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 Ca 2+ -and phosphatidylserine (PS)-dependent manner. In wild-type mice, hydroxyeicosatetraenoic acid-phospholipids (HETE-PLs) † Corresponding author. o-donnellvb@cardiff.ac.uk (V.B.O'D.); collinspw@cardiff.ac.uk (P.W.C.). * These authors contributed jointly to the work. Author contributions:Experiments were conducted by SNL, DAS, GM, RU, AOC, DF, JM, SR, VJT, AB, SF, MA, MH, KAR, CPT, JA and GK, and designed by SNL, DAS, PDG, SH, VBO, SAJ, PRT, PWC, PVJ. CLP and SO provided clinical samples. AP provided supervision and training. SNL, DAS, VBO and PWC wrote the paper. All authors edited the manuscript. Competing interests:The authors have declared that they have no competing interests. Europe PMC Funders GroupAuthor Manuscript Sci Signal. Author manuscript; available in PMC 2017 December 07. Europe PMC Funders Author ManuscriptsEurope PMC Funders Author Manuscripts 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 β2GPI (β2-glycoprotein I), 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.

Atherosclerosis and its complications are responsible for one in three global deaths. Nutraceuticals show promise in the prevention and treatment of atherosclerosis but require an indepth understanding of the mechanisms underlying their actions. A previous study showed that the omega-6 fatty acid, dihomo-γ-linolenic acid (DGLA), attenuated atherosclerosis in the apolipoprotein E deficient mouse model system. However, the mechanisms underlying such protective effects of DGLA are poorly understood and were therefore investigated. We show that DGLA attenuates chemokine-driven monocytic migration together with foam cell formation and the expression of key pro-atherogenic genes induced by three pro-inflammatory cytokines in human macrophages. The effect of DGLA on interferon-γ signaling was mediated via inhibition of signal transducer and activator of transcription-1 phosphorylation on serine 727. In relation to anti-foam cell action, DGLA inhibits modified LDL uptake by both macropinocytosis and receptor-mediated endocytosis, the latter by reduction in expression of two key scavenger receptors (SR-A and CD36), and stimulates cholesterol efflux from foam cells. DGLA also improves macrophage mitochondrial bioenergetic profile by decreasing proton leak. Gamma-linolenic acid and prostaglandin E1, upstream precursor and key metabolite respectively of DGLA, also acted in an anti-atherogenic manner. The actions of DGLA extended to other key atherosclerosis-associated cell types with attenuation of endothelial cell proliferation and migration of smooth muscle cells in response to platelet-derived growth factor. This study provides novel insights into the molecular mechanisms underlying the anti-atherogenic actions of DGLA and supports further assessments on its protective effects on plaque regression in vivo and in human trials.

BackgroundAlveolar macrophages are sentinels of the pulmonary mucosa and central to maintaining immunological homeostasis. However, their role in governing the response to allergen is not fully understood. Inappropriate responses to the inhaled environment manifest as asthma.MethodsWe utilized a mechanistic IL-13-driven model and a house dust mite allergen mucosal sensitization model of allergic airway disease to investigate the role of alveolar macrophages in regulating pulmonary inflammation.ResultsIL-13-dependent eosinophilic and Th2 inflammation was enhanced in mice depleted of alveolar macrophages using clodronate liposomes. Similarly, depletion of alveolar macrophages during house dust mite sensitization or established disease resulted in augmented Th2 immunity and increased allergen-specific IgG1 and IgE. Clodronate treatment also delayed the resolution of tissue inflammation following cessation of allergen challenge. Strikingly, tissue interstitial macrophages were elevated in alveolar macrophage-deficient mice identifying a new homeostatic relationship between different macrophage subtypes. A novel role for the macrophage-derived immunoregulatory cytokine IL-27 was identified in modulating Th2 inflammation following mucosal allergen exposure.ConclusionsIn summary, alveolar macrophages are critical regulators of Th2 immunity and their dysregulation promotes an inflammatory environment with exacerbation of allergen-induced airway pathology. Manipulating IL-27 may provide a novel therapeutic strategy for the treatment of asthma.

The inflammatory activation and recruitment of defined myeloid populations is essential for controlling the bridge between innate and adaptive immunity and shaping the immune response to microbial challenge. However, these cells exhibit significant functional heterogeneity and the inflammatory signals that differentially influence their effector characteristics are poorly characterized. In this study, we defined the phenotype of discrete subsets of effective antigen‐presenting cells (APCs) in the peritoneal cavity during peritonitis. When the functional properties of these cells were compared to inflammatory monocyte‐derived macrophages we noted differential responses to the immune‐modulatory cytokine IL‐10. In contrast to the suppressive actions of IL‐10 on inflammatory macrophages, the recruitment of APCs was relatively refractory and we found no evidence for selective inhibition of APC differentiation. This differential response of myeloid cell subsets to IL‐10 may thus have limited impact on development of potentially tissue‐damaging adaptive immune responses, while restricting the magnitude of the inflammatory response. These findings may have clinical relevance in the context of peritoneal dialysis patients, where recurrent infections are associated with immune‐mediated membrane dysfunction, treatment failure, and increased morbidity.

