Ferritin H can counteract inflammatory response in hybrid fish and its parental species after Aeromonas hydrophila infection.

Ferritin H can participate in the regulation of fish immunity. Tissue-specific analysis revealed that the highest expressions of Ferritin H in parental species were observed in spleen, while peaked level of Ferritin H mRNA in hybrid fish was observed in liver. In addition, A. hydrophila challenge could sharply enhance their Ferritin H mRNA expression in liver, kidney and spleen. To further investigate their roles in immune regulation, their Ferritin H fusion proteins were produced in vitro. Ferritin H fusion proteins could exhibit a direct binding activity to A. hydrophila and endotoxin in a dose-dependent manner, restrict dissemination of A. hydrophila to tissues and abrogate inflammatory cascades. Moreover, treatment with Ferritin H fusion proteins could reduce A. hydrophila-induced lipid peroxidation. These results indicated that Ferritin H in hybrid fish elicited a similar immune regulation of A. hydrophila-induced inflammatory signals in comparison with those of its parents.

Antibodies specific to ferritin light chain polypeptide are frequently detected in patients with immune­related pancytopenia.

Immuno-related pancytopenia (IRP) is characterized by pancytopenia resulting from bone marrow suppression or destruction mediated by auto­antibodies. In our previous study, a K562 cDNA library was established, which was used to screen for seven possible auto­antigens produced by hematopoietic cells in patients with IRP, including ferritin light chain (FTL). In the present study, FTL was expressed and purified, and the levels of the auto­antibodies specific to FTL were measured. Through ELISA, it was shown that the titer of anti­FTL antibodies was higher in patients with IRP without treatment compared with those who had recovered from IRP, those with severe aplastic anemia (SAA), those with myelodysplastic syndrome (MDS) and the healthy controls. Furthermore, the expression levels of FTL­mRNA were upregulated in patients with IRP without treatment compared with those who had recovered from IRP, those with MDS and the normal controls. The results suggest that FTL antibody expression is upregulated in patients with IRP. Detecting FTL antibodies may therefore have certain clinical value in differentiating between IRP, SAA and MDS. Furthermore, in specific patients with IRP, FTL as an auto­antigen may induce immune attack on hematopoietic stem cells.

An Apoferritin-Hemagglutinin Conjugate Vaccine with Encapsulated Nucleoprotein Antigen Peptide from Influenza Virus Confers Enhanced Cross Protection.

Naturally occurring self-assembling ferritin nanoparticles have become widely appreciated for vaccine design. In this study, an apoferritin (AFt) nanocage was used as a carrier to construct a biomimetic influenza vaccine by encapsulating a conserved internal nucleoprotein (NP) antigen peptide inside the nanocage, followed by chemically conjugating the surface antigen hemagglutinin (HA) protein on the outer surface of the AFt. Benefiting from the excellent thermal stability and thermallyassociated structural flexibility of the AFt nanocages, a novel temperature shift based encapsulation process was proposed and proved efficient for encapsulation of the NP peptides. On average, about 18 NPs were encapsulated and 1.6 HA antigens were conjugated in each of the HA-AFt+NP dual-antigen influenza vaccines. Upon immunization in mice, the HA-AFt+NP vaccine elicited both HA and NP-specific antibodies, and conferred complete protection against a lethal infection of both homologous PR8 H1N1 and heterologous A/FM/1/47 (FM1, H1N1) strains, while the HA-AFt conjugate vaccine without encapsulated NP antigen only conferred 60% protection against the FM1 H1N1 viral challenge. The potential cross-protective effect of the HA-AFt+NP vaccine was further demonstrated by significant specific hemagglutination inhibition (HAI) titers in serum of the immunized mice against heterologous A/Hong Kong/4801/2014 (H3N2) viral strain, which was about 3-fold of that induced by HA antigen and 2-fold of the HA-AFt conjugate vaccine. This biomimetic HA-AFt+NP conjugate vaccine, therefore, may represent a new strategy for developing a potential universal influenza vaccine without the need of any adjuvant, and further broaden the application of AFt nanocages in the areas of vaccine development and delivery system.

