Melanie Stables

Old and new generation lipid mediators in acute inflammation and resolution.

Originally regarded as just membrane constituents and energy storing molecules, lipids are now recognised as potent signalling molecules that regulate a multitude of cellular responses via receptor-mediated pathways, including cell growth and death, and inflammation/infection. Derived from polyunsaturated fatty acids (PUFAs), such as arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), each lipid displays unique properties, thus making their role in inflammation distinct from that of other lipids derived from the same PUFA. The diversity of their actions arises because such metabolites are synthesised via discrete enzymatic pathways and because they elicit their response via different receptors. This review will collate the bioactive lipid research to date and summarise the findings in terms of the major pathways involved in their biosynthesis and their role in inflammation and its resolution. It will include lipids derived from AA (prostanoids, leukotrienes, 5-oxo-6,8,11,14-eicosatetraenoic acid, lipoxins and epoxyeicosatrienoic acids), EPA (E-series resolvins), and DHA (D-series resolvins, protectins and maresins).

Resolution-phase macrophages possess a unique inflammatory phenotype that is controlled by cAMP

Neutralizing injurious stimuli, proinflammatory mediator catabolism, and polymorphonuclear leukocyte (PMN) clearance are determinants of inflammatory resolution. To this, we recently added innate-type lymphocyte repopulation as being central for restoring postinflammation tissue homeostasis with a role in controlling innate immune–mediated responses to secondary infection. However, although macrophages dominate resolution, their phenotype and role in restoring tissue physiology once inflammation abates are unknown. Therefore, we isolated macrophages from the resolving phase of acute inflammation and found that compared with classically activated proinflammatory M1 cells, resolution-phase macrophages (rMs) possess weaker bactericidal properties and express an alternatively activated phenotype but with elevated markers of M1 cells including inducible cyclooxygenase (COX 2) and nitric oxide synthase (iNOS). This phenotype is controlled by cAMP, which, when inhibited, transforms rM to M1 cells. Conversely, elevating cAMP in M1 cells transforms them to rMs, with implications for cAMP in the resolution of systemic inflammation. It transpires that although rMs are dispensable for clearing PMNs during self-limiting inflammation, they are essential for signaling postresolution lymphocyte repopulation via COX 2 lipids. Thus, rM macrophages are neither classically nor alternatively activated but a hybrid of both, with a role in mediating postresolution innate-lymphocyte repopulation and restoring tissue homeostasis.

Transcriptomic analyses of murine resolution-phase macrophages

Macrophages are either classically (M1) or alternatively-activated (M2). Whereas this nomenclature was generated from monocyte-derived macrophages treated in vitro with defined cytokine stimuli, the phenotype of in vivo-derived macrophages is less understood. We completed Affymetrix-based transcriptomic analysis of macrophages from the resolution phase of a zymosan-induced peritonitis. Compared with macrophages from hyperinflamed mice possessing a pro-inflammatory nature as well as naive macrophages from the uninflamed peritoneum, resolution-phase macrophages (rM) are similar to monocyte-derived dendritic cells (DCs), being CD209a positive but lacking CD11c. They are enriched for antigen processing/presentation (MHC class II [H2-Eb1, H2-Ab1, H2-Ob, H2-Aa], CD74, CD86), secrete T- and B-lymphocyte chemokines (Xcl1, Ccl5, Cxcl13) as well as factors that enhance macrophage/DC development, and promote DC/T cell synapse formation (Clec2i, Tnfsf4, Clcf1). rM are also enriched for cell cycle/proliferation genes as well as Alox15, Timd4, and Tgfb2, key systems in the termination of leukocyte trafficking and clearance of inflammatory cells. Finally, comparison with in vitro-derived M1/M2 shows that rM are neither classically nor alternatively activated but possess aspects of both definitions consistent with an immune regulatory phenotype. We propose that macrophages in situ cannot be rigidly categorized as they can express many shades of the inflammatory spectrum determined by tissue, stimulus, and phase of inflammation.

