Specialised Pro-resolving Lipid Mediators (SPMs)

Glossary

 

 

Agonist: A substance that can bind to a cell surface receptor and produce a certain action in the cell, similar to that produced by a substance.
 

Angiogenesis: Formation of blood vessels.
 

Antagonist: A compound capable of lessening the activity of another, such as a hormone, neurotransmitter, enzyme or drug..
 

Anti-inflammation: Inhibition of the inflammatory process.
 

Apoptosis: Specific form of programmed cell death, involved controlling cell developmentand growth.
 

Class switching: Switch in the lipid mediator class over time.
 

Diapedesis: The process by which formed blood elements, mainly leukocytes, pass through intact vessel walls.
 

Efferocytosis: Phagocytosis of apoptotic cells by macrophages.
 

Eicosanoids: Physiologically active substances, derived from fatty acids with 20 carbon units.
 

Oxidative: stress Biochemical imbalance favouring free radicals (reactive species) over antioxidants, which causes cell and tissue damage to the body.
 

Exudate: A mass of elements which seep out of blood vessels in the inflammatory process and are deposited in the tissue interstitium or cavities of the organism.
 

Homeostasis: A set of self-regulating phenomena that balance the composition and properties of an organism’s internal medium.
 

Resolution: Agonist action on the inflammatory process, whose purpose is to return tissues to homeostasis.
 

Transcellular synthesis: Generation of new bioactive compounds that none of the cell types involvedare capable of producing by themselves.
 

1. Inflammation: definition and its importance

1.1. Inflammation as a necessary response. Acute and chronic inflammation

The appearance of acute localised inflammation is a sign that the immune system is responding to infection or tissue injury. It is a process that enables the body to eliminate invasive organisms and repair damaged tissues1-3.

Although the inflammatory response protects the host, preventing it from becoming chronic or systemic requires complete resolution of acute inflammation (i.e. complete resolution of leukocyte infiltration andelimination of the cellular detritus) and the return of tissues to homeostasis (Fig. 1).

This is currently considered the key element in many of the predominant diseases of modern western society1-3.

 

So, progression of the inflammatory response to its natural conclusion should be induced, to favour the benefits and restorative qualities of inflammation that can otherwise be lost with anti-inflammatory therapies5.

2. The sequence and characteristics of the inflammatory process

En la respuesta inflamatoria aguda pueden distinguirse dos fases fundamentales: inicio y resolución6.

2.1. Onset of the inflammatory response

Redness, swelling, heat and pain are signs of inflammation, the body’s response to injury or infection. They are caused by increased blood flow and capillary permeability (enabling large molecules to leave the blood stream and cross the endothelial wall) and greater leukocyte movement from the blood to the damaged tissue7.

This marks the onset of the immune response, whose purpose is to eliminate invasive pathogens and toxins and then repair the tissue: cell arrival is induced by an increase in adhesion molecules (intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-selectin) on the surface of endothelial cells, permitting the binding of leukocytes and subsequent diapedesis7.

Neutrophils (polymorphonuclear cells, hence PMNs) are the first cells to appear, followed by monocytes, macrophagesand lymphocytes. A trigger at the site of infection or injury (such as a bacterial endotoxin) activates the neutrophils, macrophages and monocytes and these form proinflammatory cytokines (such as tumour necrosis factor alpha (TNF-α), interleukins (IL) 1, 6 and 8 and eicosanoids, such as prostaglandin E2 (PGE2)), nitric oxide, metalloproteins from the extracellular matrix and other mediators7. One such chemical mediatoris leukotriene B4 (LTB4), a potent chemoattractant involved in recruiting more neutrophils and leukocytes to the initially damaged area2.

2.2. Resolution of inflammation

However, this idea changed with the identification of specialised pro-resolving mediators (SPMs), whose discovery has gone on to show that resolution is not a passive process, as originally thought, but a more active process, one that is programmed and regulated by tissues6,8.

Serhan et al. have shown that, along with previously known processes, the initial phase in the inflammatory response also programmes when signalling pathway are to be activated to bring about the normal spontaneous conclusion, or resolution, of local inflammation3, as explained below.

3. Specialised pro-resolving lipid mediators (SPMs)

During the inflammatory response, biosynthesis of PUFA-derived lipid mediators increases over time. The initial mediators (prostaglandins (PGs) and leukotrienes (LTs)) are produced within seconds or minutes and regulate oedema and postcapillary events at the site after neutrophil recruitment. Over time (hours or days),leukocyte recruitment increases due to the appearance of monocytes and macrophages, thereby increasing the amounts of mediators involving these cells. This means that, initially, mostly proinflammatory mediators are produced, while SPMs appear later on, promoting resolution3,9 (Fig. 2).

Representatives of each family of the new lipid mediators (discussed in more detail below) act at different stages of resolution. Each of these compounds is anti-inflammatory when administered in vivo and can shorten the time required for resolution and recovery of tissue homeostasis. Resolution of acute inflammation specifically involves a staggered, transitory mobilisation of different fatty acid precursors in the exudate, such as AA, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). In turn, each precursor is transformed into independent families of bioactive compounds which act as local chemical mediators. This has major implications for the programming and circuit system of lipid mediators in resolution2.

