RESUMO
Macrophages in the endometrium promote receptivity and implantation by secreting proinflammatory cytokines and other factors like fractalkine (FKN). Macrophages are closely linked to regulating iron homeostasis and can modulate iron availability in the tissue microenvironment. It has been revealed that the iron metabolism of the mother is crucial in fertility. Iron metabolism is strictly controlled by hepcidin, the principal iron regulatory protein. The inflammatory cytokines can modulate hepcidin synthesis and, therefore, the iron metabolism of the endometrium. It was proven recently that FKN, a unique chemokine, is implicated in maternal-fetal communication and may contribute to endometrial receptivity and implantation. In the present study, we investigated the effect of activated THP-1 macrophages and FKN on the iron metabolism of the HEC-1A endometrial cells. We established a noncontact coculture with or without recombinant human FKN supplementation to study the impact of the macrophage-derived factors and FKN on the regulation of hepcidin synthesis and iron release and storage of endometrial cells. Based on our findings, the conditioned medium of the activated macrophages could modify hepcidin synthesis via the nuclear factor kappa-light-chain-enhancer of activated B cells, the signal transducer and activator of transcription 3, and the transferrin receptor 2/bone morphogenetic protein 6/suppressor of mothers against decapentaplegic 1/5/8 signaling pathways, and FKN could alter this effect on the endometrial cells. It was also revealed that the conditioned macrophage medium and FKN modulated the iron release and storage of HEC-1A cells. FKN signaling may be involved in the management of iron trafficking of the endometrium by the regulation of hepcidin. It can contribute to the iron supply for fetal development at the early stage of the pregnancy.
Assuntos
Quimiocina CX3CL1 , Hepcidinas , Feminino , Humanos , Quimiocina CX3CL1/metabolismo , Quimiocina CX3CL1/farmacologia , Hepcidinas/metabolismo , Endométrio/metabolismo , Macrófagos/metabolismo , Ferro/metabolismoRESUMO
Iron is a crucial element in the human body. Endometrial iron metabolism is implicated in endometrium receptivity and embryo implantation. Disturbances of the maternal as well as the endometrial iron homeostasis, such as iron deficiency, can contribute to the reduced development of the fetus and could cause an increased risk of adverse pregnancy outcomes. Fractalkine is a unique chemokine that plays a role in the communication between the mother and the fetus. It has been demonstrated that FKN is involved in the development of endometrial receptivity and embryo implantation, and it functions as a regulator of iron metabolism. In the present study, we examined the effect of FKN on the iron metabolism of HEC-1A endometrial cells in a state of iron deficiency mediated by desferrioxamine treatment. Based on the findings, FKN enhances the expression of iron metabolism-related genes in iron deficiency and modifies the iron uptake via transferrin receptor 1 and divalent metal transporter-1, and iron release via ferroportin. FKN can activate the release of iron from heme-containing proteins by elevating the level of heme oxygenase-1, contributing to the redistribution of intracellular iron content. It was revealed that the endometrium cells express both mitoferrin-1 and 2 and that their levels are not dependent on the iron availability of the cells. FKN may also contribute to maintaining mitochondrial iron homeostasis. FKN can improve the deteriorating effect of iron deficiency in HEC-1A endometrium cells, which may contribute to the development of receptivity and/or provide iron delivery towards the embryo.
