Esther G. Meyron-Holtz

The Hyperferritinemic Syndrome: macrophage activation syndrome, Still’s disease, septic shock and catastrophic antiphospholipid syndrome

BackgroundOver the last few years, accumulating data have implicated a role for ferritin as a signaling molecule and direct mediator of the immune system. Hyperferritinemia is associated with a multitude of clinical conditions and with worse prognosis in critically ill patients.DiscussionThere are four uncommon medical conditions characterized by high levels of ferritin, namely the macrophage activation syndrome (MAS), adult onset Still’s disease (AOSD), catastrophic antiphospholipid syndrome (cAPS) and septic shock, that share a similar clinical and laboratory features, and also respond to similar treatments, suggesting a common pathogenic mechanism. Ferritin is known to be a pro-inflammatory mediator inducing expression of pro-inflammatory molecules, yet it has opposing actions as a pro-inflammatory and as an immunosuppressant. We propose that the exceptionally high ferritin levels observed in these uncommon clinical conditions are not just the product of the inflammation but rather may contribute to the development of a cytokine storm.SummaryHere we review and compare four clinical conditions and the role of ferritin as an immunomodulator. We would like to propose including these four conditions under a common syndrome entity termed “Hyperferritinemic Syndrome”.

Designer Aminoglycosides that Selectively Inhibit Cytoplasmic rather than Mitochondrial Ribosomes Show Decreased Ototoxicity: a Strategy for the Treatment of Genetic Diseases

Background: Whether the mitochondrial or cytoplasmic protein synthesis inhibition predominates to aminoglycoside-induced ototoxicity is unclear. Results: The ototoxicity of an aminoglycoside correlates primarily with its ability to block mitochondrial rather than cytoplasmic protein synthesis. Conclusion: Designer aminoglycosides that selectively inhibit cytoplasmic rather than mitochondrial ribosome show decreased ototoxicity. Significance: The results are beneficial for development of aminoglycoside-based drugs to treat human genetic diseases. There is compelling evidence that aminoglycoside (AG) antibiotics can induce the mammalian ribosome to suppress disease-causing nonsense mutations and partially restore the expression of functional proteins. However, prolonged AG treatment can cause detrimental side effects in patients, including most prominently, ototoxicity. Recent mechanistic discussions have considered the relative contributions of mitochondrial and cytoplasmic protein synthesis inhibition to AG-induced ototoxicity. We show that AGs inhibit mitochondrial protein synthesis in mammalian cells and perturb cell respiration, leading to a time- and dose-dependent increase in superoxide overproduction and accumulation of free ferrous iron in mitochondria caused by oxidative damage of mitochondrial aconitase, ultimately leading to cell apoptosis via the Fenton reaction. These deleterious effects increase with the increased potency of AG to inhibit the mitochondrial rather than cytoplasmic protein synthesis, which in turn correlates with their ototoxic potential in both murine cochlear explants and the guinea pig in vivo. The deleterious effects of AGs were alleviated in synthetic derivatives specially designed for the treatment of genetic diseases caused by nonsense mutations and possessing low affinity toward mitochondrial ribosomes. This work highlights the benefit of a mechanism-based drug redesign strategy that can maximize the translational value of “readthrough therapy” while mitigating drug-induced side effects. This approach holds promise for patients suffering from genetic diseases caused by nonsense mutations.

A possible role for secreted ferritin in tissue iron distribution

Ferritin is known as a well-conserved iron detoxification and storage protein that is found in the cytosol of many prokaryotic and eukaryotic organisms. In insects and worms, ferritin has evolved into a classically secreted protein that transports iron systemically. Mammalian ferritins are found intracellularly in the cytosol, as well as in the nucleus, the endo-lysosomal compartment and the mitochondria. Extracellular ferritin is found in fluids such as serum and synovial and cerebrospinal fluids. We recently characterized the biophysical properties, secretion mechanism and cellular origin of mouse serum ferritin, which is actively secreted by a non-classical pathway involving lysosomal processing. Here, we review the data to support a hypothesis that intracellular and extracellular ferritin may play a role in intra- and intercellular redistribution of iron.

Endoplasmic reticulum anchored heme-oxygenase 1 faces the cytosol

Heme-oxygenase 1 is an endoplasmic reticulum-anchored enzyme that breaks down heme into iron, carbon monoxide and biliverdin. Heme is a hydrophobic co-factor in many proteins, including hemoglobin. Free heme is highly cytotoxic and, therefore, both heme synthesis and breakdown are tightly regulated. During turnover of heme proteins, heme is released in the phago-lysosomal compartment or the cytosol. The subcellular location of the heme-oxygenase 1 active site has not been clarified. Using constructs of heme-oxygenase 1 with fluorescent proteins, and the endogenous heme-oxygenase 1 in two variations of protease protection assays, we determined that heme-oxygenase 1 is membrane-bound and faces the cytosol in non-activated macrophages in vivo. These findings imply that in quiescent macrophages, heme breakdown products are generated in the cytosol. This facilitates iron recycling to ferroportin for iron export and to ferritin for iron storage.

