Paul C. Norris

Eicosanoid storm in infection and inflammation

Controlled immune responses to infection and injury involve complex molecular signalling networks with coordinated and often opposing actions. Eicosanoids and related bioactive lipid mediators derived from polyunsaturated fatty acids constitute a major bioactive lipid network that is among the most complex and challenging pathways to map in a physiological context. Eicosanoid signalling, similar to cytokine signalling and inflammasome formation, has primarily been viewed as a pro-inflammatory component of the innate immune response; however, recent advances in lipidomics have helped to elucidate unique eicosanoids and related docosanoids with anti-inflammatory and pro-resolution functions. This has advanced our overall understanding of the inflammatory response and its therapeutic implications. The induction of a pro-inflammatory and anti-inflammatory eicosanoid storm through the activation of inflammatory receptors by infectious agents is reviewed here.

Omega-3 fatty acids cause dramatic changes in TLR4 and purinergic eicosanoid signaling

Dietary fish oil containing ω3 fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), elicit cardioprotective and anti-inflammatory effects through unresolved mechanisms that may involve competition and inhibition at multiple levels. Here, we report the effects of arachidonic acid (AA), EPA, and DHA supplementation on membrane incorporation, phospholipase A2 catalyzed release, and eicosanoid production in RAW264.7 macrophages. Using a targeted lipidomics approach, we observed that Toll-like receptor 4 and purinergic receptor activation of supplemented cells leads to the release of 22-carbon fatty acids that potently inhibit cyclooxygenase pathways. This inhibition was able to shunt metabolism of AA to lipoxygenase pathways, augmenting leukotriene and other lipoxygenase mediator synthesis. In resident peritoneal macrophages, docosapentaenoic acid (DPA) was responsible for cyclooxygenase inhibition after EPA supplementation, offering fresh insights into how EPA exerts anti-inflammatory effects indirectly through elongation to 22-carbon DPA.

High-Throughput Lipidomic Analysis of Fatty Acid Derived Eicosanoids and N-Acylethanolamines

Fatty acid-derived eicosanoids and N-acylethanolamines (NAE) are important bioactive lipid mediators involved in numerous biological processes including cell signaling and disease progression. To facilitate research on these lipid mediators, we have developed a targeted high-throughput mass spectrometric based methodology to monitor and quantitate both eicosanoids and NAEs, and can be analyzed separately or together in series. Each methodology utilizes scheduled multiple reaction monitoring (sMRM) pairs in conjunction with a 25 min reverse-phase HPLC separation. The eicosanoid methodology monitors 141 unique metabolites and quantitative amounts can be determined for over 100 of these metabolites against standards. The analysis covers eicosanoids generated from cycloxygenase, lipoxygenase, cytochrome P450 enzymes, and those generated from non-enzymatic pathways. The NAE analysis monitors 36 metabolites and quantitative amounts can be determined for 33 of these metabolites against standards. The NAE method contains metabolites derived from saturated fatty acids, unsaturated fatty acids, and eicosanoids. The lower limit of detection for eicosanoids ranges from 0.1pg to 1pg, while NAEs ranges from 0.1pg to 1000pg. The rationale and design of the methodology is discussed.

Specificity of eicosanoid production depends on the TLR-4-stimulated macrophage phenotype

Eicosanoid metabolism differs in profile and quantity between macrophages of different tissue origin and method of elicitation, as well as between primary and immortalized macrophages after activation with inflammatory stimuli. Using a lipidomic approach, we comprehensively analyzed the eicosanoids made by murine RPMs, TGEMs, BMDM, and the macrophage‐like cell line RAW after stimulation with the TLR‐4‐specific agonist KLA. Direct correlation among total COX metabolites, COX side‐products (11‐HETE, 15‐HETE), COX‐2 mRNA, and protein at 8 h was found when comparing each cell type. Comprehensive qPCR analysis was used to compare relative transcript levels between the terminal prostanoid synthases themselves as well as between each cell type. Levels of PGE2, PGD2, and TxB2 generally correlated with enzyme transcript expression of PGES, PGDS, and TBXS, providing evidence of comparable enzyme activities. PGIS transcript was expressed only in RPM and TGEM macrophages and at an exceptionally low level, despite high metabolite production compared with other synthases. Presence of PGIS in RPM and TGEM also lowered the production of PGE2 versus PGD2 by approximately tenfold relative to BMDM and RAW cells, which lacked this enzyme. Our results demonstrate that delayed PG production depends on the maximal level of COX‐2 expression in different macrophages after TLR‐4 stimulation. Also, the same enzymes in each cell largely dictate the profile of eicosanoids produced depending on the ratios of expression between them, with the exception of PGIS, which appears to have much greater synthetic capacity and competes selectively with mPGES‐1.