Assuntos
Quimiocina CX3CL1 , Deficiências de Ferro , Gravidez , Feminino , Humanos , Quimiocina CX3CL1/genética , Quimiocina CX3CL1/metabolismo , Endométrio/metabolismo , Implantação do Embrião/fisiologia , Ferro/metabolismoRESUMO
Vitamin D3 (VD) is crucial for various cell functions, including gene regulation, antioxidant defense, and neural health. Neurodegenerative conditions are closely linked to the unfolded protein response (UPR), a mechanism reacting to endoplasmic reticulum (ER) stress. Iron metabolism is intricately associated with UPR and neurodegeneration. This study used SH-SY5Y neuroblastoma cells to investigate the relationship between UPR, iron metabolism, and VD. Different sequences of treatments (pre- and post-treatments) were applied using VD and thapsigargin (Tg), and various methods were used for evaluation, including real-time qPCR, Western blotting, ELISA, and iron content analysis. The findings indicate that VD affects UPR pathways, cytokine release, and iron-related genes, potentially offering anti-inflammatory benefits. It also influences iron transporters and storage proteins, helping to maintain cellular iron balance. Furthermore, pro-inflammatory cytokines like interleukin-6 (IL-6) and tumor necrosis factor alpha (TNFα) were impacting UPR activation in cells. VD also influenced fractalkine (CX3CL1) gene expression and secretion, suggesting its potential as a therapeutic agent for addressing neuroinflammation and iron dysregulation. This research provides insights into the intricate connections among VD, UPR, and iron metabolism in SH-SY5Y neuroblastoma cells, with implications for future investigations and potential therapeutic approaches in neurodegenerative diseases characterized by UPR dysregulation and iron accumulation.
Assuntos
Neuroblastoma , Doenças Neurodegenerativas , Humanos , Vitamina D , Neuroblastoma/tratamento farmacológico , Resposta a Proteínas não Dobradas , Estresse do Retículo Endoplasmático , Doenças Neurodegenerativas/tratamento farmacológico , Doenças Neurodegenerativas/metabolismo , Citocinas/metabolismo , Ferro/metabolismoRESUMO
Fractalkine (CX3CL1/FKN) is a unique chemokine belonging to the CX3C chemokine subclass. FKN exists in two forms: a membrane-bound form expressed by both endometrium cells and trophoblasts thought to be implicated in maternal-fetal interaction and a soluble form expressed by endometrium cells. Endometrium receptivity is crucial in embryo implantation and a complex process regulated by large numbers of proteins, e.g., cytokines, progesterone receptor (PR), SOX-17, prostaglandin receptors (PTGER2), and tissue inhibitors of metalloproteinases (TIMPs). It has also been reported that iron is important in fertility and affects the iron status of the mother. Therefore, iron availability in the embryo contributes to fertilization and pregnancy. In this study, we focused on the effect of iron deficiency on the secreted cytokines (IL-6, IL-1ß, leukocyte inhibitory factor, TGF-ß), chemokines (IL-8, FKN), and other regulatory proteins (bone morphogenic protein 2, activin, follistatin, PR, SOX-17, prostaglandin E2 receptor, TIMP2), and the modifying effect of FKN on the expression of these proteins, which may improve endometrium receptivity. Endometrial iron deficiency was mediated by desferrioxamine (DFO) treatment of HEC-1A cells. FKN was added to the cells 24 h and 48 h after DFO with or without serum for modelling the possible iron dependence of the alterations. Our findings support the hypothesis that FKN ameliorates the effects of anemia on the receptivity-related genes and proteins in HEC-1A cells by increasing the secretion of the receptivity-related cytokines via the fractalkine receptor (CX3CR1). FKN may contribute to cell proliferation and differentiation by regulating activin, follistatin, and BMP2 expressions, and to implantation by altering the protein levels of PR, SOX-17, PTGER2, and TIMP2. FKN mitigates the negative effect of iron deficiency on the receptivity-related genes and proteins of HEC-1A endometrium cells, suggesting its important role in the regulation of endometrium receptivity.