Two ferritin genes (MdFerH and MdFerL) are involved in iron homeostasis, antioxidation and immune defense in housefly Musca domestica.

Ferritin is a ubiquitous multi-subunit iron storage protein, made up of heavy chain and light chain subunits. In recent years, invertebrate ferritins have emerged as an important, yet largely underappreciated, component of host defense and antioxidant system. Here, two alternatively spliced transcripts encoding for a unique ferritin heavy chain homolog (MdFerH), and a transcript encoding for a light chain homolog (MdFerL) are cloned and characterized from Musca domestica. Comparing with MdFerH1, a fragment is absent at the 5' untranslated region of MdFerH2, where a putative iron response element is present. Amino acid sequence analysis shows that MdFerH possesses a strictly conserved ferroxidase site, while MdFerL has a putative atypical active center. Tissue distribution analysis indicates that MdFers are enriched expressed in gut. When the larvae receive diverse stimulations, including challenge by bacteria, exposure to excess Fe2+, doxorubicin or ultraviolet, the expression of MdFers is positively up-regulated in different degrees and different temporal patterns, indicating their potential roles in oxidative stress. The two mRNA isoforms of MdFerH appear to be differentially expressed in different tissues, but seem to show the similar expression patterns under diverse stress conditions. Further investigation reveals that silencing MdFers can alter the redox homeostasis, leading elevated mortalities of larvae following bacterial infection. Inspiringly, recombinant MdFerL produced in Pichia pastoris shows significant iron-chelating activity in vitro. These results suggest a pivotal role of ferritins from housefly in iron homeostasis, antibacterial immunity and redox balance.

Enhanced Detection of Infectious Pancreatic Necrosis Virus via Lateral Flow Chip and Fluorometric Biosensors Based on Self-Assembled Protein Nanoprobes.

Salmon fish farmers face remarkable problems in fish rearing and handling due to the spread of disease by infectious pancreatic necrosis virus (IPNV). Therefore, we developed a straightforward and sensitive technique to detect IPNV-based on recombinant human apoferritin heavy chain (hAFN-H) protein nanoparticles. In this study, the 24 subunits of the hAFN-H were genetically modified to express 6×His-tag and protein-G at their C-terminal site using Escherichia coli. We thus achieved a two-step signal amplifying strategy that utilizes a recombinant hAFN-H nanoprobe having a protein-G-binding site that targets the Fc region of monoclonal antibodies and a 6×His-tag that actively interacts with the functionalized Ni-NTA derivatives. In this study, we report a considerable advancement in magnetic bead-based detection systems that use Ni-NTA-Atto 550, reliably exhibiting detection limits of 1.02 TCID50/mL (50% tissue culture infective dose). Additionally, we propose a lateral flow chip-based detection method that uses the hAFN-H surface functionalized with 5 nm of the Ni-NTA-nanogold complex as a nanoprobe; the limit of detection towards IPNV was 0.88 TCID50/mL. The detection of IPNV by this recombinant hAFN-H nanoprobe was linear to virus titers in the range of 101-103 TCID50/mL.

Ferritin Light Chain Confers Protection Against Sepsis-Induced Inflammation and Organ Injury.

Despite the prevalence and recognition of its detrimental impact, clinical complications of sepsis remain a major challenge. Here, we investigated the effects of myeloid ferritin heavy chain (FtH) in regulating the pathogenic sequelae of sepsis. We demonstrate that deletion of myeloid FtH leads to protection against lipopolysaccharide-induced endotoxemia and cecal ligation and puncture (CLP)-induced model of sepsis as evidenced by reduced cytokine levels, multi-organ dysfunction and mortality. We identified that such protection is predominantly mediated by the compensatory increase in circulating ferritin (ferritin light chain; FtL) in the absence of myeloid FtH. Our in vitro and in vivo studies indicate that prior exposure to ferritin light chain restrains an otherwise dysregulated response to infection. These findings are mediated by an inhibitory action of FtL on NF-κB activation, a key signaling pathway that is implicated in the pathogenesis of sepsis. We further identified that LPS mediated activation of MAPK pathways, specifically, JNK, and ERK were also reduced with FtL pre-treatment. Taken together, our findings elucidate a crucial immunomodulatory function for circulating ferritin that challenges the traditional view of this protein as a mere marker of body iron stores. Accordingly, these findings will stimulate investigations to the adaptive nature of this protein in diverse clinical settings.

