Lipidomic and transcriptional analysis of the linoleoyl-omega-hydroxyceramide biosynthetic pathway in human psoriatic lesions.

A complex assembly of lipids including fatty acids, cholesterol, and ceramides is vital to the integrity of the mammalian epidermal barrier. The formation of this barrier requires oxidation of the substrate fatty acid, linoleic acid (LA), which is initiated by the enzyme 12R-lipoxygenase (LOX). In the epidermis, unoxidized LA is primarily found in long-chain acylceramides termed esterified omega-hydroxy sphingosine (EOS)/phytosphingosine/hydroxysphingosine (collectively EOx). The precise structure and localization of LOX-oxidized EOx in the human epidermis is unknown, as is their regulation in diseases such as psoriasis, one of the most common inflammatory diseases affecting the skin. Here, using precursor LC/MS/MS, we characterized multiple intermediates of EOx, including 9-HODE, 9,10-epoxy-13-HOME, and 9,10,13-TriHOME, in healthy human epidermis likely to be formed via the epidermal LOX pathways. The top layers of the skin contained more LA, 9-HODE, and 9,10,13-TriHOME EOSs, whereas 9,10-epoxy-13-HOME EOS was more prevalent deeper in the stratum corneum. In psoriatic lesions, levels of native EOx and free HODEs and HOMEs were significantly elevated, whereas oxidized species were generally reduced. A transcriptional network analysis of human psoriatic lesions identified significantly elevated expression of the entire biosynthetic/metabolic pathway for oxygenated ceramides, suggesting a regulatory function for EOx lipids in reconstituting epidermal integrity. The role of these new lipids in progression or resolution of psoriasis is currently unknown. We also discovered the central coordinated role of the zinc finger protein transcription factor, ZIC1, in driving the phenotype of this disease. In summary, long-chain oxygenated ceramide metabolism is dysregulated at the lipidomic level in psoriasis, likely driven by the transcriptional differences also observed, and we identified ZIC1 as a potential regulatory target for future therapeutic interventions.

The procoagulant activity of tissue factor expressed on fibroblasts is increased by tissue factor-negative extracellular vesicles.

Tissue factor (TF) is critical for the activation of blood coagulation. TF function is regulated by the amount of externalised phosphatidylserine (PS) and phosphatidylethanolamine (PE) on the surface of the cell in which it is expressed. We investigated the role PS and PE in fibroblast TF function. Fibroblasts expressed 6-9 x 104 TF molecules/cell but had low specific activity for FXa generation. We confirmed that this was associated with minimal externalized PS and PE and characterised for the first time the molecular species of PS/PE demonstrating that these differed from those found in platelets. Mechanical damage of fibroblasts, used to simulate vascular injury, increased externalized PS/PE and led to a 7-fold increase in FXa generation that was inhibited by annexin V and an anti-TF antibody. Platelet-derived extracellular vesicles (EVs), that did not express TF, supported minimal FVIIa-dependent FXa generation but substantially increased fibroblast TF activity. This enhancement in fibroblast TF activity could also be achieved using synthetic liposomes comprising 10% PS without TF. In conclusion, despite high levels of surface TF expression, healthy fibroblasts express low levels of external-facing PS and PE limiting their ability to generate FXa. Addition of platelet-derived TF-negative EVs or artificial liposomes enhanced fibroblast TF activity in a PS dependent manner. These findings contribute information about the mechanisms that control TF function in the fibroblast membrane.

A HPLC method to monitor the occurrence of lipid peroxidation in intravenous lipid emulsions used in parenteral nutrition using in-line UV and charged aerosol detection.

Parenteral Nutrition (PN) provides life sustaining support where gastrointestinal nutrition is inadequate due to disease or prematurity. Intravenous lipid emulsions (IVLEs) form a staple part of PN. Whilst the physical stability of IVLE's is relatively well known and quantified, chemical stability is an area where little testing has occurred. We report a new sensitive method for the monitoring of selected triglycerides present within two IVLEs and the detection and quantification of the peroxidation product 4-hydroxynonenal (HNE) using HPLC with in-line UV and charged aerosol detection (CAD). IVLEs used included the soy-bean oil based emulsion Intralipid® 20% and SMOFlipid® 20% (Fresenius Kabi UK), based on soy-bean, olive, fish oil and medium chain triglycerides. Assay validation gave R2 values of ≥0.99 for all selected triglyceride peaks and 4-hydroxynonenal. Inter and intra-day repeatability gave RSD values < 7.2% for CAD detection, achieving a precise and repeatable method. HNE was confirmed through internal standardisation of the HPLC method. Selected triglycerides were identified using ESI-MS with MicroTOF. This novel method permits the chemical stability of IVLEs to be quantified and monitored in respect to lipid peroxidation during storage prior to delivery to the patient, ensuring the optimal safety of IVLEs in a clinical setting.

