The view that most lipid mediators are proinflammatory arises largely from the finding that non-steroidal anti-inflammatory drugs block the biosynthesis of prostaglandins and affect renal physiology. Resolution of inflammation was widely held to be a passive process until recently, with the identification of novel biochemical pathways and lipid-derived mediators that are actively biosynthesized during resolution, which possess potent anti-inflammatory and proresolving actions. A systems lipid mediator informatics approach systematically identified new families of endogenous locally acting mediators in resolving exudates formed from omega-3-polyunsaturated fatty acids in addition to classic eicosanoids and aspirin-triggered lipoxins generated from arachidonic acid. Given their potent actions, the new chemical mediators were coined resolvins and protectins. This chapter presents recent advances in the biosynthesis and actions of these new proresolving families of mediators that have proven to be both organ protective and antifibrotic, with attention to their roles in renal systems and potential in organ regeneration.

Introduction

The inflammatory response is, in general, protective and ultimately rids tissues of both the cause and consequences of tissue injury that can accompany host defense . Acute inflammation, defined by its cardinal signs dolor, calor and rubor, may lead to chronic inflammation, scarring and eventual loss of function, if the tissue fails to completely resolve the inflammatory site . The polymorphonuclear neutrophils (PMNs) of the first line of host defense, in this context, must also exit from the inflamed tissues ( Fig. 4.1 ) in order to return to homeostasis and resolve . In recent years it has become widely appreciated that, in addition to the classic diseases associated with inflammation such as psoriasis, periodontal disease and arthritis, uncontrolled inflammation governs the pathogenesis of many other prevalent diseases including cardiovascular and cerebrovascular disease, cancer, obesity and Alzheimer’s disease . Of interest, another class of arachidonic acid-derived eicosanoids, the lipoxins (LXs) and aspirin-triggered lipoxins (ATLs), were the first mediators recognized to have both endogenous anti-inflammatory and proresolving actions .

(a) Eicosanoid mediators in inflammation and resolution. (b) Hypothetical temporal scheme and role of lipid mediators in initiation and resolution of acute inflammation.

In recent years, the Serhan laboratory has identified novel enzymatic pathways activated during the resolution phase of self-limited inflammatory responses that are initiated from precursors eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), the major n-3 fatty acids, also widely known as the omega-3 polyunsaturated fatty acids (PUFAs) or simply fish oils. These new compounds are biosynthesized in resolving exudates and possess potent actions, controlling the duration and resolution of inflammation . The term resolvins, resolution-phase interaction products, was introduced to signify that the new structures are endogenous, locally acting mediators possessing potent anti-inflammatory and immunoregulatory actions . At the cellular level, these include reducing neutrophil infiltration and regulating the cytokine–chemokine axis and reactive oxygen species, as well as lowering the magnitude of the inflammatory response . The terms protectin and specifically neuroprotectin D1 when generated in the neural tissue were introduced, given the formation and potent anti-inflammatory as well as protective actions demonstrated for the novel and potent DHA-derived 10,17-docasatriene in animal models of stroke and Alzheimer’s disease . Both families of mediators, the resolvins and protectins, are potent local-acting agonists of endogenous anti-inflammation and are proresolving mediators . The connection of these new anti-inflammatory mediators [lipoxins (LX), resolvins (Rv) and protectins (PD)] to the control of an acute inflammatory response and its timely resolution are illustrated in Fig. 4.1 .

Since the early twentieth century, omega-3 fatty acids (PUFAs) have been known to possess beneficial roles in health and organ function . At high concentrations in vitro, n-3 PUFAs decrease production of proinflammatory prostaglandins, cytokines and reactive oxygen species held to play critical roles in inflammatory diseases . Clinically relevant anti-inflammatory properties were reported with high doses of n-3 fatty acids in both rheumatoid arthritis and periodontal disease , whereas the evidence available at this time remains inconclusive for several other conditions [reviewed in Ref. ]. Of interest, cardiovascular disease was reduced with high-dose n-3 in a multicenter clinical trial . Also, blood levels of EPA and DHA were shown to reduce the risk of cardiovascular disease . These findings raised the question of what mechanism(s) underlie the many beneficial actions of n-3 PUFAs.