Ferritin H Deficiency in Myeloid Compartments Dysregulates Host Energy Metabolism and Increases Susceptibility to Mycobacterium tuberculosis Infection.

Iron is an essential factor for the growth and virulence of Mycobacterium tuberculosis (Mtb). However, little is known about the mechanisms by which the host controls iron availability during infection. Since ferritin heavy chain (FtH) is a major intracellular source of reserve iron in the host, we hypothesized that the lack of FtH would cause dysregulated iron homeostasis to exacerbate TB disease. Therefore, we used knockout mice lacking FtH in myeloid-derived cell populations to study Mtb disease progression. We found that FtH plays a critical role in protecting mice against Mtb, as evidenced by increased organ burden, extrapulmonary dissemination, and decreased survival in Fth-/- mice. Flow cytometry analysis showed that reduced levels of FtH contribute to an excessive inflammatory response to exacerbate disease. Extracellular flux analysis showed that FtH is essential for maintaining bioenergetic homeostasis through oxidative phosphorylation. In support of these findings, RNAseq and mass spectrometry analyses demonstrated an essential role for FtH in mitochondrial function and maintenance of central intermediary metabolism in vivo. Further, we show that FtH deficiency leads to iron dysregulation through the hepcidin-ferroportin axis during infection. To assess the clinical significance of our animal studies, we performed a clinicopathological analysis of iron distribution within human TB lung tissue and showed that Mtb severely disrupts iron homeostasis in distinct microanatomic locations of the human lung. We identified hemorrhage as a major source of metabolically inert iron deposition. Importantly, we observed increased iron levels in human TB lung tissue compared to healthy tissue. Overall, these findings advance our understanding of the link between iron-dependent energy metabolism and immunity and provide new insight into iron distribution within the spectrum of human pulmonary TB. These metabolic mechanisms could serve as the foundation for novel host-directed strategies.

First comparative characterization of three distinct ferritin subunits from a teleost: Evidence for immune-responsive mRNA expression and iron depriving activity of seahorse (Hippocampus abdominalis) ferritins.

Ferritins play an indispensable role in iron homeostasis through their iron-withholding function in living beings. In the current study, cDNA sequences of three distinct ferritin subunits, including a ferritin H, a ferritin M, and a ferritin L, were identified from big belly seahorse, Hippocampus abdominalis, and molecularly characterized. Complete coding sequences (CDS) of seahorse ferritin H (HaFerH), ferritin M (HaFerM), and ferritin L (HaFerL) subunits were comprised of 531, 528, and 522 base pairs (bp), respectively, which encode polypeptides of 177, 176, and 174 amino acids, respectively, with molecular masses of ∼20-21 kDa. Our in silico analyses demonstrate that these three ferritin subunits exhibit the typical characteristics of ferritin superfamily members including iron regulatory elements, domain signatures, and reactive centers. The coding sequences of HaFerH, M, and L were cloned and the corresponding proteins were overexpressed in a bacterial system. Recombinantly expressed HaFer proteins demonstrated detectable in vivo iron sequestrating (ferroxidase) activity, consistent with their putative iron binding capability. Quantification of the basal expression of these three HaFer sequences in selected tissues demonstrated a gene-specific ubiquitous spatial distribution pattern, with abundance of mRNA in HaFerM in the liver and predominant expression of HaFerH and HaFerL in blood. Interestingly, the basal expression of all three ferritin genes was found to be significantly modulated against pathogenic stress mounted by lipopolysaccharides (LPS), poly I:C, Streptococcus iniae, and Edwardsiella tarda. Collectively, our findings suggest that the three HaFer subunits may be involved in iron (II) homeostasis in big belly seahorse and that they are important in its host defense mechanisms.

