Interleukin-6 signaling drives fibrosis in unresolved inflammation.

Fibrosis in response to tissue damage or persistent inflammation is a pathological hallmark of many chronic degenerative diseases. By using a model of acute peritoneal inflammation, we have examined how repeated inflammatory activation promotes fibrotic tissue injury. In this context, fibrosis was strictly dependent on interleukin-6 (IL-6). Repeat inflammation induced IL-6-mediated T helper 1 (Th1) cell effector commitment and the emergence of STAT1 (signal transducer and activator of transcription-1) activity within the peritoneal membrane. Fibrosis was not observed in mice lacking interferon-γ (IFN-γ), STAT1, or RAG-1. Here, IFN-γ and STAT1 signaling disrupted the turnover of extracellular matrix by metalloproteases. Whereas IL-6-deficient mice resisted fibrosis, transfer of polarized Th1 cells or inhibition of MMP activity reversed this outcome. Thus, IL-6 causes compromised tissue repair by shifting acute inflammation into a more chronic profibrotic state through induction of Th1 cell responses as a consequence of recurrent inflammation.

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.

Human platelets generate phospholipid-esterified prostaglandins via cyclooxygenase-1 that are inhibited by low dose aspirin supplementation.

Oxidized phospholipids (oxPLs) generated nonenzymatically display pleiotropic biological actions in inflammation. Their generation by cellular cyclooxygenases (COXs) is currently unknown. To determine whether platelets generate prostaglandin (PG)-containing oxPLs, then characterize their structures and mechanisms of formation, we applied precursor scanning-tandem mass spectrometry to lipid extracts of agonist-activated human platelets. Thrombin, collagen, or ionophore activation stimulated generation of families of PGs comprising PGE2 and D2 attached to four phosphatidylethanolamine (PE) phospholipids (16:0p/, 18:1p/, 18:0p/, and 18:0a/). They formed within 2 to 5 min of activation in a calcium, phospholipase C, p38 MAP kinases, MEK1, cPLA2, and src tyrosine kinase-dependent manner (28.1 ± 2.3 pg/2 × 108 platelets). Unlike free PGs, they remained cell associated, suggesting an autocrine mode of action. Their generation was inhibited by in vivo aspirin supplementation (75 mg/day) or in vitro COX-1 blockade. Inhibitors of fatty acyl reesterification blocked generation significantly, while purified COX-1 was unable to directly oxidize PE in vitro. This indicates that they form in platelets via rapid esterification of COX-1 derived PGE2/D2 into PE. In summary, COX-1 in human platelets acutely mediates membrane phospholipid oxidation via formation of PG-esterified PLs in response to pathophysiological agonists.

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.

Esterified eicosanoids: generation, characterization and function.

Eicosanoids are oxidation products of C20 polyunsaturated fatty acids (e.g. arachidonic acid) that include prostaglandins, thromboxanes, leukotrienes and hydroperoxy fatty acids. They have important biological roles in vivo, including regulation of renal, cardiovascular and gastrointestinal function. Historically, eicosanoids were thought to mediate their signaling actions exclusively as free acids, however evidence is now emerging that they may also be generated attached to other functional groups including phospholipids and glycerol, and that these more complex forms are pathophysiological signaling mediators in their own right. Early studies showed that exogenously added eicosanoids could become esterified into membrane phospholipids of cells, while more recently, it was uncovered that esterified eicosanoids are formed endogenously. This review summarizes our current knowledge of this area, starting with the early discoveries documenting what is known about eicosanoid generation and their esterification, and moving on to discuss the discovery that esterified eicosanoids are generated endogenously by a number of different cell types. Recent research that is highlighting new structures and functions of these important lipid mediators will be presented. This article is part of a Special Issue entitled: Oxidized phospholipids-their properties and interactions with proteins.

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.

Loss of CD4+ T cell IL-6R expression during inflammation underlines a role for IL-6 trans signaling in the local maintenance of Th17 cells.

