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Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) can exert antidepressant, anti-inflammatory and neuroprotective properties, but the exact molecular mechanism underlying their effects is still not fully understood. We conducted both in vitro and clinical investigations to test which EPA or DHA metabolites are involved in these anti-inflammatory, neuroprotective and antidepressant effects. In vitro, we used the human hippocampal progenitor cell line HPC0A07/03C, and pre-treated cells with either EPA or DHA, followed by interleukin 1beta (IL1β), IL6 and interferon-alpha (IFN-α). Both EPA and DHA prevented the reduction in neurogenesis and the increase in apoptosis induced by these cytokines; moreover, these effects were mediated by the lipoxygenase (LOX) and cytochrome P450 (CYP450) EPA/DHA metabolites, 5-hydroxyeicosapentaenoic acid (HEPE), 4-hydroxydocosahexaenoic acid (HDHA), 18-HEPE, 20-HDHA, 17(18)-epoxyeicosatetraenoic acid (EpETE) and 19(20)-epoxydocosapentaenoic acid (EpDPA), detected here for the first time in human hippocampal neurones using mass spectrometry lipidomics of the supernatant. In fact, like EPA/DHA, co-treatment with these metabolites prevented cytokines-induced reduction in neurogenesis and apoptosis. Moreover, co-treatment with 17(18)-EpETE and 19(20)-EpDPA and the soluble epoxide hydroxylase (sEH) inhibitor, TPPU (which prevents their conversion into dihydroxyeicosatetraenoic acid (DiHETE)/ dihydroxydocosapentaenoic acid (DiHDPA) metabolites) further enhanced their neurogenic and anti-apoptotic effects. Interestingly, these findings were replicated in a sample of n = 22 patients with a DSM-IV Major Depressive Disorder, randomly assigned to treatment with either EPA (3.0 g/day) or DHA (1.4 g/day) for 12 weeks, with exactly the same LOX and CYP450 lipid metabolites increased in the plasma of these patients following treatment with their precursor, EPA or DHA, and some evidence that higher levels of these metabolites were correlated with less severe depressive symptoms. Overall, our study provides the first evidence for the relevance of LOX- and CYP450-derived EPA/DHA bioactive lipid metabolites as neuroprotective molecular targets for human hippocampal neurogenesis and depression, and highlights the importance of sEH inhibitors as potential therapeutic strategy for patients suffering from depressive symptoms.

Barrier integrity is central to the maintenance of healthy immunological homeostasis. Impaired skin barrier function is linked with enhanced allergen sensitization and the development of diseases such as atopic dermatitis (AD), which can precede the development of other allergic disorders, for example, food allergies and asthma. Epidemiological evidence indicates that children suffering from allergies have lower levels of dietary fibre-derived short-chain fatty acids (SCFA). Using an experimental model of AD-like skin inflammation, we report that a fermentable fibre-rich diet alleviates systemic allergen sensitization and disease severity. The gut-skin axis underpins this phenomenon through SCFA production, particularly butyrate, which strengthens skin barrier function by altering mitochondrial metabolism of epidermal keratinocytes and the production of key structural components. Our results demonstrate that dietary fibre and SCFA improve epidermal barrier integrity, ultimately limiting early allergen sensitization and disease development.The Graphical Abstract was designed using Servier Medical Art images ( https://smart.servier.com ).

Estimates of heritability are the first step in identifying a trait with substantial variation due to genetic factors. Large-scale genetic analyses can identify the DNA variants that influence the levels of circulating lipid species and the statistical technique Mendelian randomisation can use these DNA variants to address potential causality of these lipids in disease. We estimated the heritability of plasma eicosanoids, octadecanoids and docosanoids to identify those lipid species with substantial heritability. We analysed plasma lipid mediators in 31 White British families (196 participants) ascertained for high blood pressure and deeply clinically and biochemically phenotyped over a 25-year period. We found that the dihydroxyeicosatrienoic acid (DHET) species, 11,12-DHET and 14,15-DHET, products of arachidonic acid metabolism by cytochrome P450 (CYP) monooxygenase and soluble epoxide hydrolase (sEH), exhibited substantial heritability (h2 = 33%−37%; Padj

