, literature search, writing, paper revision and acceptance; R.G.: data interpretation, data interpretation, literature search, writing, paper revision and acceptance.

The authors declare no competing interests.

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