Assuntos
Quimiocina CX3CL1 , Deficiências de Ferro , Feminino , Humanos , Ativinas , Quimiocina CX3CL1/genética , Citocinas/genética , Desferroxamina/farmacologia , Endométrio , Folistatina , FerroRESUMO
Dietary lutein can be naturally metabolized to 3'-epilutein and 3'-oxolutein in the human body. The epimerization of lutein can happen in acidic pH, and through cooking, 3'-epilutein can be the product of the direct oxidation of lutein in the retina, which is also present in human serum. The 3'-oxolutein is the main oxidation product of lutein. Thus, the allylic oxidation of dietary lutein can result in the formation of 3'-oxolutein, which may undergo reduction either to revert to dietary lutein or epimerize to form 3'-epilutein. We focused on the effects of 3'-epilutein and 3'-oxolutein itself and on glutamate-induced neurotoxicity on SH-SY5Y human neuroblastoma cells to identify the possible alterations in oxidative stress, inflammation, antioxidant capacity, and iron metabolism that affect neurological function. ROS measurements were performed in the differently treated cells. The inflammatory state of cells was followed by TNFα, IL-6, and IL-8 cytokine ELISA measurements. The antioxidant status of the cells was determined by the total antioxidant capacity kit assay. The alterations of genes related to ferroptosis and lipid peroxidation were followed by gene expression measurements; then, thiol measurements were performed. Lutein metabolites 3'-epilutein and 3'-oxolutein differently modulated the effect of glutamate on ROS, inflammation, ferroptosis-related iron metabolism, and lipid peroxidation in SH-SY5Y cells. Our results revealed the antioxidant and anti-inflammatory features of 3'-epilutein and 3'-oxolutein as possible protective agents against glutamate-induced oxidative stress in SH-SY5Y cells, with greater efficacy in the case of 3'-epilutein.
Assuntos
Luteína , Neuroblastoma , Humanos , Antioxidantes/farmacologia , Antioxidantes/metabolismo , Ácido Glutâmico/toxicidade , Ácido Glutâmico/metabolismo , Espécies Reativas de Oxigênio , Cromatografia Líquida de Alta Pressão , Estresse Oxidativo , FerroRESUMO
Endometrium receptivity and successful implantation require a complex network of regulatory factors whom production is strictly controlled especially at the implantation window. Many regulators like steroid hormones, prostaglandins, cytokines, extracellular matrix proteins and downstream cell signalling pathways are involved in the process of embryo-endometrium interaction. Our work reveals the effect of fractalkine (FKN), a unique chemokine on progesterone receptor, SOX-17 and NRF2 expressions in HEC-1A endometrial cell line. FKN activates fractalkine receptor signalling and the expression of SOX-17 through progesterone receptor in HEC-1A endometrial cells, and as a consequence it increases endometrial receptivity. Fractalkine also activates the NRF2-Keap-1 signal transduction pathway regulating the IL-6 and IL-1ß cytokine productions, which increase endometrial receptivity, as well. The NRF2 transcription factor increases the expression of the iron exporter ferroportin in HEC-1A cells activating iron release towards JEG-3 trophoblast cells. The iron measurements show that iron content of endometrial cells decreases while heme concentration increases at FKN treatment. At the same time, the trophoblast cells show increased iron uptake and total iron content. Based on our results it seems that FKN enhances the establishment of endometrial receptivity and meanwhile it regulates the iron homeostasis of endometrium contributing to the iron availability of the trophoblast cells and the embryo.
Assuntos
Implantação do Embrião/fisiologia , Endométrio/citologia , Ferro/metabolismo , Trofoblastos/metabolismo , Linhagem Celular Tumoral , Quimiocina CX3CL1/metabolismo , Técnicas de Cocultura , Feminino , HumanosRESUMO
Fractalkine (CX3CL1) acts as a chemokine as well as a regulator of iron metabolism. Fractalkine binds CX3CR1, the fractalkine receptor on the surface of monocytes/macrophages regulating different intracellular signalling pathways such as mitogen-activated protein kinase (MAPK), phospholipase C (PLC) and NFκB contributing to the production of pro-inflammatory cytokine synthesis, and the regulation of cell growth, differentiation, proliferation and metabolism. In this study, we focused on the modulatory effects of fractalkine on the immune response and on the iron metabolism of Escherichia coli and Pseudomonas aeruginosa lipopolysaccharides (LPS) and Staphylococcus aureus lipoteichoic acid (LTA) activated THP-1 cells to get a deeper insight into the role of soluble fractalkine in the regulation of the innate immune system. Pro-inflammatory cytokine secretions of the fractalkine-treated, LPS/LTA-treated, and co-treated THP-1 cells were determined using ELISArray and ELISA measurements. We analysed the protein expression levels of signalling molecules regulated by CX3CR1 as well as hepcidin, the major iron regulatory hormone, the iron transporters, the iron storage proteins and mitochondrial iron utilization. The results showed that fractalkine treatment alone did not affect the pro-inflammatory cytokine secretion, but it was proposed to act as a regulator of the iron metabolism of THP-1 cells. In the case of two different LPS and one type of LTA with fractalkine co-treatments, fractalkine was able to alter the levels of signalling proteins (NFκB, PSTAT3, Nrf2/Keap-1) regulating the expression of pro-inflammatory cytokines as well as hepcidin, and the iron storage and utilization of the THP-1 cells.