Background: Genome-wide association studies have reproducibly linked the Orosomucoid-like 3 (ORMDL3) gene to risk of asthma. ORMDL3 regulates sphingolipid synthesis and is rhythmically expressed. There is mounting evidence supporting the role of sphingolipid metabolism in asthma pathogenesis. Since asthma is a highly rhythmic disease, we investigated whether serum sphingolipids vary by time of day in patients with and without asthma. Methods: Ten individuals with allergic asthma (AA) and 10 healthy non-smokers (HV) completed overnight visits. Blood samples were taken every 6 hours over 24hr. Targetted lipidomics analysis was performed using ultra performance liquid chromatography with tandem mass spectrometry. Results: Five ceramides (CER) at 4am and two at 4pm were significantly higher in AA than HV (p Conclusion: Sphingolipids in asthma acquire circadian rhythmicity. Further study is needed to gain mechanistic insight into the role of clock control in ORMDL3-sphingolipid pathway, which may lead to novel therapeutic approach in asthma.

Acne skin demonstrates increased transepidermal water loss (TEWL) compared with healthy skin, which may be due, in part, to altered ceramide (CER) levels. We analysed ceramides in the stratum corneum of healthy and acne skin, and studied seasonal variation over the course of a year. Using ultraperformance liquid chromatography with electrospray ionisation and tandem mass spectrometry (UPLC/ESI-MS/MS), we identified 283 ceramides. Acne-affected skin demonstrated overall lower levels of ceramides, with notable reductions in CER[NH] and CER[AH] ceramides, as well as the acylceramides CER[EOS] and CER[EOH]; these differences were more apparent in the winter months. Lower ceramide levels reflected an increase in TEWL in acne, compared with healthy skin, which partly resolves in the summer. Individual ceramide species with 18-carbon 6-hydroxysphingosine (H) bases (including CER[N(24)H(18)], CER[N(26)H(18)], CER[A(24)H(18)], CER[A(26)H(18)]) were significantly reduced in acne skin, suggesting that CER[NH] and CER[AH] species may be particularly important in a healthy skin barrier.

Solar ultraviolet radiation (UVR) exposure of human skin has beneficial and harmful effects on health, including impact on immune function, inflammation and reportedly mood, but these are not fully elucidated. Since the endocannabinoid system is implicated in many activities including mood alteration, our objective was to (i) determine and quantify circulating levels of a wide range of endocannabinoid and N-acyl ethanolamine (NAE) species (ii) evaluate whether these are modulated by cutaneous UVR exposures, as attained through repeated low level summer sunlight exposure. Wearing goggles to prevent eye exposure, 16 healthy volunteers (23–59 y; 10 light skin, phototype II, and 6 dark skin, phototype V) received the same UVR exposures (1.3 SED, 95% UVA/5% UVB) thrice weekly for 6 weeks, whilst casually dressed to expose ∼35% skin surface area. Blood samples were taken at baseline, days 1, 3 and 5 of week one, then at weekly intervals, and analysed by LC-MS/MS. Eleven endocannabinoids and NAEs were detected and quantified at baseline, with N-palmitoyl ethanolamine the most abundant (30% of total). Levels did not vary according to phototype (p > 0.05), except for the NAE docosapentaenoyl ethanolamide, which was higher in phototype II than V (p = 0.0002). Level of the endocannabinoid, 2-AG, was elevated during the UVR exposure course (p < 0.05 vs. baseline for all subjects; p < 0.01 for each phototype group), with maximum levels reached by week 2–3, while NAE species did not significantly alter. These findings suggest differential involvement of the cutaneous endocannabinoid system in low dose solar UVR responses in humans.

