globotriaosylceramide and Inflammation

Fabry disease (FD) is a lysosomal storage disorder, depending on defects in alpha-galactosidase A (GAL) activity. At the clinical level, FD shows a high phenotype variability. Among them, cardiovascular dysfunction is often recurrent or, in some cases, is the sole symptom (cardiac variant) representing the leading cause of death in Fabry patients. The existing therapies, besides specific symptomatic treatments, are mainly based on the restoration of GAL activity. Indeed, mutations of the galactosidase alpha gene (GLA) cause a reduction or lack of GAL activity leading to globotriaosylceramide (Gb3) accumulation in several organs. However, several other mechanisms are involved in FD's development and progression that could become useful targets for therapeutics. This review discusses FD's cardiovascular phenotype and the last findings on molecular mechanisms that accelerate cardiac cell damage.

Topics: alpha-Galactosidase; Animals; Cardiovascular Diseases; Fabry Disease; Humans; Inflammation; Mitochondria; Mutation; Phenotype; Trihexosylceramides

Lysosomal storage diseases are usually considered to be pathologies in which the passive deposition of unwanted materials leads to functional changes in lysosomes. Lysosomal deposition of unmetabolized glycolipid substrates stimulates the activation of pathogenic cascades, including immunological processes, and particularly the activation of inflammation. In lysosomal storage diseases, the inflammatory response is continuously being activated because the stimulus cannot be eliminated. Consequently, inflammation becomes a chronic process. Lysosomes play a role in many steps of the immune response. Leukocyte perturbation and over-expression of immune molecules have been reported in Fabry disease. Innate immunity is activated by signals originating from dendritic cells via interactions between toll-like receptors and globotriaosylceramide (Gb3) and/or globotriaosylsphingosine (lyso-Gb3). Evidence indicates that these glycolipids can activate toll-like receptors, thus triggering inflammation and fibrosis cascades. In the kidney, Gb3 deposition is associated with the increased release of transforming growth factor beta and with epithelial-to-mesenchymal cell transition, leading to the over-expression of pro-fibrotic molecules and to renal fibrosis. Interstitial fibrosis is also a typical feature of heart involvement in Fabry disease. Endomyocardial biopsies show infiltration of lymphocytes and macrophages, suggesting a role for inflammation in causing tissue damage. Inflammation is present in all tissues and may be associated with other potentially pathologic processes such as apoptosis, impaired autophagy, and increases in pro-oxidative molecules, which could all contribute synergistically to tissue damage. In Fabry disease, the activation of chronic inflammation over time leads to organ damage. Therefore, enzyme replacement therapy must be started early, before this process becomes irreversible.

Topics: Animals; Apoptosis; Autophagy; Dendritic Cells; Enzyme Replacement Therapy; Fabry Disease; Humans; Immunity, Innate; Inflammation; Kidney Diseases; Lysosomes; Mice; Toll-Like Receptors; Trihexosylceramides

Topics: Caspase 3; Caspase 7; Cell Death; Endoplasmic Reticulum; Endoplasmic Reticulum Stress; Exosomes; Humans; Inflammation; Interleukin-1beta; Interleukin-8; Leukocytes, Mononuclear; Macrophages; Mitogen-Activated Protein Kinases; Shiga Toxin 2; THP-1 Cells; Trihexosylceramides

Shiga toxin (Stx) produced by enterohemorrhagic

Topics: Animals; Brain Diseases; Cytokines; Disease Models, Animal; Enterohemorrhagic Escherichia coli; Escherichia coli Infections; Heat-Shock Response; Hemolytic-Uremic Syndrome; Inflammation; Lipopolysaccharides; Macrophages; Microglia; Rats; Rats, Wistar; Shiga Toxin 2; Trihexosylceramides

Alteration of glycosphingolipid (GSL) expression plays key roles in the pathogenesis and pathophysiology of many important human diseases, including cancer, diabetes and glycosphingolipidosis. Inflammatory processes are involved in development and progression of diabetic nephropathy, a major complication of type 2 diabetes mellitus. GSLs are known to play roles in inflammatory responses in various diseases, and levels of renal GSLs are elevated in mouse models of diabetic nephropathy; however, little is known regarding the pathophysiological role of these GSLs in this disease process. We studied proinflammatory activity of GSLs in diabetic nephropathy using spontaneously diabetic mouse strain KK. Mice were fed a high-fat diet (HFD) (60% kcal from fat) or normal diet (ND) (4.6% kcal from fat) for a period of 8 wk. HFD-feeding resulted in quantitative and qualitative changes of renal globo-series GSLs (particularly Gb3Cer), upregulation of TNF-α, and induction of renal inflammation. Gb3Cer/Gb4Cer treatment enhanced inflammatory responses via TLR4 in TLR4/MD-2 complex expressing cells, including HEK293T, mouse bone marrow-derived macrophages (BMDMs) and human monocytes. Our findings suggest that HFD-induced increase of Gb3Cer/Gb4Cer positively modulate TLR4-mediated inflammatory response, and that such GSLs play an important pathophysiological role in diabetic nephropathy.