Assuntos
Quimiocina CX3CL1 , Lipopolissacarídeos , Quimiocina CX3CL1/metabolismo , Citocinas/metabolismo , Hepcidinas/metabolismo , Ferro/metabolismo , Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/metabolismo , NF-kappa B/metabolismo , Ácidos TeicoicosRESUMO
The hormone hepcidin plays a central role in controlling iron homeostasis. Iron-mediated hepcidin synthesis is triggered via the BMP/SMAD pathway. At inflammation, mainly IL-6 pro-inflammatory cytokine mediates the regulation of hepcidin via the JAK/STAT signalling pathway. Microglial cells of the central nervous system are able to recognize a broad spectrum of pathogens via toll-like receptors and initiate inflammatory response. Although the regulation of hepcidin synthesis is well described in many tissues, little is known about the inflammation mediated hepcidin regulation in microglia. In this study, we investigated the pathways, which are involved in HAMP regulation in BV2 microglia due to inflammatory mediators and the possible relationships between the iron regulatory pathways. Our results showed that IL-6 produced by resting BV2 cells was crucial in maintaining the basal HAMP expression and hepcidin secretion. It was revealed that IL-6 neutralization decreased both STAT3 and SMAD1/5/9 phosphorylation suggesting that IL-6 proinflammatory cytokine is necessary to maintain SMAD1/5/9 activation. We revealed that IL-6 influences BMP6 and TMPRSS6 protein levels, moreover it modified TfR2 expression, as well. In this study, we revealed that BV2 microglia increased their hepcidin secretion upon IL-6 neutralization although the major regulatory pathways were inhibited. Based on our results it seems that both at inflammation and at normal condition the absence of IL-6 triggered HAMP transcription and hepcidin secretion via the NFκB pathway and possibly by the autocrine effect of TNFα cytokine on BV2 microglia.
Assuntos
Proteína Morfogenética Óssea 6/metabolismo , Hepcidinas/metabolismo , Interleucina-6/metabolismo , Proteínas de Membrana/metabolismo , Microglia/metabolismo , Receptores da Transferrina/metabolismo , Serina Endopeptidases/metabolismo , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Inflamação/induzido quimicamente , Inflamação/metabolismo , Lipopolissacarídeos/farmacologia , Camundongos , Microglia/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Proteínas Smad/metabolismo , Ácidos Teicoicos/farmacologiaRESUMO
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.
Assuntos
Parede Celular/química , Citocinas/metabolismo , Escherichia coli/química , Ferro/metabolismo , Lipopolissacarídeos/farmacologia , Pseudomonas aeruginosa/química , Staphylococcus aureus/química , Células THP-1/metabolismo , Ácidos Teicoicos/farmacologia , Transporte Biológico , Receptor 1 de Quimiocina CX3C/biossíntese , Receptor 1 de Quimiocina CX3C/genética , Quimiocina CX3CL1/metabolismo , Citocinas/biossíntese , Citosol/metabolismo , Ferritinas/biossíntese , Ferritinas/genética , Heme Oxigenase-1/biossíntese , Heme Oxigenase-1/genética , Hepcidinas/biossíntese , Hepcidinas/genética , Humanos , Mitocôndrias/metabolismo , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Oxirredutases/biossíntese , Oxirredutases/genética , RNA Mensageiro/biossíntese , RNA Neoplásico/genética , Células THP-1/efeitos dos fármacosRESUMO
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.
Assuntos
Quimiocina CX3CL1/farmacologia , Ativinas/metabolismo , Western Blotting , Proteína Morfogenética Óssea 2/metabolismo , Receptor 1 de Quimiocina CX3C/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Cocultura , Feminino , Folistatina/metabolismo , Humanos , Immunoblotting , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Quinases p38 Ativadas por Mitógeno/efeitos dos fármacosRESUMO
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.