Because the precursors to both resolvins and protectins are the essential n-3 PUFAs ( Figs 4.1–4.2, 4.4 ), their relation to dietary supplementation by n-3 PUFAs also raises new and interesting questions, given the widely appreciated notion that n-3 supplementation reduces inflammatory diseases. Resolvins and protectins are distinct chemical families that now join the lipoxins as potent agonists of endogenous anti-inflammation and are proresolving chemical mediators of interest in health and particularly renal disease. It is important to note that the biosynthesis of resolvins and protectins is stereochemically controlled via enzymatic reactions that give rise to specific chemical mediators that carry potent bioactions and require precise stereochemistry to activate specific receptors. Resolvins and protectins are, therefore, distinct from the autooxidation products obtained from DHA, EPA or other PUFAs, which may also arise in tissues via interactions with reactive oxygen radicals. These autooxidation products are complex mixtures of mono-, dihydroxy- and trihydroxy- products of PUFAs as well as specific isoprostanes and neuroisoprostanes that can serve as biomarkers of oxidative stress . This chapter summarizes recent new findings on lipoxins, resolvin and protectin biosynthesis and their actions, with a focus on their impact in renal tissues.

Transcellular biosynthesis of aspirin-triggered lipid mediators: 15-epi-lipoxins.

Biosynthesis of E-series resolvins.

Lipid Mediators: Chemical Autacoids in Inflammation and Resolution

Lipid-derived mediators are well positioned to play important role(s) as signaling molecules in inflammation and tissue regeneration because they are small molecules, local acting, rapidly generated and locally inactivated. Microbial invaders, tissue injury or surgical trauma activate the release and formation of arachidonate-derived eicosanoids. Acute inflammation is characterized by the rapid, time-dependent influx of PMNs into the site, the first line of phagocytic host defense. Proinflammatory prostaglandins and leukotriene B ₄ control local blood flow, vascular dilation and permeability changes needed at the site for leukocyte adhesion, diapedesis and recruitment . These chemical mediators are enzymatically generated via specific cyclooxygenase (COX) and lipoxygenase (LOX) pathways . As exudates form and pustules are walled off, prostaglandins mediate a number of responses including vasoconstriction, vascular permeability changes, pain, vasodilation and edema. At contained sites of self-limited inflammation, prostaglandin E ₂ (PGE ₂ ) and PGD ₂ also signal the end or resolution of inflammation by activating the transcriptional regulation of 15-lipoxygenase (LO) in human neutrophils that in turn gives rise to the temporal dissociation of eicosanoids and production of lipoxins . Hence, the prostaglandins and leukotrienes are rapidly generated, while the lipoxins are biosynthesized later in the time-course, coinciding with the onset of the resolution phase ( Fig. 4.1 ).

Lipoxins

Lipoxins were the first family of mediators identified in vivo with anti-inflammatory and proresolving actions ( Table 4.1 ) . Lipoxins are trihydroxytetraene-containing products of arachidonic acid ( Fig. 4.1 ). Their biosynthesis and actions were recently reviewed in detail [for in-depth reviews, see Special Issue on Lipoxins and Aspirin-Triggered Lipoxins and contributions within] and are discussed here in view of their role(s) in anti-inflammation and resolution. Eicosanoid class switching refers to changes in production within the arachidonate-derived family, namely the temporal change in mediator profiles from prostaglandin and leukotriene, to lipoxin. In this case, PMNs switch from leukotriene B ₄ to lipoxin production . Lipoxins, specifically LXA ₄ and LXB ₄ , as well as their aspirin-triggered forms (see below), stop or limit further PMN entry into the exudates as well as counter-regulate the main signs of inflammation ( Table 4.2 ). As new PMNs parachute into exudates, older and apoptotic PMN must be removed from the site in a timely fashion for inflammation to resolve ( Fig. 4.1 ). Once PMNs enter an exudate, they interact with other cells such as neighboring leukocytes, platelets, endothelial, mucosal epithelial and fibroblasts in their immediate vicinity and are able to engage in transcellular biosynthesis to produce LX and eventually new mediators . The process of transcellular biosynthesis is defined as the generation of new bioactive mediators that neither cell type can produce on its own. For example, human platelets on their own do not produce LX. When platelets adhere to PMNs, the resulting platelet–PMN aggregates become a major intravascular source of LX that in turn halts further PMN diapedesis and recruitment [reviewed in Ref. ]. Also, when PMNs interact with mucosal epithelial cells in the lung, oral or gastrointestinal mucosa, these PMN biosynthesize LX from precursor 15-hydroxyeicosatetraenoic acid (15-HETE) donated by interactions with mucosal epithelial cells . Hence, PMNs switch their phenotype in that they change the profile of lipid mediators that they produce depending on their local environment . Exudate PMN switch their lipid mediator phenotype compared to, for example, peripheral blood PMN, which generate predominantly LTB ₄ as their main product . During the course of inflammation and complete resolution, as discussed below, mediator switching also occurs between families of lipid mediators, namely from eicosanoids to resolvins of the E and D series as well as protectins . This progression of the exudate is dependent on the availability of substrate, which is supplied to the evolving exudates by local edema . The edema proteins, i.e. albumin, carry circulating n-3 PUFA into the exudate for the transcellular biosynthesis of resolvins and related products.