Networks of enzymatically oxidized membrane lipids support calcium-dependent coagulation factor binding to maintain hemostasis.

Blood coagulation functions as part of the innate immune system by preventing bacterial invasion, and it is critical to stopping blood loss (hemostasis). Coagulation involves the external membrane surface of activated platelets and leukocytes. Using lipidomic, genetic, biochemical, and mathematical modeling approaches, we found that enzymatically oxidized phospholipids (eoxPLs) generated by the activity of leukocyte or platelet lipoxygenases (LOXs) were required for normal hemostasis and promoted coagulation factor activities in a Ca2+- and phosphatidylserine (PS)-dependent manner. In wild-type mice, hydroxyeicosatetraenoic acid-phospholipids (HETE-PLs) enhanced coagulation and restored normal hemostasis in clotting-deficient animals genetically lacking p12-LOX or 12/15-LOX activity. Murine platelets generated 22 eoxPL species, all of which were missing in the absence of p12-LOX. Humans with the thrombotic disorder antiphospholipid syndrome (APS) had statistically significantly increased HETE-PLs in platelets and leukocytes, as well as greater HETE-PL immunoreactivity, than healthy controls. HETE-PLs enhanced membrane binding of the serum protein ß2GP1 (ß2-glycoprotein 1), an event considered central to the autoimmune reactivity responsible for APS symptoms. Correlation network analysis of 47 platelet eoxPL species in platelets from APS and control subjects identified their enzymatic origin and revealed a complex network of regulation, with the abundance of 31 p12-LOX-derived eoxPL molecules substantially increased in APS. In summary, circulating blood cells generate networks of eoxPL molecules, including HETE-PLs, which change membrane properties to enhance blood coagulation and contribute to the excessive clotting and immunoreactivity of patients with APS.

The Precise Structures and Stereochemistry of Trihydroxy-linoleates Esterified in Human and Porcine Epidermis and Their Significance in Skin Barrier Function: IMPLICATION OF AN EPOXIDE HYDROLASE IN THE TRANSFORMATIONS OF LINOLEATE.

Creation of an intact skin water barrier, a prerequisite for life on dry land, requires the lipoxygenase-catalyzed oxidation of the essential fatty acid linoleate, which is esterified to the ω-hydroxyl of an epidermis-specific ceramide. Oxidation of the linoleate moiety by lipoxygenases is proposed to facilitate enzymatic cleavage of the ester bond, releasing free ω-hydroxyceramide for covalent binding to protein, thus forming the corneocyte lipid envelope, a key component of the epidermal barrier. Herein, we report the transformations of esterified linoleate proceed beyond the initial steps of oxidation and epoxyalcohol synthesis catalyzed by the consecutive actions of 12R-LOX and epidermal LOX3. The major end product in human and porcine epidermis is a trihydroxy derivative, formed with a specificity that implicates participation of an epoxide hydrolase in converting epoxyalcohol to triol. Of the 16 possible triols arising from hydrolysis of 9,10-epoxy-13-hydroxy-octadecenoates, using LC-MS and chiral analyses, we identify and quantify specifically 9R,10S,13R-trihydroxy-11E-octadecenoate as the single major triol esterified in porcine epidermis and the same isomer with lesser amounts of its 10R diastereomer in human epidermis. The 9R,10S,13R-triol is formed by SN2 hydrolysis of the 9R,10R-epoxy-13R-hydroxy-octadecenoate product of the LOX enzymes, a reaction specificity characteristic of epoxide hydrolase. The high polarity of triol over the primary linoleate products enhances the concept that the oxidations disrupt corneocyte membrane lipids, promoting release of free ω-hydroxyceramide for covalent binding to protein and sealing of the waterproof barrier.

Human Platelets Utilize Cycloxygenase-1 to Generate Dioxolane A3, a Neutrophil-activating Eicosanoid.

