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The immune microenvironment in breast cancer (BCa) is controlled by a complex network of communication between various cell types. Here, we find that recruitment of B lymphocytes to BCa tissues is controlled via mechanisms associated with cancer cell-derived extracellular vesicles (CCD-EVs). Gene expression profiling identifies the Liver X receptor (LXR)-dependent transcriptional network as a key pathway that controls both CCD-EVs-induced migration of B cells and accumulation of B cells in BCa tissues. The increased accumulation oxysterol ligands for LXR (i.e., 25-hydroxycholesterol and 27-hydroxycholesterol) in CCD-EVs is regulated by the tetraspanin 6 (Tspan6). Tspan6 stimulates the chemoattractive potential of BCa cells for B cells in an EV- and LXR-dependent manner. These results demonstrate that tetraspanins control intercellular trafficking of oxysterols via CCD-EVs. Furthermore, tetraspanin-dependent changes in the oxysterol composition of CCD-EVs and the LXR signaling axis play a key role in specific changes in the tumor immune microenvironment.

Oxysterols play important roles in development and diseases, but can be highly challenging to analyze. To ensure satisfactory measurements, oxysterols must typically be separated with chromatography prior to detection. Here, we will devote attention to the chromatography of oxysterols, focusing on gas chromatography and liquid chromatography. We will present the role of stationary phases, mobile phases, and dimensions and geometries of particles/columns. We discuss how these parameters may affect the chromatography, regarding factors such as speed and resolution. Finally, we present some less explored avenues for separation of oxysterols.

Elevated total cholesterol in midlife has been associated with increased risk of dementia in later life. We have previously shown that low-density lipoprotein (LDL) is more oxidized in the plasma of dementia patients, although total cholesterol levels are not different from those of age-matched controls. β-Amyloid (Aβ) peptide, which accumulates in Alzheimer disease (AD), arises from the initial cleavage of amyloid precursor protein by β-secretase-1 (BACE1). BACE1 activity is regulated by membrane lipids and raft formation. Given the evidence for altered lipid metabolism in AD, we have investigated a mechanism for enhanced Aβ production by SH-SY5Y neuronal-like cells exposed to oxidized LDL (oxLDL). The viability of SH-SY5Y cells exposed to 4 μg oxLDL and 25 µM 27-hydroxycholesterol (27OH-C) was decreased significantly. Lipids, but not proteins, extracted from oxLDL were more cytotoxic than oxLDL. In parallel, the ratio of reduced glutathione (GSH) to oxidized glutathione was decreased at sublethal concentrations of lipids extracted from native and oxLDL. GSH loss was associated with an increase in acid sphingomyelinase (ASMase) activity and lipid raft formation, which could be inhibited by the ASMase inhibitor desipramine. 27OH-C and total lipids from LDL and oxLDL independently increased Aβ production by SH-SY5Y cells, and Aβ accumulation could be inhibited by desipramine and by N-acetylcysteine. These data suggest a mechanism whereby oxLDL lipids and 27OH-C can drive Aβ production by GSH depletion, ASMase-driven membrane remodeling, and BACE1 activation in neuronal cells., Graphical abstract, Highlights • Oxidized LDL lipids but not proteins oxidize SHSY-5Y cell glutathione, triggering lipid rafts. • Oxidized lipid-induced rafts promote β-secretase activity and β-amyloid secretion. • These effects are mimicked by 27-hydroxycholesterol present in oxidized LDL.

Oxysterols are oxidized derivatives of cholesterol that are formed enzymatically or via reactive oxygen species or both. Cholesterol or oxysterols ingested as food are absorbed and packed into lipoproteins that are taken up by hepatic cells. Within hepatic cells, excess cholesterol is metabolised to form bile acids. The endoplasmic reticulum acts as the main organelle in the bile acid synthesis pathway. Metabolised sterols originating from this pathway are distributed within other organelles and in the cell membrane. The alterations to membrane oxysterol:sterol ratio affects the integrity of the cell membrane. The presence of oxysterols changes membrane fluidity and receptor orientation. It is well documented that hydroxylase enzymes located in mitochondria facilitate oxysterol production via an acidic pathway. More recently, the presence of oxysterols was also reported in lysosomes. Peroxisomal deficiencies favour intracellular oxysterols accumulation. Despite the low abundance of oxysterols compared to cholesterol, the biological actions of oxysterols are numerous and important. Oxysterol levels are implicated in the pathogenesis of multiple diseases ranging from chronic inflammatory diseases (atherosclerosis, Alzheimer's disease and bowel disease), cancer and numerous neurodegenerative diseases. In this article, we review the distribution of oxysterols in sub-cellular organelles and in biological fluids.