The cause of chronic inflammation in ulcerative colitis (UC) is incompletely understood. Here we tested the hypothesis that an excessive acute inflammatory response to bacteria contributes to the pathogenesis. Acute inflammatory responses were provoked in vivo in UC patients and healthy controls by intradermal inoculation with bacteria. Vascular responses were quantified by laser Doppler. Inflammatory exudates were recovered in superimposed suction blisters and cells measured by polychromatic flow cytometry, cytokines by multiplex array, and inflammatory lipids by mass spectrometry. Vascular responses in UC patients were heightened at 24h after bacterial injection (p=0·03), and remained abnormally high at 48h (p=0·0005) and this amplified response was seen in UC with Gram-positive as well as Gram-negative organisms (p=0·01). The cellular infiltrate over the injection site, composed largely of neutrophils at 4 hours a was greater in UC (p=0·002). At 48h, the increased numbers of cells in UC were composed of neutrophils (p=0·001) and CD4 lymphocytes (p=0·001). The exaggerated inflammation in UC was not a cytokine-driven phenomenon. Exaggerated onset was normalised in patients taking 5-aminosalicylates, accompanied by increased concentrations of hydroxy fatty acids 9-oxo-octadecadienoic acid (OxoODE; p=0·05) and 13-OxoODE (p=0·01) in resolving exudates. In vitro, these compounds suppressed macrophage inflammatory cytokine secretion through PPARγ (pSummaryWysoczanski and colleagues demonstrate that the inflammatory response to injected bacteria is exaggerated and prolonged in ulcerative colitis. This disordered inflammation appears to be associated with increased secretion of PGE2. 5-aminosalicylate drugs, which are used to treat this condition, normalize inflammation and PGE2 secretion, and appear to work through PPARγ

Nutritional supplementation with fish oil or ω-3 (n-3) polyunsaturated fatty acids (PUFAs) has potential benefits for skin inflammation. Although the differential metabolism of the main n-3PUFA eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) could lead to distinct activities, there are no clinical studies comparing their relative efficacy in human skin. Following a 10-wk oral supplementation of healthy volunteers and using mass spectrometry-based lipidomics, we found that n-3PUFA mainly affected the epidermal mediator lipidome. EPA was more efficient than DHA in reducing production of arachidonic acid–derived lipids, and both n-3PUFA lowered N-acyl ethanolamines. In UV radiation–challenged skin (3 times the minimum erythemal dose), EPA attenuated the production of proinflammatory lipids, whereas DHA abrogated the migration of Langerhans cells, as assessed by immunohistochemistry. Interestingly, n-3PUFA increased the infiltration of CD4+ and CD8+ T cells but did not alter the erythemal response, either the sunburn threshold or the resolution of erythema, as assessed by spectrophotometric hemoglobin index readings. As EPA and DHA differentially impact cutaneous inflammation through changes in the network of epidermal lipids and dendritic and infiltrating immune cells, they should be considered separately when designing interventions for cutaneous disease.—Kendall, A. C., Pilkington, S. M., Murphy, S. A., Del Carratore, F., Sunarwidhi, A. L., Kiezel-Tsugunova, M., Urquhart, P., Watson, R. E. B., Breitling, R., Rhodes, L. E., Nicolaou, A. Dynamics of the human skin mediator lipidome in response to dietary ω-3 fatty acid supplementation.

