Lipidomic profiling of influenza infection identifies mediators that induce and resolve inflammation.

Bioactive lipid mediators play a crucial role in the induction and resolution of inflammation. To elucidate their involvement during influenza infection, liquid chromatography/mass spectrometry lipidomic profiling of 141 lipid species was performed on a mouse influenza model using two viruses of significantly different pathogenicity. Infection by the low-pathogenicity strain X31/H3N2 induced a proinflammatory response followed by a distinct anti-inflammatory response; infection by the high-pathogenicity strain PR8/H1N1 resulted in overlapping pro- and anti-inflammatory states. Integration of the large-scale lipid measurements with targeted gene expression data demonstrated that 5-lipoxygenase metabolites correlated with the pathogenic phase of the infection, whereas 12/15-lipoxygenase metabolites were associated with the resolution phase. Hydroxylated linoleic acid, specifically the ratio of 13- to 9-hydroxyoctadecadienoic acid, was identified as a potential biomarker for immune status during an active infection. Importantly, some of the findings from the animal model were recapitulated in studies of human nasopharyngeal lavages obtained during the 2009-2011 influenza seasons.

NCoR repression of LXRs restricts macrophage biosynthesis of insulin-sensitizing omega 3 fatty acids.

Macrophage-mediated inflammation is a major contributor to obesity-associated insulin resistance. The corepressor NCoR interacts with inflammatory pathway genes in macrophages, suggesting that its removal would result in increased activity of inflammatory responses. Surprisingly, we find that macrophage-specific deletion of NCoR instead results in an anti-inflammatory phenotype along with robust systemic insulin sensitization in obese mice. We present evidence that derepression of LXRs contributes to this paradoxical anti-inflammatory phenotype by causing increased expression of genes that direct biosynthesis of palmitoleic acid and ω3 fatty acids. Remarkably, the increased ω3 fatty acid levels primarily inhibit NF-κB-dependent inflammatory responses by uncoupling NF-κB binding and enhancer/promoter histone acetylation from subsequent steps required for proinflammatory gene activation. This provides a mechanism for the in vivo anti-inflammatory insulin-sensitive phenotype observed in mice with macrophage-specific deletion of NCoR. Therapeutic methods to harness this mechanism could lead to a new approach to insulin-sensitizing therapies.

Lipidomics of phospholipase A2 reveals exquisite specificity in macrophages.

Phospholipase A2 (PLA2) constitutes a superfamily of enzymes that hydrolyze phospholipids at their sn-2 fatty acyl position. Our laboratory has demonstrated that PLA2 enzymes regulate membrane remodeling and cell signaling by their specificity toward their phospholipid substrates at the molecular level. Recent in vitro studies show that each type of PLA2, including Group IVA cytosolic PLA2 (cPLA2), Group V secreted PLA2 (sPLA2), Group VIA calcium independent PLA2 (iPLA2) and Group VIIA lipoprotein-associated PLA2, also known as platelet-activating factor acetyl hydrolase, can discriminate exquisitely between fatty acids at the sn-2 position. Thus, these enzymes regulate the production of diverse PUFA precursors of inflammatory metabolites. We now determined PLA2 specificity in macrophage cells grown in cell culture, where the amounts and localization of the phospholipid substrates play a role in which specific phospholipids are hydrolyzed by each enzyme type. We used PLA2 stereospecific inhibitors in tandem with a novel UPLC-MS/MS-based lipidomics platform to quantify more than a thousand unique phospholipid molecular species demonstrating cPLA2, sPLA2, and iPLA2 activity and specificity toward the phospholipids in living cells. The observed specificity follows the in vitro capability of the enzymes and can reflect the enrichment of certain phospholipid species in specific membrane locations where particular PLA2's associate. For assaying, we target 20:4-PI for cPLA2, 22:6-PG for sPLA2, and 18:2-PC for iPLA2. These new results provide great insight into the physiological role of PLA2 enzymes in cell membrane remodeling and could shed light on how PLA2 enzymes underpin inflammation and other lipid-related diseases.

Lipid storage disease in 4 sibling superb birds-of-paradise (Lophorina superba).