Assuntos
Quimiocina CX3CL1/farmacologia , Hepcidinas/metabolismo , Ferro/metabolismo , Microglia/metabolismo , Animais , Receptor 1 de Quimiocina CX3C/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Regulação para Baixo/efeitos dos fármacos , Endocitose/efeitos dos fármacos , Ferritinas/metabolismo , Humanos , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Interleucina-6/biossíntese , Interleucina-6/genética , Proteínas de Membrana/metabolismo , Camundongos , Microglia/efeitos dos fármacos , Mitocôndrias/metabolismo , Modelos Biológicos , NF-kappa B/metabolismo , Fosfotirosina/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Serina Endopeptidases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , alfa 1-Antitripsina/metabolismoRESUMO
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.
Assuntos
Quelantes/toxicidade , Cuprizona/toxicidade , Ferro/metabolismo , Animais , Corpo Caloso/efeitos dos fármacos , Corpo Caloso/metabolismo , Homeostase , Metabolismo dos Lipídeos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
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.
Assuntos
Mediadores da Inflamação/metabolismo , Ferro/metabolismo , Lipopolissacarídeos/metabolismo , Microglia/metabolismo , Ácidos Teicoicos/farmacologia , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/metabolismo , Linhagem Celular Tumoral , Citocinas/genética , Citocinas/metabolismo , Expressão Gênica , Regulação da Expressão Gênica , Hepcidinas/genética , Hepcidinas/metabolismo , Humanos , Lipopolissacarídeos/farmacologia , Neurônios/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Treg abnormalities have been implicated in the pathogenesis of systemic sclerosis (SSc). Treg subpopulations and their cytokines, IL-10 and TGF-ß in the peripheral blood of early stage SSc patients were investigated. We hypothesized that epigenetically regulated methylation of the FOXP3 promoter and enhancer regions are altered in Tregs of SSc patients, which might be involved in the T cell imbalance. CD4+CD25+Foxp3+CD127- Treg cells were significantly elevated in patients with diffuse cutaneous SSc and in patients with anti-Scl-70/RNA-Pol-III autoantibody positivity and with lung fibrosis. Increased CD62L+ Treg cells were present in all SSc subgroups. The production of immunosuppressive cytokines by both CD127- and CD62L+ Tregs was diminished. We observed reduced methylation of Treg specific FOXP3 enhancer regions, and elevated FOXP3 gene expression in active SSc cases with negative correlation in the frequency of CD62L+IL-10+ Tregs. Our data indicate an inappropriate distribution and cytokine production of Treg cells in early form SSc.
Assuntos
Escleroderma Sistêmico/imunologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , Adulto , Idoso , Anticorpos Antinucleares/imunologia , Metilação de DNA , DNA Topoisomerases Tipo I , Epigênese Genética , Feminino , Fatores de Transcrição Forkhead/genética , Expressão Gênica , Regulação da Expressão Gênica , Humanos , Interleucina-10/imunologia , Pessoa de Meia-Idade , Proteínas Nucleares/imunologia , Regiões Promotoras Genéticas , Fibrose Pulmonar/etiologia , Fibrose Pulmonar/imunologia , RNA Polimerase III/imunologia , Esclerodermia Difusa/complicações , Esclerodermia Difusa/imunologia , Esclerodermia Limitada/complicações , Esclerodermia Limitada/imunologia , Escleroderma Sistêmico/complicações , Fator de Crescimento Transformador beta/imunologiaRESUMO