Healthy skin depends on a unique lipid profile to form a barrier that confers protection and prevents excessive water loss, aids cell‐cell communication, and regulates cutaneous homeostasis and inflammation. Alterations in the cutaneous lipid profile can have severe consequences for skin health and have been implicated in numerous inflammatory skin conditions. Thus, skin lipidomics is increasingly of interest, and recent developments in mass spectrometry‐based analytical technologies can deliver in‐depth investigation of cutaneous lipids, providing insight into their role and mechanism of action. The choice of tissue sampling technique and analytical approach depends on the location and chemistry of the lipid of interest. Lipidomics can be conducted by various mass spectrometry approaches, including different chromatography and ionisation techniques. Targeted mass spectrometry is a sensitive approach for measuring low‐abundance signaling lipids, such as eicosanoids, endocannabinoids, and ceramides. This approach requires specific extraction, chromatography and mass spectrometry protocols to quantitate the lipid targets. Untargeted mass spectrometry reveals global changes and allows analysis of hundreds of complex lipids across a range of lipid classes, including phospholipids, glycerophospholipids, cholesteryl esters and sphingolipids. Mass spectrometry lipid imaging, including matrix‐assisted laser desorption ionisation mass spectrometry and desorption electrospray ionisation mass spectrometry, can reveal information about abundance and anatomical distribution of lipids within a single skin sample. Skin lipidomics can provide qualitative and quantitative data on hundreds of biologically‐relevant lipid species with different properties and activities, all found within a single skin sample, and support translational studies exploring the involvement of lipids in skin health and disease.

While resolution is believed to switch inflammation off and return the affected tissues back to homeostasis, we found that it triggers a prolonged phase of immune suppression. During resolution of acute inflammation (∼72h) CD4+ and CD8+ lymphocytes as well as natural killer (NK) cells migrated into tissues secreting IFNγ from day 6. IFNγ carried out two distinct functions: firstly, through IP-10, IFNγ indirectly triggered monocyte infiltration, which differentiated into macrophages (moMϕs). Secondly, moMϕs were directly acted upon by IFNγ to express microsomal prostaglandin E synthase-1 (mPGES-1) alongside cyclooxygenase (COX 1) resulting in sustained prostaglandin (PG)E2 synthesis peaking at day 14 and lasting for several weeks. PGE2 suppressed innate immune responses to bacterial infection opening a prolonged window of postresolution infectious opportunity within the host. At the same time, it also differentially modulated multiple aspect of the adaptive immune system including suppressing lymphocyte function whilst also generating myeloid-derived suppressor cells, the net effect of which was impaired uptake/presentation of exogenous antigens. Therefore, we have defined a novel sequence of post-resolution events starting with IFNγ and culminating in sustained PGE2, which we propose dampens the propensity to develop autoimmune responses to endogenous antigens at the cost of local tissue infection.

Human skin has a distinct profile of fatty acids and related bioactive lipid mediators that regulate many aspects of epidermal and dermal homeostasis, including immune and inflammatory reactions. Sebum lipids act as effective antimicrobial agents, shape immune cell communications and contribute to the epidermal lipidome. The essential fatty acid linoleic acid is crucial for the structure of the epidermal barrier, while polyunsaturated fatty acids act as precursors to eicosanoids, octadecanoids and docosanoids through cyclooxygenase, lipoxygenase and cytochrome P450 monooxygenase-mediated reactions, and endocannabinoids and N-acyl ethanolamines. Cross-communication between these families of bioactive lipids suggests that their cutaneous activities should be considered as part of a wider metabolic network that can be targeted to maintain skin health, control inflammation and improve skin pathologies.

Low heart rate variability (HRV) predicts sudden cardiac death. Long-chain (LC)n-3 PUFA (C20–C22) status is positively associated with HRV. This cross-sectional study investigated whether vegans aged 40–70 years (n23), whose diets are naturally free from EPA (20 : 5n-3) and DHA (22 : 6n-3), have lower HRV compared with omnivores (n24). Proportions of LCn-3 PUFA in erythrocyte membranes, plasma fatty acids and concentrations of plasma LCn-3 PUFA-derived lipid mediators were significantly lower in vegans. Day-time interbeat intervals (IBI), adjusted for physical activity, age, BMI and sex, were significantly shorter in vegans compared with omnivores (mean difference −67 ms; 95 % CI −130, −3·4,PP=0·039). Conversely, vegans presented with decreased 8 h day-time HRV: mean adjusted difference in SDNN −20 ms; 95 % CI −37, −3,P=0·021, with no differences during nocturnal sleep. Day-time parameters of beat-to-beat HRV (root of the mean of the sum of the squares of differences between adjacent normal-to-normal intervals, percentage of adjacent normal-to-normal intervals that differ by >50 % and high-frequency power) were similarly lower in vegans, with no differences during sleep. In conclusion, vegans have higher 24 h SDNN, but lower day-time HRV and shorter day-time IBI relative to comparable omnivores. Vegans may have reduced availability of precursor markers for pro-resolving lipid mediators; it remains to be determined whether there is a direct link with impaired cardiac function in populations with low-n-3 status.