Phospholipase A2 regulates eicosanoid class switching during inflammasome activation

Significance Group IVA cytosolic phospholipase A2 (GIVA cPLA2) is widely viewed as the primary enzyme responsible for inflammatory arachidonic acid (AA) release and with high specificity. Our results demonstrate dual, phase-specific release of AA and 15-hydroxyeicosatetraenoic (15-HETE) acid by GIVA cPLA2 in primary and immortalized macrophages during a receptor-mediated program required for complete inflammatory commitment. These dual actions by GIVA cPLA2 were necessary for biosynthesis of proresolving lipoxins, providing a unique, upstream example of an enzyme linked to both the initiation and resolution of inflammation. Further, our results demonstrate a single-cell mechanism of lipoxin synthesis that is more efficient than the established transcellular biosynthetic mechanisms, underscoring the importance of enzyme coupling and the possibility of proresolution therapies at the membrane level. Initiation and resolution of inflammation are considered to be tightly connected processes. Lipoxins (LX) are proresolution lipid mediators that inhibit phlogistic neutrophil recruitment and promote wound-healing macrophage recruitment in humans via potent and specific signaling through the LXA4 receptor (ALX). One model of lipoxin biosynthesis involves sequential metabolism of arachidonic acid by two cell types expressing a combined transcellular metabolon. It is currently unclear how lipoxins are efficiently formed from precursors or if they are directly generated after receptor-mediated inflammatory commitment. Here, we provide evidence for a pathway by which lipoxins are generated in macrophages as a consequence of sequential activation of toll-like receptor 4 (TLR4), a receptor for endotoxin, and P2X7, a purinergic receptor for extracellular ATP. Initial activation of TLR4 results in accumulation of the cyclooxygenase-2–derived lipoxin precursor 15-hydroxyeicosatetraenoic acid (15-HETE) in esterified form within membrane phospholipids, which can be enhanced by aspirin (ASA) treatment. Subsequent activation of P2X7 results in efficient hydrolysis of 15-HETE from membrane phospholipids by group IVA cytosolic phospholipase A2, and its conversion to bioactive lipoxins by 5-lipoxygenase. Our results demonstrate how a single immune cell can store a proresolving lipid precursor and then release it for bioactive maturation and secretion, conceptually similar to the production and inflammasome-dependent maturation of the proinflammatory IL-1 family cytokines. These findings provide evidence for receptor-specific and combinatorial control of pro- and anti-inflammatory eicosanoid biosynthesis, and potential avenues to modulate inflammatory indices without inhibiting downstream eicosanoid pathways.

Novel proresolving and tissue-regenerative resolvin and protectin sulfido-conjugated pathways

