alexa Immunoresolving Lipid Mediators and Resolution of Inflammation in Aging | OMICS International
ISSN: 2167-7182
Journal of Gerontology & Geriatric Research
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Immunoresolving Lipid Mediators and Resolution of Inflammation in Aging

Antonio Recchiuti*
Department of Clinical and Experimental Sciences, “G. d’Annunzio” University of Chieti and Center of Excellence on Aging (CeSI), Italy
Corresponding Author : Antonio Recchiuti
Department of Experimental and Clinical Science and Center of Excellence on Aging (Ce.S.I.)
University “G. D’Annunzio” School of Medicine
Via dei Vestini 31, 66013 Chieti, Italy
Tel: +39-0871-541464
Fax: +39-0871-541482L
E-mail: [email protected]
Received January 21, 2014; Accepted March 10, 2014; Published March 12, 2014
Citation: Recchiuti A (2014) Immunoresolving Lipid Mediators and Resolution of Inflammation in Aging. J Gerontol Geriat Res 3:151. doi:10.4172/2167-7182.1000151
Copyright: © 2014 Recchiuti A. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Abstract

Unresolved inflammation is associated with several widely recurrent aging-associated diseases such as arthritis, periodontitis, metabolic disorders, atherosclerosis, and neurodegeneration. Endogenous mechanisms that curtail excessive inflammation and prompt its timely resolution are of considerable interest. In recent years, previously unrecognized chemical mediators derived from polyunsaturated fatty acids were identified as endogenous specialized pro-resolving lipid mediators (SPM) that control both the magnitude and duration of acute inflammation and activate resolution. Lipoxins (LX), resolvins (Rv), protectins (PD), and maresins (Mar) are possess distinct chemical structures, bind to specific G-protein coupled receptors (GPCRs) in a stereospecific manner, and regulate biological pathways to promote resolution in several pre-clinical experimental settings of age-related inflammatory diseases. This review highlights the biosynthesis of SPM and cellular mechanisms that underscore their beneficial bioactions in the regulation of acute inflammation in age-related diseases. The elucidation of these mechanisms operating in vivo to keep acute inflammation under physiologic boundaries and stimulate resolution opened many new opportunities in resolution pharmacology to target aging-associated chronic inflammatory pathologies.

Keywords
Neurodegeneration; Inflammatory pathologies; Polymorphonuclear neutrophils
Abbreviations
AA: Arachidonic Acid (5Z, 8Z, 11Z, 14Z - eicosatetraenoic acid); ALX/FPR2: Lipoxin A4 Receptor/Formyl peptide Receptor 2; COX: Cyclooxygenase; DHA: Docosahexaenoic Acid (4Z, 7Z, 10Z, 13Z, 16Z, 19Z - docosahexaenoic acid); EPA: Eicosapentaenoic Acid (5Z, 8Z, 11Z, 14Z, 17Z- eicosapentaenoic acid); GPR32/DRV1: G-Protein Coupled Receptor 32/Resolvin D1 Receptor 1; IκB: Nuclear Factor of κ Light Polypeptide Gene Enhancer in B-cells inhibitor; IL-Interleukin; LM: Lipid Mediator; LO: Lipoxygenase; LX: Lipoxin; Mar: Maresin; (N)PD: (Neuro)Protectin; PG: Prostaglandin; PUFA: Polyunsaturated Fatty Acid; Rv: Resolvin; SPM: Specialized Pro-Resolving Mediator
Acute Inflammation: A Protective Host Response that May Turn into Harm
Acute inflammation is a defensive physiological response occurring in vascularized tissues following injuries or infections [1]. At histological levels, the “cardinal signs” of inflammation were described by the Roman physician Celsus (1st century BC) and are rubor (redness), tumor (swelling), calor (heat), and dolor (pain). These are the macroscopic events tightly regulated at molecular and cellular level in tissues. Edema is one of the earliest event in acute inflammation, arising from increased vascular permeability of the microcirculation (Figure 1). Next, leukocytes, mainly polymorphonuclear neutrophils (PMN), are recruited at sites of inflammation, transmigrate blood vessels linings, and accumulate in the inflamed site to eradicate the cause of inflammation (Figure 1). In experimental acute inflammation monocytes enter the inflammatory site and differentiate into macrophages (MФs). These latter are pivots in initiating the resolution and the return to homeostasis, i.e. the subsidence of the inflammation and restoration to the previous normal condition, mainly by clearing microbes, cellular debris, and apoptotic cells through non phlogistic phagocytosis (termed efferocytosis) [2-4] and by promoting tissue repair (Figure 1). In order to maintain the host in a healthy status, both the initiation of acute inflammation and its resolution must be efficient. Indeed, impaired acute inflammation will not provide defense against pathogens early post infection [5], while non-resolving inflammation can cause further damage to the host and lead to loss of function, a common feature of many human pathologies including arthritis, asthma, cancers, and cardiovascular diseases [6-8]. Therefore, it is not how often or how extensive an acute inflammatory reaction starts, but how effective and quickly it resolves that determines whether inflammation is detrimental or favorable to the host. Given the high occurrence of inflammation-related diseases in aging, understanding how acute inflammation resolves is of extreme interest.