Background: A subset of T-cells expresses the B-cell marker CD20 and in rheumatoid arthritis secretes Interleukin (IL)-17. IL-17 secreting T-cells (Th17) have also been implicated in the inflammatory response in the central nervous system in multiple sclerosis (MS) and may be a potential target for elimination by biologic therapeutics. ScFvRit:sFasL comprises of a rituximab-derived antibody fragment scFvRit genetically fused to human soluble FasL that specifically eliminated T-cells.Objective: To determine the presence and phenotype of CD20+T-cells in blood and brain of MS patients. Second, to determine whether scFvRit:sFasL can selectively eliminate CD20 + T-cells. After CD20-selective binding, scFvRit:sFasL is designed to trigger FasL-mediated activation-induced cell death of T-cells, but not B-cells.Methods: Flow cytometry and immunohistochemistry were used to screen for CD20+inflammatory T-cells in MS blood and brain tissue. ScFvRit:sFasL pro-apoptotic activity was evaluated by Annexin-V/PI staining followed by flow cytometry assessment.Results: Peripheral blood (n=11) and chronic but not active lesions of MS patient brains (n=5) contained CD20+inflammatory T-cells. Activated CD20+T-cells were predominantly CD4+and secreted both IL-17 and INF-gamma. ScFvRit:sFasL triggered CD20-restricted FasL-mediated activation-induced cell death in peripheral blood CD20 +T-cells, but not CD20+B-cells.Conclusion: CD20+ inflammatory T-cells are present in blood and chronic brain lesions of MS patients. ScFvRit:sFasL selectively eliminated CD20+T-cells and may eliminate pathogenic T-cells without B-cell depletion. (C) 2014 Elsevier B.V. All rights reserved.

Hyperbaric oxygen (HBO) therapy is an effective treatment for diabetic chronic wounds. HBO reduces inflammation and accelerates wound healing, by mechanisms that remain unclear. Here we examined a mechanism by which HBO may reduce neutrophil recruitment, through changes in endothelial and neutrophil adhesion molecule expression and function. Human umbilical vein endothelial cells and neutrophils were exposed to selected chronic wound conditions, comprising hypoxia in the presence of lipopolysaccharide and tumor necrosis factor-alpha, and then treated with HBO. We observed neutrophil adhesion to endothelial cells following treatment with chronic wound conditions, which was reversed by HBO treatment. This was partly explained by reduced expression of endothelial intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 by HBO. No changes in neutrophil adhesion molecule expression (CD18, CD11b, CD62L, CD31) were observed following HBO treatment. However, HBO decreased hydrogen peroxide generation by neutrophils, and induced nitrous oxide-related protein modifications. The transnitrosating agent S-nitroso-L-cysteine ethyl ester (600 μM) also reduced neutrophil adhesion to human umbilical vein endothelial cell monolayers, and the iNOS inhibitor 1400 W (10 μM) and HgCl2, which promotes the decomposition of S-nitrosothiols (1 mM), reversed the effect of HBO, suggesting that S-nitrosation may inhibit neutrophil-endothelial cell adhesion. This study indicates that HBO could reduce inflammation in wounds through reduced neutrophil recruitment, mediated by S-nitrosation.