Pedigree analysis, clinical, gross, microscopic, ultrastructural, and lipidomic findings in 4 female superb bird-of-paradise (SBOP, Lophorina superba) siblings led to the diagnosis of a primary inherited glycerolipid storage disease. These birds were the offspring of a related breeding pair (inbreeding coefficient = 0.1797) and are the only known SBOPs to display this constellation of lesions. The birds ranged from 0.75 to 4.3 years of age at the time of death. Two birds were euthanized and 1 died naturally due to the disease, and 1 died of head trauma with no prior clinical signs. Macroscopic findings included hepatomegaly and pallor (4/4), cardiac and renal pallor (2/4), and coelomic effusion (1/4). Microscopic examination found marked tissue distortion due to cytoplasmic lipid vacuoles in hepatocytes (4/4), cardiomyocytes (4/4), renal tubular epithelial cells (4/4), parathyroid gland principal cells (2/2), exocrine pancreatic cells (3/3), and the glandular cells of the ventriculus and proventriculus (3/3). Ultrastructurally, the lipids were deposited in single to coalescing or fused droplets lined by an inconspicuous or discontinuous monolayer membrane. Lipidomic profiling found that the cytoplasmic lipid deposits were primarily composed of triacylglycerols. Future work, including sequencing of the SBOP genome and genotyping, will be required to definitively determine the underlying genetic mechanism of this disease.

PPARα exacerbates necroptosis, leading to increased mortality in postinfluenza bacterial superinfection.

Patients infected with influenza are at high risk of secondary bacterial infection, which is a major proximate cause of morbidity and mortality. We have shown that in mice, prior infection with influenza results in increased inflammation and mortality upon Staphylococcus aureus infection, recapitulating the human disease. Lipidomic profiling of the lungs of superinfected mice revealed an increase in CYP450 metabolites during lethal superinfection. These lipids are endogenous ligands for the nuclear receptor PPARα, and we demonstrate that Ppara-/- mice are less susceptible to superinfection than wild-type mice. PPARα is an inhibitor of NFκB activation, and transcriptional profiling of cells isolated by bronchoalveolar lavage confirmed that influenza infection inhibits NFκB, thereby dampening proinflammatory and prosurvival signals. Furthermore, network analysis indicated an increase in necrotic cell death in the lungs of superinfected mice compared to mice infected with S. aureus alone. Consistent with this, we observed reduced NFκB-mediated inflammation and cell survival signaling in cells isolated from the lungs of superinfected mice. The kinase RIPK3 is required to induce necrotic cell death and is strongly induced in cells isolated from the lungs of superinfected mice compared to mice infected with S. aureus alone. Genetic and pharmacological perturbations demonstrated that PPARα mediates RIPK3-dependent necroptosis and that this pathway plays a central role in mortality following superinfection. Thus, we have identified a molecular circuit in which infection with influenza induces CYP450 metabolites that activate PPARα, leading to increased necrotic cell death in the lung which correlates with the excess mortality observed in superinfection.

Influence of Dietary n-3 Long Chain Polyunsaturated Fatty Acid Intake on Oxylipins in Erythrocytes of Women with Rheumatoid Arthritis.

Oxylipins derived from n-3 fatty acids are suggested as the link between these fatty acids and reduced inflammation. The aim of the present study was to explore the effect of a randomized controlled cross-over intervention on oxylipin patterns in erythrocytes. Twenty-three women with rheumatoid arthritis completed 2 × 11-weeks exchanging one cooked meal per day, 5 days a week, for a meal including 75 g blue mussels (source for n-3 fatty acids) or 75 g meat. Erythrocyte oxylipins were quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The results were analyzed with multivariate data analysis. Orthogonal projections to latent structures (OPLS) with effect projections and with discriminant analysis were performed to compare the two diets' effects on oxylipins. Wilcoxon signed rank test was used to test pre and post values for each dietary period as well as post blue-mussel vs. post meat. The blue-mussel diet led to significant changes in a few oxylipins from the precursor fatty acids arachidonic acid and dihomo-É£-linolenic acid. Despite significant changes in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) and free EPA in erythrocytes in the mussel group, no concurrent changes in their oxylipins were seen. Further research is needed to study the link between n-3 fatty-acid intake, blood oxylipins, and inflammation.