Local mediators orchestrate the host response to both sterile and infectious challenge and resolution. Recent evidence demonstrates that maresin sulfido‐conjugates actively resolve acute inflammation and promote tissue regeneration. In this report, we investigated self‐limited infectious exudates for novel bioactive chemical signals in tissue regeneration and resolution. By use of spleens from Escherichia coli infected mice, self‐resolving infectious exudates, human spleens, and blood from patients with sepsis, we identified 2 new families of potent molecules. Characterization of their physical properties and isotope tracking demonstrated that the bioactive structures contained a docosahexaenoate backbone and sulfido‐conjugated triene or tetraene double‐bond systems. Activated human phagocytes converted 17‐hydro(peroxy)‐4Z,7Z,10Z,13Z,15E,19Z‐docosahexaenoic acid to these bioactive molecules. Regeneration of injured planaria was accelerated with nanomolar amounts of 16‐glutathionyl, 17‐hydroxy‐4Z,7Z,10,12,14,19Z‐docosahexaenoic acid and 16‐cysteinylglycinyl, 17‐hydroxy‐4Z,7Z,10,12,14,19Z‐docosahexaenoic acid (Protectin sulfido‐conjugates) or 8‐glutathionyl, 7,17‐dihydroxy‐4Z,9, 11,13Z,15E,19Z‐docosahexaenoic acid and 8‐cysteinylglycinyl, 7,17‐dihydroxy‐4Z,9,11,13Z, 15E,19Z‐docosahexaenoic acid (Resolvin sulfido‐conjugates). Each protectin and resolvin sulfido‐conjugate dose dependently (0.1‐10 nM) stimulated human macrophage bacterial phagocytosis, phagolysosomal acidification, and efferocytosis. Together, these results identify 2 novel pathways and provide evidence for structural elucidation of new resolution moduli. These resolvin and protectin conjugates identified in mice and human infected tissues control host responses promoting catabasis.—Dalli, J., Ramon, S., Norris, P. C., Colas, R. A., Serhan, C. N. Novel proresolving and tissue‐regenerative resolvin and protectin sulfido‐conjugated pathways. FASEB J. 29, 2120‐2136 (2015). www.fasebj.org

Identification and Profiling of Specialized Pro-Resolving Mediators in Human Tears by Lipid Mediator Metabolomics

Specialized pro-resolving mediators (SPM), e.g. Resolvin D1, Protectin D1, Lipoxin A₄, and Resolvin E1 have each shown to be active in ocular models reducing inflammation. In general, SPMs have specific agonist functions that stimulate resolution of infection and inflammation in animal disease models. The presence and quantity of SPM in human emotional tears is of interest. Here, utilizing a targeted LC-MS-MS metabololipidomics based approach we document the identification of pro-inflammatory (Prostaglandins and Leukotriene B₄) and pro-resolving lipid mediators (D-series Resolvins, Protectin D1, and Lipoxin A₄) in human emotional tears from 12 healthy individuals. SPMs from the Maresin family (Maresin 1 and Maresin 2) were not present in these samples. Principal Component Analysis (PCA) revealed gender differences in the production of specific mediators within these tear samples as the SPMs were essentially absent in these female donors. These results indicate that specific SPM signatures are present in human emotional tears at concentrations known to be bioactive. Moreover, they will help to further appreciate the mechanisms of production and action of SPMs in the eye, as well as their physiologic roles in human ocular disease resolution.

Systematic analysis of rat 12/15-lipoxygenase enzymes reveals critical role for spinal eLOX3 hepoxilin synthase activity in inflammatory hyperalgesia

Previously, we observed significant increases in spinal 12‐lipoxygenase (LOX) metabolites, in particular, hepoxilins, which contribute to peripheral inflammation‐induced tactile allodynia. However, the enzymatic sources of hepoxilin synthase (HXS) activity in rats remain elusive. Therefore, we overexpressed each of the 6 rat 12/15‐LOX enzymes in HEK‐293T cells and measured by LC‐MS/MS the formation of HXB3, 12‐HETE, 8‐HETE, and 15‐HETE from arachidonic acid (AA) at baseline and in the presence of LOX inhibitors (NDGA, AA‐861, CDC, baicalein, and PD146176) vs. vehicle‐treated and mock‐transfected controls. We detected the following primary intrinsic activities: 12‐LOX (Alox12, Alox15), 15‐LOX (Alox15b), and HXS (Alox12, Alox15). Similar to human and mouse orthologs, proteins encoded by rat Alox12b and Alox12e possessed minimal 12‐LOX activity with AA as substrate, while eLOX3 (encoded by Aloxe3) exhibited HXS without 12‐LOX activity when coexpressed with Alox12b or supplemented with 12‐HpETE. CDC potently inhibited HXS and 12‐LOX activity in vitro (relative IC50s: CDC, ~0.5 and 0.8 μM, respectively) and carrageenan‐evoked tactile allodynia in vivo. Notably, peripheral inflammation significantly increased spinal eLOX3; intrathecal pretreatment with either siRNA targeting Aloxe3 or an eLOX3‐selective antibody attenuated the associated allodynia. These findings implicate spinal eLOX3‐mediated hepoxilin synthesis in inflammatory hyperesthesia and underscore the importance of developing more selective 12‐LOX/HXS inhibitors.—Gregus, A. M., Dumlao, D. S., Wei, S. C., Norris, P. C., Catella, L. C., Meyerstein, F. G., Buczynski, M. W., Steinauer, J. J., Fitzsimmons, B. L., Yaksh, T. L., Dennis, E. A. Systematic analysis of rat 12/15‐lipoxygenase enzymes reveals critical role for spinal eLOX3 hepoxilin synthase activity in inflammatory hyperalgesia. FASEB J. 27, 1939–1949 (2013). www.fasebj.org