The skin is the primary barrier from the outside environment, protecting the host from injury, infectious pathogens, water loss and solar ultraviolet radiation. In this role, it is supported by a highly organized system comprising elements of innate and adaptive immunity, responsive to inflammatory stimuli. The cutaneous immune system is regulated by mediators such as cytokines and bioactive lipids that can initiate rapid immune responses with controlled inflammation, followed by efficient resolution. However, when immune responses are inadequate or mounted against non-infectious agents, these mediators contribute to skin pathologies involving unresolved or chronic inflammation. Skin is characterized by active lipid metabolism and fatty acids play crucial roles both in terms of structural integrity and functionality, in particular when transformed to bioactive mediators. Eicosanoids, endocannabinoids and sphingolipids are such key bioactive lipids, intimately involved in skin biology, inflammation and immunity. We discuss their origins, role and influence over various cells of the epidermis, dermis and cutaneous immune system and examine their function in examples of inflammatory skin conditions. We focus on psoriasis, atopic and contact dermatitis, acne vulgaris, wound healing and photodermatology that demonstrate dysregulation of bioactive lipid metabolism and examine ways of using this insight to inform novel therapeutics.

BackgroundSodium lauryl sulfate (SLS) and ultraviolet radiation (UVR) are two commonly encountered cutaneous inflammatory stimuli. Differing histopathological and clinical features implicate involvement of alternative inflammatory pathways; bioactive lipid mediators (eicosanoids, endocannabinoids and sphingolipids) are likely candidates for regulation of the divergent inflammatory responses.ObjectivesTo assess comprehensively bioactive lipid involvement in SLS- and UVR-induced inflammatory responses, to provide a better understanding of bioactive lipid mediator pathways in irritant inflammation.MethodsButtock skin from 10 healthy volunteers was treated with two minimal erythema doses of UVR (275–380 nm, peak 305 nm) or an SLS dose optimized for each individual, to produce a comparable, moderate erythema. Punch biopsies were taken 24 h postchallenge and from untreated skin, and separated into dermis and epidermis. Lipids [including 15 prostanoids, 15 hydroxy fatty acids (HFAs), nine endocannabinoids and related N-acyl ethanolamides (NAE), and 21 sphingolipids] were extracted and quantified using liquid chromatography–tandem mass spectrometry.ResultsIncreased epidermal NAE and HFA expression was observed in response to SLS but not UVR-induced low-level inflammation. Significant changes following SLS treatment included augmented levels of NAE, possessing proinflammatory and some reported anti-inflammatory properties, with 3·7-fold (P = 0·02) and threefold (P = 0·01) increased expression of palmitoyl and stearoyl ethanolamides, respectively, in addition to 1·9-fold (P = 0·02) increased expression of 12-hydroxyeicosatetraenoic acid.ConclusionsThe differential bioactive lipid upregulation implicates their involvement in skin irritant responses, potentially reflecting roles in inflammatory cell recruitment and subsequent resolution of inflammation, giving scope for new treatment approaches to irritant dermatitis.

The thrombopoietin receptor (MPL) has been shown to be mutated (MPL W515L) in myelofibrosis and thrombocytosis yet new approaches to treat this disorder are still required. We have previously shown that transcriptome and proteomic effects do not correlate well in oncogene-mediated leukemogenesis. We therefore investigated the effects of MPL W515L using proteomics. The consequences of MPL W515L expression on over 3300 nuclear and 3500 cytoplasmic proteins were assessed using relative quantification mass spectrometry. We demonstrate that MPL W515L expression markedly modulates the CXCL12/CXCR4/CD45 pathway associated with stem and progenitor cell chemotactic movement. We also demonstrated that MPL W515L expressing cells displayed increased chemokinesis which required the MPL W515L-mediated dysregulation of MYC expression via phosphorylation of the RNA transport protein THOC5 on tyrosine 225. In addition MPL W515L expression induced TGFβ secretion which is linked to sphingosine 1-phosphate production and the increased chemokinesis. These studies identify several pathways which offer potential targets for therapeutic intervention in the treatment of MPL W515L-driven malignancy. We validate our approach by showing that CD34+ cells from MPL W515L positive patients display increased chemokinesis and that treatment with a combination of MYC and sphingosine kinase inhibitors leads to the preferential killing of MPL W515L expressing cells.