Microbial infections or tissue damage precipitate an acute inflammatory response in peripheral vascularized tissues, characterized by edema, exudate formation, and leukocyte infiltration. Polymorphonuclear leukocytes (PMN) are among the first leukocytes that infiltrate and fight the pathogenic noxa through engulfment and phagocytosis. Their apoptosis followed by removal initiate tissue resolution. Monocytes that enter the inflamed site as second wave and differentiate into pro-resolving macrophages (MФs) are master cells in resolution. Activated leukocytes release MPs that can promote resolution. Ancient physicians defined resolving exudates pus bonum et laudabile (“good and laudable pus”) as soon as they recognized that it anticipated the resolution of infections and healing of wounds. Today, we appreciate that exudates, in addition to deliver leukocytes in inflamed tissues, also carries bio-precursors for lipoxins, E-series Resolvins, D-series Resolvins, Protectins (neuroprotectin D1), and Maresins, collectively known as Specialized Pro-resolving lipid Mediators (SPM). SPM work to keep the inflammatory response within physiological boundaries and accelerate the return to homeostasis, providing novel opportunities of therapeutics and treatment of human diseases. For more details on the historical steps that changed our understanding and concepts on the process of resolution of acute inflammation.
Resolution of Inflammation: An Active Process Regulated by Specific Chemical Mediators
Resolution has been well described by pathologists more than 100 years ago as the time when the number of neutrophils infiltrating the inflamed tissue is decreasing [1]. This process has been traditionally considered passive, simply due to the attenuation/dissipation of proinflammatory signals. Pioneer work from dr. Serhan et al. [9-12] and from many others worldwide [4,13-16] have demonstrated that resolution of inflammation is instead an active process orchestrated by specific chemical mediators that turn on biochemical pathways and MФ functions to enable the return to homeostasis. Among them, endogenous lipid mediators (LM) biosynthesized from essential Polyunsaturated Fatty Acids (PUFA) play essential roles in resolution acting as “resolution agonists” to a) keep inflammation under physiological boundaries preventing excessive PMN infiltration and b) expedite the complete return to homeostasis stimulating efferocytosis of MФ (Figure 1). Therefore, they represent a new genus of specialized pro-resolving lipid mediators (SPM) [17,18] or immunoresolvents since they act by finely regulating immune processes to promote resolution and the return to homeostasis [19-21]. The SPM genus include lipoxins (LX), resolvins (Rv), protectins (PD), and maresins (Mar) that are enzymatically biosynthesized by lipoxygenase (LO)- driven pathways from Arachidonic Acid (AA), Eicosapentaenoic Acid (EPA), or Docosahexaenoic Acid (DHA) that rapidly appear in exudates and are made available for the conversion into immunoresolvent [22] (Figure 2). In human system, both resident and blood cells contribute to the biosynthesis of SPM, which can be detected intact, at pico- to nanogram levels, in biological fluids [23,24] as well as in tissues in basal conditions and in response to stimuli such as physical exercise [25,26], inflammation [27], or vascular damage [28]. In addition to the LO-pathway, a distinct biochemical route for the biosynthesis of SPM is operative in the vasculature of inflammatory loci. This is initiated by aspirin, a derivative of salicilates, upon acetylation of cyclooxygenase- (COX) 2. The covalent modification of COX-2 shifts the enzyme activity from endoperoxydase into a LO–like, initiating the biosynthesis of epimeric forms of SPM, such as 15R-epi- LXA4 coined “Aspirin Triggered Lipoxin” (ATL) [29]. Notably, ATL, produced in vivo in human subjects taking aspirin [30], proved to be responsible for the local anti-inflammatory actions of low-dose aspirin [30]. Hence, in addition to block formation, aspirin impinges on resolution by triggering the biosynthesis SPM (Figure 2) [31].
In order to define resolution in unbiased, quantitative terms, mathematical resolution indices were introduced by Bannenberg et al. determining the cellular changes in exudates following an acute inflammatory stimulus (namely zymosan A particles from S. cerevisiae, a Toll-like receptor activator). Resolution indices encompass: Tmax, i.e., time point of maximum PMN infiltration (Ψmax); T50, time necessary to achieve 50% reduction in PMN number (Ψ50) from Ψmax; resolution interval (Ri = T50 -Tmax); time interval between Tmax and T50 [32]. The introduction of resolution indices permits the evaluation of pro-resolution bioactions of endogenous chemical mediators or pharmacological agents in pre-clinical models of inflammatory diseases [33-35].
Other chemical mediators are involved in endogenous resolution pathways to switch off leukocyte infiltration and restore homeostasis. Among them are proteins such as the glucocorticoid-induced annexin (Anx) A1 and galectins, which tune the inflammatory response and bring about homeostasis (for recent reviews see refs [36,37]. Furthermore, recent results demonstrate that small inhibitors of cyclin-dependent kinases [3,38] and histone deacetylases [39] can promote resolution by inducing PMN apoptosis and stimulating their prompt removal by MФs, indicating that resolution can be pharmacologically targeted. The appreciation of resolution as a programmed process governed by specific chemical mediators offers opportunities in the uncharted terrain of resolution pharmacology, namely harnessing endogenous controllers of inflammation as therapeutics or biotemplates for new drugs to treat inflammation-related diseases [18,40]. For example, AnxA1 served as model for the generation of peptides [41,42] and engineered nanoparticles [43] that dampen inflammation and protect from tissue damage. Likewise, several LX stable analogs obtained by organic synthesis proved to have anti-inflammatory and organ protective activities [44-46] and results by Norling et al. demonstrate the efficacy of human neutrophil-derived nanoparticles carrying a benzo-LX analog in reducing peritoneal and joint inflammation [47]. Moreover, RX-10045, a synthetic resolvin analog formulated by Resolvyx Pharmaceuticals for Inc. for topical application, proved safe and effective in reducing the severity of dry eye syndrome in a phase II placebo controlled clinical trial (see http://clinicaltrials.gov. Entry Identifier: NCT00799552) and have moved forward to phase III clinical trial with Celtic Therapeutics. Finally, a recent placebocontrolled, randomized, comparative study demonstrated that a LX analog significantly ameliorates clinical parameters of juvenile eczema [48], further translating results from pre-clinical models to humans and establishing the effectiveness of SPM-based pro-resolution pharmacology.