Eicosanoids are important mediators of fever, pain, and inflammation that modulate cell signaling during acute and chronic disease. We show by using lipidomics that thrombin-activated human platelets generate a new type of eicosanoid that both stimulates and primes human neutrophil integrin (Mac-1) expression, in response to formylmethionylleucylphenylalanine. Detailed characterization proposes a dioxolane structure, 8-hydroxy-9,11-dioxolane eicosatetraenoic acid (dioxolane A3, DXA3). The lipid is generated in nanogram amounts by platelets from endogenous arachidonate during physiological activation, with inhibition by aspirin in vitro or in vivo, implicating cyclooxygenase-1 (COX). Pharmacological and genetic studies on human/murine platelets revealed that DXA3 formation requires protease-activated receptors 1 and 4, cytosolic phospholipase A2 (cPLA2), Src tyrosine kinases, p38 MAPK, phospholipase C, and intracellular calcium. From data generated by purified COX isoforms and chemical oxidation, we propose that DXA3 is generated by release of an intermediate from the active site followed by oxygenation at C8. In summary, a new neutrophil-activating platelet-derived lipid generated by COX-1 is presented that can activate or prime human neutrophils, suggesting a role in innate immunity and acute inflammation.

Skin Metabolites Define a New Paradigm in the Localization of Skin Tropic Memory T Cells.

The localization of memory T cells to human skin is essential for long-term immune surveillance and the maintenance of barrier integrity. The expression of CCR8 during naive T cell activation is controlled by skin-specific factors derived from epidermal keratinocytes and not by resident dendritic cells. In this study, we show that the CCR8-inducing factors are heat stable and protease resistant and include the vitamin D3 metabolite 1α,25-dihydroxyvitamin D3 and PGE2. The effect of either metabolite alone on CCR8 expression was weak, whereas their combination resulted in robust CCR8 expression. Elevation of intracellular cAMP was essential because PGE2 could be substituted with the adenylyl cyclase agonist forskolin, and CCR8 expression was sensitive to protein kinase A inhibition. For effective induction, exposure of naive T cells to these epidermal factors needed to occur either prior to or during T cell activation even though CCR8 was only detected 4-5 d later in proliferating T cells. The importance of tissue environments in maintaining cellular immune surveillance networks within distinct healthy tissues provides a paradigm shift in adaptive immunity. Epidermal-derived vitamin D3 metabolites and PGs provide an essential cue for the localization of CCR8(+) immune surveillance T cells within healthy human skin.

A novel role for 12/15-lipoxygenase in regulating autophagy.

12/15-Lipoxygenase (LOX) enzymatically generates oxidized phospholipids in monocytes and macrophages. Herein, we show that cells deficient in 12/15-LOX contain defective mitochondria and numerous cytoplasmic vacuoles containing electron dense material, indicating defects in autophagy or membrane processing, However, both LC3 expression and lipidation were normal both basally and on chloroquine treatment. A LOX-derived oxidized phospholipid, 12-hydroxyeicosatetraenoic acid-phosphatidylethanolamine (12-HETE-PE) was found to be a preferred substrate for yeast Atg8 lipidation, versus native PE, while both native and oxidized PE were effective substrates for LC3 lipidation. Last, phospholipidomics demonstrated altered levels of several phospholipid classes. Thus, we show that oxidized phospholipids generated by 12/15-LOX can act as substrates for key proteins required for effective autophagy and that cells deficient in this enzyme show evidence of autophagic dysfunction. The data functionally link phospholipid oxidation with autophagy for the first time.

Identification and quantification of aminophospholipid molecular species on the surface of apoptotic and activated cells.

This protocol measures externalization of aminophospholipids (APLs) to the outside of the plasma membrane using mass spectrometry (MS). APL externalization occurs in numerous events, and it is relevant for transplant medicine, immunity and cancer. In this protocol, externalized APLs are chemically modified by using a cell-impermeable reagent (sulfo-NHS-biotin), and then they are isolated via a liquid:liquid extraction and quantified by reverse-phase liquid chromatography tandem MS (LC-MS/MS) against in-house-generated standards. This protocol describes a complementary method to existing assays that are not quantitative (e.g., annexin V flow cytometry), and it is applicable to the study of membrane reorganization in all cell types during apoptosis (e.g., during development, cancer, psychiatric disorders and other conditions, aging, vesiculation and cell division). The protocol takes ∼2-4 d, including the generation of standards.