The CD68(+)/H-ferritin(+) cells colonize the lymph nodes of the patients with adult onset Still's disease and are associated with increased extracellular level of H-ferritin in the same tissue: correlation with disease severity and implication for pathogenesis.

In this work, we aimed to evaluate the levels of ferritin enriched in H subunits (H-ferritin) and ferritin enriched in L subunits (L-ferritin) and the cells expressing these two molecules in the lymph node (LN) biopsies obtained from adult-onset Still's disease (AOSD) patients, and the possible correlation among these data and the severity of the disease. Ten patients with AOSD underwent LN biopsy. All the samples were stained by immunofluorescence. A statistical analysis was performed to estimate the possible correlation among both H-ferritin and L-ferritin tissue expression and the clinical picture of the disease. Furthermore, the same analysis was performed to evaluate the possible correlation among the number of CD68(+)/H-ferritin(+) or CD68(+)/L-ferritin(+) cells and the clinical picture. Immunofluorescence analysis demonstrated an increased tissue H-ferritin expression in the LNs of AOSD patients. This increased expression correlated with the severity of the disease. An increased number of CD68 macrophages expressing H-ferritin was observed in the LN samples of our patients. Furthermore, we observed that the number of CD68(+)/H-ferritin(+) cells correlated significantly with the severity of the clinical picture. Our data showed an imbalance between the levels of H- and L-ferritin in LNs of AOSD patients and the evidence of an increased number of CD68(+)/H-ferritin(+) cells in the same organs. Furthermore, a correlation among both the tissue H-ferritin levels and the CD68(+)/H-ferritin(+) cells and the clinical picture was observed.

CD11b is protective in complement-mediated immune complex glomerulonephritis.

In chronic serum sickness, glomerular immune complexes form, yet C57BL/6 mice do not develop glomerulonephritis unless complement factor H (CfH) is absent, indicating the relevance of complement regulation. Complement receptor 3 (CD11b) and Fcγ receptors on leukocytes, and CfH on platelets, can bind immune complexes. Here we induced immune complex-mediated glomerulonephritis in CfH(-/-) mice chimeric for wild-type, CfH(-/-), CD11b(-/-), or FcRγ(-/-) bone marrow stem cells. Glomerulonephritis was worse in CD11b(-/-) chimeras compared with all others, whereas disease in FcRγ(-/-) and wild-type chimeras was comparable. Disease tracked strongly with humoral immune responses, but not glomerular immune complex deposits. Interstitial inflammation with M1 macrophages strongly correlated with glomerulonephritis scores. CD11b(-/-) chimeras had significantly more M1 macrophages and CD4(+) T cells. The renal dendritic cell populations originating from bone marrow-derived CD11c(+) cells were similar in all experimental groups. CD11b(+) cells bearing colony-stimulating factor 1 receptor were present in kidneys, including CD11b(-/-) chimeras; these cells correlated negatively with glomerulonephritis scores. Thus, experimental immune complex-mediated glomerulonephritis is associated with accumulation of M1 macrophages and CD4(+) T cells in kidneys and functional renal insufficiency. Hence, CD11b on mononuclear cells is instrumental in generating an anti-inflammatory response in the inflamed kidney.

Association of ferritin antibodies with Takayasu arteritis.

Takayasu arteritis (TA) is difficult to diagnose because diagnostic biomarkers have not yet been established. In a previous study, we detected autoantibodies against the human ferritin heavy chain protein (HFC) in the sera of patients with giant cell arteritis (GCA) and/or polymyalgia rheumatica (PMR). The aim of this study is to evaluate the frequency of autoantibodies against HFC in TA. We established seven ELISA assays for the detection of autoantibodies against HFC. We used the full-length recombinant HFC expressed in Escherichia coli or one of six different HFC peptides as autoantigens: 1-18Aa (98.8 % purity), 19-45Aa (98.8 % purity), 52-78Aa (98.3 % purity), 79-104Aa (98.8 % purity), 105-143Aa (98.4 % purity) and 145-183Aa (98.5 % purity). We collected sera from 48 patients with TA, 36 patients with systemic lupus erythematosus (SLE), 35 patients with arteriosclerosis, 133 patients with febrile diseases, which are known to generate unspecific autoantibodies, and 50 blood donors, which served as controls. The best results were obtained using the ferritin peptides as antigens. By combining the results from the different ELISAs that detect autoantibodies against the HFC peptides 19-44A, 79-104A and 105-144A, we were able to detect ferritin peptide antibodies in 30/48 (62 %) of the TA patients. The frequency was lower than in early GCA and PMR (previous study showed up to 92 %). Positive results were observed in 0/50 (0 %) of the control blood donors, 10/36 (28 %) of the SLE patients, 4/35 (11 %) of the arteriosclerosis patients and 27/133 (20 %) of the fever patients. Considering the lack of biomarkers for TA, autoantibodies against HFC peptides could act as useful markers for TA.