4. Actions of the SPMs, resolution agonists

As the protective inflammatory response develops, this enables damaged tissues to be repaired and invasive organisms eliminated. After a microbial infection or tissue injury, proinflammatory lipid mediators (such asLTs and PGs) are released and this initiates a series of signal cascades in order to destroy pathogens and repair damaged tissue3. Ideally, the process is self-limiting and promotes complete resolution and the return to homeostasis. A more detailed description of the sequence of pro-resolving events would be: arrest of neutrophilinfiltration; switching off of proinflammatory pathways (eicosanoids and cytokines), phagocytosis, efferocytosisand return to homeostasis, with the regeneration of the damaged tissue.

At the cellular level, the main resolution events involving SPMs are:

4.1. Arrest of neutrophil infiltration

SPMs stimulate resolution of acute inflammation by halting the excessive arrival of neutrophils in the inflamedtissues10. The effect is to limit neutrophil-mediated tissue injury1.

4.2. Efferocytosis

Stimulation of efferocytosis6, with the intervention of mediators such as maresins (MaR), by pro-resolving macrophages is another distinctive event in the action of SPMs compared to traditional anti-inflammatories, as it permits the elimination of cell detritus, apoptotic neutrophils and pathogens. This action by pro-resolving macrophages is essential for resolving inflammation and preventing it from becoming chronic10.

4.3. Lipid mediator class switching (Fig. 3)

  • Class switch: As exudate develops, the PG- and LT-producing signalling pathways then switch on the active production of the processes that transcribe enzymes required for the genesis of another class ofeicosanoid, also generated from AA: lipoxins (LXs)3 (see the section ‘SPM families’). LXs actively promote resolution of inflammation by regulating the entry of new neutrophils into inflamed areas and organs with reperfusion injury; reducing vascular permeability, while stimulating monocyte infiltration, which seems to be essential for wounds to heal; and stimulating macrophage uptake of apoptotic neutrophils.This lipid mediator class switching in the eicosanoid family, from proinflammatory to anti-inflammatory eicosanoids (i.e. PG and LT to LX), is an active process and one that highlights how leukocytes can trigger a spontaneous resolution response to acute inflammation2. This is especially important, as it represents a crucial change in mediator profile and the onset of resolution, preventing inflammation from perpetuating, thereby causing chronic conditions and diseases.
  • Substrate switch: During the course of the acute inflammatory response, mediators switch not only class but also substrate to form new families of chemical mediators. AA-derived mediators vary towardsuse of ω-3 PUFA in this process to generate resolvins (Rv) and protectins (PD)2 (see the section ‘SPMfamilies’).

4.4. Return to tissue homeostasis

As explained above, although the inflammatory response protects the host, acute inflammation needs to be completely resolved and tissue returned to homeostasis if progression to chronic and systemic inflammationis to be avoided1-3.

This means that SPMs act as inflammation resolution agonists, controlling the duration and magnitude of acute inflammation11.

Inflammation has to be actively regulated to bring about its resolution and conserve tissue (and, consequently, organ) homeostasis12.

5. Resolution vs. anti-inflammation

Pharmacological treatment for inflammation with anti-inflammatories can impede the inflammation resolution process because it usually involves inhibiting the enzymes involved in producing proinflammatory mediators; however, these same enzymes are also required for the synthesis of SPMs, which are responsible for resolution of inflammation. Anti-inflammatory therapy can block or inhibit proinflammatory mediators, but this is not the same as resolution. Indeed, without inflammation there is no resolution. Cyclooxygenase (COX) inhibitors prevent PG biosynthesis but prolong the interval to resolution, so tissue remains inflamed for longer. A pro-resolving action involves agonist action via target cells and/or critical events which favour resolution andthe return of tissue to homeostasis13.

For instance, after irregular resistance exercise, synthesis of proinflammatory PG and LT peaks about 1-2hours later; in addition, levels of the pro-resolving mediators LXA4, LXB4, RvE1 and RvD1 rise during thefirst 3 hours, followed 24 hours later by 15-HETE and a PD1 isomer. Ibuprofen blocks PG responses to exercise, but also inhibits COX-1 and COX-2, thereby reducing the pro-resolving lipid mediator response as well.This means that anti-inflammatory drugs could delay and/or reduce the natural resolution of the inflammatory response14.

Anti-inflammatories have an antagonistic effect (on enzymes or receptors), while SPMs are agonists and stimulate receptors in different cell populations15.

Furthermore, SPMs are not immunosuppressants, unlike many currently used anti-inflammatory drugs1.

Recognising that resolution of inflammation is proactive opens up alternative therapeutic paradigms based on resolution of the acute phase of inflammation and preventing the onset of chronic inflammation3.

5.1 The inflammation resolution mechanism and aspirin

As shown by Serhan et al. over the last two decades, the mechanism for the resolution of inflammation also explains how aspirin works. Aspirin has a unique characteristic with regard to inflammation: it boosts resolution by contributing to the synthesis of forms of ‘aspirin-triggered’ (AT) SPMs2,13. Different series of pro-resolving lipid mediators are generated, depending on the substrate and whether aspirin is present. Biochemical synthesisof these compounds is enzymatically mediated and produces stereospecific isomers (stereoisomers) that interact with highly selective receptors4.

The LXs and their ‘AT’ forms are generated from AA, while LX is biosynthesised via interactions between5-lipooxygenase (5-LOX) and 12-LOX or 15-LOX. Another very similar form, 15-epimer-LX, is generated through interactions between 5-LOX and aspirin-acetylated COX-2 (COX-2 expression increases during acute inflammation). COX-2 converts AA into 15(R)-HETE instead of PG. 15(R)-HETE serves as a substratefor LOX-2 in its transformation into 15(R)-LXA4. It is worth pointing out that 15(R)-LX is approximatelytwice as potent as 15(S)-LX4.