The importance of the lipoxygenase-hepoxilin pathway in the mammalian epidermal barrier.

This review covers the background to discovery of the two key lipoxygenases (LOX) involved in epidermal barrier function, 12R-LOX and eLOX3, and our current views on their functioning. In the outer epidermis, their consecutive actions oxidize linoleic acid esterified in ω-hydroxy-ceramide to a hepoxilin-related derivative. The relevant background to hepoxilin and trioxilin biochemistry is briefly reviewed. We outline the evidence that linoleate in the ceramide is the natural substrate of the two LOX enzymes and our proposal for its importance in construction of the epidermal water barrier. Our hypothesis is that the oxidation promotes hydrolysis of the oxidized linoleate moiety from the ceramide. The resulting free ω-hydroxyl of the ω-hydroxyceramide is covalently bound to proteins on the surface of the corneocytes to form the corneocyte lipid envelope, a key barrier component. Understanding the role of the LOX enzymes and their hepoxilin products should provide rational approaches to ameliorative therapy for a number of the congenital ichthyoses involving compromised barrier function. This article is part of a Special Issue entitled The Important Role of Lipids in the Epidermis and their Role in the Formation and Maintenance of the Cutaneous Barrier. Guest Editors: Kenneth R. Feingold and Peter Elias.

Characterization of platelet aminophospholipid externalization reveals fatty acids as molecular determinants that regulate coagulation.

Aminophospholipid (APL) trafficking across the plasma membrane is a key event in cell activation, apoptosis, and aging and is required for clearance of dying cells and coagulation. Currently the phospholipid molecular species externalized are unknown. Using a lipidomic method, we show that thrombin, collagen, or ionophore-activated human platelets externalize two phosphatidylserines (PSs) and five phosphatidylethanolamines (PEs). Four percent of the total cellular PE/PS pool (∼300 ng/2 × 10(8) cells, thrombin), is externalized via calcium mobilization and protease-activated receptors-1 and -4, and 48% is contained in microparticles. Apoptosis and energy depletion (aging) externalized the same APLs in a calcium-dependent manner, and all stimuli externalized oxidized phospholipids, termed hydroxyeicosatetraenoic acid-PEs. Transmembrane protein-16F (TMEM-16F), the protein mutated in Scott syndrome, was required for PE/PS externalization during thrombin activation and energy depletion, but not apoptosis. Platelet-specific APLs optimally supported tissue factor-dependent coagulation in human plasma, vs. APL with longer or shorter fatty acyl chains. This finding demonstrates fatty acids as molecular determinants of APL that regulate hemostasis. Thus, the molecular species of externalized APL during platelet activation, apoptosis, and energy depletion were characterized, and their ability to support coagulation revealed. The findings have therapeutic implications for bleeding disorders and transfusion therapy. The assay could be applied to other cell events characterized by APL externalization, including cell division and vesiculation.

Steric analysis of epoxyalcohol and trihydroxy derivatives of 9-hydroperoxy-linoleic acid from hematin and enzymatic synthesis.

We characterize the allylic epoxyalcohols and their trihydroxy hydrolysis products generated from 9R- and 9S-hydroperoxy-octadecenoic acid (HPODE) under non-enzymatic conditions, reaction with hematin and subsequent acid hydrolysis, and enzymatic conditions, incubation with Beta vulgaris containing a hydroperoxide isomerase and epoxide hydrolase. The products were resolved by HPLC and the regio and stereo-chemistry of the transformations were determined through a combination of (1)H NMR and GC-MS analysis of dimethoxypropane derivatives. Four trihydroxy isomers were identified upon mild acid hydrolysis of 9S,10S-trans-epoxy-11E-13S-hydroxyoctadecenoate: 9S,10R,13S, 9S,12R,13S, 9S,10S,13S and 9S,12S,13S-trihydroxy-octadecenoic acids, in the ratio 40:26:22:12. We also identified a prominent δ-ketol rearrangement product from the hydrolysis as mainly the 9-hydroxy-10E-13-oxo isomer. Short incubation (5 min) of 9R- and 9S-HPODE with B. vulgaris extract yielded the 9R- and 9S-hydroxy-10E-12R,13S-cis-epoxy products respectively. Longer incubation (60 min) gave one specific hydrolysis product via epoxide hydrolase, the 9R/S,12S,13S-trihydroxyoctadecenoate. These studies provide a practical approach for the isolation and characterization of allylic epoxy alcohol and trihydroxy products using a combination of HPLC, GC-MS and (1)H NMR.