Hyperbaric oxygen (HBO) therapy involves the inhalation of 100% oxygen, whilst inside a chamber at greater than atmospheric pressure. It is an effective treatment for chronic diabetic wounds, although the molecular mechanisms involved remain unclear. We hypothesised that HBO could alter inflammatory gene expression in human endothelial cells via a reactive oxygen/nitrogen species-mediated pathway. Endothelial cells were exposed to a chronic wound model comprising hypoxia (2% O(2) at 1 atmosphere absolute (ATA); PO(2) ~2 kPa) in the presence of lipopolysaccharide and TNF-α for 24h, then treated with HBO for 90 min (97.5% O(2) at 2.4 ATA; PO(2) ~237 kPa). 5h post-HBO, 19 genes involved in adhesion, angiogenesis, inflammation and oxidative stress were downregulated. Notably, only angiogenin gene expression, which promotes both angiogenesis and nitric oxide production (reflected by increased eNOS protein expression in this study), was upregulated. This led to a decrease in endothelial IL-8 mRNA and protein, which could help alleviate inflammatory processes during chronic wound healing. This was no longer evident 22.5h post-HBO, demonstrating the importance of daily exposures in HBO treatment protocols. These studies indicate that elevated oxygen transiently regulates inflammatory gene expression in endothelial cells, which may enhance chronic wound healing.

Oxidation of polyunsaturated fatty acids (PUFA) through enzymatic or non-enzymatic free radical-mediated reactions can yield an array of lipid metabolites including eicosanoids, octadecanoids, docosanoids and related species. In mammals, these oxygenated PUFA mediators play prominent roles in the physiological and pathological regulation of many key biological processes in the cardiovascular, renal, reproductive and other systems including their pivotal contribution to inflammation. Mass spectrometry-based technology platforms have revolutionized our ability to analyze the complex mixture of lipid mediators found in biological samples, with increased numbers of metabolites that can be simultaneously quantified from a single sample in few analytical steps. The recent development of high-sensitivity and high-throughput analytical tools for lipid mediators affords a broader view of these oxygenated PUFA species, and facilitates research into their role in health and disease. In this review, we illustrate current analytical approaches for a high-throughput lipidomic analysis of eicosanoids and related mediators in biological samples. This article is part of a Special Issue entitled "Oxygenated metabolism of PUFA: analysis and biological relevance."

Hyperbaric oxygen has proven to be a useful treatment for chronic wounds. However, therapeutic conditions vary between treatment centers, and we wished to investigate the effects of different treatment pressures on cells under inflammatory conditions. Endothelial cells were exposed to a chronic wound model comprising hypoxia (2% O2 at 1 atmosphere absolute (atm abs); PO2 approximately 2 kPa) in the presence of 0.5 microg/ml lipopolysaccharide and 1 ng/ml TNF-alpha for 24 hours, then treated with normobaric oxygen (NBO2; 95%O2/5%CO2 at 1.0 atm abs; PO2 approximately 96.3 kPa), hyperbaric oxygen (HBO2) at 1.5 atm abs (1.5HBO2; 96.7%O2/3.3%CO2 at 1.5 atm abs; PO2 approximately 147 kPa), and HBO2 at 2.4 atm abs (2.4HBO2; 97.9%O2/2.1%CO2 at 2.4 atm abs; PO2 approximately 238 kPa). The mRNA expression of 92 inflammatory genes was then analyzed, and we identified changes in genes involved in adhesion molecule expression, angiogenesis and tissue remodeling, intracellular signaling, and cellular oxygen responses and redox signaling. We noted differences in expression between different treatment pressures, highlighting the need for further research into the use of different therapeutic protocols in the treatment of inflammatory conditions such as chronic wounds.