Effects of Low-Dose Aspirin on Acute Inflammatory Responses in Humans

Aspirin is a unique nonsteroidal anti-inflammatory drug; at high doses (aspirinhigh, 1g), it is anti-inflammatory stemming from the inhibition of cyclooxygenase and proinflammatory signaling pathways including NF-κB, but is cardioprotective at lower doses (aspirinlow, 75 mg). The latter arises from the inhibition of thromboxane (Tx) B2, a prothrombotic eicosanoid also implicated in polymorphonuclear leukocyte trafficking. As a result, aspirinlow is widely used as a primary and secondary preventative against vascular disease. Despite this and its ability to synthesize proresolution 15-epi-lipoxin A4 it is not known whether aspirinlow is anti-inflammatory in humans. To address this, we generated skin blisters by topically applying cantharidin on the forearm of healthy male volunteers, causing an acute inflammatory response including dermal edema formation and leukocyte trafficking. Although not affecting blister fluid volume, aspirinlow (75 mg, oral, once daily/10 days) reduced polymorphonuclear leukocyte and macrophage accumulation independent of NF-κB-regulated gene expression and inhibition of conventional prostanoids. However, aspirinlow triggered 15-epi-lipoxin A4 synthesis and up-regulated its receptor (FPRL1, ALX). From complimentary in vitro experiments, we propose that 15-epi-lipoxin A4 exerts its protective effects by triggering antiadhesive NO, thereby dampening leukocyte/endothelial cell interaction and subsequent extravascular leukocyte migration. Since similar findings were obtained from murine zymosan-induced peritonitis, we suggest that aspirinlow possesses the ability to inhibit mammalian innate immune-mediated responses. This highlights 15-epi-lipoxin A4 as a novel anti-inflammatory working through a defined receptor and suggests that mimicking its mode of action represents a new approach to treating inflammation-driven diseases.

Sex differences in resident immune cell phenotype underlie more efficient acute inflammatory responses in female mice

Females are protected against mortality arising from severe sepsis; however, the precise mechanisms that confer this survival advantage in females over males are unclear. Resident leukocytes in resting tissues have a significant influence on circulating cytokine levels and recruitment of blood leukocytes during acute inflammatory responses. Whether the phenotype of resident leukocytes is distinct in females is unknown. In the present study, we show that the numbers of leukocytes occupying the naive peritoneal and pleural cavities is higher in female than in male mice and rats, comprising more T and B lymphocytes and macrophages. The altered immune cell composition of the female peritoneum is controlled by elevated tissue chemokine expression. Female resident macrophages also exhibit greater TLR expression and enhanced phagocytosis and NADPH oxidase-mediated bacterial killing. However, macrophage-derived cytokine production is diminished by proportionally more resident immunomodulatory CD4+ T lymphocytes. Ovarian hormones regulate macrophage phenotype, function, and numbers, but have no significant impact on T-lymphocyte populations in females. We have identified a fundamental sex difference in phenotype of resident leukocytes. We propose that the distinct resident leukocyte population in females allows aggressive recognition and elimination of diverse infectious stimuli without recruitment of circulating neutrophils or excessive cytokine production.

Dichotomy in duration and severity of acute inflammatory responses in humans arising from differentially expressed proresolution pathways