Automated Annotation of Sphingolipids Including Accurate Identification of Hydroxylation Sites Using MSn Data.

Sphingolipids constitute a heterogeneous lipid category that is involved in many key cellular functions. For high-throughput analyses of sphingolipids, tandem mass spectrometry (MS/MS) is the method of choice, offering sufficient sensitivity, structural information, and quantitative precision for detecting hundreds to thousands of species simultaneously. While glycerolipids and phospholipids are predominantly non-hydroxylated, sphingolipids are typically dihydroxylated. However, species containing one or three hydroxylation sites can be detected frequently. This variability in the number of hydroxylation sites on the sphingolipid long-chain base and the fatty acyl moiety produces many more isobaric species and fragments than for other lipid categories. Due to this complexity, the automated annotation of sphingolipid species is challenging, and incorrect annotations are common. In this study, we present an extension of the Lipid Data Analyzer (LDA) "decision rule set" concept that considers the structural characteristics that are specific for this lipid category. To address the challenges inherent to automated annotation of sphingolipid structures from MS/MS data, we first developed decision rule sets using spectra from authentic standards and then tested the applicability on biological samples including murine brain and human plasma. A benchmark test based on the murine brain samples revealed a highly improved annotation quality as measured by sensitivity and reliability. The results of this benchmark test combined with the easy extensibility of the software to other (sphingo)lipid classes and the capability to detect and correctly annotate novel sphingolipid species make LDA broadly applicable to automated sphingolipid analysis, especially in high-throughput settings.

Deciphering lipid structures based on platform-independent decision rules.

We achieve automated and reliable annotation of lipid species and their molecular structures in high-throughput data from chromatography-coupled tandem mass spectrometry using decision rule sets embedded in Lipid Data Analyzer (LDA; http://genome.tugraz.at/lda2). Using various low- and high-resolution mass spectrometry instruments with several collision energies, we proved the method's platform independence. We propose that the software's reliability, flexibility, and ability to identify novel lipid molecular species may now render current state-of-the-art lipid libraries obsolete.

AMP-activated protein kinase activation ameliorates eicosanoid dysregulation in high-fat-induced kidney disease in mice.

High-fat diet (HFD) causes renal lipotoxicity that is ameliorated with AMP-activated protein kinase (AMPK) activation. Although bioactive eicosanoids increase with HFD and are essential in regulation of renal disease, their role in the inflammatory response to HFD-induced kidney disease and their modulation by AMPK activation remain unexplored. In a mouse model, we explored the effects of HFD on eicosanoid synthesis and the role of AMPK activation in ameliorating these changes. We used targeted lipidomic profiling with quantitative MS to determine PUFA and eicosanoid content in kidneys, urine, and renal arterial and venous circulation. HFD increased phospholipase expression as well as the total and free pro-inflammatory arachidonic acid (AA) and anti-inflammatory DHA in kidneys. Consistent with the parent PUFA levels, the AA- and DHA-derived lipoxygenase (LOX), cytochrome P450, and nonenzymatic degradation (NE) metabolites increased in kidneys with HFD, while EPA-derived LOX and NE metabolites decreased. Conversely, treatment with 5-aminoimidazole-4-carboxamide-1-ß-D-furanosyl 5'-monophosphate (AICAR), an AMPK activator, reduced the free AA and DHA content and the DHA-derived metabolites in kidney. Interestingly, kidney and circulating AA, AA metabolites, EPA-derived LOX, and NE metabolites are increased with HFD; whereas, DHA metabolites are increased in kidney in contrast to their decreased circulating levels with HFD. Together, these changes showcase HFD-induced pro- and anti-inflammatory eicosanoid dysregulation and highlight the role of AMPK in correcting HFD-induced dysregulated eicosanoid pathways.

Pro- and anti-inflammatory eicosanoids in psoriatic arthritis.