Molecular profile and functional characterization of the ferritin H subunit from rock bream (Oplegnathus fasciatus), revealing its putative role in host antioxidant and immune defense.

Ferritins are iron binding proteins made out of 24 subunits, involved in iron homeostasis and metabolism in cellular environments. Here, we sought to identify and functionally characterize a one type of subunits of ferritin (ferritin H-like subunit) from rock bream (Oplegnathus fasciatus; RbFerH). The complete coding sequence of RbFerH was 531 bp in length, encoding a 177-amino acid protein with a predicted molecular mass of 20.8 kDa. The deduced protein structure possessed the domain architecture characteristic of known ferritin H subunits, including metal ligands for iron binding, a ferroxidase center, and two iron-binding region signatures. As expected, the 5' untranslated region of the RbFerH cDNA sequence contained a putative iron response element region, a characteristic regulatory element involved in its translation. The RbFerH gene comprised 5 exons and 4 introns spanning a 4195 bp region. Overexpressed recombinant RbFerH protein demonstrated prominent Fe(II) ion depriving activity, bacteriostatic properties, and protective effects against oxidative double-stranded DNA damage. Using quantitative polymerase chain reaction (qPCR), we found that RbFerH was expressed ubiquitously in the majority of physiologically important tissues in rock bream. A greater abundance of the mRNA transcripts were detected in blood and liver tissues. Upon administering different microbial pathogens and pathogen-derived mitogens, RbFerH transcription was markedly elevated in the blood of rock bream. Taken together, our findings suggest that RbFerH acts as a potent iron sequestrator in rock bream and may actively participate in antimicrobial as well as antioxidative defense.

Ferritin heavy chain in triple negative breast cancer: a favorable prognostic marker that relates to a cluster of differentiation 8 positive (CD8+) effector T-cell response.

Ferritin heavy chain (FTH1) is a 21-kDa subunit of the ferritin complex, known for its role in iron metabolism, and which has recently been identified as a favorable prognostic protein for triple negative breast cancer (TNBC) patients. Currently, it is not well understood how FTH1 contributes to an anti-tumor response. Here, we explored whether expression and cellular compartmentalization of FTH1 correlates to an effective immune response in TNBC patients. Analysis of the tumor tissue transcriptome, complemented with in silico pathway analysis, revealed that FTH1 was an integral part of an immunomodulatory network of cytokine signaling, adaptive immunity, and cell death. These findings were confirmed using mass spectrometry (MS)-derived proteomic data, and immunohistochemical staining of tissue microarrays. We observed that FTH1 is localized in both the cytoplasm and/or nucleus of cancer cells. However, high cytoplasmic (c) FTH1 was associated with favorable prognosis (Log-rank p = 0.001), whereas nuclear (n) FTH1 staining was associated with adverse prognosis (Log-rank p = 0.019). cFTH1 staining significantly correlated with total FTH1 expression in TNBC tissue samples, as measured by MS analysis (Rs = 0.473, p = 0.0007), but nFTH1 staining did not (Rs = 0.197, p = 0.1801). Notably, IFN γ-producing CD8+ effector T cells, but not CD4+ T cells, were preferentially enriched in tumors with high expression of cFTH1 (p = 0.02). Collectively, our data provide evidence toward new immune regulatory properties of FTH1 in TNBC, which may facilitate development of novel therapeutic targets.