Similarly, Rvs, PDs and various AT epimers (e.g. the AT form of RvD1, AT-RvD1) are derived from the acids5Z,8Z,11Z,14Z,17Z- EPA and 4Z,7Z,10Z,13Z,16Z,19Z-DHA4. It should be stressed that Rvs and PDs arecompletely different from EPA and DHA oxidation products, which are not specific and may also be present during inflammation4.

The result is that aspirin favours the resolution process.

6. SPM families

While studying the acute inflammatory response in mouse exudate to try and find a link between ω-3 PUFA and endogenous anti-inflammation, Serhan et al. (2000) identified a series of novel oxygenated products formed enzymatically from the main PUFAs (Fig. 4) that serve as precursors to compounds that have potent actions for resolving inflammatory exudate2.

SPMs are generated through complex metabolic pathways that involve a variety of cell types, a process known as transcellular synthesis. This process is defined as the generation of new bioactive compounds that the celltypes involved are incapable of producing on their own. For instance, human platelets alone cannot produce LX, but when they adhere to neutrophils, the association provides an important intravascular source of LX,which in turn arrests neutrophil diapedesis and recruitment13.

The different SPMs do not have equivalent anti-inflammatory and pro-resolving actions. Each SPM has exclusive structure and functions, acting at different moments in the resolution process1,16.

6.1. Enzymatic oxygenation of arachidonic acid (AA)

This process generates both proinflammatory mediators and pro-resolving lipid mediators.

6.1.1. Lipoxins (LX)

LXs were the first SPMs to be identified. They are synthesised from AA when leukocytes interact with mucouscells (the gastrointestinal tract epithelium or bronchial tissue) and in blood vessels when leukocytes interact with platelets15. Their in vivo actions are listed in Table 1.

6.2. Enzymatic oxygenation of ω-3 PUFA

This process generates both proinflammatory mediators and pro-resolving lipid mediators.

This process generates (Fig. 5)3:

  • E-series resolvins from EPA: RvE1 and RvE2
  • D-series resolvins from DHA: RvD1, RvD2, RvD3, RvD4, RvD5, RvD6
  • Protectins (or neuroprotectins) from DHA: PD1/NPD1
  • Maresins, from DHA: MaR1

Not only have the Rvs, PDs and MaRs been identified, but so have peptide conjugates of these SPMs that favour the elimination of bacteria and tissue regeneration. The terms used to name these series are: maresin conjugates in tissue regeneration (MCTR); resolvin conjugates in tissue regeneration (RCTR); and protectin conjugates in tissue regeneration (PCTR). Their complex structures and potent actions highlight the importance of the intermediate steps in SPM biosynthesis and function1.

6.2.1. Resolvins

Rvs were first isolated from the inflammatory exudate of mice that had been treated with aspirin and EPA/ DHA17 and were also generated in vitro by co-incubating human epithelial cells and neutrophils4. The biosynthesis of resolvins starts in the endothelial cells. They are synthesised from EPA (E-series, RvE) and DHA (D-series RvD). As with LXs, they are also produced by the COX-2 pathway in the presence of aspirin-triggered forms. Rvs have potent anti-inflammatory and immuno regulatory effects that include blocking production of proinflammatory mediators (chemokines and cytokines) and leukocyte traffic to the infection site and the elimination of neutrophils from mucous surfaces. They act by limiting neutrophil transendothelial migration in vitro and infiltration in vivo, but they also favour proinflammatory chemokine clearance, recruitment and phagocytosis of monocytes and lymphatic phagocyte elimination17.

E-series resolvins (Fig. 6)

RvE1 forms spontaneously and its levels rise in healthy subjects who take aspirin and/or EPA4.

In an experimental periodontitis model (with rabbits) administration of RvE1 led to the complete recovery of damaged tissues, including bone, and normalisation of inflammation markers, including C-reactive proteinand IL-1β4.

RvE1 and LXA4 prevent pathological inflammation, but also regulate physiological inflammation that is beneficial to the host, as in wound healing4.

RvE2 is synthesised by human neutrophils in larger quantities than RvE14.

D-series resolvins (Fig. 7)

D-series resolvins were also originally isolated from the inflammatory exudate of mice, who in this case had been treated with aspirin and DHA4.

Resolvins as immune system regulators

Resolvin target cells18:

  • Neutrophils. Neutrophils are the first line of defence, migrating to infection or injury sites; transendothelial migration is a pivotal event in neutrophil recruitment. RvE1 inhibits LTB4-stimulated neutrophil migration. Rvs also favour the actions of neutrophils: RvE1 favours neutrophil phagocytosis of Candida albicans while RvE2 also favours neutrophil phagocytosis of Escherichia coli. These actions are important for limiting the invasion of pathogens and resolving inflammation.
  • Dendritic cells. Dendritic cells play an essential role in innate immunity and the onset of acquired immunity. In their immature state, they are distributed in tissues in contact with the exterior (such as mucous surfaces or skin). During inflammation, they migrate to inflamed peripheral tissues, where they capture antigens. After maturing, they migrate to the lymph nodes, where they activate naive T-cells and provide the cytokines required for their proliferation and differentiation. RvE1 can block these latter processes (dendritic cells exposed to RvE1 and pathogens remain at the inflammation site instead of migrating to the lymph nodes) and also favour T-cell apoptosis.
  • Macrophages. The key histological event in tissue resolution is eliminating apoptotic neutrophils and tissue detritus. RvE1 helps resolve inflammation by activating macrophages to eliminate apoptotic cells and detritus from the infection sites and RvD2 stimulates resolution by activating macrophages to eliminate bacteria.
  • Platelets. Platelets play an important role in blood clotting, wound healing and inflammation. Platelet- leukocyte and platelet-endothelial cell interactions represent the connection between thrombosis and inflammation. RvE1 blocks platelet aggregation, except when this is induced by collagen; this suggests that they act to block excessive platelet aggregation but not collagen-induced physiological coagulation.
  • Leukocyte-endothelial cell interactions. Leukocyte recruitment is a key element in acute inflammation. Rvs modulate the expression of adhesion molecules in leukocytes and the production of anti-adhesive mediators by endothelial cells.

6.2.2. Protectins (Fig. 8.)

In humans, PDs are synthesised in mononuclear peripheral blood cells4 and glial cells15. They are much more potent than DHA and show specific tissue bioactivity4. They reduce cytokine expression and, like the Rvs, also stop neutrophil infiltration. In addition, they reduce T-cell migration and TNF and interferon-γ signalling and promote T-cell apoptosis4.

In Alzheimer’s disease, DHA, PD1/NPD1 and 15-LOX levels in the hippocampus are selectively lowered.This would suggest a possible mechanism for the drop in neuroprotection associated with the disorder, i.e. less inhibition of apoptosis and the subsequent increase in neuronal death4.

Retinal pigment epithelial cells generate PD1/NPD1 in the presence of oxidative stress, permitting up-regulationof anti-apoptotic proteins, down-regulation of pro-apoptotic proteins and A2E-mediated apoptosis oftoxic metabolites, all of which protects them from apoptosis induced by oxidative stress and cell ageing4.

In an experimental asthma model, PD1/NPD1 administration reduced the response to allergenic stimuli (hyperreactivityof the airways and eosinophil- and T-cell-mediated inflammation)4.

6.2.3. Maresins (Fig. 9)

MaRs are produced by macrophages from DHA and have potent pro-resolving and tissue homeostasis actions. Maresin 1 (MaR1) was the first to be identified6.

Table 2 and Figure 10 show the known actions of resolvins, protectins and maresins.

7. Identification of resolvins and other SPMs in human tissues

Newly developed identification methods have found Rv and other SPMs in human tissues (Fig. 11) from healthy subjects, in human diseases16 and in breast milk19 (the high content of pro-resolving and anti-inflammatorymediators and their precursors suggests they play an important role in the immunity of newborns, which is a possible reason why breast milk is superior to infant formulas), but also in planarians, (marineflatworms), tunicates (tubular invertebrates found on the sea bed), frogs, mice16, trout and salmon1. Thisshows that evolution has conserved the SPMs, the structures that control potent bioactions in the mainsystems and organs1.

8. SPMs vs. long-chain omega-3 polyunsaturated fatty acids

Unlike EPA and DHA, ω-3 PUFA-derived SPMs can produce biological activity at nanomolar concentrations12 and have specific receptors that are not activated by their omega-3 precursors.

9. SPM receptors

The selectivity and specificity of the pro-resolution system could also be due to that fact that each SPM has its own receptors, known as specific G-protein coupled receptors (GPCR), which produce rapid intracellular signalling and long-term actions1. SPMs exert their potent actions by activating GPCRs9. Because these GPCR samplify intracellular signals, low doses of SPMs are enough for them to stop inflammation and promote resolution6.

In humans, some such specific receptors have been found in neutrophils, monocytes, T-cells, macrophages, synovial cells, fibroblasts and intestinal epithelial cells, and their mRNA is also found in the spleen, lung, placentaand liver6.

10. SPMs as resolution biomarkers

SPMs have great potential as biomarkers:

Measuring their levels in patients with infections could provide information on the status of inflammation resolution which in turn would help stratify patients and assess the efficacy of treatments16.

In patients with cystic fibrosis, resolvin D1 (RvD1) concentrations in plasma and sputum can be used as peripheral biomarkers for the degree of resolution of inflammation in the lungs20.

11. Chronic inflammation and disease

There is an obvious association between pathological inflammatory response and inflammatory diseases, but now we also know that it is involved in other disorders7. Some of the most difficult diseases to treat are associate dwith excessive, uncontrolled or chronic inflammation (this relationship was previously unknown), including: cardiovascular disease, rheumatoid arthritis, gum disease, asthma, diabetes, inflammatory bowel disease, cancer, age-related macular degeneration and Alzheimer’s disease. Although the involvement of inflammation in the onset of these diseases is well established, we do not fully know how it specifically contributes to theirpathogenesis1-3.

12. SPMs in different disorders

The identification of SPMs has opened up new paradigms in understanding and therapeutic possibilities for diseases such as cardiovascular disease, rheumatoid arthritis, gum disease, asthma, diabetes, inflammatory bowel disease, age-related macular degeneration, Alzheimer’s disease3 and cancer1.

12.1. Allergies

Allergies are a reaction of the immune system to substances that are harmless to most other people. Normally,the immune system combats germs, but in most allergic reactions it responds to a false alarm. People with allergies tend to be sensitive to more than one thing and substances that commonly cause reactions are pollen, dust mites, mould spores, animal dander, certain foods, some insect bites and certain drugs.