Importantly, resolution is not synonymous of anti-inflammation. This is because, in order to be considered a “pro-resolver” a chemical entity, in addition to serve as “stop signals” for leukocyte trafficking and other cardinal signs of inflammation (e.g. swelling, pain), must stimulate efferocytosis by MФ, favor the antibacterial activities, and promote tissue repair. Along these lines, while COX and LO inhibitors reduce some of the cellular events of the inflammatory reaction (e.g. edema formation, PMN recruitment, and pain), they dramatically impairs resolution [33,49]. In contrast, aspirin and glucocorticoids act synergistically with endogenous pro-resolution pathways [13].
Complete resolution also requires the clearance of microparticles (MPs) shed by activated or apoptotic cells in inflammatory loci from plasma membranes. MPs are now recognized as “specialized shuttles” of bioactive molecules with important roles in inflammation and resolution. Indeed, a subset of PMN-derived MPs that exert antiinflammatory actions was identified [42,46]. Furthermore, Norling et al. developed, from human PMN, novel nano-proresolving medicines (NPRMs) containing SPM that proved bioactive in reducing acute inflammation in vivo, expediting resolution, and promoting wound healing [50]. Additional biological properties of NPRMs have recently been demonstrated in a modified focal microfluidic chamber [51] on isolated human leukocytes, further confirming the possibility to exploit NPRMs for delivering endogenous pro-resolution mediators therapeutically.
Biosynthesis of SPM from PUFA
The identification SPM was achieved using a self-limited or naturally resolving acute inflammation model in vivo and a systems approach [11,52] and reviewed [17]. For this, the murine dorsal air pouch was ideal because it permitted isolation of inflammatory exudates for cellular analyses, proteomics, [11,52] and direct LMlipidomics of bioactive products, as well as their inactive precursors and further metabolites, during a self-limited acute inflammation, i.e., the natural means by which inflammation returns to homeostasis. With this systems approach it was possible to establish the local and temporal dissociation of LM biosynthesis [32] from onset to resolution phase of inflammation. Indeed, in this setting, the eicosanoid biosynthesis underwent a “class switch” with the deactivation of the biosynthesis of pro-inflammatory leukotrienes (LT) and the initiation of LX and Rv production [12]. LM lipidomics using liquid chromatographytandem mass spectrometry (LC-MS/MS) coupled with informatics permit profiling of closely related compounds and identification of new molecules. Retrograde, both biogenic and total organic, synthesis allows the complete elucidation of chemical structure, stereochemistry, and physical properties, along with the recapitulation of the in vivo biosynthetic pathway [19,53,54]. The matching/identification of LM is usually carried out with at least two different instruments and/ or solvent systems and the criteria to identify a known LM are the following: a) LC retention time should match by coelution with the LM authentic standard; b) UV chromophore should match the synthetic and authentic LM (i.e., λmax and band shape); as well as c) > 6 diagnostic ions of tandem MS/MS spectrum. Recently, a new set of SPM derived from DHA has been identified with targeted LM metabolomics [21], providing new mechanisms for the beneficial actions of PUFAs. The next paragraph will illustrate biosynthetic routes and chemical properties of the main SPM.
Lipoxins
LXs are “lipoxygenase interaction products” derived from the enzymatic conversion of arachidonic acid (AA) via trancellular biosynthesis during cell-cell interactions occurring during inflammation [55]. LXA4 and B4 were the first SPM identified by Serhan et al. [9,10]. Although LXs were identified in 1980s in the Samuelsson laboratory [9], their potent bioactions were uncovered some years later when it became clear that they act as “stop signals” of further PMN infiltration [56] and as potent stimuli for the non-phlogistic recruitment of monocytes [57] and MФ efferocytosis [58] (recently reviewed in [40]). In humans, sequential oxygenation of AA by 15-LO and 5-LO, followed by enzymatic hydrolysis, leads to the biosynthesis of LXA4 and B4 in mucosal tissues, such as airways, gastrointestinal tract, and oral cavity [27,59,60] (Figure 2) [61-63]. Blood vessels represent a second site for LX biosynthesis, with the conversion of 5-LO-derived LTA4 into LXA4 and B4 by 12-LO in platelets [64,65].
ATL: The first aspirin triggered SPM
The ATL synthetic pathway is initiated by aspirin by acetylation of COX-2 which renders the enzyme capable of converting AA into 15R-HETE, the substrate of leukocyte 5-LO for the biosynthesis of positional isomers of LXA4, named 15R-epi- LXA4 [29]. This observation proved, for the first time, that aspirin has aspirin has the unique capability, among non-steroidal anti-inflammatory drugs (NSAID), to “jump start” resolution by its ability to trigger endogenous biosynthesis of so called “aspirin triggered” LX (ATL) (Figure 2). In keeping with this, ATL is produced in vivo in humans taking aspirin [30] and mediates the local anti-inflammatory actions of lowdose aspirin in healthy individuals [31]. Interestingly, studies from Birnbaum et al. demonstrate that atorvastatin, a widely used lipidlowering drug, promotes the myocardial generation of 15R -LXA4 via S-nitrosylation of COX-2 [63]. Furthermore, Gutierrez et al. recently showed that pioglitazone, an insulin-sensitizing agent, elevates plasma levels of 15-epi-LXA4 [64], providing further mechanisms for the beneficial actions of this drug [65].