Suppression of immunoglobulin production in human peripheral blood mononuclear cells by monocytes via secretion of heavy-chain ferritin.

In vitro antigen stimulation of peripheral blood mononuclear cells (PBMCs) does not induce immunoglobulin (Ig) production. However, pretreatment of PBMCs with l-leucyl-l-leucine methyl ester (LLME) prior to in vitro stimulation removes the suppression of Ig production. In the present study, we attempted to identify the target cells of LLME and determine the mechanisms by which Ig production in PBMCs is suppressed. We found that CD14(+) monocytes are involved in the suppression of Ig production in PBMCs. Furthermore, we confirmed that heavy-chain ferritin derived from CD14(+) monocytes suppresses Ig production in PBMCs, possibly through iron sequestration.

Epitope mapping of antibodies against ferritin heavy chain in giant cell arteritis and polymyalgia rheumatica.

OBJECTIVES: In a previous study we found an association between antibodies against the human ferritin heavy chain (HFC) protein and giant cell arteritis (GCA) and/or polymyalgia rheumatica (PMR), especially in GCA/PMR patients prior to glucocorticoid treatment. Antibodies against the N-terminal part of ferritin were present in 92% of untreated patients, 69% of patients with disease flare, and 13% of patients in remission. These antibodies appeared to be markers for the early detection of a disease complex usually diagnosed with considerable delay. Our aim in this study was to optimize the diagnostic test by epitope mapping of antibodies against HFC using peptide antigens in enzyme-linked immunosorbent assays (ELISAs). METHOD: We evaluated serum samples from a selected group of GCA/PMR patients in whom the sensitivity of antibodies against the N-terminal ferritin peptide was only 35%. Patients with late-onset rheumatoid arthritis (LORA), patients with fever, patients with granulomatosis with polyangiitis (GPA), patients without any autoimmune disease at age > 65 years, and blood donors served as controls. RESULTS: By combining different ELISAs we were able to increase the frequency of human ferritin peptide antibodies in GCA/PMR (p < 0.0001) without significantly altering the false-positive rate (FPR) of the diagnostic test. The frequency of antibodies against human ferritin peptide increased from 53% to 74% in GCA/PMR patients with disease flare, from 29% to 40% in GCA/PMR patients in partial remission, and from 8% to 45% in GCA/PMR patients in complete remission. CONCLUSIONS: The potential diagnostic test for GCA/PMR can be improved by combining three human ferritin peptide antibodies.

Ferritin H is a novel marker of early erythroid precursors and macrophages.

AIMS: Macrophages play a critical role in iron homeostasis by recycling iron from red cells and storing it in ferritin, an iron storage protein. The recycled iron is delivered to erythroid precursors for erythropoiesis. In this study, we aimed to determine whether ferritin is highly expressed in macrophages and erythroid precursors, and whether it can be used as a marker for these two cell types. METHODS AND RESULTS: A ferritin monoclonal antibody was developed, and immunohistochemistry was performed. In normal bone marrows, ferritin antibody stained early erythroid precursors and macrophages. In contrast, myeloid cells, lymphoid cells and megakaryocytes lacked ferritin expression. In leukaemic bone marrows, ferritin was selectively expressed in erythroid blasts (M6), whereas all other blasts were negative. In lymph nodes, ferritin was highly and specifically expressed in macrophages, whereas lymphocytes completely lacked ferritin expression. In non-haematopoietic tissues, ferritin antibody highlighted alveolar macrophages in the lung, as well as sinus macrophages in the liver and spleen. CONCLUSIONS: We conclude that ferritin is a novel and reliable marker for macrophages and early erythroid precursors, and may be of clinical utility in the diagnosis of diseases associated with these two cell types.

Association of ferritin autoantibodies with giant cell arteritis/polymyalgia rheumatica.