Lipoxins (Lxs) and aspirin-triggered epi-Lxs (15-epi-LxA4) act through the ALX/FPRL1 receptor to block leukocyte trafficking, dampen cytokine/chemokine synthesis, and enhance phagocytic clearance of apoptotic leukocytes—key requisites for inflammatory resolution. Although studies using primarily inbred rodents have highlighted resolution as an active event, little is known about the role resolution pathways play in controlling the duration/profile of inflammatory responses in humans. To examine this, we found two types of responders to cantharidin-induced skin blisters in male healthy volunteers: those with immediate leukocyte accumulation and cytokine/chemokine synthesis followed by early resolution and a second group whose inflammation increased gradually over time followed by delayed resolution. In early resolvers, blister 15-epi-LxA4 and leukocyte ALX were low, but increased as inflammation abated. In contrast, in delayed resolvers, 15-epi-LxA4 and ALX were high early in the response but waned as inflammation progressed. Elevating 15-epi-LxA4 in early resolvers using aspirin increased blister leukocyte ALX but reduced cytokines/chemokines as well as polymorphonuclear leukocyte and macrophage numbers. These findings show that two phenotypes exist in humans with respect to inflammation severity/longevity controlled by proresolution mediators, namely 15-epi-LxA4. These data have implications for understanding the etiology of chronic inflammation and future directions in antiinflammatory therapy.

Resolution of acute inflammation bridges the gap between innate and adaptive immunity.

Acute inflammation is traditionally characterized by polymorphonuclear leukocytes (PMN) influx followed by phagocytosing macrophage (Mφs) that clear injurious stimuli leading to resolution and tissue homeostasis. However, using the peritoneal cavity, we found that although innate immune-mediated responses to low-dose zymosan or bacteria resolve within days, these stimuli, but not hyperinflammatory stimuli, trigger a previously overlooked second wave of leukocyte influx into tissues that persists for weeks. These cells comprise distinct populations of tissue-resident Mφs (resMφs), Ly6c(hi) monocyte-derived Mφs (moMφs), monocyte-derived dendritic cells (moDCs), and myeloid-derived suppressor cells (MDSCs). Postresolution mononuclear phagocytes were observed alongside lymph node expansion and increased numbers of blood and peritoneal memory T and B lymphocytes. The resMφs and moMφs triggered FoxP3 expression within CD4 cells, whereas moDCs drive T-cell proliferation. The resMφs preferentially clear apoptotic PMNs and migrate to lymph nodes to bring about their contraction in an inducible nitric oxide synthase-dependent manner. Finally, moMφs remain in tissues for months postresolution, alongside altered numbers of T cells collectively dictating the magnitude of subsequent acute inflammatory reactions. These data challenge the prevailing idea that resolution leads back to homeostasis and asserts that resolution acts as a bridge between innate and adaptive immunity, as well as tissue reprogramming.

Priming innate immune responses to infection by cyclooxygenase inhibition kills antibiotic-susceptible and -resistant bacteria

Inhibition of cyclooxygenase (COX)-derived prostaglandins (PGs) by nonsteroidal anti-inflammatory drugs (NSAIDs) mediates leukocyte killing of bacteria. However, the relative contribution of COX1 versus COX2 to this process, as well as the mechanisms controlling it in mouse and humans, are unknown. Indeed, the potential of NSAIDs to facilitate leukocyte killing of drug-resistant bacteria warrants investigation. Therefore, we carried out a series of experiments in mice and humans, finding that COX1 is the predominant isoform active in PG synthesis during infection and that its prophylactic or therapeutic inhibition primes leukocytes to kill bacteria by increasing phagocytic uptake and reactive oxygen intermediate-mediated killing in a cyclic adenosine monophosphate (cAMP)-dependent manner. Moreover, NSAIDs enhance bacterial killing in humans, exerting an additive effect when used in combination with antibiotics. Finally, NSAIDs, through the inhibition of COX prime the innate immune system to mediate bacterial clearance of penicillin-resistant Streptococcus pneumoniae serotype 19A, a well-recognized vaccine escape serotype of particular concern given its increasing prevalence and multi-antibiotic resistance. Therefore, these data underline the importance of lipid mediators in host responses to infection and the potential of inhibitors of PG signaling pathways as adjunctive therapies, particularly in the con-text of antibiotic resistance.