E-series resolvins
The essential roles of omega-3 PUFA EPA in health were already evident in 1929 [66] and ω-3 proved beneficial effects in human diseases including potential antithrombotic, immunoregulatory, and antiinflammatory properties [67,68]. Also, the Gruppo Italiano per lo Studio della Sopravvivenza nell’Infarto Miocardico-Prevenzione trial reported a significant∼ decrease in cardiovascular death in >11,000 patients surviving myocardial infarction taking 1 g of ω-3 PUFA daily along with recommended preventive treatments including aspirin [69]. However, the mechanisms offered for explaining their beneficial actions (e.g., preventing conversion of AA to pro-inflammatory and prothrombotic eicosanoids; serving as an alternate substrate for the 5-series LTs that are less potent than 4-series LTs; conversion by COX to 3-series prostanoids that also maintain antithrombotic actions) [67,68,70] have not been generally accepted due to the lack of molecular evidence in vivo and the high concentrations of ω-3 PUFA required in vitro to achieve putative “beneficial actions”. To address the molecular basis for anti-inflammatory properties of ω-3 fatty acids, an unbiased LC-MS/MS-based informatics approach was developed to identify novel mediators generated from ω-3 precursors during acute inflammation in vivo. Using this approach, EPA was found to be enzymatically converted into novel potent LMs coined resolvins (an acronym of resolution phase interaction products) because a) they are produced during cell-cell interactions occurring in the resolution phase of acute inflammatory response; (b) “stop” further neutrophil entry to sites of inflammation, and (c) reduce exudates [11,32,52,71,72]. EPAderived E-series Rv are endogenously biosynthesized in vivo in resolving murine exudates and in isolated human cells by isolated cells (e.g. endothelial cells -leukocyte interaction) and in whole blood (vide infra). The complete stereochemistry of first member of this family, RvE1, has been established as 5S,12R,18R-trihydoxy-6Z,8E,10E,14Z,16E-EPA [73]. In vascular endothelial cells, aspirin acetylated COX-2 converts EPA into 18R-Hydro(peroxy)-Eicosapentaenoic acid (HEPE), which is rapidly taken up by activated leukocytes (e.g., PMN) and further metabolized into RvE1 (Figure 2). Interestingly, chiral HPLC analysis indicated that the 18R-HEPE isomer was dominant to its epimer 18S-HEPE in human plasma from healthy volunteers taking EPA, whereas human subjects who were administered aspirin before EPA had more 18S- than 18R-HEPE. These results indicate that aspirin might promote 18S-HEPE production as well as 18R-HEPE from ingested EPA [74]. Notably, 18S-HEPE can also be converted to RvE1 and RvE2 by human recombinant 5-LO and LTA4 hydrolase, a LTB4- synthesizing enzymes [74], and RvE1 is also produced via cytochrome P450-driven oxygenation of EPA [11] and by Candida albicans [75]. RvE2 (5S, 18-dihydroxy-EPE) is biosynthesized in resolving exudates and in human whole blood via reduction of 5S-hydroperoxy, 18-hydroxy-EPE, an intermediate in the biosynthetic pathway of RvE1 [76-78]. Conversely, novel EPA-derived SPM, namely 18R-RvE3 (17R, 18R-dihydroxy-5Z, 8Z, 11Z, 13E, and 15E-EPE) and epimeric 17R, 18S-RvE3, that possess potent anti-inflammatory actions both in vitro and in vivo are biosynthesized via 12/15-LO by eosinophils [79,80].
D-series resolvins
Earlier LC-MS/MS-based analyses of resolving exudates from mice given DHA and aspirin provided the first evidence for the formation of novel endogenous 17-hydroxy-containing mediators [53]. Recapitulation of biosynthetic pathways using isolated human cells and recombinant enzymes established potential origins of novel compounds isolated from resolving exudates in vivo. Indeed, hypoxic human endothelial cells COX-2 converted DHA to 13-hydroxy-DHA that switched with ASA to 17R-HDHA that can be transformed to di- and trihydroxy products by human PMN. These compounds were termed “aspirin triggered” D-series resolvins [52]. Remarkably, in the absence of aspirin, D-series resolvins carrying the 17S-hydroxy group were identified in murine exudates and isolated human cells [52,71]. The enzymatic processes leading to the formation of 17S- and 17R-RvD1 are shown in Figure 2. Following the complete organic synthesis, the stereochemistry of 17S-, 17R-RvD1, and RvD2 were established as 7S, 8R,17S-trihydroxy-4Z,9E,11E,13Z,15E,19Z-docosahexaenoic acid (17S-RvD1), 7S, 8R, 17R-trihydroxy-4Z,9E,11E,13Z,15E,19Zdocosahexaenoic acid (17R-RvD1) [53], and 7S, 16R, 17S-trihydroxy- 4Z, 8E, 10Z, 12E, 14E, 19Z-docosahexaenoic acid (RvD2) [81]. Additional members of this family have identified (RvD3-RvD6). Each of these arises by similar biosynthetic routes, but has distinct chemical structures and potentially additional bioactions that are now being unveiled [20,82].
(Neuro) protectins
In addition to D-series Rvs, DHA also serves as precursor of a new family of LM characterized by a conjugated triene system and two alcohol groups called protectins (PD), in view of their protective actions in neural tissues within the immune system, while the prefix neuroprotectin gives the tissue localization and site of action. The structure of the founding member of this family, PD1, was first disclosed in a report on the isolation and elucidation of resolvins [52,71] and its complete stereochemistry later established as 10R,17S-dihydroxydocosa- 4Z,7Z,11E,13E,15Z,19Z-hexaenoic acid [72]. In addition to PD1, several stereo- and positional isomers that also possess lower bioactivity than PD1 were identified in human and mouse tissues. These include 10S, 17S-diHDHA, 4S,17S-diHDHA, 7S,17S-diHDHA, and 22-hydrox-10,17S-docosatriene (a putative inactivation product of PD1) [53,72]. Finally, a novel aspirin triggered COX-2 driven pathway that biosynthesize the 17R-epimeric form of PD1 from DHA has been reported [83] (Figure 3). The total organic synthesis and complete streochemical assignment of AT-PD1 (10R,17R- dihydroxy-docosa- 4Z,7Z,11E,13E,15Z,19Z-hexaenoic acid) were recently achieved [84].