IL-6 responses are classically orchestrated via a membrane-bound IL-6R (CD126) alpha subunit (classical IL-6R signaling) or through a soluble form of this cognate receptor (IL-6 trans signaling). Appraisal of IL-6R expression on human and mouse T cells emphasized that IL-6R expression is closely linked with that of CCR7 and CD62L. In this regard, infiltrating effector T cells from clinical and experimental peritonitis episodes lose IL-6R expression, and anti-CD3/CD28 Ab costimulation of peripheral T cells in vitro leads to a downregulation in IL-6R expression. Consequently, IL-6 signaling through membrane-bound IL-6R seems to be limited to naive or central memory T cell populations. Loss of IL-6R expression by activated T cells further suggests that these effector cells might still retain IL-6 responsiveness via IL-6 trans signaling. Using IL-6R-deficient mice and recombinant tools that modulate the capacity of IL-6 to signal via its soluble receptor, we report that local control of IL-6 trans signaling regulates the effector characteristics of the T cell infiltrate and promotes the maintenance of IL-17A-secreting CD4(+) T cells. Therefore, we concluded that classical IL-6R signaling in naive or central memory CD4(+) T cells is required to steer their effector characteristics, whereas local regulation of soluble IL-6R activity might serve to maintain the cytokine profile of the Th cell infiltrate. Therefore, the activation status of a T cell population is linked with an alteration in IL-6 responsiveness.

Contribution of individual histidines to prion protein copper binding.

The prion protein is well-established as a copper binding protein. The N-terminus of the protein contains an octameric repeat region with each of the four repeats containing a histidine. The N-terminus has two additional histidines distal to the repeat region that has been commonly known as the fifth site. While binding of copper by the protein has been extensively studied, the contribution of each histidine to copper binding in the full-length protein has not. Here we used a battery of mutants of the recombinant mouse prion protein to assess copper binding with both isothermal titration calorimetry and cyclic voltammetry. The findings indicate that there is extensive cooperativity between different binding sites in the protein. The two highest-affinity binding events occur at the fifth site and at the octameric repeat region. However, the first binding is that to the octameric repeat region. Subsequent binding events after the two initial binding events have lower affinities within the octameric repeat region.

Oncostatin M receptor-beta signaling limits monocytic cell recruitment in acute inflammation.

Although the IL-6-related cytokine oncostatin M (OSM) affects processes associated with disease progression, the specific function of OSM in the face of an inflammatory challenge remains unclear. In this report, a peritoneal model of acute inflammation was used to define the influence of OSM on chemokine-mediated leukocyte recruitment. When compared with wild-type and IL-6-deficient mice, peritoneal inflammation in oncostatin M receptor-beta-deficient (OSMR-KO) mice resulted in enhanced monocytic cell trafficking. In contrast to IL-6-deficient mice, OSMR-KO mice displayed no difference in neutrophil and lymphocyte migration. Subsequent in vitro studies using human peritoneal mesothelial cells and an in vivo appraisal of inflammatory chemokine expression after peritoneal inflammation identified OSM as a prominent regulator of CCL5 expression. Specifically, OSM inhibited IL-1beta-mediated NF-kappaB activity and CCL5 expression in human mesothelial cells. This was substantiated in vivo where peritoneal inflammation in OSMR-KO mice resulted in a temporal increase in both CCL5 secretion and NF-kappaB activation. These findings suggest that IL-6 and OSM individually affect the profile of leukocyte trafficking, and they point to a hitherto unidentified interplay between OSM signaling and the inflammatory activation of NF-kappaB.

Enzymatic lipid oxidation by eosinophils propagates coagulation, hemostasis, and thrombotic disease.

Blood coagulation is essential for physiological hemostasis but simultaneously contributes to thrombotic disease. However, molecular and cellular events controlling initiation and propagation of coagulation are still incompletely understood. In this study, we demonstrate an unexpected role of eosinophils during plasmatic coagulation, hemostasis, and thrombosis. Using a large-scale epidemiological approach, we identified eosinophil cationic protein as an independent and predictive risk factor for thrombotic events in humans. Concurrent experiments showed that eosinophils contributed to intravascular thrombosis by exhibiting a strong endogenous thrombin-generation capacity that relied on the enzymatic generation and active provision of a procoagulant phospholipid surface enriched in 12/15-lipoxygenase-derived hydroxyeicosatetraenoic acid-phosphatidylethanolamines. Our findings reveal a previously unrecognized role of eosinophils and enzymatic lipid oxidation as regulatory elements that facilitate both hemostasis and thrombosis in response to vascular injury, thus identifying promising new targets for the treatment of thrombotic disease.