NLRP3 Inflammasome Deficiency Protects against Microbial Sepsis via Increased Lipoxin B4 Synthesis

Rationale: Sepsis, a life‐threatening organ dysfunction caused by a dysregulated host response to infection, is a major public health concern with high mortality and morbidity. Although inflammatory responses triggered by infection are crucial for host defense against invading microbes, the excessive inflammation often causes tissue damage leading to organ dysfunction. Resolution of inflammation, an active immune process mediated by endogenous lipid mediators (LMs), is important to maintain host homeostasis. Objectives: We sought to determine the role of the nucleotide‐binding domain, leucine‐rich repeat‐containing receptor, pyrin domain‐containing‐3 (NLRP3) inflammasome in polymicrobial sepsis and regulation of LM biosynthesis. Methods: We performed cecal ligation and puncture (CLP) using mice lacking NLRP3 inflammasome‐associated molecules to assess mortality. Inflammation was evaluated by using biologic fluids including plasma, bronchoalveolar, and peritoneal lavage fluid. Local acting LMs in peritoneal lavage fluid from polymicrobacterial septic mice were assessed by mass spectrometry‐based metabololipidomics. Measurements and Main Results: Genetic deficiency of NLRP3 inhibited inflammatory responses and enhanced survival of CLP‐induced septic mice. NLRP3 deficiency reduced proinflammatory LMs and increased proresolving LM, lipoxin B4 (LXB4) in septic mice, and in macrophages stimulated with LPS and ATP. Activation of the NLRP3 inflammasome induced caspase‐7 cleavage and pyroptosis. Caspase‐7 deficiency similarly reduced inflammation and mortality in CLP‐induced sepsis, and increased LXB4 production in vivo and in vitro. Exogenous application of LXB4 reduced inflammation, pyroptosis, and mortality of mice after CLP. Conclusions: Genetic deficiency of NLRP3 promoted resolution of inflammation in polymicrobial sepsis by relieving caspase‐7‐dependent repression of LXB4 biosynthesis, and increased survival potentially via LXB4 production and inhibition of proinflammatory cytokines.

Resolvin D3 multi-level proresolving actions are host protective during infection ☆

Resolution of infection and inflammation is governed by innate immune cells. The resolvin family of n-3 mediators produced by resolving exudates stimulates clearance of neutrophils and attenuates pro-inflammatory signals. Using metabololipidomics, endogenous resolvin D3 (RvD3) was identified in self-resolving exudates during active E. coli infection. Through a new, independent synthetic route for RvD3, we matched endogenous and synthetic RvD3 and determined that RvD3 (ng doses) potently reduced the resolution interval (Ri) by ~4.5h during E. coli peritonitis after administration at peak inflammation (Tmax=12h) and increased leukocyte phagocytosis of E. coli and neutrophils as well as reduced proinflammatory cytokines, chemokines, MMP-2 and MMP-9. At pM-nM concentrations, RvD3 also enhanced human macrophage efferocytosis and bacterial phagocytosis, increased neutrophil bacterial phagocytosis and intracellular ROS generation, and reduced human platelet-PMN aggregation. These results provide additional evidence for potent RvD3 immunoresolvent actions in host defense, host protection and antimicrobial defense.