SPMs generated from EPA and DHA counter-regulate eosinophilic inflammation of the airways and favour resolution of inflammation. A number of epidemiological and observational studies suggest consumption offish (a source of EPA and DHA) during pregnancy and childhood has a protective effect, preventing and reducing allergy rates and atopic disorders (asthma, wheezing, eczema, hay fever, sensitisation to certain food sand positive skin tests) later on in children’s lives. We can now explain these effects at the molecular level, and SPMs play a key role21.

12.2. Coronary artery disease

In patients with coronary artery disease, levels of certain SPMs (RvD1, RvD2, RvD3, RvD5 and RvE1) are lowor non-existent compared to healthy subjects, which could favour the progression of chronic vascular inflammationand a predisposition to coronary atherosclerosis and thrombosis. Supplementation with certain SPM sincreases their plasma concentration10.

12.3. Rheumatoid arthritis and osteoarthritis

Uncontrolled inflammation produces tissue fibrosis and damage. A good example of tissue damage caused byexcessive leukocyte build-up is arthritis (in patients with rheumatoid arthritis, the number of neutrophils inthe synovial fluid exceeds one billion/day22 and the MaR metabolic pathway has also been identified in thefluid23). Therefore, mechanisms that stop neutrophil infiltration in inflammation sites and efferocytosis are very important17. Specific SPMs shorten the resolution of inflammation interval, limiting neutrophil recruitmentand stimulating efferocytosis by macrophages and elimination of bacteria, all of which are required for tissue repair and regeneration6.

In addition, interrupting class switching has also been observed to have harmful consequences on mouse models of arthritis6.

Two mouse arthritis models have confirmed the anti-inflammatory and protective roles of tissues from the12/15-LOX and LXA4 axis in regulating TNFα. This is also the initial LOX in the synthesis of D-series Rvand PD11.

In murine arthritis, RvD1 and 17-HDHA reduce pain and tissue damage and have proved more potent than steroids or analgesic treatment9. In a mouse model of rheumatoid arthritis, administration of 100 ng/day of17R-RvD1 significantly reduced the severity of the arthritis, cachexia, oedema in the hind legs and leukocyte infiltration, while also shortening the remission interval. The metabolic lipidomic profile of arthritic joints showed that 17R-RvD1 significantly reduced PGE2 synthesis and increased SPM levels, while molecular analyses indicated that 17R-RvD1 favoured expression of the genes associated with cartilage matrix synthesis24.

12.4. Asthma

Asthma is a chronic inflammatory disease of the airways. It is characterised by infiltration of eosinophils andT-cells that produce cytokines and lipid mediators which, in turn contribute to the pathogenesis of the disease. In a mouse asthma model, intravenous administration of RvE1 shortened the resolution interval (i.e. the timetaken for inflammatory cells to drop by 50% from peak inflammation), lowered production of proinflammatory cytokines and improved the mucosity and response hypercapacity of the airways, both of which are typical symptoms of asthma18.

In humans, while PD1 and 17-HDHA have been identified in exhaled breath of healthy individuals, they have only been found at trace levels in subjects with clinical asthma exacerbation. An LXA4 deficiency has also been observed in the latter25.

Lipidomic analyses have revealed dysregulation of the 15-LOX pathway in the eosinophils of patients with severe asthma: a surprisingly large drop in PD1 biosynthesis has been observed, while levels of 5-HETE(5-LOX-dependent AA metabolite) are similar to those of healthy subjects, suggesting that the 15-LOX pathwayis selectively dysregulated (Fig. 12)21

AT-RvD1 has been shown to have anti-inflammatory effects in human bronchial epithelial cells stimulated with lipopolysaccharides or Dermatophagoides pteronyssinus (found in domestic dust mites, it is one of the mostimportant allergens, along with lipopolysaccharides), which opens up new strategic therapeutic possibilities forcontrolling airway inflammation26.

12.5. Cancer

As long ago as 1860, Virchow noted the relationship between cancer and inflammation, after he observed inflammatory cells in biopsies of tumour tissues. Inflammatory stimuli (chronic infections, inhalation of pollutants,smoking and obesity) are known to harm DNA and cause somatic mutations and carcinogenesis.Around the tumour, inflammatory cells speed up progression of the cancer, metastasis and immune responsesto radiotherapy, chemotherapy and immunotherapy. Thus, targeting this area represents a reasonable line totake in the treatment of cancer. Already, endogenous SPMs have been applied to various cancer models, with promising results27.

In animal models, the observed anti-cancer actions of SPMs are targeted directly at the tumour cells (e.g.inhibiting proliferation and migration), the area around the tumour (e.g. inhibiting angiogenesis) and pre-cancerous lesions (e.g. favouring resolution of colitis)27.

The growth of tumours is closely related to inflammation, so therapeutic objectives which target the inflammatory environment of the tumour offer a potential new direction for cancer research27.

12.6. Eczema

In paediatric patients with eczema, an LXA4 derivative improved the severity of symptoms with the same efficacy as a topical steroid; patients’ quality of life also improved without causing secondary effects9.

12.7. Alzheimer’s disease

Alzheimer’s disease is associated with low levels of PD1/NPD1 and other SPMs (LXA4 and RvD1 in the cerebrospinalfluid and the hippocampus, correlating to Mini-Mental State Examination scores of these patients)9.

