Our data show selective survival of tTregs and elevated production of immunosuppressive cytokines by Treg cells after GC treatment, which may contribute to the immunosuppressive effects of GCs.

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.

Fractalkine (CX3CL1) is a potent inflammatory mediator of the central nervous system, which is expressed by neurons and regulates microglial functions by binding to fractalkine receptor (CX3CR1). It has been demonstrated that neuroinflammation plays an important role in iron accumulation of the brain leading to neuronal cell death. The major regulator of iron homeostasis is the peptide hormone hepcidin. Hepcidin expression is triggered by inflammatory conditions, which may contribute to the neuronal iron accumulation. In the present study, we established a bilaminar co-culture system of differentiated SH-SY5Y cells and BV-2 microglia as a neuronal model to examine the effect of soluble fractalkine on iron homeostasis of microglia and SH-SY5Y cells. We determined the hepcidin expression of fractalkine-treated microglia which showed significant elevation. We examined the relation between increased hepcidin secretion, the known hepcidin regulators and the signalling pathways controlled by fractalkine receptor. Our data revealed that TMPRSS6 and alpha 1-antitrypsin levels decreased due to fractalkine treatment, as well as the activity of NFκB pathway and the tyrosine phosphorylation of STAT5 factor. Moreover, fractalkine-induced hepcidin production of microglia initiated ferroportin internalisation of SH-SY5Y cells, which contributed to iron accumulation of neurons. Our results demonstrate that soluble form of fractalkine regulates hepcidin expression of BV-2 cells through fractalkine-mediated CX3CR1 internalisation. Moreover, fractalkine indirectly contributes to the iron accumulation of SH-SY5Y cells by activating ferroportin internalisation and by triggering the expressions of divalent metal transporter-1, ferritin heavy chain and mitochondrial ferritin.

No abstract

Lutein is a tetraterpene carotenoid, which has been reported as an important antioxidant and it is widely used as a supplement. Oxidative stress participates in many human diseases, including different types of neurodegenerative disorders. Microglia, the primary immune effector cells in the central nervous system, are implicated in these disorders by producing harmful substances such as reactive oxygen species (ROS). The protective mechanisms which scavenge ROS include enzymes and antioxidant substances. The protective effects of different carotenoids against oxidative stress have been described previously. Our study focuses on the effects of lutein on antioxidant enzymes, cytokines and iron metabolism under stress conditions in BV-2 microglia. We performed cell culture experiments: BV-2 cells were treated with lutein and/or with H2O2; the latter was used for inducing oxidative stress in microglial cells. Real-time PCR was performed for gene expression analyses of antioxidant enzymes, and ELISA was used for the detection of pro- and anti-inflammatory cytokines. Our results show that the application of lutein suppressed the H2O2-induced ROS (10′: 7.5 ng + 10 µM H2O2p = 0.0002; 10 ng/µL + 10 µM H2O2p = 0.0007), influenced iron utilization and changed the anti-inflammatory and pro-inflammatory cytokine secretions in BV-2 cells. Lutein increased the IL-10 secretions compared to control (24 h: 7.5 ng/µL p = 0.0274; 10 ng/µL p = 0.0008) and to 10 µM H2O2-treated cells (24 h: 7.5 ng/µL + H2O2p = 0.0003; 10 ng/µL + H2O2p = 0.0003), while it decreased the TNFα secretions compared to H2O2 treated cells (24 h: 7.5 ng/µL + H2O2p < 0.0001; 10 ng/µL + H2O2p < 0.0001). These results contribute to understanding the effects of lutein, which may help in preventing or suppressing ROS-mediated microglia activation, which is related to neuronal degeneration in oxidative stress scenario.

One of the models to investigate the distinct mechanisms contributing to neurodegeneration in multiple sclerosis is based on cuprizone (CZ) intoxication. CZ is toxic to mature oligodendrocytes and produces demyelination within the central nervous system but does not cause direct neuronal damage. The CZ model is suitable for better understanding the molecular mechanism of de- and remyelination processes of oligodendrocytes. CZ is a copper chelating agent and it also affects the iron metabolism in brain and liver tissues. To determine the early effect of CZ treatment on iron homeostasis regulation, cytosolic and mitochondrial iron storage, as well as some lipid metabolism genes, we investigated the expression of respective iron homeostasis and lipid metabolism genes of the corpus callosum (CC) and the liver after short-term CZ administration. In the present study C57BL/6 male mice aged four weeks were fed with standard rodent food premixed with 0.2 w/w% CZ for two or eight days. The major findings of our experiments are that short-term CZ treatment causes significant changes in iron metabolism regulation as well as in the expression of myelin and lipid synthesis-related genes, even before apparent demyelination occurs. Both in the CC and the liver the iron uptake, utilization and storage are modified, though not always the same way or to the same extent in the two organs. Understanding the role of iron in short-term and long-term CZ intoxication could provide a partial explanation of the discrepant signs of acute and chronic MS. These could contribute to understanding the development of multiple sclerosis and might provide a possible drug target.

Embryo implantation is a complex process regulated by a network of biological molecules. Recently, it has been described that fractalkine (CX3CL1, FKN) might have an important role in the feto–maternal interaction during gestation since the trophoblast cells express fractalkine receptor (CX3CR1) and the endometrium cells secrete fractalkine. CX3CR1 controls three major signalling pathways, PLC-PKC pathway, PI3K/AKT/NFκB pathway and Ras-mitogen-activated protein kinases (MAPK) pathways regulating proliferation, growth, migration and apoptosis. In this study, we focused on the molecular mechanisms of FKN treatment influencing the expression of implantation-related genes in trophoblast cells (JEG-3) both in mono-and in co-culture models. Our results reveal that FKN acted in a concentration and time dependent manner on JEG-3 cells. FKN seemed to operate as a positive regulator of implantation via changing the action of progesterone receptor (PR), activin receptor and bone morphogenetic protein receptor (BMPR). FKN modified also the expression of matrix metalloproteinase 2 and 9 controlling invasion. The presence of HEC-1A endometrial cells in the co-culture contributed to the effect of fractalkine on JEG-3 cells regulating implantation. The results suggest that FKN may contribute to the successful attachment and implantation of embryo.

No abstract