BACKGROUND: The hypothalamus of the central nervous system is implicated in the development of diabetes due to its glucose-sensing function. Dysregulation of the hypothalamic glucose-sensing neurons leads to abnormal glucose metabolism. It has been described that fractalkine (FKN) is involved in the development of hypothalamic inflammation, which may be one of the underlying causes of a diabetic condition. Moreover, iron may play a role in the pathogenesis of diabetes via the regulation of hepcidin, the iron regulatory hormone synthesis. MicroRNAs (miRNAs) are short non-coding molecules working as key regulators of gene expression, usually by inhibiting translation. Hypothalamic miRNAs are supposed to have a role in the control of energy balance by acting as regulators of hypothalamic glucose metabolism via influencing translation. METHODS: Using a miRNA array, we analysed the expression of diabetes, inflammation, and iron metabolism related miRNAs in the hypothalamus of a streptozotocin-induced rat type 1 diabetes model. Determination of the effect of miRNAs altered by STZ treatment on the target genes was carried out at protein level. RESULTS: We found 18 miRNAs with altered expression levels in the hypothalamus of the STZ-treated animals, which act as the regulators of mRNAs involved in glucose metabolism, pro-inflammatory cytokine synthesis, and iron homeostasis suggesting a link between these processes in diabetes. The alterations in the expression level of these miRNAs could modify hypothalamic glucose sensing, tolerance, uptake, and phosphorylation by affecting the stability of hexokinase-2, insulin receptor, leptin receptor, glucokinase, GLUT4, insulin-like growth factor receptor 1, and phosphoenolpyruvate carboxykinase mRNA molecules. Additional miRNAs were found to be altered resulting in the elevation of FKN protein. The miRNA, mRNA, and protein analyses of the diabetic hypothalamus revealed that the iron import, export, and iron storage were all influenced by miRNAs suggesting the disturbance of hypothalamic iron homeostasis. CONCLUSION: It can be supposed that glucose metabolism, inflammation, and iron homeostasis of the hypothalamus are linked via the altered expression of common miRNAs as well as the increased expression of FKN, which contribute to the imbalance of energy homeostasis, the synthesis of pro-inflammatory cytokines, and the iron accumulation of the hypothalamus. The results raise the possibility that FKN could be a potential target of new therapies targeting both inflammation and iron disturbances in diabetic conditions.
RESUMO
The xanthophyll carotenoid lutein has been widely used as supplementation due to its protective effects in light-induced oxidative stress. Its antioxidant and anti-inflammatory features suggest that it has a neuroprotective role as well. Glutamate is a major excitatory neurotransmitter in the central nervous system (CNS), which plays a key role in regulating brain function. Excess accumulation of intracellular glutamate accelerates an increase in the concentration of reactive oxygen species (ROS) in neurons leading to glutamate neurotoxicity. In this study, we focused on the effects of glutamate on SH-SY5Y neuroblastoma cells to identify the possible alterations in oxidative stress, inflammation, and iron metabolism that affect the neurological function itself and in the presence of antioxidant lutein. First, ROS measurements were performed, and then catalase (CAT) and Superoxide Dismutase (SOD) enzyme activity were determined by enzyme activity assay kits. The ELISA technique was used to detect proinflammatory TNFα, IL-6, and IL-8 cytokine secretions. Alterations in iron uptake, storage, and release were followed by gene expression measurements and Western blotting. Total iron level detections were performed by a ferrozine-based iron detection method, and a heme assay kit was used for heme measurements. The gene expression toward lipid-peroxidation was determined by RT-PCR. Our results show glutamate changes ROS, inflammation, and antioxidant enzyme activity, modulate iron accumulation, and may initiate lipid peroxidation in SH-SY5Y cells. Meanwhile, lutein attenuates the glutamate-induced effects on ROS, inflammation, iron metabolism, and lipid peroxidation. According to our findings, lutein could be a beneficial, supportive treatment in neurodegenerative disorders.
RESUMO
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.