Genetic modification of iron metabolism in mice affects the gut microbiota

The composition of the gut microbiota is affected by environmental factors as well as host genetics. Iron is one of the important elements essential for bacterial growth, thus we hypothesized that changes in host iron homeostasis, may affect the luminal iron content of the gut and thereby the composition of intestinal bacteria. The iron regulatory protein 2 (Irp2) and one of the genes mutated in hereditary hemochromatosis Hfe , are both proteins involved in the regulation of systemic iron homeostasis. To test our hypothesis, fecal metal content and a selected spectrum of the fecal microbiota were analyzed from Hfe−/−, Irp2−/− and their wild type control mice. Elevated levels of iron as well as other minerals in feces of Irp2−/− mice compared to wild type and Hfe−/− mice were observed. Interestingly significant variation in the general fecal-bacterial population-patterns was observed between Irp2−/− and Hfe−/− mice. Furthermore the relative abundance of five species, mainly lactic acid bacteria, was significantly different among the mouse lines. Lactobacillus (L.) murinus and L.intestinalis were highly abundant in Irp2−/− mice, Enterococcus faecium species cluster and a species most similar to Olsenella were highly abundant in Hfe-/- mice and L. johnsonii was highly abundant in the wild type mice. These results suggest that deletion of iron metabolism genes in the mouse host affects the composition of its intestinal bacteria. Further studying the relationship between gut microbiota and genetic mutations affecting systemic iron metabolism in human should lead to clinical implications.

Cardiac Protection by Preconditioning Is Generated via an Iron-Signal Created by Proteasomal Degradation of Iron Proteins

Ischemia associated injury of the myocardium is caused by oxidative damage during reperfusion. Myocardial protection by ischemic preconditioning (IPC) was shown to be mediated by a transient ‘iron-signal’ that leads to the accumulation of apoferritin and sequestration of reactive iron released during the ischemia. Here we identified the source of this ‘iron signal’ and evaluated its role in the mechanisms of cardiac protection by hypoxic preconditioning. Rat hearts were retrogradely perfused and the effect of proteasomal and lysosomal protease inhibitors on ferritin levels were measured. The iron-signal was abolished, ferritin levels were not increased and cardiac protection was diminished by inhibition of the proteasome prior to IPC. Similarly, double amounts of ferritin and better recovery after ex vivo ischemia-and-reperfusion (I/R) were found in hearts from in vivo hypoxia pre-conditioned animals. IPC followed by normoxic perfusion for 30 min (‘delay’) prior to I/R caused a reduced ferritin accumulation at the end of the ischemia phase and reduced protection. Full restoration of the IPC-mediated cardiac protection was achieved by employing lysosomal inhibitors during the ‘delay’. In conclusion, proteasomal protein degradation of iron-proteins causes the generation of the ‘iron-signal’ by IPC, ensuing de-novo apoferritin synthesis and thus, sequestering reactive iron. Lysosomal proteases are involved in subsequent ferritin breakdown as revealed by the use of specific pathway inhibitors during the ‘delay’. We suggest that proteasomal iron-protein degradation is a stress response causing an expeditious cytosolic iron release thus, altering iron homeostasis to protect the myocardium during I/R, while lysosomal ferritin degradation is part of housekeeping iron homeostasis.

Alginate encapsulated cells secreting Fas‐ligand reduce lymphoma carcinogenicity

Fas ligand (CD95L/APO‐1) is considered as a potent anti‐tumor agent due to its mediated cell death properties. We have designed a polymeric microencapsulation system, which encapsulates soluble FasL secreting cells. The encapsulated cells continuously release soluble FasL (sFasL) at the tumor site, while the device protects the encapsulated cells from the host immune system. The potential and efficacy of this system are demonstrated in vitro and in vivo for tumor inhibition. Polymeric microcapsules composed of Alginate Poly‐l‐lysine were optimized to encapsulate L5 secreting sFasL cells. The expression and anti‐tumor activities of the sFasL were confirmed in vitro and tumor inhibition was studied in vivo in SCID mice bearing subcutaneous lymphoma tumors. In vitro, sFasL secreted by the encapsulated L5‐sFasL cells was biologically active, inhibited proliferation and induced apoptotic cell death in Fas sensitive tumor cells. Mice injected with encapsulated L5‐sFasL cells on the day of tumor injection or 10 days after tumor injection showed significant reduction in tumor volume, of 87% and 95%, respectively. Our findings show that encapsulated cells expressing sFasL can be used as a local device and efficiently suppress malignant Fas sensitive tumors with no side effects. (Cancer Sci 2012; 103: 116–124)