MaR1 (which favours neuronal survival and elimination of Aβ42 from the microglia and limits inflammation of the microglia) is lower in patients with Alzheimer’s disease, suggesting it plays a role in the pathogenesis of the disease. Consequently, correcting its deficiency through SPM supplementation could be a promising therapeutic strategy5.

12.8. Inflammatory bowel disease

Inflammatory bowel diseases are characterised by an abnormal mucosa response to normally non-pathogenicbacteria, inflammation of the colon associated with loss of barrier function, leukocytosis and proinflammatorygene expression. Prior intraperitoneal administration of RvE1 increased survival rates, reduced weight lossand improved histology, while also reducing the neutrophil count in mucosa tissues and proinflammatory geneexpression18.

12.9. Gum disease

Periodontitis is a local inflammation characterised by neutrophil-mediated tissue injury, followed by the developmentof chronic injury. As with other osteolytic inflammatory diseases (such as arthritis), current tissueengineering procedures are limited. There is a critical need for new regenerative therapies, as current ones areunable to control inflammation during regeneration. In rabbit periodontitis models, inflammation resolutionagonists (including LXA4 and RvE1) prevent bone loss and facilitate periodontal regeneration28.

In rabbits with P. gingivalis-induced periodontitis, topical application of RvE1 reduced neutrophil infiltration, tissue damage and osteoclast production; it also restored lost bone and improved the clinical parameters of gum disease18.

In Hanford miniature swine with induced periodontitis, administration of a lipoxin analogue (bLXA4) dramatically reduced inflammatory cell infiltration and increased lost connective tissue regeneration and new bone formation28.

12.10. Chronic obstructive pulmonary disease

In chronic obstructive pulmonary disease (COPD), pro-resolution signalling and the metabolic pathways inpulmonary tissue are negatively affected, suggesting that SPM supplementation could reduce the development of emphysema by controlling chronic inflammation. Corticosteroids, which are habitually used to control acute bouts of COPD, block the production of proinflammatory mediators, which could favour the inflammatory disease. In a mouse model, RvD1 administration was associated with a reduction in emphysema caused by tobacco smoke, along with reductions in inflammation, oxidative stress and cellular death29.

12.11. Multiple sclerosis

Multiple sclerosis is a progressive disease of the central nervous system that causes multiple lesions in the formof disseminated plaques to the myelin that coats neuron axons and constitutes white matter. It produces a variety of symptoms, including paralysis of the lower limbs, tingling and loss of sensitivity, among others. Multiple sclerosis is one of many diseases involving chronic inflammation30. Mass spectrometry has detected low SPM levels among these patients1,9.

12.12. Cystic fibrosis

Cystic fibrosis is a hereditary disease caused by deficient function of the exocrine glands. It is characterised by signs of chronic pulmonary disease and pancreatic dysfunction.

Recent studies suggest that patients with cystic fibrosis have a lower capacity to synthesise SPMs, contributing to the development and duration of inflammation20. Progressive lung disease is the main cause of morbidity and mortality in patients with cystic fibrosis. Their lungs suffer a high pathogenic bacteria load which leads tochronic neutrophil infiltration and the release of neutrophil elastase and other harmful products. Abnormalities in innate immune system responses, producing chronic inflammation and contributing to the pathogenesis of the disease, have also been identified. These abnormalities lead to lower production of anti-inflammatory lipid mediators and an increase in proinflammatory chemokines. Consequently, using SPMs to treat the diseaseis a promising possibility20.

Lung function is better in cystic fibrosis patients with higher concentrations of RvD1 in the sputum16 (thusRvD1 titres can be used as biomarkers for the resolution status of these patients)20 and there is a correlation between RvD1 levels and biomarkers such as IL-1β and IL-8 in sputum20.

12.13. Infections

SPMs are a potential therapeutic agent against a variety of pathogens (an extremely interesting possibility, given the rise of resistance). They can reprogramme the immune response and also seem to have additive effects when administered together with currently used drugs, including antibiotics, where they have permitted an experimental reduction in dose. This all suggests that new therapies for infections based on resolution couldbe developed16.

 

In the experimental situations listed below, a relationship has been observed between the degree of infection, or its results, and the presence of different SPMs. In some cases their use as therapeutic agents has also been studied, with promising results:

  • Peritoneal infections due to E. coli16.
  • Airway infections due to bacteria (E. coli, Pseudomonas aeruginosa, Streptococcus pneumoniae, nontypeable Haemophilus influenza, Cryptococcus neoformans) and viruses (respiratory syncytial virus, flu virus)16.
  • Skin and eye infections due to bacteria (Staphylococcus aureus, P. aeruginosa) and virus (herpes simplexvirus 1)16.
  • Topical RvE1 reduces the severity of gum disease in rabbits, favouring elimination of Porphyromonas gingivalis9.
  • Parasitical gastrointestinal tract infections (Toxoplasma gondii, Schistosoma japonicum)16.
  • Parasitical infections of the central nervous system (cerebral malaria initiated by Plasmodium infection)16.
  • Fungal infections (Candida)9.

In patients with tuberculosis, production of RvD is up-regulated and circulating levels of LXA4 are higher inactive infections compared to patients with latent infections16; in patients with sepsis, the lipid mediator profilecorrelates with the results16; and a relationship has also been observed between SPM levels and results in patientswith cystic fibrosis (in whom P. aeruginosa infections are a major cause of death)16,20.