Proresolving Pathways, Essential Fatty Acids and Resolution Indices

Prostaglandins such as PGE ₂ (produced by both COX-1 and COX-2) are generated in the initial phase of inflammation ( Fig. 4.1 ) and have a dual role in stimulating resolution . Signaling pathways leading to prostaglandin E ₂ and D ₂ actively switch on the transcription of enzymes (15-LOX type 1) required for the generation of lipoxins as well as PUFA-derived resolvins and protectins . Selective COX-2 inhibition, by blocking production of PGE ₂ and PGD ₂ , delays the onset of resolution . Hence, COX-2 has a role in both the initiation of acute inflammatory response and the resolution phase. Lipoxins promote resolution by limiting the further recruitment of PMN to sites of inflammation and decreasing reperfusion or reflow tissue injury [reviewed in Ref. ]. Lipoxins also reduce vascular permeability and promote non-phlogistic (i.e. non-inflammatory) recruitment of monocytes and stimulate clearance of apoptotic neutrophils via macrophages ( Table 4.2 ). Thus, a key process in resolution is the temporal “switch” or transition in the lipid mediator profiles from pro- to anti-inflammatory eicosanoids at sites of inflammation, which has direct implications for the treatment of inflammatory diseases. Drugs that disrupt this switch may have unwanted side-effects in resolution, as do inhibitors of COX and LOX .

COX-2 also plays a key role in the biosynthesis of prostaglandin D ₂ , which is a precursor to the cyclopentenones ( Fig. 4.1 ). These include prostaglandin J ₂ , an activator of peroxisome proliferator-activated receptor-γ (PPAR-γ). This property has led to the proposal that cyclopentenone prostaglandins can be anti-inflammatory via regulation of nuclear factor-κB (NF-κB) [reviewed in Refs ]. These studies also bring to light the multifunctional role of COX-2 in initiation versus termination of acute inflammation and the induction of proresolving lipid mediators.

Resolution of acute inflammation in murine models involves the appearance in exudates of EPA and DHA, which follow the appearance and accumulation of unesterified arachidonate . Precursors are transformed via enzymatic mechanisms to bioactive compounds such as lipoxins, resolvins and protectins that regulate the duration and magnitude of inflammation, namely shorten the period of neutrophil infiltration and initiate clearance of apoptotic PMNs ( Table 4.3 ). LX, Rv and PD1 also increase the expression of CCR5 receptors on T cells and aging PMNs, which enhances the clearance of local chemokines from the inflammatory site . Apoptotic neutrophils are then phagocytosed by macrophages , leading to neutrophil clearance and release of anti-inflammatory and reparative cytokines such as transforming growth factor-β ₁ (TGF-β ₁ ) .

A set of resolution indices was introduced as a quantitative means for assessing the major resolution parameters and the impact of specific agents within active resolution . These indices permit quantitative assessment of the impact of agents in resolution and the ability to compare their impact in resolution in an unbiased fashion. With these resolution indices defined, specific lipid mediators (e.g. RvE1, LX and PD1) were pinpointed to promote resolution via specific and separate mechanisms. When grossly viewed as the same outcome, namely anti-inflammation, each of these mediators is anti-inflammatory . In addition, it is now clear that anti-inflammation and proresolution are not the same processes . These measurable indices are useful tools for evaluating the molecular basis of therapeutic interventions in disease models where inflammation–resolution is a component as well as identifying when and where agents may be resolution toxic .

Aspirin is Resolution Friendly

Hippocrates advocated the use of willow bark in treatment of pain of childbirth and fever. The bark was later found to contain antipyretic substances . Salicylates were isolated many years later. In 1899 in Berlin, aspirin was launched on the trademark list in the German Patent Office; Felix Hoffman working at Bayer added the acetyl group to the structure with the goal of enhancing salicylate uptake and actions. Today, aspirin is still one of the most widely used non-steroidal anti-inflammatory drugs. Aspirin’s many clinical benefits are still unfolding at the cellular and molecular level and in many clinical studies. Although it is clear that aspirin inhibits prostaglandin and thromboxane formation by acetylating and blocking the catalytic activity of COX-1 and hence is a major mechanism in anti-inflammatory and antithrombotic therapy , aspirin’s well-appreciated ability to limit leukocyte traffic into sites of inflammation, the key mechanism in reducing leukocyte infiltration during inflammation, remained to be established.

