Lipoxin

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A lipoxin (LX or Lx), an acronym for lipoxygenase interaction product, is a bioactive autacoid metabolite of arachidonic acid made by various cell types. They are categorized as nonclassic eicosanoids and members of the specialized pro-resolving mediator (SPM) family of polyunsaturated fatty acid (PUFA) metabolites. Like other SPMs, LXs form during an inflammatory response and act to resolve it. The first lipoxins identified were lipoxin A₄ (LXA₄) and lipoxin B₄ (LXB₄), followed by their respective epimers, the epi-lipoxins 15-epi-LXA₄ and 15-epi-LXB₄.

History

LXA₄ and LXB₄ were first described by Charles Serhan, Mats Hamberg, and Bengt Samuelsson in 1984. They reported that human blood neutrophils, when stimulated, make these two lipoxins and that neutrophils, when stimulated by either of the LXs, mounted superoxide anion (O₂^&minus;) generation and degranulation responses. Both responses are considered to be pro-inflammatory in that, while aimed at neutralizing invading pathogens and digesting foreign material, can contribute to damaging host tissues and thereby prolonging and promoting further inflammation. Subsequent studies, however, found that these lipoxins, as well as their epimers, epi-LXA₄ and LXB₄, act primarily to dampen and resolve inflammation, i.e. they are anti-inflammatory cell signaling agents.

Biochemistry

Lipoxins are derived enzymatically from arachidonic acid, an ω−6 fatty acid. Structurally, they are defined as arachidonic acid metabolites that contain three hydroxyl residues (also termed hydroxy residues) and four double bonds. This structural definition distinguishes them from other specialized pro-resolving mediators (SPMs), such as the resolvins, neuroprotectins, and maresins. All of these SPMs have activities and functions similar, although not necessarily identical, to the lipoxins.

Synthesis

Formation of LXs is conserved across a broad range of animal species from fish to humans. Biosynthesis of the LXs requires two separate enzymatic attacks on arachidonic acid (AA). One attack involves attachment of a hydroperoxy (-O-OH) residue to carbon 15, conversion of this species to a 14,15-epoxide, and the resolution of this epoxide to form either 14,15-dihydroxy-eicosatetraenoate or 15-hydroxy-eicosatetraenoate products. This step is catalyzed by enzymes with 15-lipoxygenase activity, which in humans includes ALOX15, ALOX12, aspirin-treated cyclooxygenase 2, and cytochrome P450s of the microsomal, mitochondrial, or bacterial subclasses. ALOX15B may also conduct this metabolism. The other enzyme attack point forms a 5,6-epoxide which is resolved to either 5,6-dihydroxy-eicosatetraenoate or 5-hydroxy eicosatetraenoate products; this step catalyzed by 5-lipoxygenase (ALOX5). Accordingly, these double oxygenations yield either 5,6,15-trihydroxy- or 5,14,15-trihydroxy-eicosatetraenoates. The double oxygenations may be conducted within a single cell type which possesses ALOX5 and an enzyme with 15-lipoxygenase activity or, alternatively, by two different cell types, each of which possesses one of these enzyme activities. In the latter transcellular biosynthetic pathway, one cell type forms either the 5,6-dihydroxy-, 5-hydroxy-, 14,15-dihydroxy- or a 15-hydroxy-eicosatetraenoate, and then passes this intermediate to a second cell type, which metabolizes it to the final LX product. For example, LXs are formed by platelets which, lacking ALOX5, cannot synthesize them. Rather, neutrophils form the 5,6-epoxide leukotriene A₄ (LTA₄) via ALOX5, and pass it to platelets that then reduce it to a 5,6-dihydroxy-eicosateteraenoate product and further metabolize it through ALOX12 to form the 15-hydroxy product, LXA₄.

Further metabolism

LXs are rapidly metabolized, mainly by macrophages, to inactive products by being oxidized at carbon 15 to form 15-keto (also termed 15-oxo) LX products by a 15-hydroxyprostaglandin dehydrogenase; 15-oxo-LXA₄ may be further metabolized to 13,14-dihydro-LXA₄ by an oxidoreductase. 15-Epi-LXA₄ and 15-epi-LXB₄ are more resistant to the dehydrogenation enzyme than their LX epimers. The role of these pathways in limiting or contributing to the activity of the LXs has not been fully evaluated.

Endocannabinoid system

The anti-inflammatory lipid lipoxin A₄ is an endogenous allosteric enhancer of the CB1 cannabinoid receptor. Lipoxin A₄ enhances the affinity of anandamide at this receptor to exert cannabimimetic effects in the brain, by allosterically enhancing AEA signaling and thereby potentiating the effects of this endocannabinoid both in vitro and in vivo. In addition to this, LXA₄ displays a CB1 receptor-dependent protective effect against β-amyloid-induced spatial memory impairment in mice.

