Distinct Effects of Escherichia coli,Pseudomonas aeruginosa and Staphylococcus aureus Cell Wall Component-Induced Inflammation on the Iron Metabolism of THP-1 Cells

Macrophages are essential immune cells of the innate immune system. They participate in the development and regulation of inflammation. Macrophages play a fundamental role in fighting against bacterial infections by phagocytosis of bacteria, and they also have a specific role in immunomodulation by secreting pro-inflammatory cytokines. In bacterial infection, macrophages decrease the serum iron concentration by removing iron from the blood, acting as one of the most important regulatory cells of iron homeostasis. We examined whether the Gram-positive and Gram-negative cell wall components from various bacterial strains affect the cytokine production and iron transport, storage and utilization of THP-1 monocytes in different ways. We found that S. aureus lipoteichoic acid (LTA) was less effective in activating pro-inflammatory cytokine expression that may related to its effect on fractalkine production. LTA-treated cells increased iron uptake through divalent metal transporter-1, but did not elevate the expression of cytosolic and mitochondrial iron storage proteins, suggesting that the cells maintained iron efflux via the ferroportin iron exporter. E. coli and P. aeruginosa lipopolysaccharides (LPSs) acted similarly on THP-1 cells, but the rates of the alterations of the examined proteins were different. E. coli LPS was more effective in increasing the pro-inflammatory cytokine production, meanwhile it caused less dramatic alterations in iron metabolism. P. aeruginosa LPS-treated cells produced a smaller amount of pro-inflammatory cytokines, but caused remarkable elevation of both cytosolic and mitochondrial iron storage proteins and intracellular iron content compared to E. coli LPS. These results prove that LPS molecules from different bacterial sources alter diverse molecular mechanisms in macrophages that prepossess the outcome of the bacterial infection.

Large protein organelles form a new iron sequestration system with high storage capacity

Iron storage proteins are essential for cellular iron homeostasis and redox balance. Ferritin proteins are the major storage units for bioavailable forms of iron. Some organisms lack ferritins, and it is not known how they store iron. Encapsulins, a class of protein-based organelles, have recently been implicated in microbial iron and redox metabolism. Here, we report the structural and mechanistic characterization of a 42 nm two-component encapsulin-based iron storage compartment from Quasibacillus thermotolerans. Using cryo-electron microscopy and x-ray crystallography, we reveal the assembly principles of a thermostable T = 4 shell topology and its catalytic ferroxidase cargo and show interactions underlying cargo-shell co-assembly. This compartment has an exceptionally large iron storage capacity storing over 23,000 iron atoms. Our results reveal a new approach for survival in diverse habitats with limited or fluctuating iron availability via an iron storage system able to store 10 to 20 times more iron than ferritin.

Structure and function of a 9.6 megadalton bacterial iron storage compartment

Iron storage proteins are essential for maintaining intracellular iron homeostasis and redox balance. Iron is generally stored in a soluble and bioavailable form inside ferritin protein compartments. However, some organisms do not encode ferritins and thus rely on alternative storage strategies. Encapsulins, a class of protein-based organelles, have recently been implicated in microbial iron and redox metabolism. Here, we report the structural and mechanistic characterization of a 42 nm two-component encapsulin-based iron storage compartment from Quasibacillus thermotolerans. Using cryo-electron microscopy and x-ray crystallography, we reveal the assembly principles of a thermostable T = 4 shell topology and its catalytic ferroxidase cargo. We show that the cargo-loaded compartment has an exceptionally large iron storage capacity storing over 23,000 iron atoms. These results form the basis for understanding alternate microbial strategies for dealing with the essential element iron.

Effect of Inflammatory Mediators Lipopolysaccharide and Lipoteichoic Acid on Iron Metabolism of Differentiated SH-SY5Y Cells Alters in the Presence of BV-2 Microglia

Lipopolysaccharide (LPS) and lipoteichoic acid (LTA), the Gram-negative and the Gram-positive bacterial cell wall components are important mediators of neuroinflammation in sepsis. LPS and LTA are potent activators of microglial cells which induce the production of various pro-inflammatory cytokines. It has been demonstrated that disturbance of iron homeostasis of the brain is one of the underlying causes of neuronal cell death but the mechanisms contributing to this process are still questionable. In the present study, we established monocultures of differentiated SH-SY5Y cells and co-cultures of differentiated SH-SY5Y cells and BV-2 microglia as neuronal model systems to selectively examine the effect of inflammatory mediators LPS and LTA on iron homeostasis of SH-SY5Y cells both in mono- and co-cultures. We monitored the IL-6 and TNFα secretions of the treated cells and determined the mRNA and protein levels of iron importers (transferrin receptor-1 and divalent metal transporter-1), and iron storing genes (ferritin heavy chain and mitochondrial ferritin). Moreover, we examined the relation between hepcidin secretion and intracellular iron content. Our data revealed that LPS and LTA triggered distinct responses in SH-SY5Y cells by differently changing the expressions of iron uptake, as well as cytosolic and mitochondrial iron storage proteins. Moreover, they increased the total iron contents of the cells but at different rates. The presence of BV-2 microglial cells influenced the reactions of SH-SY5Y cells on both LPS and LTA treatments: iron uptake and iron storage, as well as the neuronal cytokine production have been modulated. Our results demonstrate that BV-2 cells alter the iron metabolism of SH-SY5Y cells, they contribute to the iron accumulation of SH-SY5Y cells by manipulating the effects of LTA and LPS proving that microglia are important regulators of neuronal iron metabolism at neuroinflammation.

The importance of eukaryotic ferritins in iron handling and cytoprotection

Ferritins, the main intracellular iron storage proteins, have been studied for over 60 years, mainly focusing on the mammalian ones. This allowed the elucidation of the structure of these proteins and the mechanisms regulating their iron incorporation and mineralization. However, ferritin is present in most, although not all, eukaryotic cells, comprising monocellular and multicellular invertebrates and vertebrates. The aim of this review is to provide an update on the general properties of ferritins that are common to various eukaryotic phyla (except plants), and to give an overview on the structure, function and regulation of ferritins. An update on the animal models that were used to characterize H, L and mitochondrial ferritins is also provided. The data show that ferritin structure is highly conserved among different phyla. It exerts an important cytoprotective function against oxidative damage and plays a role in innate immunity, where it also contributes to prevent parenchymal tissue from the cytotoxicity of pro-inflammatory agonists released by the activation of the immune response activation. Less clear are the properties of the secretory ferritins expressed by insects and molluscs, which may be important for understanding the role played by serum ferritin in mammals.

Iron binding to human heavy-chain ferritin

Maxi-ferritins are ubiquitous iron-storage proteins with a common cage architecture made up of 24 identical subunits of five α-helices that drive iron biomineralization through catalytic iron(II) oxidation occurring at oxidoreductase sites (OS). Structures of iron-bound human H ferritin were solved at high resolution by freezing ferritin crystals at different time intervals after exposure to a ferrous salt. Multiple binding sites were identified that define the iron path from the entry ion channels to the oxidoreductase sites. Similar data are available for another vertebrate ferritin: the M protein fromRana catesbeiana. A comparative analysis of the iron sites in the two proteins identifies new reaction intermediates and underlines clear differences in the pattern of ligands that define the additional iron sites that precede the oxidoreductase binding sites along this path. Stopped-flow kinetics assays revealed that human H ferritin has different levels of activity compared with itsR. catesbeianacounterpart. The role of the different pattern of transient iron-binding sites in the OS is discussed with respect to the observed differences in activity across the species.