Maresins
Macrophages have pivotal tasks in restoring homeostasis [2] and are main SPM-synthesizing cells during this active process. For example, MФ ingestion of apoptotic PMN concomitantly initiate tissue resolution [15,16] and the biosynthesis of LXA4, RvE1, and PD1, but not LTB4 [33,85]. Along these lines, maresins are a new family of SPM produced by MФs identified with LM-metabolomics [86]. A 12/15-LO-dependent biochemical pathway converts DHA into 14-hydroxydocosaexaenoic acid (HDHA), which is rapidly converted by isolated MФ into a new set of products, whose molecular structure was established [54] and recently confirmed [19]. Macrophage 12-LO converts this 14-HDHA intermediate into a 13,14-epoxide precursor of 7,14-dihydroxydocosa-4Z,8E,10E,12Z,16Z,19Z-hexaenoic acid, named maresin (from macrophage mediator in resolving inflammation) 1 (MaR1) (Figure 2) [54]. The complete stereochemical assignment and total organic synthesis of Mar1 have been achieved [19] and double bond geometry and chirality of 13,14-epoxy DHA elucidated together with novel bioactions [87]. These include inhibition of proinflammatory LTB4 biosynthesis by LTA4 hydrolase as well AA conversion by 12-LO and regulation of M1/M2 macrophage phenotype (88). In addition to MaR1, a double dioxygenation product, namely 7S, 14S-dihydroxydocosa-4Z,8E,10Z,12E,16Z,19Z-hexaenoic acid (7S,14S-diHDHA), formed by consecutive lipoxygenation of 14- HDHA, was also identified and proved bioactive but less potent than MaR1 in stimulating efferocytosis with human cells [19,54].
GPCRs for SPM in Anti-Inflammation and Resolution
By the definition, SPM: a) are generated within the resolution phase of inflammation; b) limit leukocyte infiltration; c) enhance phagocytic activity of pro-resolving MФ to remove apoptotic cells and/or microbes; d) stimulate the clearance of PMN from mucosal surfaces and their anti-microbial actions. If a LM carries each of these bioactivities, then it falls into the category of SPM. Beside these general actions, each SPM possesses additional peculiar activities (Table 1). For instance, RvD1 and E1 oppositely regulate interferon γ, LXA4, and IL-5 in airway exudates during the resolution of allergic inflammation [88] and reviewed in and [89]. In isolated cell systems and experimental models of inflammation and resolution, SPM proved to be active in the nano- to sub micromolar or nano- to low microgram dose range and to act through specific G-protein coupled receptors (GPCRs) in a stereospecific manner (Table 1). Given the important protective function of acute inflammation against external or internal dangers and the need to prevent this reaction from becoming uncontrolled, it is not surprisingly that SPM have some overlapping immunoresolving actions. Further, the sites of biosynthesis for each SPM and the degree of cell distribution of their GPCRs may account for their selectivity and specificity within resolution programs.
The first evidence for receptor-mediated actions of LXA4 arises from studies by Nigham et al. that demonstrated stimulation of rapid lipid remodeling and pertussis toxin (PTX)-sensitive release of AA in PMN treated with LXA4 [89]. Specific, reversible, and stereoselective binding of synthetic (11,12-3H)-LXA4 to intact human PMN (with a Kd ~ 0.5 nM) further confirmed the involvement of membrane receptor, likely belonging to the GPCR superfamily, in LXA4 bioactions [90,91]. Screening of cDNA clones from differentiated HL60 human cells identified formyl peptide receptor like-1, an homologue of formyl receptor, as putative LXA4 GPCR [92]. This receptor has been renamed ALX/FPR2 in light of its high affinity for LXA4 [93] and is highly expressed in myeloid cells and at a lower extent in lymphocytes, dendritic cells, and resident cells [94]. Orthologous genes of the human ALX/FPR2 have been identified in rodents [95,96]. In addition to LXA4, ALX/FPR2 is activated by the glucocorticoid-induced protein AnxA1 and its N-terminal peptides [13], representing the prototype of GPCR able to coordinate anti-inflammatory and pro-resolving activities of both lipid and peptide ligands. While earlier studies demonstrated that radio-labeled 15-epi- LXA4 binds at cysteinyl LT receptor 1 (CysLT1) with equal affinity to LTD4, providing additional molecular mechanisms for ATL dampering CysLT signals in the vasculature [97], genetic manipulation of ALX/FPR2 and its orthologue in mice has provided evidence for the essential role of this GPCR in mediating LX actions. Indeed, myeloid-specific overexpression of human ALX/ FPR2 in mice resulted in increased sensitivity to suboptimal doses (10 ng/mouse) of the ligand Kstable analog, with >50% reduction in PMN infiltration to zymosan-induced peritonitis compared with <10% reduction in nontransgenic littermates [98]. On the contrary, ALX/FPR2 nullified mice displayed an exacerbated inflammation and delayed resolution phenotype and did not respond to endogenous and synthetic ligands [99]. More strikingly, ATL amounts and ALX/FPR2 expression levels dictate both the magnitude and duration of acute inflammation in humans [100]. Hence, mechanisms that regulate its levels in tissues are of wide interest. In this regard, Simiele et al. recently unraveled the molecular basis of ALX/FPR2 transcriptional machinery, with the identification of the core promoter sequence, the elucidation of transcription factors and epigenetic mechanisms that regulate promoter activity, and the identification of the first inheritable SNP that impairs promoter activity in individuals at high cardiovascular risk [101].