Ferritin polarization and iron transport across monolayer epithelial barriers in mammals

Epithelial barriers are found in many tissues such as the intestine, kidney and brain where they separate the external environment from the body or a specific compartment from its periphery. Due to the tight junctions that connect epithelial barrier-cells (EBCs), the transport of compounds takes place nearly exclusively across the apical or basolateral membrane, the cell-body and the opposite membrane of the polarized EBC, and is regulated on numerous levels including barrier-specific adapted trafficking-machineries. Iron is an essential element but toxic at excess. Therefore, all iron-requiring organisms tightly regulate iron concentrations on systemic and cellular levels. In contrast to most cell types that control just their own iron homeostasis, EBCs also regulate homeostasis of the compartment they enclose or the body as a whole. Iron is transported across EBCs by specialized transporters such as the transferrin receptor and ferroportin. Recently, the iron storage protein ferritin was also attributed a role in the regulation of systemic iron homeostasis and we gathered evidence from the literature and original data that ferritin is polarized in EBC, suggesting also a role for ferritin in iron trafficking across EBCs.

Ferritin is secreted via 2 distinct nonclassical vesicular pathways

Ferritin turnover plays a major role in tissue iron homeostasis, and ferritin malfunction is associated with impaired iron homeostasis and neurodegenerative diseases. In most eukaryotes, ferritin is considered an intracellular protein that stores iron in a nontoxic and bioavailable form. In insects, ferritin is a classically secreted protein and plays a major role in systemic iron distribution. Mammalian ferritin lacks the signal peptide for classical endoplasmic reticulum-Golgi secretion but is found in serum and is secreted via a nonclassical lysosomal secretion pathway. This study applied bioinformatics and biochemical tools, alongside a protein trafficking mouse models, to characterize the mechanisms of ferritin secretion. Ferritin trafficking via the classical secretion pathway was ruled out, and a 2:1 distribution of intracellular ferritin between membrane-bound compartments and the cytosol was observed, suggesting a role for ferritin in the vesicular compartments of the cell. Focusing on nonclassical secretion, we analyzed mouse models of impaired endolysosomal trafficking and found that ferritin secretion was decreased by a BLOC-1 mutation but increased by BLOC-2, BLOC-3, and Rab27A mutations of the cellular trafficking machinery, suggesting multiple export routes. A 13-amino-acid motif unique to ferritins that lack the secretion signal peptide was identified on the BC-loop of both subunits and plays a role in the regulation of ferritin secretion. Finally, we provide evidence that secretion of iron-rich ferritin was mediated via the multivesicular body-exosome pathway. These results enhance our understanding of the mechanism of ferritin secretion, which is an important piece in the puzzle of tissue iron homeostasis.

Digestive fate of dietary carrageenan: Evidence of interference with digestive proteolysis and disruption of gut epithelial function

Scope: The objective of this study was to interrogate two mechanisms by which commercial Carrageenans (E407) (CGN) may adversely affect human health: (i) Through modification of gastric proteolysis and (ii) Through affecting gut epithelial structure and function. Methods and results: Three commercial CGN samples with distinct zeta‐potentials (stable at the pH range of 3–7 and varied with physiological levels of CaCl2) were mixed with milk, soy or egg protein isolates, then subjected to a semi‐dynamic in vitro digestion model and analyzed by SDS‐PAGE. This revealed varying levels of interference with gastric digestive proteolysis and a significant decrease in pepsin activity. Further, a Caco‐2 cell model was used to explore various effects of physiologically digested CGN (pdCGN) on various epithelial cell functions and characteristics. Samples of pdCGN (0.005–0.5 mg/mL) affected the epithelial barrier function, including redistribution of the tight‐junction protein Zonula Occludens (Zo)‐1, changes in cellular F‐actin architecture and increased monolayer permeability to the transfer of macromolecules. Moreover, pdCGN induced elevation in the levels of the pro‐inflammatory IL‐8 receptor CXCR1. Conclusion: This work raises the possibility that CGN may reduce protein and peptide bioaccessibility, disrupt normal epithelial function, promote intestinal inflammation, and consequently compromise consumer health.