Along these lines, aspirin turns on production of the body’s own endogenous anti-inflammatory lipid mediators, namely aspirin-triggered lipoxins, as well as resolvins (see below). These novel lipid mediators actively reduce inflammation ( Tables 4.1 and 4.2 ). As depicted in Fig. 4.2 , this action of aspirin involves cell–cell interactions, for example between COX-2-bearing cells (vascular endothelial cells or epithelial cells) and leukocytes . By acetylating COX-2, aspirin redirects COX-2’s catalytic activity away from generating the intermediate for prostaglandins and thromboxanes towards producing 15 R -HETE. The COX-2 enzyme with this modification remains catalytically active. This product of vascular endothelial cells and a wide range of other cells are converted to 15-epi-LXA ₄ by leukocyte 5-lipoxygenase, termed aspirin-triggered 15-epi-lipoxin A ₄ (ATL). ATL shares actions with LXA ₄ , and appears to be longer acting, resisting rapid dehydrogenation in vivo ( Table 4.2 ). It is important to point out that the role of PMNs is well appreciated in the pathogenesis of rheumatoid arthritis , a concept that was recently reaffirmed . Aspirin also increases the hepatic biosynthesis of 15-epi-LX via enhancing p450 production of 15R-HETE in a COX-2-independent pathway in rats , which is also likely to contribute to ATL plasma levels in healthy individuals taking low-dose aspirin and in local sites of inflammation .

EPA and DHA are each converted via aspirin-acetylated COX-2 to generate bioactive epimers of resolvins and protectins (see below). Thus, aspirin is unique among anti-inflammatory drugs in that it not only blocks proinflammatory prostaglandins but also jump-starts resolution by enabling the local production of endogenous epimers of resolution-phase mediators that share characteristic features with their counterparts in terms of reducing inflammation and PMN-mediated injury, which are major components in many human diseases.

LXA ₄ and ATL display counter-regulatory roles in animal models of disease ( Table 4.4 ), possess local organ-specific functions, and modulate leukotriene formation and their activities. The protective actions of LXA ₄ and ATL are ligand-receptor dependent. The principal LXA ₄ receptor first described in neutrophils by Fiore et al. is a G-protein-coupled receptor (GPCR) now designated FPR2/ALX, or ALX/FPR2 when the ligand is a lipoxin . ALX/FPR2 binds LXA ₄ with high affinity (subnanomolar K _d ); it also binds several peptide ligands, and receptor activation can result in either proinflammatory or anti-inflammatory responses . Krishnamoorthy et al. recently found that LXA ₄ and the resolvin RvD1 (see below) can interact with another GPCR, the “orphan” GPR32 . It is noteworthy that, although LXA ₄ and LXB ₄ share many biological activities, LXB ₄ does not bind FPR2/ALX and the LXB ₄ receptor has not yet been identified. LXA ₄ also displays partial antagonism at a subclass of cysteinyl LT receptors (CysLTs), which partly accounts for its counter-regulatory impact on LTD ₄ responses . Cross-talk between FPR2/ALX and tyrosine kinase growth factor receptors [platelet-derived growth factor (PDGF), epidermal growth factor (EGF), vascular endothelial growth factor-2 (VEGF-2)] also demonstrates a mechanism by which LXA ₄ and ATL regulate responses, such as proliferation, angiogenesis and fibrosis . LXA ₄ has been shown to bind the nuclear aryl hydrocarbon receptor (AhR) in dendritic cells, although far higher concentrations of LXA ₄ are needed for activation of this receptor compared to the GPCR . Transgenic overexpression of human ALX/FPR2 leads to decreased PMN infiltration with endogenous LXA ₄ . Furthermore, ALX/FPR2 is upregulated by glucocorticoids ( Table 4.1 ) and activated by glucocorticoid-induced ligand such as annexin-1 and annexin-derived peptides . Diminished LX and ATL levels are associated with the pathophysiology of several human diseases . LXA ₄ and ATL regulate tumor necrosis factor-α (TNF-α)-directed neutrophil actions and stimulate IL-4 in exudates, and thus regulate endogenous mediators in the pathogenesis of inflammatory conditions such as periodontal disease . In many human diseases, LX production appears to be deficient compared to leukotrienes. These include cardiovascular , asthma , kidney inflammation , cystic fibrosis , gastrointestinal and periodontal disease ( Table 4.4 ). Designed metabolically stable analogs of LXs and ATLs are useful tools in examining the role(s) and local actions of lipoxins in vivo . Administration of LX stable analogs in animal models protects from tissue damage and inflammation and enhances resolution . Identification of these anti-inflammatory actions of LXs and ATLs provided strong evidence for the existence of endogenous anti-inflammatory mediators derived from arachidonic acid. Along with reducing PMN influx , redirecting chemokines and cytokines , and reducing pain , LX and ATL have the ability to stimulate the removal of apoptotic PMNs by macrophages in vitro and at sites of inflammation in vivo . This proresolving agonist activity is shared by ( Table 4.1 ) annexin 1 and glucocorticoids , and accelerates the return of the tissue to homeostasis ( Tables 4.1 and 4.2 ).