At least two GPCRs are involved in mediating RvE1 actions, namely ChemR23 and BLT1 [73,102]. [3H]-RvE1 bound to ChemR23 transfectants with high affinity (Kd=11.3 ± 5.4 nM) and stereoselectivity [73]. Also, the synthetic peptide fragment (YHSFFFPGQFAFS) derived from human chemerin that was earlier reported to be a ligand for this same receptor [103] displaced [3H]-RvE1 binding by ~70% when tested at 10 μM concentration, suggesting that RvE1 and chemerin share recognition sites on ChemR23 [73,104]. [3H]-RvE1 specific binding was also demonstrated with membrane fractions isolated from human PMN (Kd of ~50 nM) and was displaced by homoligand RvE1 (Ki ~ 34 nM), LTB4 (Ki =0.08 nM) and LTB4 receptor 1 (BLT1) selective antagonist U-75302 (Ki =1.5 nM), but not by the chemerin peptide [102]. These results strikingly demonstrated that RvE1 binding sites are pharmacologically distinct from ChemR23 on human PMN and prompted to investigate whether RvE1 binds to LTB4 receptors. In these studies, [3H]-RvE1 also gave high affinity binding to recombinant BLT1 (Kd ~ 45 nM) that was competed by unlabeled LTB4 (Ki =3 nM). In contrast, BLT2-overexpressing cells did not show [3H]- RvE1 binding at concentrations up to 10 nM. These results clearly demonstrated that RvE1 binds to BLT1 on human PMN and acts as a partial agonist to attenuate LTB4 in coming signals in both mouse and human leukocytes [102]. Human ChemR23 is expressed in brain, kidney, cardiovascular, gastrointestinal, and myeloid tissues [73]. More recently, direct evidence for ligand-receptor interactions of RvE1 and its epimer 18S-RvE1 was provided using ChemR23 and BLT1 β-arrestin cells with EC50 (~ 6.3 pM) lower than that obtained with RvE1 (~0.14 nM). 18S-RvE1 also antagonized LTB4-mediated BLT1 activation with higher potency and efficacy than RvE1 in BLT1 β-arrestin cells [74]. Hence, RvE1 and 18S-RvE1 can share the same site(s) of specific binding to human ChemR23 as well as BLT1.
RvE2 exerts potent and cell-specific bioactions on human leukocytes [77,78]. Recently, tritium-labeled [3H]-RvE2 was synthesized and gave comparable Kd (~ 25 nM) with other SPM in isolated human PMN. In addition, using ChemR23 and BLT1 β-arrestin cells RvE2 was found to share, at least in part, receptors with RvE1 [74].
RvD1 also exerts specific bioactivities on human PMN (e.g., PTXsensitive reduction of F-actin polymerization), did not stimulate Ca2+ release, and did not activate cAMP in human PMN [105]. [3H]-RvD1 prepared by catalytic hydrogenation of synthetic [13,14]-acetylenic RvD1 methyl ester specifically bound to human PMN with high affinity (Kd ~ 0.17 nM) and was displaced by cold RvD1 (100%) and LXA4 (~ 60%), but not the AnxA1-derived Ac2-12 peptide [105]. [3H]-RvD1 also showed specific binding with human monocytes [105]. Screening of phylogenetically related GPCR linked to inflammation and chemotaxis in NF-κB-responsive engineered cells demonstrated that RvD1 significantly reduced TNF-α-induced NF-κB activation in cells overexpressing either the lipoxin receptor ALX/FPR2 and the orphan, GPR32, but not other GPCRs (e.g. BLT1, BLT2, CB1, GPR-1, FPR, and ChemR23) [105]. Moreover, RvD1 dose-dependently activated ALX/FPR2 and GPR32 in recombinant β-arrestin cells with EC50 in the low picomolar range (EC50 ~ 1.2 pM for ALX/FPR2; 8.8 pM for GPR32) [105]. In comparison, at equimolar concentrations ATRvD1, RvD1-carboxy-methyl ester, and a metabolically stable analog 17 (R/S)-methyl RvD1-ME, activated both ALX/FPR2 and GPR32 with similar potencies and EC50, whereas the biosynthetic precursor native DHA was not active with GPR32 and ALX/FPR2 [106]. Hence, RvD1, AT-RvD1, and the derivatives carboxy methyl ester and 17(R/S)-RvD1 directly activate ALX/FPR2 and GPR32. Studies with genetically engineered mice and selective receptor antagonists or blocking antibodies confirmed the ALX/FPR2 and GPR32 dependency of immunoresolving actions of RvD1 [31,39,107-114], which involves regulatory mechanisms on transcription factors, microRNAs, and select genes [108]. Human GPR32 was identified in peripheral blood leukocytes and arterial and venous tissues using a cDNA array. It is mostly abundant on PMN, monocytes and macrophages and is also present on vascular endothelial cells [105]. The murine ortholog of GPR32 is currently unknown whereas it exists in chimpanzees. Regulatory mechanisms of GPR32 are unknown, while those of ALX/FPR2 have recently been uncovered [101]. Although specific receptors for RvD2, RvD3 and RvD4 have not yet been uncovered, the stereoselective actions of RvD2 were inhibited by petussis toxin [81], implicating the involvement of GPCRs. More recently, Chiang et al. showed activation of RvD1-receptor GPR32 is activated by RvD5 with the recombinant human GPR32 [82].
Specific binding of tritium-labeled (N) PD1 was demonstrated with both retinal pigment cells (RPE) and human PMN (Kd ~ 30 pmol/mg of cell protein), although, at high concentration of radio-ligand (> 10 nM), non-specific binding was evident, likely because of the highly hydrophobic nature of this compound. Also, in competition studies, the free acid form of cold (N)PD1 showed 90-100% displacement of radio-labeled (N)PD1, while other structurally related omega-3 fatty acid-derived compounds gave only minimal or no displacement [115].
