Patients with advanced cirrhosis experience frequent infections leading to sepsis, which carries high mortality. While innate immune dysfunction underlies this vulnerability, the precise cause remains elusive. We found prostaglandin (PGE 2 ) elevated in acutely decompensated (AD) patients at immunosuppressive levels. Plasma from AD and end-stage liver disease (ESLD) patients suppressed macrophage cytokine secretion and bacteria killing in a PGE 2 receptor-dependent manner, effects not seen in stable cirrhosis. Mouse models (bile duct ligation and CCL4-liver injury) also demonstrated elevated PGE 2 , which when inhibited completely restored immune competence and survival following infection. Importantly, albumin binds/inactivates PGE 2 resulting in greater PGE 2 bioavailability. This results in enhanced immunosuppressive effects of AD plasma in patients with low albumin levels. Administering albumin to AD patients reversed immunosuppressive properties of their plasma; protective effects recapitulated in rodent survival studies. Thus, elevated PGE 2 combined with hypoalbuminemia mediates immunosuppression in AD and ESLD patients, which can be reversed with albumin. Cyclooxygenase (COX)-derived lipid mediators have broad immunosuppressive effects12-15 that could explain the aetiology of infection susceptibility in cirrhosis patients. Thus, we used a number of in vitro and in vivo assays using plasma from patients with AD and ESLD derived from clinical trials to investigate the role of bioactive lipid mediators in immunosuppression as well as animal models of liver injury for survival analyses. ResultsProstaglandin E 2 (PGE 2 ) is elevated in patients with acute decompensation at levels that are immunosuppressive via its effect on the EP2/3 receptor ESI/LC-MS/MS analysis of acutely decompensated patient plasma (day 1-2 of hospital admission) demonstrated significantly elevated PGE 2 , PGF 2 α, 5-and 15-HETE compared to HV ( Figure 1A and supplementary Figure 1E-G). However, only PGE 2 dampened TNFα release from LPS-stimulated human monocyte-derived macrophages when pre-treated with the mean concentrations observed in AD patients (0.1ng/ml) ( Figure 1B).Human monocyte-derived macrophages were incubated with culture media supplemented with 25% (vol./vol.) plasma from AD patients (see Table 1 for clinical characteristics).Compared to macrophages treated with media supplemented with HV plasma, AD plasma caused a significant decrease in LPS-stimulated TNFα that was reversed by pre-incubating Europe PMC Funders Author ManuscriptsEurope PMC Funders Author Manuscripts macrophages with the E-prostanoid (EP) 1-3/D-prostanoid (DP) 1 receptor antagonist, AH6809 ( Figure 1C). Additionally, macrophages were incubated with E. coli in the presence/absence of HV or AD plasma. Compared to macrophages treated with HV plasma, those with AD plasma exhibited reduced bacterial killing, an effect reversed by pretreatment with AH6809 (50μM) ( Figure 1D). AH6809 had no direct bactericidal effect while cell viability was unaffected b...

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Sunburn is a commonly occurring acute inflammatory process, with dermal vasodilatation and leukocyte infiltration as central features. Ultraviolet (UV) B-induced hydrolysis of membrane phospholipids releases polyunsaturated fatty acids, and their subsequent metabolism by cyclooxygenases (COXs) and lipoxygenases (LOXs) may produce potent eicosanoid mediators modulating different stages of the inflammation. Our objective was to identify candidate eicosanoids formed during the sunburn reaction in relation to its clinical and histological course. We exposed skin of healthy humans (n=32) to UVB and, for 72 h, examined expression of proinflammatory and anti-inflammatory eicosanoids using LC/ESI-MS/MS, and examined immunohistochemical expression of COX-2, 12-LOX, 15-LOX, and leukocyte markers, while quantifying clinical erythema. We show that vasodilatory prostaglandins (PGs) PGE2, PGF2α, and PGE3 accompany the erythema in the first 24–48 h, associated with increased COX-2 expression at 24 h. Novel, potent leukocyte chemoattractants 11-, 12-, and 8-monohydroxy-eicosatetraenoic acid (HETE) are elevated from 4 to 72 h, in association with peak dermal neutrophil influx at 24 h, and increased dermal CD3+ lymphocytes and 12- and 15-LOX expression from 24 to 72 h. Anti-inflammatory metabolite 15-HETE shows later expression, peaking at 72 h. Sunburn is characterized by overlapping sequential profiles of increases in COX products followed by LOX products that may regulate subsequent events and ultimately its resolution.—Rhodes, L. E., Gledhill, K., Masoodi, M., Haylett, A. K., Brownrigg, M., Thody, A. J., Tobin, D. J., Nicolaou, A. The sunburn response in human skin is characterized by sequential eicosanoid profiles that may mediate its early and late phases.

Prostanoids are potent mediators of many physiological and pathophysiological processes. Of the many analytical methodologies used for their qualitative and quantitative analysis, electrospray tandem mass spectrometry coupled to liquid chromatography (ESI-LC-MS/MS) offers a rapid, sensitive and versatile system applicable to lipidomic analyses. We have developed an ESI-LC-MS/MS assay for twenty-seven mediators including prostaglandins, prostacyclines, thromboxanes, dihydroprostaglandins and isoprostanes. The assay was liner over the concentration range 1-100 pg/μL. The limits of detection and quantitation were 0.5-50 pg and 2-100 pg respectively, whilst recoveries were from 83-116 % depending on the metabolite. The assay can be applied to profiling prostanoids produced by a variety of biological fluids and extracts including brain, liver, plasma and urine, facilitating thus our understanding of the role of these lipid mediators in health and disease, as well as assisting in drug development.

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.

Lipid mediators are produced from the oxidation of polyunsaturated fatty acids through enzymatic and free radical-mediated reactions. When subject to oxygenation via cyclooxygenases, lipoxygenases, and cytochrome P450 monooxygenases, polyunsaturated fatty acids give rise to an array of metabolites including eicosanoids, docosanoids, and octadecanoids. These potent bioactive lipids are involved in many biochemical and signaling pathways, with inflammation being of particular importance. Moreover, because they are produced by more than one pathway and substrate, and are present in a variety of biological milieus, their analysis is not always possible with conventional assays. Liquid chromatography coupled to electrospray mass spectrometry offers a versatile and sensitive approach for the analysis of bioactive lipids, allowing specific and accurate quantitation of multiple species present in the same sample. Here we explain the principles of this approach to mediator lipidomics and present detailed protocols for the assay of enzymatically produced oxygenated metabolites of polyunsaturated fatty acids that can be tailored to answer biological questions or facilitate assessment of nutritional and pharmacological interventions.

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