Hemostatic defects are treated using coagulation factors; however, clot formation also requires a procoagulant phospholipid (PL) surface. Here, we show that innate immune cell-derived enzymatically oxidized phospholipids (eoxPL) termed hydroxyeicosatetraenoic acid-phospholipids (HETE-PLs) restore hemostasis in human and murine conditions of pathological bleeding. HETE-PLs abolished blood loss in murine hemophilia A and enhanced coagulation in factor VIII- (FVIII-), FIX-, and FX-deficient human plasma . HETE-PLs were decreased in platelets from patients after cardiopulmonary bypass (CPB). To explore molecular mechanisms, the ability of eoxPL to stimulate individual isolated coagulation factor/cofactor complexes was tested in vitro. Extrinsic tenase (FVIIa/tissue factor [TF]), intrinsic tenase (FVIIIa/FIXa), and prothrombinase (FVa/FXa) all were enhanced by both HETE-PEs and HETE-PCs, suggesting a common mechanism involving the fatty acid moiety. In plasma, 9-, 15-, and 12-HETE-PLs were more effective than 5-, 11-, or 8-HETE-PLs, indicating positional isomer specificity. Coagulation was enhanced at lower lipid/factor ratios, consistent with a more concentrated area for protein binding. Surface plasmon resonance confirmed binding of FII and FX to HETE-PEs. HETE-PEs increased membrane curvature and thickness, but not surface charge or homogeneity, possibly suggesting increased accessibility to cations/factors. In summary, innate immune-derived eoxPL enhance calcium-dependent coagulation factor function, and their potential utility in bleeding disorders is proposed.

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Type 2-associated goblet cell hyperplasia and mucus hypersecretion are well known features of asthma. 15-Lipoxygenase-1 (15LO1) is induced by the type 2 cytokine IL-13 in human airway epithelial cells (HAECs) in vitro and is increased in fresh asthmatic HAECs ex vivo. 15LO1 generates a variety of products, including 15-hydroxyeicosatetraenoic acid (15-HETE), 15-HETE-phosphatidylethanolamine (15-HETE-PE), and 13-hydroxyoctadecadienoic acid (13-HODE). In this study, we investigated the 15LO1 metabolite profile at baseline and after IL-13 treatment, as well as its influence on goblet cell differentiation in HAECs. Primary HAECs obtained from bronchial brushings of asthmatic and healthy subjects were cultured under air-liquid interface culture supplemented with arachidonic acid and linoleic acid (10 μM each) and exposed to IL-13 for 7 days. Short interfering RNA transfection and 15LO1 inhibition were applied to suppress 15LO1 expression and activity. IL-13 stimulation induced expression of 15LO1 and preferentially generated 15-HETE-PE in vitro, both of which persisted after removal of IL-13. 15LO1 inhibition (by short interfering RNA and chemical inhibitor) decreased IL-13-induced forkhead box protein A3 (FOXA3) expression and enhanced FOXA2 expression. These changes were associated with reductions in both mucin 5AC and periostin. Exogenous 15-HETE-PE stimulation (alone) recapitulated IL-13-induced FOXA3, mucin 5AC, and periostin expression. The results of this study confirm the central importance of 15LO1 and its primary product, 15-HETE-PE, for epithelial cell remodeling in HAECs.

The alarm cytokine interleukin‐1β (IL‐1β) is a potent activator of the inflammatory cascade following pathogen recognition. IL‐1β production typically requires two signals: first, priming by recognition of pathogen‐associated molecular patterns leads to the production of immature pro‐IL‐1β; subsequently, inflammasome activation by a secondary signal allows cleavage and maturation of IL‐1β from its pro‐form. However, despite the important role of IL‐1β in controlling local and systemic inflammation, its overall regulation is still not fully understood. Here we demonstrate that peritoneal tissue‐resident macrophages use an active inhibitory pathway, to suppress IL‐1β processing, which can otherwise occur in the absence of a second signal. Programming by the transcription factor Gata6 controls the expression of prostacyclin synthase, which is required for prostacyclin production after lipopolysaccharide stimulation and optimal induction of IL‐10. In the absence of secondary signal, IL‐10 potently inhibits IL‐1β processing, providing a previously unrecognized control of IL‐1β in tissue‐resident macrophages.