Oxidation and S-nitrosylation of cysteinyl thiols (Cys-SH) to sulfenic (Cys-SOH), sulfinic (Cys-SO2H), sulfonic acids (Cys-SO3H), disulphides and S-nitrosothiols are suggested as important post-translational modifications that can activate or deactivate the function of many proteins. Non-enzymatic post-translational modifications to cysteinyl thiols have been implicated in a wide variety of physiological and pathophysiological states but have been difficult to monitor in a physiological setting because of a lack of experimental tools. The purpose of this review is to bring together the approaches that have been developed for stably trapping cysteine either in its reduced or oxidised forms for enrichment and or subsequent mass spectrometric analysis. These tools are providing insight into potential targets for post-translational modifications to cysteine modification in vivo. This article is part of a Special Issue entitled: Special Issue: Posttranslational Protein modifications in biology and Medicine. © 2013.

T cell receptor signalling requires a reduced cell surface protein environment and increased reactive oxygen species (ROS) inhibit activation. The main objective of this study was to identify sulphenated proteins on the membrane of T cells at rest, during activation and under oxidising conditions. Naive CD4+ T cells were activated using anti-CD3/CD28 antibodies in the presence or absence H2O2 (20 µM) for 24 h. Activated CD4+ T cells in the presence of H2O2 show a significant decrease in CD25 expression by twofold and IL-2 secretion (0.67±0.14ng/ml versus 1.16±0.09ng/ml). NAC (10 mM) did not reverse the effect of H2O2 on IL-2 or CD25 after 24 h. CD4+ T cell-membrane proteins were isolated using MEM-PR kit after T cell activation with or without H2O2 and sulphenated proteins were captured by a dimedone based technique. Captured proteins were identified by LC-MS/MS and the table below shows the number of sulphenated proteins; Activation alone did not affect the number of sulphenated membrane proteins. Activation in the presence of H2O2, which associated with lower intracellular calcium flux, show a reduction in number of sulphenated proteins possibly due to disulphide bond formation.

Oxidized lipids have been recently recognized as important mediators that are involved in a wide range of cellular functions and stress responses. These lipid species are formed transiently and present in low concentrations in biological systems which creates challenges to detection techniques. Therefore, high quality of methods must be employed to improve understanding of the roles oxidised lipids. The recent development of high sensitivity mass spectrometry tools has enabled the field of lipidomics to reach a level that was impossible years ago. Due to a lack of bioinformatics tools to support high-throughput identification of previously unconsidered, oxidized lipids, the detection of oxidized lipids is mostly performed by targeted approaches. This approach can significantly improve the sensitivity for detection in limited biological sample volumes. In our laboratory, we have established mass spectrometry based targeted methods to analyse oxidised cholesterol (oxysterols), oxidised phospholipids and isoprostane species. Using these techniques, we have demonstrated discrete oxidised lipids fingerprints in plasma during health and chronic inflammatory diseases such as chronic kidney disease, Alzheimer's disease and hypercholesterolaemia (Dias et. al., Free Rad Biol 2018).

Alzheimer's disease (AD) aetiology is complex with gene and environmental risk factor interaction. Our recent studies have confirmed that oxLDL is higher in vascular dementia patients and correlated with cognitive function. We hypothesised that endothelial cells of the blood-brain barrier are critical mediators of systemic nutrient effects within the brain. Therefore we have studied the effect of 27 hydroxycholesterol and F2alpha isoprostane on microvascular endothelial cell redox state, inflammatory cytokines and regulatory microRNA (miR) profile. We showed that lipids from patients with dementia or hypercholesterolaemia release directional inflammatory molecular signatures via an endothelial redox state-dependent mechanism. miR expression in endothelial cells with and without oxidised lipid treatment was undertaken using an Agilent DNA microarray scanner and microarray data was analysed using GeneSpring GX software. Upregulated miR showed an increase in oxidative stress and inflammatory pathways. Downregulated pathways included growth factor signalling. Using qPCR, we determined that miR-144 and 146 which are anti-inflammatory and redox regulating modulators were decreased by oxidised lipids. A neurotrophic factor-targeting miR was increased in expression. These data highlight that oxidised lipids have important regulatory effects on endothelial microvascular cell function.

