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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

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.

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.

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."

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.