Topics: Animals; Diabetes Mellitus, Type 2; Diabetic Nephropathies; Diet, High-Fat; Disease Models, Animal; Disease Progression; Glycosphingolipids; HEK293 Cells; Humans; Inflammation; Kidney; Macrophages; Male; Mice; Signal Transduction; Toll-Like Receptor 4; Trihexosylceramides; Tumor Necrosis Factor-alpha

Fabry disease (FD) is an X-linked lysosomal storage disorder associated with pain triggered by heat or febrile infections. We modelled this condition by measuring the cytokine expression of peripheral blood mononuclear cells (PBMC) from FD patients in vitro upon stimulation with heat and lipopolysaccharide (LPS). We enrolled 67 FD patients and 37 healthy controls. We isolated PBMC, assessed their gene expression of selected pro- and anti-inflammatory cytokines, incubated them with heat, LPS, globotriaosylceramide (Gb3), and tumor necrosis factor-α (TNF), and measured TNF secretion in the supernatant and intracellular Gb3 accumulation, respectively. We found increased TNF, interleukin (IL-)1β, and toll-like receptor 4 (TLR4) gene expression in FD men (p < .05 to p < .01). TNF and IL-10 were higher, and IL-4 was lower in the subgroup of FD men with pain compared to controls (p < .05 to p < .01). Hereby, TNF was only increased in FD men with pain and classical mutations (p < .05) compared to those without pain. PBMC from FD patients secreted more TNF upon stimulation with LPS (p < .01) than control PBMC. Incubation with Gb3 and an additional α-galactosidase A inhibitor did not further increase TNF secretion, but incubation with TNF greatly increased the Gb3 load in FD PBMC compared to controls (p < .01). Also, LPS incubation and heat challenge (40 °C) increased Gb3 accumulation in PBMC of patients compared to baseline (p < .05 each), while no alterations were observed in control PBMC. Our data show that TNF holds a crucial role in the pathophysiology of FD associated pain, which may open a novel perspective for analgesic treatment in FD pain.

Topics: Adolescent; Adult; Aged; Case-Control Studies; Cytokines; Fabry Disease; Female; Gene Expression; Hot Temperature; Humans; Inflammation; Leukocytes, Mononuclear; Lipopolysaccharides; Male; Middle Aged; Trihexosylceramides; Tumor Necrosis Factor-alpha; Young Adult

Shiga toxin (Stx)-mediated immune responses, including the production of the proinflammatory cytokines tumor necrosis-α (TNF-α) and interleukin-1β (IL-1β), may exacerbate vascular damage and accelerate lethality. However, the immune signaling pathway activated in response to Stx is not well understood. Here, we demonstrate that enzymatically active Stx, which leads to ribotoxic stress, triggers NLRP3 inflammasome-dependent caspase-1 activation and IL-1β secretion in differentiated macrophage-like THP-1 (D-THP-1) cells. The treatment of cells with a chemical inhibitor of glycosphingolipid biosynthesis, which suppresses the expression of the Stx receptor globotriaosylceramide and subsequent endocytosis of the toxin, substantially blocked activation of the NLRP3 inflammasome and processing of caspase-1 and IL-1β. Processing and release of both caspase-1 and IL-1β were significantly reduced or abolished in Stx-intoxicated D-THP-1 cells in which the expression of NLRP3 or ASC was stably knocked down. Furthermore, Stx mediated the activation of caspases involved in apoptosis in an NLRP3- or ASC-dependent manner. In Stx-intoxicated cells, the NLRP3 inflammasome triggered the activation of caspase-8/3, leading to the initiation of apoptosis, in addition to caspase-1-dependent pyroptotic cell death. Taken together, these results suggest that Stxs trigger the NLRP3 inflammasome pathway to release proinflammatory IL-1β as well as to promote apoptotic cell death.