New Perspectives on Aspirin and the Endogenous Control of Acute Inflammatory Resolution

Aspirin is unique among the nonsteroidal anti-inflammatory drugs in that it has both anti-inflammatory as well as cardio-protective properties. The cardio-protective properties arise form its judicious inhibition of platelet-derived thromboxane A2 over prostacyclin, while its anti-inflammatory effects of aspirin stem from its well-established inhibition of prostaglandin (PG) synthesis within inflamed tissues. Thus aspirin and the other NSAIDs have popularised the notion of inhibiting PG biosynthesis as a common anti-inflammatory strategy based on the erroneous premise that all eicosanoids are generally detrimental to inflammation. However, our fascination with aspirin has shown a more affable side to lipid mediators based on our increasing interest in the endogenous control of acute inflammation and in factors that mediate its resolution. Epi-lipoxins (epi-LXs), for instance, are produced from aspirins acetylation of inducible cyclooxygenase 2 (COX-2) and together with Resolvins represent an increasingly important family of immuno-regulatory and potentially cardio-protective lipid mediators. Aspirin is beginning to teach us what nature knew all along — that not all lipid mediators are bad. It seems that while some eicosanoids are pathogenic in a variety of diseases, others are unarguable protective. In this review we will re-count aspirins colorful history, discuss its traditional mode of action and the controversies associated therewith, as well as highlight some of the new pathways in inflammation and the cardiovascular systems that aspirin has recently revealed.

CYP450-derived oxylipins mediate inflammatory resolution

Significance A number of lipid mediators are known to contribute to inflammatory resolution. Fatty acid metabolites of cytochrome P450 (CYP) enzymes are found in abundance; however, their roles in inflammatory resolution are not known. Targeted lipidomics revealed that CYP450-epoxy-oxylipins were present during acute inflammation and inflammatory resolution. Using mice lacking soluble epoxide hydrolase, the major metabolizing pathway for CYP450-derived fatty acid mediators, and CYP450 epoxygenase inhibition specifically during resolution, we show that CYP450-derived lipids dramatically limit the accumulation of inflammatory monocytes during resolution. Moreover, all cells of the monocyte lineage examined showed a dramatic alteration in their proresolution phenotype following epoxygenase inhibition. These findings demonstrate that the CYP450-epoxy-oxylipins pathway has a critical role in monocyte lineage recruitment and resolution activity during inflammatory resolution. Resolution of inflammation has emerged as an active process in immunobiology, with cells of the mononuclear phagocyte system being critical in mediating efferocytosis and wound debridement and bridging the gap between innate and adaptive immunity. Here we investigated the roles of cytochrome P450 (CYP)-derived epoxy-oxylipins in a well-characterized model of sterile resolving peritonitis in the mouse. Epoxy-oxylipins were produced in a biphasic manner during the peaks of acute (4 h) and resolution phases (24–48 h) of the response. The epoxygenase inhibitor SKF525A (epoxI) given at 24 h selectively inhibited arachidonic acid- and linoleic acid-derived CYP450-epoxy-oxlipins and resulted in a dramatic influx in monocytes. The epoxI-recruited monocytes were strongly GR1+, Ly6chi, CCR2hi, CCL2hi, and CX3CR1lo. In addition, expression of F4/80 and the recruitment of T cells, B cells, and dendritic cells were suppressed. sEH (Ephx2)−/− mice, which have elevated epoxy-oxylipins, demonstrated opposing effects to epoxI-treated mice: reduced Ly6chi monocytes and elevated F4/80hi macrophages and B, T, and dendritic cells. Ly6chi and Ly6clo monocytes, resident macrophages, and recruited dendritic cells all showed a dramatic change in their resolution signature following in vivo epoxI treatment. Markers of macrophage differentiation CD11b, MerTK, and CD103 were reduced, and monocyte-derived macrophages and resident macrophages ex vivo showed greatly impaired phagocytosis of zymosan and efferocytosis of apoptotic thymocytes following epoxI treatment. These findings demonstrate that epoxy-oxylipins have a critical role in monocyte lineage recruitment and activity to promote inflammatory resolution and represent a previously unidentified internal regulatory system governing the establishment of adaptive immunity.