INTRODUCTION: Eicosanoids are biological lipids that serve as both activators and suppressors of inflammation. Eicosanoid pathways are implicated in synovitis and joint destruction in inflammatory arthritis, yet they might also have a protective function, underscoring the need for a comprehensive understanding of how eicosanoid pathways might be imbalanced. Until recently, sensitive and scalable methods for detecting and quantifying a high number of eicosanoids have not been available. OBJECTIVE: Here, we intend to describe a detailed eicosanoid profiling in patients with psoriatic arthritis (PsA) and evaluate correlations with parameters of disease activity. METHODS: Forty-one patients with PsA, all of whom satisfied the CASPAR classification criteria for PsA, were studied. Outcomes reflecting the activity of peripheral arthritis as well as skin psoriasis, Disease Activity Score (DAS)28, Clinical Disease Index (CDAI) and Body Surface Area (BSA) were assessed. Serum eicosanoids were determined by LC-MS, and the correlation between metabolite levels and disease scores was evaluated. RESULTS: Sixty-six eicosanoids were identified by reverse-phase LC/MS. Certain eicosanoids species including several pro-inflammatory eicosanoids such as PGE2, HXB3 or 6,15-dk,dh,PGF1a correlated with joint disease score. Several eicosapentaenoic acid (EPA)-derived eicosanoids, which associate with anti-inflammatory properties, such as 11-HEPE, 12-HEPE and 15-HEPE, correlated with DAS28 (Disease Activity Score) and CDAI (Clinical Disease Activity Index) as well. Of interest, resolvin D1, a DHA-derived anti-inflammatory eicosanoid, was down-regulated in patients with high disease activity. CONCLUSION: Both pro- and anti-inflammatory eicosanoids were associated with joint disease score, potentially representing pathways of harm as well as benefit. Further studies are needed to determine whether these eicosanoid species might also play a role in the pathogenesis of joint inflammation in PsA.

Quantitative determination of esterified eicosanoids and related oxygenated metabolites after base hydrolysis.

Eicosanoids and related metabolites (oxylipins) possess potent signaling properties, elicit numerous important physiologic responses, and serve as biomarkers of disease. In addition to their presence in free form, a considerable portion of these bioactive lipids is esterified to complex lipids in cell membranes and plasma lipoproteins. We developed a rapid and sensitive method for the analysis of esterified oxylipins using alkaline hydrolysis to release them followed by ultra-performance LC coupled with mass spectrometric analysis. Detailed evaluation of the data revealed that several oxylipins are susceptible to alkaline-induced degradation. Nevertheless, of the 136 metabolites we examined, 56 were reproducibly recovered after alkaline hydrolysis. We classified those metabolites that were resistant to alkaline-induced degradation and applied this methodology to quantify metabolite levels in a macrophage cell model and in plasma of healthy subjects. After alkaline hydrolysis of lipids, 34 metabolites could be detected and quantified in resting and activated macrophages, and 38 metabolites were recovered from human plasma at levels that were substantially greater than in free form. By carefully selecting internal standards and taking the observed experimental limitations into account, we established a robust method that can be reliably employed for the measurement of esterified oxylipins in biological samples.

Failure to up-regulate transcription of genes necessary for muscle adaptation underlies limb girdle muscular dystrophy 2A (calpainopathy).

Limb girdle muscular dystrophy 2A is due to loss-of-function mutations in the Calpain 3 (CAPN3) gene. Our previous data suggest that CAPN3 helps to maintain the integrity of the triad complex in skeletal muscle. In Capn3 knock-out mice (C3KO), Ca2+ release and Ca2+/calmodulin kinase II (CaMKII) signaling are attenuated. We hypothesized that calpainopathy may result from a failure to transmit loading-induced Ca2+-mediated signals, necessary to up-regulate expression of muscle adaptation genes. To test this hypothesis, we compared transcriptomes of muscles from wild type (WT) and C3KO mice subjected to endurance exercise. In WT mice, exercise induces a gene signature that includes myofibrillar, mitochondrial and oxidative lipid metabolism genes, necessary for muscle adaptation. C3KO muscles fail to activate the same gene signature. Furthermore, in agreement with the aberrant transcriptional profile, we observe a commensurate functional defect in lipid metabolism whereby C3KO muscles fail to release fatty acids from stored triacylglycerol. In conjunction with the defects in oxidative metabolism, C3KO mice demonstrate reduced exercise endurance. Failure to up-regulate genes in C3KO muscles is due, in part, to decreased levels of PGC1α, a transcriptional co-regulator that orchestrates the muscle adaptation response. Destabilization of PGC1α is attributable to decreased p38 MAPK activation via diminished CaMKII signaling. Thus, we elucidate a pathway downstream of Ca2+-mediated CaMKII activation that is dysfunctional in C3KO mice, leading to reduced transcription of genes involved in muscle adaptation. These studies identify a novel mechanism of muscular dystrophy: a blunted transcriptional response to muscle loading resulting in chronic failure to adapt and remodel.