Oxidative stress has been implicated in the pathogenesis of many neurodegenerative diseases including Alzheimer’s disease. The transcription factor, Nrf2 (nuclear factor E2-related factor 2) that binds to the antioxidant responsive element (ARE) activates a battery of genes encoding enzymes and factors essential for neuronal survival. We have investigated the hypothesis that a downstream product of cyclooxygenase(COX-2), 15-deoxy-delta (12, 14)-prostagland in J2 (15d-PGJ2) has protective effects by activating the Nrf2 pathway during oxidative stress.Human neuroblastoma cells (SHSY5Y) were differentiated intoneuronal-like cells as described previously (Gimenez-Cassina et al.,2006). SHSY5Y cells were co-treated with 10 mM buthionine sulfoximine (BSO) 7 10 mM 15d-PGJ2. Cell viability was measured by MTT assay and cellular glutathione (GSH) levels were measured after treating cells for0.5-24 hours by GSH recycling assay. Cellular Nrf2 levels were determined by immunoblotting. IL-6 levels were measured by ELISA.15d-PGJ2 alone lowered GSH levels 30min after the treatment(12.870.64 nmol/mg protein) and returned to untreated control levels at 16hours (28.173.6 nmol/mg protein; Po0.01). Compared to intracellular GSH levels in untreated cells (27.871.8 nmol/mg protein) BSO treatment alone significantly decreased GSH (9.672.1 nmol/mg protein;Po0.001) but co-incubation with 15d-PGJ2 for 24 hours prevented the depletion elicited by BSO(21.372.7 nmol/mg protein). Compared to untreated cells BSO treatment decrease dIL-6 secretion (from 0.941.6ng/ml to 0.6971.3ng/ml) and total Nrf2 protein levels (by21%). Co-incubation with15d-PGJ2 for 24 hours with BSO did not change IL-6(0.6771.4ng/ml) or total Nrf2 level at any time point. This study suggests that neuronal toxicity resulting from glutathione depletion canbere stored by the induction of Nrf2-ARE pathway and the role of the Nrf2 signalling merits further investigation in neurodegenerative diseases.

During ageing an altered redox balance has been observed in both intracellular and extracellular compartments, primarily due to glutathione depletion and metabolic stress. Maintaining redox homeostasis is important for controlling proliferation and apoptosis in response to specific stimuli for a variety of cells. For T cells, the ability to generate specific response to antigen is dependent on the oxidation state of cell surface and cytoplasmic protein-thiols. Here we describe the effects of depleting intracellular glutathione concentration for T cell exofacial expression of thioredoxin 1 and IL-2 production, and have determined the distribution of Trx1 with ageing. Using buthionine sulfoximine to deplete intracellular glutathione in Jurkat T cells we show using Western blotting that cell surface thioredoxin-1 is lowered and that the response to the lectin phytohaemagglutinin measured by ELISA as IL-2 production is also decreased. Using flow cytometry we show that the distribution of Trx1 on primary CD4+ T cells is age-dependent, with lower surface Trx1 expression and greater variability of surface expression observed with age. Together these data suggest that a relationship exists between the intracellular redox compartment and exofacial surface. Redox imbalance may be important for impaired T cell function during ageing.

Elevated cholesterol in mid-life has been associated with increased risk of dementia in later life. We have previously shown that low density lipoprotein (LDL) is more oxidised in the plasma of dementia patients although total cholesterol levels remained unchanged [1]. We have investigated the hypothesis that amyloid beta production and neurodegeneration can be driven by oxidised lipids derived from LDL following the loss of blood brain barrier integrity with ageing. Therefore, we have investigated amyloid beta formation in SHSY5Y cells treated with LDL, minimally modified (ox) LDL, and lipids extracted from both forms of LDL. LDL-treated SHSY-5Y cell viability was not significantly decreased with up to 8 μg LDL/2 × 104 cells compared to untreated cells. However, 8 μg oxLDL protein/2 × 104 cells decreased the cell viability significantly by 33.7% (P < 0.05). A more significant decrease in cell viability was observed when treating cells with extracted lipids from 8 μg of LDL (by 32.7%; P < 0.01) and oxLDL (by 41%; P < 0.01). In parallel, the ratio of reduced to oxidised GSH was decreased; GSH concentrations were significantly decreased following treatment with 0.8 μg/ml oxLD-L (7.35 ± 0.58;P < 0.01), 1.6 μg/ml (5.27 ± 0.23; P < 0.001) and 4 μg/ml (5.31 ± 0.31; P < 0.001). This decrease in redox potential was associated with an increase acid sphingomyelinase activity and lipid raft formation which could be inhibited by desipramine; SHSY5Y cells treated with oxLDL, and lipids from LDL and oxLDL for 16 h showed significantly increased acid sphingomyelinase activity (5.32 ± 0.35; P < 0.05, 5.21 ± 0.6; P < 0.05, and 5.58 ± 0.44; P < 0.01, respectively) compared to control cells (2.96 ± 0.34). As amyloid beta production is driven by the activity of beta secretase and its association with lipid rafts, we investigated whether lipids from ox-LDL can influence amyloid beta by SHSY-5Y cells in the presence of oxLDL. Using ELISA and Western blot, we confirmed that secretion of amyloid beta oligomers is increased by SHSY-5Y cells in the presence of oxLDL lipids. These data suggest a mechanism whereby LDL, and more significantly oxLDL lipids, can drive amyloid beta production and cytotoxicity in neuronal cells. [1] Li L, Willets RS, Polidori MC, Stahl W, Nelles G, Sies H, Griffiths HR. Oxidative LDL modification is increased in vascular dementia and is inversely associated with cognitive performance. Free Radic Res. 2010 Mar; 44(3): 241–8.