Statins and Anti-inflammatory Lipid Mediators

The mechanisms of the well-recognized anti-inflammatory action of statins are of considerable interest. Statins (e.g. atorvastatin) and pioglitazone regulate the production of S -nitrosylated COX-2 . The S -nitrosylated COX-2 produces 15 R -HETE, which is converted by 5-lipoxygenase to 15-epi-LXA ₄ . The finding that statins regulate the production of 15-epi-lipoxin A ₄ suggests that the anti-inflammatory actions of statins are directly mediated by endogenous production of 15-epi-lipoxin A ₄ . These widely used drugs aspirin and statins thus have in common a unique ability to trigger the endogenous production of 15-epi-LXA ₄ . Further studies by Birnbaum et al. show that, when COX-2 is both acetylated and S -nitrosylated, the enzyme is inactive, providing potential adverse interactions among statins, thiazolidinediones and high-dose aspirin. It is likely that this mechanism will also affect the biosynthesis of the aspirin-triggered forms of the resolvins and protectins.

Lipoxins and Heme Oxygenase-1 System

Lipoxins exert several direct actions on endothelial cells that are protective and in line with their role in resolution. In this regard, lipoxins stimulate prostacyclin generation by endothelial cells and stimulate nitric oxide (NO) production by vascular endothelial cells . Aspirin acetylation of COX-2 generates 15-epi-lipoxins that in turn stimulate the production of NO by endothelial nitric oxide synthase (eNOS). Aspirin, in either eNOS or inducible nitric oxide synthase (iNOS) knockouts, is not anti-inflammatory in interleukin-1β (IL-1β)-induced murine peritonitis. Both aspirin and 15-epi-LXA ₄ had reduced effects on endothelial cell adherence from eNOS and iNOS knockouts compared to wild-type . This suggests that aspirin triggers the production of 15-epi-LXA ₄ , which increases NO synthesis through both eNOS and iNOS. These findings suggest a tight regulation between the generation of 15-epi-LXA ₄ and the production of vascular-derived NO. Also, aspirin-triggered lipoxins and lipoxins block VEGF-stimulated angiogenesis and migration of endothelial cells . Aspirin induces heme oxygenase-1 (HO-1) expression in endothelial cells, which is increased by ATLa in a concentration- and time-dependent fashion in human endothelial cells . The induction of HO-1 by LX and ATL appears to regulate, in part, the organ-protective actions observed with lipoxins. In a murine model of sepsis, treatment with ATLa spares lung tissues from inflammatory damage .

Resolvins are Novel Endogenous Mediators: 18 R E-Series and 17 R D-Series Resolvins

In view of the role of LX in resolution and reported beneficial actions of omega-3 in humans, it was of interest to determine whether specialized lipid mediators are involved in the resolution of self-limited inflammation. The mouse air pouch was selected for systematic analysis because acute inflammation and exudate formation are spontaneously resolved in this dorsal skin cavity, which permitted kinetic analysis of chemical mediators and leukocyte traffic. The novel lipid mediators produced from EPA were first isolated from resolving exudates that proved to contain 18 R- hydroeicosapentaenoic acid (18R-HEPE) as well as several other related bioactive compounds . The first bioactive product isolated from exudates, coined resolvin E1, reduced inflammation ( Fig. 4.4 ) and blocked human PMN transendothelial migration . Structural elucidation was carried out together with both gas chromatography–mass spectrometry (GC-MS) and tandem mass spectrometry (MS-MS)-based lipidomic analysis of bioactive fractions obtained following extraction and reverse-phase high-performance liquid chromatography (RP-HPLC). The basic structure of this potent bioactive product in resolving exudates proved to be 5,12,18 R -trihydroxyeicosapentaenoic acid . Databases were constructed containing known and theoretical fragments produced by MS-MS of putative lipid mediators. These databases were systematically researched in a stepwise fashion using ultraviolet (UV) chromophores and MS-MS spectra, then LC retention times to identify the basic structures of new compounds in the inflammatory exudates. These procedures required constructing algorithms used together with library software for mass spectral analyses . Following assessment of potential bioactions, the complete stereochemistry of the potent bioactive and related isomers was confirmed by total organic synthesis .

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