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.

Inactivation of the ferroptosis regulator Gpx4 triggers acute renal failure in mice.

Ferroptosis is a non-apoptotic form of cell death induced by small molecules in specific tumour types, and in engineered cells overexpressing oncogenic RAS. Yet, its relevance in non-transformed cells and tissues is unexplored and remains enigmatic. Here, we provide direct genetic evidence that the knockout of glutathione peroxidase 4 (Gpx4) causes cell death in a pathologically relevant form of ferroptosis. Using inducible Gpx4(-/-) mice, we elucidate an essential role for the glutathione/Gpx4 axis in preventing lipid-oxidation-induced acute renal failure and associated death. We furthermore systematically evaluated a library of small molecules for possible ferroptosis inhibitors, leading to the discovery of a potent spiroquinoxalinamine derivative called Liproxstatin-1, which is able to suppress ferroptosis in cells, in Gpx4(-/-) mice, and in a pre-clinical model of ischaemia/reperfusion-induced hepatic damage. In sum, we demonstrate that ferroptosis is a pervasive and dynamic form of cell death, which, when impeded, promises substantial cytoprotection.

AMPA receptor activation promotes non-amyloidogenic amyloid precursor protein processing and suppresses neuronal amyloid-ß production.

Soluble oligomeric amyloid ß peptide (Aß) generated from processing of the amyloid precursor protein (APP) plays a central role in the pathogenesis of Alzheimer's Disease (AD) and through actions at glutamatergic synapses affects excitability and plasticity. The physiological control of APP processing is not fully understood but stimulation of synaptic NMDA receptors (NMDAR) can suppress Aß levels through an ERK-dependent increase in α-secretase activity. AMPA-type glutamate receptors (AMPAR) couple to ERK phosphorylation independently of NMDAR activation raising the possibility that stimulation of AMPAR might similarly promote non-amyloidogenic APP processing. We have tested this hypothesis by investigating whether AMPAR directly regulate APP processing in cultured mouse cortical neurons, by analyzing APP C-terminal fragments (CTFs), soluble APP (sAPP), Aß levels, and cleavage of an APP-GAL4 reporter protein. We report that direct stimulation of AMPAR increases non-amyloidogenic α-secretase-mediated APP processing and inhibits Aß production. Processing was blocked by the matrix metalloproteinase inhibitor TAPI-1 but was only partially dependent on Ca(2+) influx and ERK activity. AMPAR can therefore, be added to the repertoire of receptors that couple to non-amyloidogenic APP processing at glutamatergic synapses and thus pharmacological targeting of AMPAR could potentially influence the development and progression of Aß pathology in AD.

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.

An optical sensor for reactive oxygen species: encapsulation of functionalised silica nanoparticles into silicate nanoprobes to reduce fluorophore leaching.

Sol-gel nanoprobes, also known as Photonic Explorer for Bioanalysis with Biologically Localised Embedding (PEBBLE), capable of performing in-vitro intracellular monitoring of reactive oxygen species have been developed using a modified form of 5(6)-carboxyfluorescein diacetate. A sol-gel matrix was selected for the design of the probes as it is photostable, optically transparent and chemically inert, and to minimise leaching of the dye from the porous matrix it was covalently immobilised to silica nanoparticles (15 nm). Using this approach, 0.1% of the dye was found to leach over a typical analysis time of 5 h and minimal photobleaching was observed. In addition, the nanoprobes were shown to respond to hydrogen peroxide, hydroxyl anions, nitric oxide, peroxynitrile and superoxide anions, obtaining limits of detection of 2.2, 1.1, 3.2, 1.1 and 1.1 nM respectively. The nanoprobes were subsequently introduced into bovine oviducts using a lipid transfection reagent (Escort IV) and fluorescence was observed.