OBJECTIVES: Polymyalgia rheumatica (PMR) and giant cell arteritis (GCA) are relatively common inflammatory disorders. Establishing the diagnosis however may be difficult, since so far no specific biomarkers of the disorders are available. METHODS: As a screening procedure, the authors used protein arrays for the detection of new autoantigens in GCA and PMR. The results of the protein array were confirmed by different ELISAs detecting IgG antibodies against the human ferritin heavy chain, N-terminal 27 amino acids of the human ferritin heavy chain or the homologous peptide of Staphylococcus epidermidis. Sera of patients with only GCA (n=64), only PMR (n=47) and both PMR and GCA (n=31) were used. RESULTS: In the ELISA using the human ferritin peptide, the sensitivity of IgG antibodies against ferritin was 92% in 36 GCA and/or PMR patients before initiation of treatment, 22/32 (69%) in patients with disease flares and 64/117 (55%) in the total cohort including treated and inactive patients. In controls, the false positive rate was 11/38 (29%) in systemic lupus erythematosus, 1/36 (3%) in rheumatoid arthritis, 0/31 (0%) in late onset rheumatoid arthritis, 3/46 (6.5%) in B-non-Hodgkin's lymphoma and 1/100 (1%) in blood donors. In the ELISA using the ferritin peptide of S epidermidis, 89% of 27 patients with untreated GCA and PMR were positive. CONCLUSION: Antibodies against the ferritin peptide were present in up to 92% of untreated, active GCA and PMR patients. They can be useful as a diagnostic marker of PMR and GCA.

Expression of the H-subunit and L-subunit of ferritin in bone marrow macrophages and cells of the erythron during cellular immune activation.

BACKGROUND: The expression of the two types of ferritin subunits, the H-subunit and L-subunit, has been shown to be differentially regulated by cytokines. The primary aim of the present study was to quantitatively measure the expression of the H-subunit and L-subunit of ferritin in bone marrow macrophages and cells of the erythron in patients with chronic T-helper cell type-1 immune stimulation. METHODS: The expression of the H-subunit and L-subunit of ferritin in bone marrow macrophages and cells of the erythron was quantitatively evaluated by post-embedding immunolocalisation with immunogold transmission electron microscopy. RESULTS: The present study showed up-regulation of the H-subunit of ferritin in the bone marrow macrophage in patients with pronounced cellular immune activation (94.7±37.3 counts/µm(2); n=31 vs 72.4±34.0 counts/µm(2); n=13, p-value=0.037). CONCLUSION: This supports a possible role for H-subunit rich ferritins in the hypoferraemia of chronic disease.

Polarization dictates iron handling by inflammatory and alternatively activated macrophages.

BACKGROUND: Macrophages play a key role in iron homeostasis. In peripheral tissues, they are known to polarize into classically activated (or M1) macrophages and alternatively activated (or M2) macrophages. Little is known on whether the polarization program influences the ability of macrophages to store or recycle iron and the molecular machinery involved in the processes. DESIGN AND METHODS: Inflammatory/M1 and alternatively activated/M2 macrophages were propagated in vitro from mouse bone-marrow precursors and polarized in the presence of recombinant interferon-γ or interleukin-4. We characterized and compared their ability to handle radioactive iron, the characteristics of the intracellular iron pools and the expression of molecules involved in internalization, storage and export of the metal. Moreover we verified the influence of iron on the relative ability of polarized macrophages to activate antigen-specific T cells. RESULTS: M1 macrophages have low iron regulatory protein 1 and 2 binding activity, express high levels of ferritin H, low levels of transferrin receptor 1 and internalize--albeit with low efficiency -iron only when its extracellular concentration is high. In contrast, M2 macrophages have high iron regulatory protein binding activity, express low levels of ferritin H and high levels of transferrin receptor 1. M2 macrophages have a larger intracellular labile iron pool, effectively take up and spontaneously release iron at low concentrations and have limited storage ability. Iron export correlates with the expression of ferroportin, which is higher in M2 macrophages. M1 and M2 cells activate antigen-specific, MHC class II-restricted T cells. In the absence of the metal, only M1 macrophages are effective. CONCLUSIONS: Cytokines that drive macrophage polarization ultimately control iron handling, leading to the differentiation of macrophages into a subset which has a relatively sealed intracellular iron content (M1) or into a subset endowed with the ability to recycle the metal (M2).