Topics: Calcium-Binding Proteins; CARD Signaling Adaptor Proteins; Carrier Proteins; Caspase 1; Caspase 3; Caspase 8; Cell Line, Tumor; Cytoskeletal Proteins; DNA-Binding Proteins; Enzyme Activation; Glycosphingolipids; Humans; Inflammation; Interleukin-1beta; NLR Family, Pyrin Domain-Containing 3 Protein; Pyroptosis; RNA Interference; RNA, Small Interfering; Shiga Toxins; Shiga-Toxigenic Escherichia coli; Signal Transduction; Trihexosylceramides; Tumor Necrosis Factor-alpha

Symptomatic or asymptomatic Shiga toxin producing Escherichia coli (STEC) infections during early pregnancy may cause maternal or fetal damage mediated by Shiga toxin type 2 (Stx2). The aim of this study is to elucidate the mechanisms responsible for early pregnancy loss in rats treated with Stx2.. Sprague Dawley pregnant rats were intraperitoneally injected at day 8 of gestation with a sublethal dose (0.5 ng of Stx2/g of total body weight, 250 μl) of purified Stx2. Control rats were injected with the same volume of PBS. The expression of globotriaosylceramide (Gb3) glycosphingolipid receptor for Stx2 was evaluated by thin-layer chromatography (TLC). Regions of hypoxia in decidual tissue were determined by pimonidazole immunohistochemistry and vascular endothelial growth factor (VEGF) expression by Western blot and immunohistochemistry. Tumor necrosis factor-alpha (TNF-α) levels in serum and decidual tissue were evaluated by ELISA. Serum progesterone levels were determined by RIA.. Decidual tissue from both, control and Stx2-treated rats showed similar expression of Gb3 receptor. Intrauterine growth restriction was observed in Stx2-treated rats, associated with hypoxia and an increase of decidual TNF-α levels. Decrease of serum progesterone levels and decidual VEGF expression were also demonstrated.. Our findings indicate that Stx2 reaches the uteroplacental unit, binds Gb3 and triggers damage in decidual tissue. Poor oxygen supply accompanied with damage in the uteroplacental unit and inflammation could be responsible for the early pregnancy loss. Decrease in the pregnancy protective factors, serum progesterone and local VEGF, may contribute to the pregnancy loss.

Topics: Abortion, Spontaneous; Animals; Female; Hypoxia; Inflammation; Pregnancy; Rats; Rats, Sprague-Dawley; Shiga Toxin 2; Trihexosylceramides; Tumor Necrosis Factor-alpha; Vascular Endothelial Growth Factor A

Fabry disease is a lysosomal storage disease belonging to the group of sphingolipidoses. In Fabry disease there is accumulation of mainly globotriaosylceramide due to deficiency of the lysosomal enzyme α-galactosidase A. The lysosome is an important compartment for the activity of invariant natural killer T (iNKT) cells. iNKT cells are lipid-specific T cells that were shown to be important in infection, autoimmunity and tumor surveillance. In several mouse models of lysosomal storage disorders there is a decrease in iNKT cell numbers. Furthermore, alterations on iNKT cell subsets have been recently described in the Fabry disease mouse model. Herein, we analyzed iNKT cells and their subsets in Fabry disease patients. Although there were no differences in the percentage of iNKT cells between Fabry disease patients and control subjects, Fabry disease patients presented a reduction in the iNKT CD4(+) cells accompanied by an increase in the iNKT DN cells. Since iNKT cell subsets produce different quantities of pro-inflammatory and anti-inflammatory cytokines, we analyzed IFN-γ and IL-4 production by iNKT cells of Fabry disease patients and mice. We found a significant reduction in the production of IL-4 by mice splenic iNKT cells and human iNKT cell subsets, but no significant alterations in the production of IFN-γ. Altogether, our results suggest a bias towards a pro-inflammatory phenotype in Fabry disease iNKT cells.

Topics: alpha-Galactosidase; Animals; CD4-Positive T-Lymphocytes; Fabry Disease; Humans; Inflammation; Interferon-gamma; Interleukin-4; Leukocytes, Mononuclear; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Mice, Knockout; Natural Killer T-Cells; Trihexosylceramides