Hyperbaric oxygen (HBO) therapy is an effective treatment for diabetic chronic wounds. HBO reduces inflammation and accelerates wound healing, by mechanisms that remain unclear. Here we examined a mechanism by which HBO may reduce neutrophil recruitment, through changes in endothelial and neutrophil adhesion molecule expression and function. Human umbilical vein endothelial cells and neutrophils were exposed to selected chronic wound conditions, comprising hypoxia in the presence of lipopolysaccharide and tumor necrosis factor-alpha, and then treated with HBO. We observed neutrophil adhesion to endothelial cells following treatment with chronic wound conditions, which was reversed by HBO treatment. This was partly explained by reduced expression of endothelial intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 by HBO. No changes in neutrophil adhesion molecule expression (CD18, CD11b, CD62L, CD31) were observed following HBO treatment. However, HBO decreased hydrogen peroxide generation by neutrophils, and induced nitrous oxide-related protein modifications. The transnitrosating agent S-nitroso-L-cysteine ethyl ester (600 μM) also reduced neutrophil adhesion to human umbilical vein endothelial cell monolayers, and the iNOS inhibitor 1400 W (10 μM) and HgCl2, which promotes the decomposition of S-nitrosothiols (1 mM), reversed the effect of HBO, suggesting that S-nitrosation may inhibit neutrophil-endothelial cell adhesion. This study indicates that HBO could reduce inflammation in wounds through reduced neutrophil recruitment, mediated by S-nitrosation.

The skin produces bioactive lipids that participate in physiological and pathological states, including homeostasis, induction, propagation, and resolution of inflammation. However, comprehension of the cutaneous lipid complement, and contribution to differing roles of the epidermal and dermal compartments, remains incomplete. We assessed the profiles of eicosanoids, endocannabinoids, N-acyl ethanolamides, and sphingolipids, in human dermis, epidermis, and suction blister fluid. We identified 18 prostanoids, 12 hydroxy-fatty acids, 9 endocannabinoids and N-acyl ethanolamides, and 21 non-hydroxylated ceramides and sphingoid bases, several demonstrating significantly different expression in the tissues assayed. The array of dermal and epidermal fatty acids was reflected in the lipid mediators produced, whereas similarities between lipid profiles in blister fluid and epidermis indicated a primarily epidermal origin of suction blister fluid. Supplementation with omega-3 fatty acids ex vivo showed that their action is mediated through perturbation of existing species and formation of other anti-inflammatory lipids. These findings demonstrate the diversity of lipid mediators involved in maintaining tissue homeostasis in resting skin and hint at their contribution to signaling, cross-support, and functions of different skin compartments. Profiling lipid mediators in biopsies and suction blister fluid can support studies investigating cutaneous inflammatory responses, dietary manipulation, and skin diseases lacking biomarkers and therapeutic targets.

Introduction: Rheumatoid arthritis (RA) is considered a T cell driven autoimmune disease, therefore, the ability of B cell depleting biologics, e.g., anti-CD20 antibodies, to alleviate RA is unclear. This study examined the proportions of IL-17-secreting lymphocytes in the blood of healthy subjects and RA patients and determined if Th17 cells belong to a CD20+ subset of T cells.Methods: Fluorescence-activated cell sorting and confocal microscopy verified CD3, CD4/CD8 and CD20-staining of T cells. IL-17 secretion was determined using a commercial assay.Results: In healthy subjects and RA patients blood, the median percentage of total CD20+ lymphocytes was similar (7.5%; n = 6 and 10.3%; n = 9, respectively) and comprised predominantly of B cells (similar to 86%). However, 2-4% of CD3+ T cells from both healthy subjects (n = 7) and RA (n = 8) individuals co-expressed CD20. The peripheral blood of healthy subjects contained few IL-17-secreting CD20+ T cells (Conclusions: In the blood of RA patients, a greater proportion of Th17 cells are of a CD20+ phenotype compared to healthy individuals. These cells may represent an additional target for anti-CD20 therapies.