Immunoresolving Actions of SPM in Aging-Related Diseases
Lipoxins and ATL
Lipoxins and ATL represent the prototype of immunoresolvents biosynthesized from AA during a lipid mediator (LM) class switching characteristic of self-contained acute inflammatory reactions [12,116]. They were the first class of PUFA-derived autacoids identified to carry dual anti-inflammatory and pro-resolution action, e.g., reducing vascular permeability and inhibiting PMN recruitment to inflammatory loci while stimulating monocytes/MФs in a non phlogistic manner [57]. LXs undergo in vivo rapid inactivation primarily through prostaglandin dehydrogenase-mediated oxidation and reduction [117]. Therefore, several synthetic stable analogs were designed to resist in vivo catabolism and proved to carry potent actions in vivo and in vitro [44-46,118,119] (Tables 1 and 2). Intriguingly, these anti-inflammatory and pro-resolving bioactions do not involve cell toxicity or immunosuppresion, but rather a fine tuning of immune processes. For instance, LXA4, ATL, stable analogs reduce PMN infiltration and prevents connective tissue and bone loss in a rabbit model of ligature-induced periodontitis [120]. They also attenuate the severity of experimental colitis (diminishing weight loss, inflammation and immune dysfunction) [121], asthma (dampening airway hyperresponsiveness and pulmonary inflammation) [122,123], and cystic fibrosis (decreasing neutrophilic inflammation, pulmonary bacterial burden and disease severity) [124]. Notably, these, and many others, chronic inflammatory diseases present evidence for defective LXmediated pro-resolution mediators and mechanisms [125-128].
Unresolved inflammation is also a hallmark of metabolic diseases, such as obesity and diabetes, and has pathophysiological roles in disease-associated multi-organ dysfunction [129]. Ex vivo studies with adipose tissues explanted from aging mice demonstrated that LXA4 increases the expression of molecules critical in insulin sensitivity (e.g., the glucose transporter GLUT-4 and IRS-1), restores insulin sensitivity in the tissue, and decreases major pro-inflammatory cytokines such as IL-6 while increasing the pro-resolving IL-10 [130]. These studies also demonstrated that LXA4 increases MФ-mediated glucose uptake in vitro [130]. Finally, results from Borgeson, Docherty et al. demonstrate that LXA4 and a synthetic analog modulate inflammation and tissue degeneration in experimental hind-limb ischemia/reperfusion injury [46,129] and diabetic renal fibrosis [131], providing further areas of investigation for pro-resolution therapies in chronic inflammatory diseases.
E-series resolvins
Resolvins of the E-series encompasses several molecules. Among them, RvE1 was the first isolated and studied in depth. RvE1 displayed potent stereoselective actions in vivo and with isolated cells (Tables 1 and 2). At nanomolar levels in vitro, RvE1 strikingly reduced human PMN transendothelial migration, dendritic cell migration and interleukin (IL)-12 production [52,73]. In many pre-clinical models of diseases RvE1 displays potent counterregulatory actions that protect against leukocyte-mediated tissue injury and excessive proinflammatory responses. For instance, administration of RvE1 in rabbit and mouse models of periodontitis reduces PMN infiltration, prevents loss of connective tissue and bone, and promotes tissue regeneration [104,132,133]. Furthermore, RvE1 protects against oxygen-induced retinopathy [134] and dry-eye syndrome [135,136], a common disorder of the tear film affecting a significant percentage of the old population [137]. Also, recently RvE1 proved to protect myocardium from ischemia-reperfusion injury reducing the size of infarct area [138]. Interestingly, among SPM, RvE1 carries peculiar, ChemR23- mediated, bioactions on leukocytes and platelets, reducing leukocyte rolling to venules in vivo, regulating adhesion molecules, and blocking adenosine diphosphate-induced aggregation and signaling, which are pivotal steps in thrombus formation [106,139]. Together, these findings provide evidence for specific, GPCR-mediated mechanisms that may account for some of the cardioprotective actions noted with dietary supplementation with EPA together with low-dose aspirin [69].
D-series resolvins
RvD1 and AT-RvD1 are potent regulators of inflammatory responses both in human and murine cells. For instance, they stop PMN transendothelial and transepithelial migration [52], and regulate endotoxins-induced cytokine production by MФs [140]. In murine zymosan-induced acute peritonitis, RvD1 lowered the Ψmax and shortened the Ri by ~ 4 h demonstrating the ability to expedite the onset of resolution [106]. Furthermore, microRNA expression analyses from exudate cells demonstrated that RvD1 controls a specific set of pro-resolving miRNAs miR-21, miR-146b, miR-208a, and miR- 219 in vivo in a time- and GPCR-dependent manner as part of its immunoresolving actions [106]. Indeed, target mRNAs for the RvD1- GPCR-regulated miRNAs included genes of the NF-kB activation pathway (e.g. IkB kinase and tumor necrosis factor receptor-associated factor 6), cytokines and chemokines (e.g., IL-8, 10, 12, interferon-α and β), programmed cell death 4, a tumor suppressor molecule that acts as a translational repressor of IL-10 [141], and 5-LO, a pivotal enzyme for the biosynthesis of LT and SPM. Interestingly, in experimental renal fibrosis, LXA4 attenuated the production of pro-fibrotic proteins (e.g., fibronectin, N-cadherin, thrombospondin, and the notch ligand jagged-1) in cultured human proximal tubular epithelial cells via upregulation of microRNA let-7c, further indicating the involvement of miRs in the SPM-triggered protective actions in mammals [142]. Along this line, results from Fredman et al., Li et al. [141,142] demonstrate that delayed resolution of acute peritonitis, triggered by high doses of zymosan A, dysregulates pro-resolving microRNA and lipid mediator profiles, namely with decreased miR-219 expression along with increased LTB4 and decreased SPM production [143]. Finally, recent studies reveal that miR-466l was temporally regulated in murine exudate leukocytes and controls prostanoid and immunoresolvent biosynthesis during resolution [144], providing novel evidence that miRs play roles in endogenous SPM-driven resolution pathways, whose failure can contribute to the development of chronic inflammation and diseases.