Fabry disease is an X-linked lysosomal disorder (LD) due to deficiency of the enzyme α-galactosidase A (αGal), which leads to the accumulation of neutral glycosphingolipids, mainly globotriaosylceramide (Gb3). Several mechanisms contribute to the diverse physiopathological alterations observed in this disease, and it has been suggested that an underlying proinflammatory state could play a significant role. The aim of this study is to investigate the presence of a proinflammatory state in the different subsets of peripheral blood mononuclear cells (PBMC) and to understand the mechanisms that contribute to its onset and perpetuation. We have shown that cultured PBMC from Fabry patients present a higher proinflammatory cytokine expression and production. Moreover, we determined that among PBMC, dendritic cells and monocytes present a basal proinflammatory cytokine production profile, which is further exacerbated with an inflammatory stimulus. Finally we established that normal, monocyte-derived dendritic cells and macrophages display the same proinflammatory profile when cultured in the presence of Gb3 and an inhibitor of αGal. Furthermore, this effect can be abolished using a TLR4 blocking antibody, indicating that TLR4 is necessary in the process. In summary, our results demonstrate the presence of a proinflammatory state involving two key subsets of innate immunity, and provide direct evidence of Gb3 having a proinflammatory role, likely mediated by TLR4, a finding that could help in the understanding of the underlying causes of the inflammatory pathogenesis of Fabry disease.

Topics: Adolescent; Adult; Aged; alpha-Galactosidase; Child; Child, Preschool; Cytokines; Dendritic Cells; Fabry Disease; Female; Humans; Inflammation; Leukocytes, Mononuclear; Macrophages; Male; Middle Aged; Toll-Like Receptor 4; Trihexosylceramides

Fabry disease is an X-linked inborn error of glycosphingolipid catabolism due to deficient activity of α-galactosidase A that leads to accumulation of the enzyme substrates, mainly globotriaosylceramide (Gb3), in body fluids and lysosomes of many cell types. Some pathophysiology hypotheses are intimately linked to reactive species production and inflammation, but until this moment there is no in vivo study about it. Hence, the aim of this study was to investigate oxidative stress parameters, pro-inflammatory cytokines and Gb3 levels in Fabry patients under treatment with enzyme replacement therapy (ERT) and finally to establish a possible relation between them. We analyzed urine and blood samples of patients under ERT (n=14) and healthy age-matched controls (n=14). Patients presented decreased levels of antioxidant defenses, assessed by reduced glutathione (GSH), glutathione peroxidase (GPx) activity and increased superoxide dismutase/catalase (SOD/CAT) ratio in erythrocytes. Concerning to the damage to biomolecules (lipids and proteins), we found that plasma levels of malondialdehyde (MDA) and protein carbonyl groups and di-tyrosine (di-Tyr) in urine were increased in patients. The pro-inflammatory cytokines IL-6 and TNF-α were also increased in patients. Urinary Gb3 levels were positively correlated with the plasma levels of IL-6, carbonyl groups and MDA. IL-6 levels were directly correlated with di-Tyr and inversely correlated with GPx activity. This data suggest that pro-inflammatory and pro-oxidant states occur, are correlated and seem to be induced by Gb3 in Fabry patients.

Topics: Adult; alpha-Galactosidase; Antioxidants; Catalase; Enzyme Replacement Therapy; Erythrocytes; Fabry Disease; Female; Glutathione; Glutathione Peroxidase; Humans; Inflammation; Interleukin-6; Male; Malondialdehyde; Middle Aged; Oxidative Stress; Reactive Oxygen Species; Superoxide Dismutase; Trihexosylceramides; Tumor Necrosis Factor-alpha; Tyrosine; Young Adult

Daphnetin extracted from Daphne odora Var. marginata contains coumarin compounds, which possess properties of analgesic and anti-inflammatory effects. In this study, we investigated the therapeutic effect of daphnetin on anti-arthritis and its role on the balance of Tregs and Th17, using a collagen-induced arthritis rat model. Collagen-induced arthritis rats were treated with daphnetin for 21 days. The therapeutic effects of daphnetin were evaluated by clinical symptoms and histopathology. The levels of Th17-, Treg-, Th2-, Th1-type cytokines in serum were determined by ELISA. The expression levels of related receptors RORγt, NF-κB, Foxp3 and CD77 in joint tissues were detected by immunohistochemistry. Our results showed that administration of daphnetin significantly alleviated the severity of the arthritis, as evidenced by the reduction of arthritis scores, suppression of the infiltration of inflammatory cells and prevention of synovial hyperplasia, thereby resulting in the joint destruction in the arthritis rats. Additionally, daphnetin treatment reduced the serum level of Th17-, Th2- and Th1-type in collagen-induced arthritis rats. Correspondingly, the expression of RORγt, NF-κB and CD77 in joint tissue of collagen-induced arthritis rats was remarkably decreased, while the expression of Foxp3 and IL-10 was remarkably increased after being administered with daphnetin. Collectively, this study demonstrated that administration of daphnetin attenuated the clinical symptoms and pathological destruction of arthritis joints. The therapeutic effects were associated with the up-regulation of Tregs, down-regulation of Th17-, Th2- and Th1-type cell responses. The results provide novel evidence that daphnetin has therapeutic effects on autoimmune arthritis through modulating the balance of Tregs and Th17.