Harmonizing lipidomics: NIST interlaboratory comparison exercise for lipidomics using SRM 1950-Metabolites in Frozen Human Plasma.

As the lipidomics field continues to advance, self-evaluation within the community is critical. Here, we performed an interlaboratory comparison exercise for lipidomics using Standard Reference Material (SRM) 1950-Metabolites in Frozen Human Plasma, a commercially available reference material. The interlaboratory study comprised 31 diverse laboratories, with each laboratory using a different lipidomics workflow. A total of 1,527 unique lipids were measured across all laboratories and consensus location estimates and associated uncertainties were determined for 339 of these lipids measured at the sum composition level by five or more participating laboratories. These evaluated lipids detected in SRM 1950 serve as community-wide benchmarks for intra- and interlaboratory quality control and method validation. These analyses were performed using nonstandardized laboratory-independent workflows. The consensus locations were also compared with a previous examination of SRM 1950 by the LIPID MAPS consortium. While the central theme of the interlaboratory study was to provide values to help harmonize lipids, lipid mediators, and precursor measurements across the community, it was also initiated to stimulate a discussion regarding areas in need of improvement.

Polyunsaturated fatty acid metabolites as novel lipidomic biomarkers for noninvasive diagnosis of nonalcoholic steatohepatitis.

Lipotoxicity is a key mechanism thought to be responsible for the progression of nonalcoholic fatty liver (NAFL) to nonalcoholic steatohepatitis (NASH). Noninvasive diagnosis of NASH is a major unmet clinical need, and we hypothesized that PUFA metabolites, in particular arachidonic acid (AA)-derived eicosanoids, in plasma would differentiate patients with NAFL from those with NASH. Therefore, we aimed to assess the differences in the plasma eicosanoid lipidomic profile between patients with biopsy-proven NAFL versus NASH versus normal controls without nonalcoholic fatty liver disease (NAFLD; based on MRI fat fraction <5%). We carried out a cross-sectional analysis of a prospective nested case-control study including 10 patients with biopsy-proven NAFL, 9 patients with biopsy-proven NASH, and 10 non-NAFLD MRI-phenotyped normal controls. We quantitatively compared plasma eicosanoid and other PUFA metabolite levels between NAFL versus NASH versus normal controls. Utilizing a uniquely well-characterized cohort, we demonstrated that plasma eicosanoid and other PUFA metabolite profiling can differentiate between NAFL and NASH. The top candidate as a single biomarker for differentiating NAFL from NASH was 11,12-dihydroxy-eicosatrienoic acid (11,12-diHETrE) with an area under the receiver operating characteristic curve (AUROC) of 1. In addition, we also found a panel including 13,14-dihydro-15-keto prostaglandin D2 (dhk PGD2) and 20-carboxy arachidonic acid (20-COOH AA) that demonstrated an AUROC of 1. This proof-of-concept study provides early evidence that 11,12-diHETrE, dhk PGD2, and 20-COOH AA are the leading eicosanoid candidate biomarkers for the noninvasive diagnosis of NASH.

Biomarkers of NAFLD progression: a lipidomics approach to an epidemic.