RESUMO
OBJECTIVE: Functional disturbances in regulatory T cells (Treg) have been described in autoimmune diseases, and their potential therapeutic use is intensively studied. Our goal was to investigate the influence of glucocorticoid hormone on the in vitro differentiation of Treg cells from thymic and splenic CD4+ T cells under different conditions to establish methods for generating stable and functionally suppressive iTregs for future use in adoptive transfer experiments. METHODS: Thymic and splenic CD4+ T lymphocytes were isolated from 3 to 4 week-old control and in vivo dexamethasone (DX) pretreated BALB/c mice using magnetic bead negative selection, followed by CD25 positive selection. The cells were cultured with anti-CD3/CD28 beads and IL-2 in the presence or absence of TGFß and/or DX for 3-6 days. Multiparametric flow cytometry was performed using CD4, CD25, CD8, TGFß (LAP) cell surface and Foxp3, IL-4, IL-10, IL-17 and IFNγ intracellular staining. Quantitative RT-PCR was performed to measure IL-10, TGFß cytokine and Foxp3 mRNA levels. RESULTS: Differentiation of thymus-derived CD4+ cells in vitro into iTreg cells was most effective (24-25%) when anti-CD3/CD28 beads, IL-2, and TGFß were present. Splenic CD4+ T cell expansion under same conditions resulted in a higher (44-45%) iTreg cell ratio that further increased (up to 50% Treg) in the presence of DX. Elevated immunosuppressive cytokine (IL-10 and TGFß) production by iTregs could be measured both at protein and mRNA levels without elevation of Th1/Th2 or Th17 cytokine production. We got the highest iTreg ratio (74%) and TGFß production when CD4+CD25+ splenic T cells were stimulated in the presence of TGFß. In vivo 4 days DX pretreatment resulted in enhanced in vitro expansion and Foxp3 expression of thymus-derived iTregs and decreased differentiation of spleen-derived iTreg cells. In these Tregs the relative expression of IL-10 mRNA significantly decreased under all in vitro stimulation conditions, while TGFß mRNA level did not change. CONCLUSION: DX promotes the expansion of thymic and splenic Treg cells, and enhances Foxp3+ expression and the production of immunosuppressive cytokines IL-10 and TGFß in vitro. In vivo pretreatment of mice with DX inhibited the immunosuppressive cytokine production of in vitro differentiated Treg cells. We hypothesize that patients receiving GC therapy may need special attention prior to in vitro expansion and transplantation of Treg cells.
Assuntos
Citocinas/metabolismo , Glucocorticoides/metabolismo , Imunomodulação , Baço/citologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Timo/citologia , Animais , Biomarcadores , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Células Cultivadas , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Glucocorticoides/farmacologia , Imunomodulação/efeitos dos fármacos , Imunofenotipagem , Camundongos , Baço/imunologia , Baço/metabolismo , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Linfócitos T Reguladores/efeitos dos fármacos , Timo/imunologia , Timo/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta/farmacologiaRESUMO
OBJECTIVE: Despite the fact that glucocorticoids (GC) are important therapeutic tools, their effects on regulatory T cells (Treg) are not well defined. The aim of our work was to investigate how GCs influence in vivo the thymic (tTreg) and peripheral Treg (pTreg) differentiation, survival and cytokine production. METHODS: Tregs were detected with flow cytometry in lymphatic organs of 4-6 weeks old BALB/c mice after repeated (2-4days), high-dose in vivo GC treatment using CD4/CD25 cell surface and Foxp3/IL-10/TGFß/glucocorticoid receptor (GR) intracellular staining. Cytokine, Foxp3, and GR mRNA levels of sorted CD4+CD25high T cells were analyzed using RT-PCR. Foxp3 and GR localization in Treg cells was investigated with confocal microscopy. RESULTS: GC treatment of mice resulted in increased relative tTreg frequency in the thymus, which was due to decreased total thymocyte numbers with unchanged absolute tTreg cell count. In contrast the relative pTreg cell ratio in secondary lymphatic organs decreased or showed no changes after GC treatment, while the absolute number of pTregs decreased. Elevated intracellular IL-10+ and TGFß+ tTreg and pTreg ratios were measured in GC-treated animals, accompanied with elevated Foxp3 mRNA expression. In addition, GC treatment caused increased TGFß and IL-35 mRNA expression in CD4+CD25high+ splenic and elevated IL-10 mRNA level in thymic tTregs. GR expression of thymic tTreg cells was lower than in pTregs. GC treatment caused an opposite change in GR levels, elevating GR in tTregs but decreasing it in pTregs. We observed a nuclear localization of GR in both tTregs and pTregs, which showed high colocalization (â¼60%) with Foxp3 transcription factor. These data suggest an interaction of these two transcription factors with further increase due to GC treatment in splenic pTregs. CONCLUSION: 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.