12.14. Acute ischaemic kidney injury

Ischaemic kidney injury is characterised by tubular epithelial injury and inflammation. Renal ischaemia followed by reperfusion triggered endogenous RvD1 formation in kidney tissue and exogenous administration of DHA favoured a reduction in neutrophil infiltration in the kidney and plasma creatinine concentrations; it also limited interstitial collagen deposition, providing protection from fibrosis18.

12.15. Aspiration pneumonia

Aspiration of stomach content causes inflammation of the airway parenchyma and a neutrophil-dependent injury in the lungs, also creating a predisposition to bacterial infection as the mucosal defence mechanisms are interrupted. In an experimental model, intravenous RvE1 administered before inducing aspiration and E. coliinfection reduced the build-up of neutrophils in the lungs by 55% and favoured the elimination of E. coli18.

12.16. Neuroinflammation and pain

In ischaemic stroke in mice, Rvs, PDs and their aspirin-triggered forms (endogenous and synthetic) down-regulate excess leukocyte infiltration and reduce local neuronal injury and COX-2, IL-1β and NFkB induction. Giventhat DHA is a precursor it could therefore be a potential protector9.

Inflammation can cause persistent pain. Synthetic LXs can reduce pain in mouse models and each SPMhas a pain-reducing effect, exerted by acting on different targets (as has been shown for RvE1 and RvD1 incentral and peripheral inflammatory pain). Administered intrathecally, RvE1 is a more potent analgesic thanmorphine or a COX-2 inhibitor. MaR1 reduces neuropathic pain caused by chemotherapy in mice and otherSPMs reduce pain caused by induced arthritis9.

Systemic prophylaxis with aspirin-triggered RvD1 (AT-RvD1) reduced post-surgery cognitive deterioration in a mouse model, protecting against neuronal dysfunction after the operation. Given that cognitive decline after major surgery is an important healthcare problem, transferring this action of AT-RvD1 to humans is a very promising option9.

12.17. Obesity

MaR1 promotes resolution of inflammation in the adipose tissue of diet-induced obese (DIO) mice, showing a reduction in macrophage infiltration and expression of inflammation markers, together with an increase in adiponectin expression31.

White adipose tissue plays an important endocrinal role in balancing metabolic homeostasis. The low-grade, yet chronic, inflammation that characterises obesity is associated with the development of insulin resistance, type 2 diabetes and nonalcoholic fatty liver disease. Recent data suggest that persistent inflammation in the adipose tissue of obese subjects is probably due to a failure in the resolution capacity of the tissue. This is probably the result of an intrinsic in capacity of the adipose tissue in obese subjects to generate adequate SPMsor due to an imbalance in proinflammatory and pro-resolving mediator levels (Fig. 13). Therefore, strategies that increase local SPM concentrations, for instance by exogenously administrating SPMs, have significant potential in the treatment of this condition and could prevent the associated metabolic disorders (with the added advantage over conventional anti-inflammatory drugs that they do not affect the host’s immune systemor cause secondary effects)32.

In obese women, three months’ supplementation with ω-3 PUFA was associated with anti-inflammatory andpro-resolving responses: a drop in the concentration of various cytokines, adhesion molecules and proinflammatory proteins and an increase in pro-resolving mediator levels. This correlated to high expression of the ALOX5 gene, which encodes the enzyme that produces numerous pro-resolving mediators. This suggests thatω-3 PUFA could be useful in low-grade obesity-associated inflammation33.

Differences in the profiles of SPMs (including RvD1, RvD2, PD 1, LXA4 and various biosynthetic pathway markers for RvD1 and PD1(17-HDHA), RvE1 (18-HEPE) and MaR1 (14-HDHA)) and ‘classic eicosanoids’(PGE2, PGD2, PGF2α, LTB4, 5-HETE, 12-HETE and 15-HETE) have been found between the subcutaneous adipose tissue of patients with terminal peripheral vascular disease and control subjects. Furthermore, individuals with peripheral vascular disease show a marked deficiency in PD1 and 17-HDHA levels in their subcutaneous adipose tissue, whereas they have high levels of SPMs in their perivascular adipose tissue, suggesting a greater capacity for resolution in these fatty deposits. Furthermore, eicosanoid and SPM levels are also higher in subcutaneous adipose tissue surrounding wounds on the feet 32.

12.18. Peritonitis

In an experimental peritonitis model, both RvE1 and RvE2 reduced neutrophil infiltration and RvE1 favoured leukocyte clearance18.

12.19. Organ regeneration and wound healing

Exogenous LXA4 stimulates re-epithelialisation of the cornea and exogenous RvE1, RvD1 and RvD2 stimulate the healing of skin wounds, reducing neutrophil infiltration and stimulating re-epithelialisation when applied topically to such wounds. Exogenous RvD1 and RvD2 also stimulate healing of diabetic wounds9.

In diabetes, the complex programmed wound healing process is affected. One of the most clinically important manifestations of diabetes is deficient wound healing (accompanied by tissue necrosis and infection). Due toperipheral nephropathies and macrovascular and microvascular disease, diabetics often have wounds on their extremities. Insufficient blood supply increases susceptibility to wounds and also hinders healing. Diabetic wounds are usually characterised by an excessive build-up of leukocytes, indicating that dysfunction in the secells and in the action of phagocytes could be a key factor in the slow healing of diabetic wounds. In mouse models, local application of RvD1 significantly reduces apoptotic cells and macrophages, leading to improved wound closure and the formation of granulation tissue34.