The spectrum of nonalcoholic fatty liver disease (NAFLD) includes steatosis, nonalcoholic steatohepatitis (NASH), and cirrhosis. Recognition and timely diagnosis of these different stages, particularly NASH, is important for both potential reversibility and limitation of complications. Liver biopsy remains the clinical standard for definitive diagnosis. Diagnostic tools minimizing the need for invasive procedures or that add information to histologic data are important in novel management strategies for the growing epidemic of NAFLD. We describe an "omics" approach to detecting a reproducible signature of lipid metabolites, aqueous intracellular metabolites, SNPs, and mRNA transcripts in a double-blinded study of patients with different stages of NAFLD that involves profiling liver biopsies, plasma, and urine samples. Using linear discriminant analysis, a panel of 20 plasma metabolites that includes glycerophospholipids, sphingolipids, sterols, and various aqueous small molecular weight components involved in cellular metabolic pathways, can be used to differentiate between NASH and steatosis. This identification of differential biomolecular signatures has the potential to improve clinical diagnosis and facilitate therapeutic intervention of NAFLD.

Modeling of eicosanoid fluxes reveals functional coupling between cyclooxygenases and terminal synthases.

Eicosanoids, including prostaglandins (PG) and leukotrienes, are lipid mediators derived from arachidonic acid. A quantitative and biochemical level understanding of eicosanoid metabolism would aid in understanding the mechanisms that govern inflammatory processes. Here, we present a combined experimental and computational approach to understanding the biochemical basis of eicosanoid metabolism in macrophages. Lipidomic and transcriptomic measurements and analyses reveal temporal and dynamic changes of the eicosanoid metabolic network in mouse bone marrow-derived macrophages (BMDM) upon stimulation of the Toll-like receptor 4 with Kdo2-Lipid A (KLA) and stimulation of the P2X7 purinergic receptor with adenosine 5'-triphosphate. Kinetic models were developed for the cyclooxygenase (COX) and lipoxygenase branches of arachidonic acid metabolism, and then the rate constants were estimated with a data set from ATP-stimulated BMDM, using a two-step matrix-based approach employing a constrained least-squares method followed by nonlinear optimization. The robustness of the model was validated through parametric sensitivity, uncertainty analysis, and predicting an independent dataset from KLA-primed ATP-stimulated BMDM by allowing the parameters to vary within the uncertainty range of the calculated parameters. We analyzed the functional coupling between COX isozymes and terminal enzymes by developing a PGH2-divided model. This provided evidence for the functional coupling between COX-2 and PGE2 synthase, between COX-1/COX-2 and PGD2 synthase, and also between COX-1 and thromboxane A2 synthase. Further, these functional couplings were experimentally validated using COX-1 and COX-2 selective inhibitors. The resulting fluxomics analysis demonstrates that the "multi-omics" systems biology approach can define the complex machinery of eicosanoid networks.

Three-dimensional enhanced lipidomics analysis combining UPLC, differential ion mobility spectrometry, and mass spectrometric separation strategies.

Phospholipids serve as central structural components in cellular membranes and as potent mediators in numerous signaling pathways. There are six main classes of naturally occurring phospholipids distinguished by their distinct polar head groups that contain many unique molecular species with distinct fatty acid composition. Phospholipid molecular species are often expressed as isobaric species that are denoted by the phospholipid class and the total number of carbon atoms and double bonds contained in the esterified fatty acyl groups (e.g., phosphatidylcholine 34:2). Techniques to separate these molecules exist, and each has positive and negative attributes. Hydrophilic interaction liquid chromatography uses polar bonded silica to separate lipids by polar head group but not by specific molecular species. Reversed phase (RP) chromatography can separate by fatty acyl chain composition but not by polar head group. Herein we describe a new strategy called differential ion mobility spectrometry (DMS), which separates phospholipid classes by their polar head group. Combining DMS with current LC methods enhances phospholipid separation by increasing resolution, specificity, and signal-to-noise ratio. Additional application of specialized information-dependent acquisition methodologies along with RP chromatography allows full isobaric resolution, identification, and compositional characterization of specific phospholipids at the molecular level.

Targeted proteomics of the eicosanoid biosynthetic pathway completes an integrated genomics-proteomics-metabolomics picture of cellular metabolism.