The role of unresolved inflammation, triggered by alterations in the nutrient sensing and regulation mechanisms, in obesity, insulin resistance, and type 2 diabetes (T2D) is widely appreciated [129]. The beneficial effects of SPM from omega-3 RvE1 and PD1 in preclinical models of obesity and diabetes have been shown in ob/ob mice, in which both omega-3-enriched diet and RvE1 administration increased expression of genes involved in glucose transport (e.g., GLUT-4) insulin signaling (e.g., IRS-1), and insulin sensitivity (e.g., PPARγ) [145]. Further studies also revealed that RvD1 improves insulin sensitivity, reduces the pro-inflammatory phenotype of adipose tissue macrophages [111], and promotes the repair of diabetic wounds in leptin-receptor deficient mice [40]. Interestingly, RvD1 also enhances the resolution of inflammation in adipose tissue by skewing MФs towards an anti-inflammatory/pro-resolution phenotype, with decreased pro-inflammatory adipokines in parallel with increased expression of anti-inflammatory genes [146]. Therefore, these results suggest that stimulating resolution with the endogenous immunoresolvent RvD1 could provide a novel therapeutic strategy for treating inflammation-related complications of obesity and obesityinduced diabetes.
Neuroprotectins
Biosynthesis of (N) PD1 occurs in neural tissues in response to injury, ischemia-reperfusion, and exposure to β-amyloid peptides from DHA, the most abundant omega-3 PUFA in nervous tissue and retina [83,147,148]. Consistent with its main site of biosynthesis, (N) PD1 carry exquisite tissue-protective and anti-inflammatory action within the brain and the eye (Tables 1 and 2) where inflammation plays key pathophysiological roles in degenerative and ischemic illnesses. For instance, (N) PD1 blocks PMN transmigration across endothelial cells in a stereospecific manner [72] and reduces leukocyte infiltration in animal models of Herpes Virus Simplex-induced stromal keratitis [149]. Interestingly, intracerebroventricular infusion of 17S-HpDHA, a 12/15-LO product of DHA and precursor of SPM, increased levels of (N)PD1 in hippocampus and attenuated neuroinflammation initiated by endotoxins at least in part via its conversion to SPM [150]. In addition, (N) PD1 reduces leukocyte accumulation, NF-κB activation, and COX-2 induction, as well as the size of damaged areas in rats following experimental stroke [83,151], providing novel therapeutic strategies for treating ischemic episodes in old patients. Alzheimer’s disease (AD) is the major cause of dementia in elderly and is determined by accumulation of amyloid Aβ plaques in the brain, which triggers neuroinflammation [152]. In this setting, (N) PD1 is a potent counter-regulator of the inflammatory response in hippocampus of AD mice and primary human neurons. In particular, (N) PD1 reduces expression of Aβ-42 -triggered expression of pro-inflammatory genes, suppresses Aβ42 peptide shedding by β-secretase-1, shifting the β-amyloid breakdown towards a non amyloidogenic pathway, and protects neurons from apoptosis [153]. Interestingly, recent studies by Medeiros et al. demonstrate that aspirin-triggered 15-epi-LXA4 also ameliorates AD symptoms in mice, reducing pro-inflammatory mediators and stimulating clearance of Aβ deposits by specialized microglial cells [154]. Together, these results highlight the important protective functions of SPM in nervous system and prompt to investigate their actions in other neurodegenerative diseases.
Maresins
Consistent with general SPM actions, both Mar1 and 7S, 14S-diHDHA reduce PMN infiltration in inflamed tissues and enhance MФ phagocytosis (Table 1). In addition they carry potent, stereospecific actions linked to analgesia and organ repair (Table 2). In particular, Mar1 reduces capsaicin-induced transient receptor potential V1 currents in dissociated primary sensory in neurons in a PTX-sensitive manner and spontaneous pain behaviors (i.e., flinching/ licking) in mice [19].
Further, Mar1, biosynthesized by brown planaria in response to wound, accelerates the repair of damaged tissue [19]. Since chronic inflammation, pain, and tissue degeneration are common signs of aging-related diseases and can cause disability and discomfort, these findings on anti-inflammatory, pain-relieving, and regenerative actions of Mar1 are intriguing.
Summation and Conclusions
In summation, the acute inflammatory response is a highly coordinated defensive response and complete resolution is its ideal outcome, whereas unresolved inflammation plays causative roles in chronic, degenerative and metabolic diseases. Resolution of inflammation is an active process governed in part by specialized immunoresolvent lipid-derived chemical mediators or SPM. SPM act in vivo and in vitro to promote the return to homeostasis and their bioactions are highly stereospecific, GPCR-mediated, and exerted at low doses. Results from the first human clinical trial with a resolvin analog are striking and can open new opportunities for resolution pharmacology. It is therefore envisageable that more human trials will be launched in the near future that will help to test the notion that stimulating resolution can improve the way we treat age-related chronic inflammatory diseases.
“Nunc autem visum est mihi de senectute aliquid ad te conscribere”
(Now, I consider appropriate to write for you something about the old age)
Marcus Tullius Cicero (Cato Maior De Senectute, 44 BC)
To my wife and my family.
Acknowledgement
The author is supported by the European Union Seventh Framework Programme [FP7/2007-2013] under grant agreement n° 294187 FP7-PEOPLECIG- 2011 (to A.R.).
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