Studies with diabetic mice, used as a model for delayed wound healing in humans, have shown that treatment with RvD1 increases the wound healing rate and favours the formation of granulation tissue (in the context ofdiabetes, wound healing time is critical if secondary infection is to be prevented). RvD2 prevents tissue necrosis in mouse burn models. It prevents thrombosis and neutrophil sequestration and favours microvascular accessto the dermis in the healing process. Furthermore, MaR1 may also play a key role in the tissue regeneration process: it directly stimulates tissue regeneration in surgical wounds of the brown planarian and is biosynthesised during the tissue regeneration process (Fig. 14)25,34.

In diabetic wounds of obese diabetic mice (a model that permits time-dependent analysis of leukocyte infiltration, apoptosis and phagocyte elimination), DHA to RvD1 conversion markers were attenuated and local application of RvD1 accelerated closure and reduced the build-up of apoptotic cells and macrophages inthe wounds. Type 2 diabetes alters the resolution of inflammation, but it seems that this alteration can be corrected by RvD1, which also restores deficient macrophage phagocytosis (which could reduce build-up ofapoptotic/necrotic cells and microbes in chronically inflamed tissues)35. Thus, RvD1 could be used to improve wound healing in diabetic patients36.

12.20. Dry eye syndrome

Topical application of Rv1 has been shown to reduce inflammation in a mouse model of dry eye syndrome. Furthermore, DHA in conjunction with a pigment epithelium-derived factor speeds up corneal nerve regenerationafter damage caused by cornea surgery (an approach that could be transferred to patients with neurotrophickeratitis and dry eye after refractive surgery), helps restore sensitivity and reduces signs of dry eyeand the rose bengal-stained area. The corneal epithelium expresses 15-LOX, which converts DHA into PD1/NPD1; thus logically this could be related to its beneficial effect on dry eye35.

In another mouse model of dry eye (induced by systemic scopolamine and exposure to an air current and low humidity 16 h/day for 5 days), topical administration of RvE1 improved the stained corneal areas by 80% compared to controls and maintained goblet cell density, compared to a 20% reduction in the control group37.In a phase 2 clinical trial, subjects with dry eye syndrome who took a synthetic RvE1 analogue experienced statistically significant dose-dependent improvements in the signs of dry eye compared to placebo. The compound was well tolerated when applied topically35.

12.21. Metabolic syndrome

Metabolic syndrome is the term given to the presence of three or more of a group of conditions that place the individual at risk of developing heart disease and type 2 diabetes: arterial hypertension, high blood sugar, high triglyceride blood levels, low HDL blood levels and excess fat around the waist.

Recent evidence shows that failure in resolution and SPM biosynthesis contributes to chronic inflammation in the context of excessive nutrients and that resolution agonists such as SPMs improve clinically relevant aspectsin metabolic syndrome34.

Immune system effector cells (including neutrophils and macrophages) are sensitive to changes in nutrition and a dysregulated systemic metabolism leads to their chronic activation and inflammatory signalling. Furthermore, inflammatory signalling also occurs in non-immune cells (such as endothelial cells and adipocytes) during periods of nutrient stress. Chronic inflammation is associated with diseases that involve systemic metabolism alterations, such as obesity, diabetes and atherosclerosis. Thus, while persistent metabolic dysfunction leads to inflammation, the latter can in turn affect metabolic homeostasis34.

The breakdown in resolution of inflammation seems to be related to hypercholesterolemia and high levels ofnon-esterified fatty acids and triglycerides, which are all related to metabolic syndrome34. Furthermore, low serum levels of LXA4 and higher amounts of abdominal visceral fat are independent predictors for the risk of developing the syndrome32. Resolution agonists (e.g. Rvs) could help break the vicious circle between these disorders and chronic inflammation.

13. Supplementation with SPMs

Supplementation with SPMs improves the body’s natural capacity to resolve inflammation when it has beencompromised by illness, environmental factors, ageing and a diet low in ω-3 PUFA.

13.1. Preserving and increasing SPM fractions in natural oils containing ω-3 LC-PUFA40

Advanced technology is used to produce standardised concentration levels of SPMs (and their precursors) infish oil from anchovies, sardines, tuna and mackerel from the Pacific and Atlantic Oceans. These SPMs remainstable for 24 months under normal storage conditions.

13.1.1. Process

The separation of crude, refined or concentrated oil containing ω-3 LC-PUFA into a range of fractions meansthe most suitable ones can then be selected to produce oils containing higher concentrations of at least oneSPM or SPM precursor or a given combination of SPMs or their precursors.

Separation and extraction methods are used to fraction the oils, as the quantities of SPMs and their precursorsin natural oils differ depending on their source. Different methods produce different compositions of SPMsand their precursors from the variety of oils used to make the end product. The most effective methods forobtaining oils rich in SPMs and their precursors are supercritical fluid extraction (SFE) and supercritical fluidchromatography (SFC), using carbon dioxide as a solvent.

13.1.2. Quantifying SPMs and their precursors

The suitability of fish oil for fractioning, with the aim of obtaining the desired combination, can be assessedor calibrated by measuring the presence of SPM precursors.The usefulness of the fractions can be established by determining anti-inflammatory or resolution-stimulating activity in those that contain higher concentrations of SPM precursors.

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