Novel keto-phospholipids are generated by monocytes and macrophages, detected in cystic fibrosis, and activate peroxisome proliferator-activated receptor-γ.

12/15-Lipoxygenases (LOXs) in monocytes and macrophages generate novel phospholipid-esterified eicosanoids. Here, we report the generation of two additional families of related lipids comprising 15-ketoeicosatetraenoic acid (KETE) attached to four phosphatidylethanolamines (PEs). The lipids are generated basally by 15-LOX in IL-4-stimulated monocytes, are elevated on calcium mobilization, and are detected at increased levels in bronchoalveolar lavage fluid from cystic fibrosis patients (3.6 ng/ml of lavage). Murine peritoneal macrophages generate 12-KETE-PEs, which are absent in 12/15-LOX-deficient mice. Inhibition of 15-prostaglandin dehydrogenase prevents their formation from exogenous 15-hydroxyeicosatetraenoic acid-PE in human monocytes. Both human and murine cells also generated analogous hydroperoxyeicosatetraenoic acid-PEs. The electrophilic reactivity of KETE-PEs is shown by their Michael addition to glutathione and cysteine. Lastly, both 15-hydroxyeicosatetraenoic acid-PE and 15-KETE-PE activated peroxisome proliferator-activated receptor-γ reporter activity in macrophages in a dose-dependent manner. In summary, we demonstrate novel peroxisome proliferator-activated receptor-γ-activating oxidized phospholipids generated enzymatically by LOX and 15-prostaglandin dehydrogenase in primary monocytic cells and in a human Th2-related lung disease. The lipids are a new family of bioactive mediators from the 12/15-LOX pathway that may contribute to its known anti-inflammatory actions in vivo.

Oxidized phospholipid signaling in immune cells.

Oxidized phospholipids (oxPLs) that can be generated either enzymatically or non-enzymatically are fast becoming recognized as important signaling mediators of the immune system. Hundreds of structures exist, but only a small fraction have been studied in detail. Their known activities include regulation of adhesion molecule expression, pro-coagulant activity and inhibition of Toll-like receptor signaling, and several have been detected in models of human and animal disease. In this review, the most studied structures of oxPLs will be summarized, along with descriptions of their known biological actions. Subsequently, the focus will be on the more recently described forms generated acutely by lipoxygenases (LOX) in human and murine immune cells.

In vivo response of GsdmA3Dfl/+ mice to topically applied anti-psoriatic agents: effects on epidermal thickness, as determined by optical coherence tomography and H&E staining.

This study evaluated in vivo the potential of optical coherence tomography (OCT) to determine changes in thickness of the epidermis in response to the topically applied anti-psoriatics betamethasone dipropionate (BD), salicylic acid (SA) and also fish oil (FO). GsdmA3Dfl/+ mice have an inflammatory hair loss phenotype that includes hyperproliferation and epidermal thickening, hence a potential psoriasis model. Changes in epidermal thickness were evaluated over a period of 10 days, with the mice treated with combined BD + SA, FO + SA and BD + FO + SA. The data were validated with conventional measurement using H&E staining coupled with microscopy. Initial baseline measurement revealed an average epidermal thickness of 26.92 ± 1.17 µm. After 10 days of treatment with BD, the average epidermal thickness was reduced by 38.8% (P = 0.0001), and inversely, treatment with FO resulted in an unexpected 105% increase (P = 0.0001) in epidermal thickness. Combined BD + FO treatment did not cause any significant change (P = 0.3755) and may further indicate opposing effects on keratinocyte proliferation. The data obtained using OCT were statistically the same as those obtained by H&E/microscopy (P = 0.4325), supporting a greater role for OCT in dermatological studies, while also allowing a reduction in the number of animals used in such studies as sacrifice at individual timepoints is not necessary.

Nitroarachidonic acid, a novel peroxidase inhibitor of prostaglandin endoperoxide H synthases 1 and 2.

