Pulmonary hypertension (PH) is a life-threatening disease featuring pulmonary vessel remodelling and perivascular inflammation. The effect, if any, of eosinophils (EOS) on the development of PH remains unclear.
EOS infiltration and chemotaxis were investigated in peripheral blood and lung tissues from pulmonary arterial hypertension (PAH) patients without allergic history and from sugen/hypoxia-induced PH mice. The role of EOS deficiency in PH development was investigated using GATA1-deletion (ΔdblGATA) mice and anti-interleukin 5 antibody-treated mice and rats. Ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was conducted to identify the critical oxylipin molecule(s) produced by EOS. Culture supernatants and lysates of EOS were collected to explore the mechanisms in co-culture cell experiments.
There was a lower percentage of EOS in peripheral blood but higher infiltration in lung tissues from PAH patients and PH mice. PAH/PH lungs showed increased EOS-related chemokine expression, mainly C-C motif chemokine ligand 11 derived from adventitial fibroblasts. EOS deficiency aggravated PH in rodents, accompanied by increased neutrophil and monocyte/macrophage infiltration. EOS highly expressed arachidonate 15-lipoxygenase (ALOX15). 14-hydroxy docosahexaenoic acid (14-HDHA) and 17-HDHA were critical downstream oxylipins produced by EOS, which showed anti-inflammatory effects on recruitment of neutrophils and monocytes/macrophages through N-formyl peptide receptor 2. They also repressed pulmonary artery smooth muscle cell (PASMC) proliferation by activating peroxisome proliferator-activated receptor γ and blunting Stat3 phosphorylation.
In PH development without external stimuli, peripheral blood exhibits a low EOS level. EOS play a protective role by suppressing perivascular inflammation and maintaining PASMC homeostasis via 14/17-HDHA.
Copyright ©The authors 2023.

Our previous results showed that the specialized pro-resolving mediator (SPM) Resolvin D1 (RvD1) promotes resolution of inflammation in salivary glands in non-obese diabetic (NOD)/ShiLtJ, a mouse model for Sjögren's syndrome (SS). Additionally, mice lacking the RvD1 receptor ALX/FPR2 show defective innate and adaptive immune responses in salivary glands. Particularly, ALX/FPR2 KO mice exhibit exacerbated inflammation in their salivary glands in response to systemic LPS treatment. Moreover, female ALX/FPR2 KO mice show increased autoantibody production and loss of salivary gland function with age. Together, these studies suggest that an underlying SPM dysregulation could be contributing to SS progression. Therefore, we investigated whether SPM production is altered in NOD/ShiLtJ using metabololipidomics and enzyme-linked immunosorbent assay (ELISA). Our results demonstrate that SPM levels were broadly elevated in plasma collected from NOD/ShiLtJ female mice after disease onset, whereas these drastic changes did not occur in male mice. Moreover, gene expression of enzymes involved in SPM biosynthesis were altered in submandibular glands (SMG) from NOD/ShiLtJ female mice after disease onset, with 5-LOX and 12/15-LOX being downregulated and upregulated, respectively. Despite this dysregulation, the abundances of the SPM products of these enzymes (ie, RvD1 and RvD2) were unaltered in freshly isolated SMG cells suggesting that other cell populations (eg, lymphocytes) may be responsible for the overabundance of SPMs that we observed. The elevation of SPMs noted here appeared to be sex mediated, meaning that it was observed only in one sex (females). Given that SS primarily affects females (roughly 90% of diagnosed cases), these results may provide some insights into the mechanisms underlying the observed sexual dimorphism.
© 2020 Federation of American Societies for Experimental Biology.

Bioactive lipids, including oxylipins, endocannabinoids, and related compounds may function as specific biochemical markers of certain aspects of inflammation. However, the postprandial responsiveness of these compounds is largely unknown; therefore, changes in the circulating oxylipin and endocannabinoid metabolome in response to a challenge meal were investigated at six occasions in a subject who freely modified her usual diet. The dietary change, and especially the challenge meal itself, represented a modification of precursor fatty acid status, with expectedly subtle effects on bioactive lipid levels. To detect even the slightest alteration, highly sensitive ultra-performance liquid chromatography (UPLC) coupled to electrospray ionization (ESI) tandem mass spectrometry (MS/MS) methods for bioactive lipid profiling was employed. A previously validated UPLC-ESI-MS/MS method for profiling the endocannabinoid metabolome was used, while validation of an UPLC-ESI-MS/MS method for oxylipin analysis was performed with acceptable outcomes for a majority of the parameters according to the US Food and Drug Administration guidelines for linearity (0.9938 < R2 < 0.9996), limit of detection (0.0005-2.1 pg on column), limit of quantification (0.0005-4.2 pg on column), inter- and intraday accuracy (85-115%) and precision (< 5%), recovery (40-109%) and stability (40-105%). Forty-seven of fifty-two bioactive lipids were detected in plasma samples at fasting and in the postprandial state (0.5, 1, and 3 hours after the meal). Multivariate analysis showed a significant shift of bioactive lipid profiles in the postprandial state due to inclusion of dairy products in the diet, which was in line with univariate analysis revealing seven compounds (NAGly, 9-HODE, 13-oxo-ODE, 9(10)-EpOME, 12(13)-EpOME, 20-HETE, and 11,12-DHET) that were significantly different between background diets in the postprandial state (but not at fasting). The only change in baseline levels at fasting was displayed by TXB2. Furthermore, postprandial responsiveness was detected for seven compounds (POEA, SEA, 9(10)-DiHOME, 12(13)-DiHOME, 13-oxo-ODE, 9-HODE, and 13-HODE). Hence, the data confirm that the UPLC-ESI-MS/MS method performance was sufficient to detect i) a shift, in the current case most notably in the postprandial bioactive lipid metabolome, caused by changes in diet and ii) responsiveness to a challenge meal for a subset of the oxylipin and endocannabinoid metabolome. To summarize, we have shown proof-of-concept of our UPLC-ESI-MS/MS bioactive lipid protocols for the purpose of monitoring subtle shifts, and thereby useful to address lipid-mediated postprandial inflammation.

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.
Copyright © 2012 Elsevier Inc. All rights reserved.

Phospholipid-esterified oxylipins include newly described families of bioactive lipids generated by lipoxygenases in immune cells. Until now, assays for their quantitation were not well developed or widely available. Here, we describe a mass spectrometric protocol that enables accurate measurement of several esterified oxylipins--in particular hydro(pero)xyeicosatetraenoic acids, hydroxyoctadecadienoic acids, hydroxydocosahexaenoic acids and keto-eicosatetraenoic acids--attached to either phosphatidylethanolamine or phosphatidylcholine. Lipids are isolated from cells or tissue using a liquid-phase organic extraction, then analyzed by HPLC-tandem mass spectrometry (LC/MS/MS) in multiple reaction-monitoring mode. The protocol can simultaneously monitor up to 23 species. Generation of standards takes ∼2 d. Following this, extraction of 30 samples takes ∼3 h, with LC/MS/MS run time of 50 min per sample.