Topics: Animals; Arthritis, Experimental; Autoimmune Diseases; Cattle; Collagen Type II; Cytokines; Daphne; Disease Progression; Down-Regulation; Female; Forkhead Transcription Factors; Inflammation; NF-kappa B; Nuclear Receptor Subfamily 1, Group F, Member 3; Rats; Rats, Wistar; T-Lymphocytes, Regulatory; Th1 Cells; Th17 Cells; Th2 Cells; Trihexosylceramides; Umbelliferones; Up-Regulation

Shiga toxins (Stxs) are the major agents responsible for hemorrhagic colitis and hemolytic-uremic syndrome (HUS) during infections caused by Stx-producing Escherichia coli (STEC) such as serotype O157:H7. Central nervous system (CNS) involvement is an important determinant of mortality in diarrhea associated-HUS. It has been suggested that vascular endothelial injuries caused by Stxs play a crucial role in the development of the disease. The current study investigates the relationship between the cytotoxic effects of Stxs and inflammatory responses in a rabbit brain treated with Stx2.. In a rabbit model treated with purified Stx2 or PBS(-), we examined the expression of the Stx receptor globotriaosylceramide (Gb3)/CD77 in the CNS and microglial activation using immunohistochemistry. The relationship between inflammatory responses and neuronal cell death was analyzed by the following methods: real time quantitative reverse transcriptase (RT)-polymerase chain reaction (PCR) to determine the expression levels of pro-inflammatory cytokines, and the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) method to detect apoptotic changes.. Gb3/CD77 expression was detected in endothelial cells but not in neurons or glial cells. In the spinal cord gray matter, significant levels of Gb3/CD77 expression were observed. Severe endothelial injury and microvascular thrombosis resulted in extensive necrotic infarction, which led to acute neuronal damage. Conversely, in the brain, Stx receptor expression was much lower. The observed neuropathology was less severe. However, neuronal apoptosis was observed at the onset of neurological symptoms, and the number of apoptotic cells significantly increased in the brain at a later stage, several days after onset. Microglial activation was observed, and tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta mRNA in the CNS parenchyma was significantly up-regulated. There was significant overexpression of TNF-alpha transcripts in the brain.. This study indicates that Stx2 may not directly damage neural cells, but rather inflammatory responses occur in the brain parenchyma in response to primary injury by Stx2 in vascular endothelial cells expressing Gb3/CD77. These findings suggest that neuroinflammation may play a critical role in neurodegenerative processes during STEC infection and that anti-inflammatory intervention may have therapeutic potential.

Topics: Animals; Apoptosis; Central Nervous System Diseases; Disease Models, Animal; Dose-Response Relationship, Drug; Endothelium, Vascular; Inflammation; Interleukin-1beta; Male; Neuroglia; Neurons; Rabbits; RNA, Messenger; Shiga Toxin 2; Trihexosylceramides; Tumor Necrosis Factor-alpha; Up-Regulation

Neurologic abnormalities are among the most serious extraintestinal complications of infection with Shiga toxin (Stx)-producing bacteria. Histopathologic examination of tissues from patients with extraintestinal sequelae suggested that Stxs damage endothelial cells. It is shown here that human brain microvascular endothelial cells (HBMECs) are relatively resistant to purified Stxs (50% cytotoxic doses [CD50s] >/=10 microgram/mL). Pretreatment of HBMECs with tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, n-butyric acid, or a cAMP analogue resulted in a 103- to 104-fold decrease in CD50 values and a 2- to 4-fold increase in fluoresceinated Stx binding to HBMECs. Treatment of HBMECs with lipopolysaccharides did not significantly alter cytotoxicity or toxin binding. TNF-alpha and IL-1beta treatment was associated with the increased HBMEC expression of the toxin-binding glycolipid globotriaosylceramide. HBMECs did not produce IL-1beta and produced only trace amounts of TNF-alpha when stimulated with purified Stx1 in vitro.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Adolescent; Bacterial Toxins; Cell Survival; Cerebral Cortex; Cerebrovascular Circulation; Cytotoxins; Endothelium, Vascular; Glycolipids; Humans; Inflammation; Interleukin-1; Lipopolysaccharides; Male; Microcirculation; Recombinant Proteins; Shiga Toxins; Shigella; Trihexosylceramides; Tumor Necrosis Factor-alpha