Prostaglandin endoperoxide H synthase (PGHS) catalyzes the oxidation of arachidonate to prostaglandin H(2). We have previously synthesized and chemically characterized nitroarachidonic acid (AANO(2)), a novel anti-inflammatory signaling mediator. Herein, the interaction of AANO(2) with PGHS was analyzed. AANO(2) inhibited oxygenase activity of PGHS-1 but not PGHS-2. AANO(2) exhibited time- and concentration-dependent inhibition of peroxidase activity in both PGHS-1 and -2. The plot of k(obs) versus AANO(2) concentrations showed a hyperbolic function with k(inact) = 0.045 s(-1) and K(i)(*app) = 0.019 µM for PGHS-1 and k(inact) = 0.057 s(-1) and K(i)(*app) = 0.020 µM for PGHS-2. Kinetic analysis suggests that inactivation of PGHS by AANO(2) involves two sequential steps: an initial reversible binding event (described by K(i)) followed by a practically irreversible event (K(i)(*app)) leading to an inactivated enzyme. Inactivation was associated with irreversible disruption of heme binding to the protein. The inhibitory effects of AANO(2) were selective because other nitro-fatty acids tested, such as nitrooleic acid and nitrolinoleic acid, were unable to inhibit enzyme activity. In activated human platelets, AANO(2) significantly decreased PGHS-1-dependent thromboxane B(2) formation in parallel with a decrease in platelet aggregation, thus confirming the biological relevance of this novel inhibitory pathway.

Esterified eicosanoids are acutely generated by 5-lipoxygenase in primary human neutrophils and in human and murine infection.

5-Lipoxygenase (5-LOX) plays key roles in infection and allergic responses. Herein, four 5-LOX-derived lipids comprising 5-hydroxyeicosatetraenoic acid (HETE) attached to phospholipids (PLs), either phosphatidylethanolamine (PE) or phosphatidylcholine (18:0p/5-HETE-PE, 18:1p/5-HETE-PE, 16:0p/5-HETE-PE, and 16:0a/5-HETE-PC), were identified in primary human neutrophils. They formed within 2 minutes in response to serum-opsonized Staphylococcus epidermidis or f-methionine-leucine-phenylalanine, with priming by lipopolysaccharide, granulocyte macrophage colony-stimulating factor, or cytochalasin D. Levels generated were similar to free 5-HETE (0.37 ± 0.14 ng vs 0.55 ± 0.18 ng/10(6) cells, esterified vs free 5-HETE, respectively). They remained cell associated, localizing to nuclear and extranuclear membrane, and were formed by fast esterification of newly synthesized free 5-HETE. Generation also required Ca(2+), phospholipase C, cytosolic and secretory phospholipase A(2), 5-LOX activating protein, and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1. 5-HETE-PLs were detected in murine S epidermidis peritonitis, paralleling neutrophil influx, and in effluent from Gram-positive human bacterial peritonitis. Formation of neutrophil extracellular traps was significantly enhanced by 5-LOX inhibition but attenuated by HETE-PE, whereas 5-HETE-PE enhanced superoxide and interleukin-8 generation. Thus, new molecular species of oxidized PL formed by human neutrophils during bacterial infection are identified and characterized.

Delineating immune-mediated mechanisms underlying hair follicle destruction in the mouse mutant defolliculated.

Defolliculated (Gsdma3(Dfl)/+) mice have a hair loss phenotype that involves an aberrant hair cycle, altered sebaceous gland differentiation with reduced sebum production, chronic inflammation, and ultimately the loss of the hair follicle. Hair loss in these mice is similar to that seen in primary cicatricial, or scarring alopecias in which immune targeting of hair follicle stem cells has been proposed as a key factor resulting in permanent hair follicle destruction. In this study we examine the mechanism of hair loss in GsdmA3(Dfl)/+ mice. Aberrant expression patterns of stem cell markers during the hair cycle, in addition to aberrant behavior of the melanocytes leading to ectopic pigmentation of the hair follicle and epidermis, indicated the stem cell niche was not maintained. An autoimmune mechanism was excluded by crossing the mice with rag1-/- mice. However, large numbers of macrophages and increased expression of ICAM-1 were still present and may be involved either directly or indirectly in the hair loss. Reverse transcriptase-PCR (RT-PCR) and immunohistochemistry of sebaceous gland differentiation markers revealed reduced peroxisome proliferator-activated receptor-γ (PPARγ), a potential cause of reduced sebum production, as well as the potential involvement of the innate immune system in the hair loss. As reduced PPARγ expression has recently been implicated as a cause for lichen planopilaris, these mice may be useful for testing therapies.

Quantitative assays for esterified oxylipins generated by immune cells.

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.