Eicosanoids constitute a diverse class of bioactive lipid mediators that are produced from arachidonic acid and play critical roles in cell signaling and inflammatory aspects of numerous diseases. We have previously quantified eicosanoid metabolite production in RAW264.7 macrophage cells in response to Toll-like receptor 4 signaling and analyzed the levels of transcripts coding for the enzymes involved in the eicosanoid metabolite biosynthetic pathways. We now report the quantification of changes in protein levels under similar experimental conditions in RAW264.7 macrophages by multiple reaction monitoring mass spectrometry, an accurate targeted protein quantification method. The data complete the first fully integrated genomic, proteomic, and metabolomic analysis of the eicosanoid biochemical pathway.

Homeocurvature adaptation of phospholipids to pressure in deep-sea invertebrates.

Hydrostatic pressure increases with depth in the ocean, but little is known about the molecular bases of biological pressure tolerance. We describe a mode of pressure adaptation in comb jellies (ctenophores) that also constrains these animals' depth range. Structural analysis of deep-sea ctenophore lipids shows that they form a nonbilayer phase at pressures under which the phase is not typically stable. Lipidomics and all-atom simulations identified phospholipids with strong negative spontaneous curvature, including plasmalogens, as a hallmark of deep-adapted membranes that causes this phase behavior. Synthesis of plasmalogens enhanced pressure tolerance in Escherichia coli, whereas low-curvature lipids had the opposite effect. Imaging of ctenophore tissues indicated that the disintegration of deep-sea animals when decompressed could be driven by a phase transition in their phospholipid membranes.

Cholesterol-dependent LXR transcription factor activity represses pronociceptive effects of estrogen in sensory neurons and pain induced by myelin basic protein fragments.

Background: A bioactive myelin basic protein (MBP) fragment, comprising MBP84-104, is released in sciatic nerve after chronic constriction injury (CCI). Intraneural injection (IN) of MBP84-104 in an intact sciatic nerve is sufficient to induce persistent neuropathic pain-like behavior via robust transcriptional remodeling at the injection site and ipsilateral dorsal root ganglia (DRG) and spinal cord. The sex (female)-specific pronociceptive activity of MBP84-104 associates with sex-specific changes in cholesterol metabolism and activation of estrogen receptor (ESR)1 signaling. Methods: In male and female normal and post-CCI rat sciatic nerves, we assessed: (i) cholesterol precursor and metabolite levels by lipidomics; (ii) MBP84-104 interactors by mass spectrometry of MBP84-104 pull-down; and (iii) liver X receptor (LXR)α protein expression by immunoblotting. To test the effect of LXRα stimulation on IN MBP84-104-induced mechanical hypersensitivity, the LXRα expression was confirmed along the segmental neuraxis, in DRG and spinal cord, followed by von Frey testing of the effect of intrathecally administered synthetic LXR agonist, GW3965. In cultured male and female rat DRGs exposed to MBP84-104 and/or estrogen treatments, transcriptional effect of LXR stimulation by GW3965 was assessed on downstream cholesterol transporter Abc, interleukin (IL)-6, and pronociceptive Cacna2d1 gene expression. Results: CCI regulated LXRα ligand and receptor levels in nerves of both sexes, with cholesterol precursors, desmosterol and 7-DHC, and oxysterol elevated in females relative to males. MBP84-104 interacted with nuclear receptor coactivator (Ncoa)1, known to activate LXRα, injury-specific in nerves of both sexes. LXR stimulation suppressed ESR1-induced IL-6 and Cacna2d1 expression in cultured DRGs of both sexes and attenuated MBP84-104-induced pain in females. Conclusion: The injury-released bioactive MBP fragments induce pronociceptive changes by selective inactivation of nuclear transcription factors, including LXRα. By Ncoa1 sequestration, bioactive MBP fragments render LXRα function to counteract pronociceptive activity of estrogen/ESR1 in sensory neurons. This effect of MBP fragments is prevalent in females due to high circulating estrogen levels in females relative to males. Restoring LXR activity presents a promising therapeutic strategy in management of neuropathic pain induced by bioactive MBP.