Lipoxin analogs

Relatively stable, i.e. metabolically resistant, synthetic analogs of LXs and aspirin-triggered 15-epi-LXA₄s can mimic many of the desirable anti-inflammatory, "pro-resolution" actions of native LXs and are being tested for clinical use. Structurally, these LX analogs often mimic the LXs in being or closely resembling a 20-carbon trihydroxy fatty acid, but are resistant to 15-hydroxyprostaglandin dehydrogenase metabolic inactivation by having a bulky or other structural modification near their 15-hydroxy residues.

LXA₄ and 15-epi-LXA₄ are high-affinity receptor ligands for and activators of the FPR2 receptor. FPR2, which is now termed the ALX, ALX/FPR, or ALX/FPR2 receptor, is a G protein coupled receptor initially identified as a receptor for the leukocyte chemotactic factor, N-formylmethionine-leucyl-phenylalanine (FMLP), based on its amino acid sequence similarity to the known FMLP receptor, FPR1. At least six homologues of this receptor are found in mice. ALX/FPR is a promiscuous (i.e. interacting with diverse ligands) receptor that binds and is activated by other ligands including: a) various N-formyl oligopeptides that, like FMLP, are either released by microbes and mitochondria or are analogs of those released by microbes and mitochondria; b) microbe-derived non-formyl oligopeptides; c) certain polypeptides that are associated with the development of chronic amyloidosis and/or inflammation including serum amyloid A (SAA) proteins, a 42-amino acid peptide form amyloid beta termed Aβ42, humanin, and a cleaved soluble fragment (amino acids 274–388) from the urokinase receptor; and d) other SPMs including resolvins RvD1, RvD2, RvD5, AT-RvD1, and RvD3 (see Specialized pro-resolving mediators).

LXA₄ and 15-epi-LXA₄ inhibit chemotaxis, transmigration, superoxide generation, NF-κB activation, and/or generation of pro-inflammatory cytokines (e.g. IL8, IL13, IL12, and IL5) by neutrophils, eosinophils, monocytes, innate lymphoid cells, and/or macrophages, as well as suppress proliferation and production of IgM and IgG antibodies by B lymphocytes. These actions appear to involve stimulating anti-inflammatory signaling pathways, but also blocking the actions of other ALX/FPR ligands which simulate pro-inflammatory pathways. Transgenic mice made to overexpress ALX/FPR exhibit markedly reduced inflammatory responses to diverse insults.

By mechanisms yet to be clearly identified, the two LXs also: a) stimulate the bacteria-killing capacity of leukocytes and airway epithelial cells; b) block production of the pro-inflammatory cytokine, TNFα, while increasing production of the anti-inflammatory cytokine, CCR5 by T lymphocytes; c)' enhance the ability of monocytes and macrophages to phagocytos (i.e. ingest) and thereby remove potentially injurious apoptotic neutrophils and eosinophils from inflammatory sites (see Efferocytosis) either by direct effecting these cells or by stimulating NK cells to do so; d) cause various cell types to reduce production of pro-inflammatory reactive oxygen species and expression of cell adhesion molecules and increase production of the platelet inhibitor, PGI2 and the vasodilator, nitric oxide; e) inhibit production of pro-inflammatory cytokines by mesangial cells, fibroblasts, and other pro-inflammatory cell types; and f) reduce perception of pain due to inflammation. Metabolically resistant structural analogs of LXB₄ and 15-epi-LXA₄ inhibit formation of peroxynitrite (i.e. ONOO⁻) to attenuate the mobilization of NFκB and AP-1 transcription factors by reducing their accumulation in the nucleus of neutrophils, monocytes, and lymphocytes; NFκB and AP-1 increase expression of pro-inflammatory genes. The two LXBs also trigger activation of Suppressor of cytokine signaling proteins (see SOCS proteins) which, in turn, inhibit activation of STAT protein transcription factors which up-regulate many genes making pro-inflammatory products. (CysLT1 and ATX/FPR2 have an amino acid sequence identity of 47%.

At higher concentrations (>30 nmole/liter), LXA₄ binds to AHR, the arylhydrocarbon receptor; following this binding, AHR enters the nucleus, where it joins with AhR nuclear translocator (ARNT). The AHR/ARNT complex binds to xenobiotic response elements to activate transcription of genes, most of which are involved primarily in xenobiotic metabolism. These genes include SOCS2 (i.e. suppressor of cytokine signaling 2), CYP1A1, CYP1A2, CYP1B1, glutathione S-transferase Ya subunit, quinone oxidoreductase, UDP-glucuronosyltransferase and aldehyde dehydrogenase 3 family, member A1. This LXA₄ activity has been demonstrated only in murine cells.

LXA₄ binds to and activates estrogen receptor alpha, with an IC50 of 46nM. LXA₄ and ATLa were shown to activate transcriptional and functional (alkaline phosphatase and proliferation) responses via ERa in human endometrial epithelial cells in vitro and in mouse uterine tissue in vivo. Interestingly, LXA₄ also demonstrated antiestrogenic potential, significantly attenuating E2-induced activity. In a mouse model of endometriosis physiologically relevant concentrations of ATLa caused a reduction in lesion size and impacted the production of inflammatory mediators. Molecules regulated via ERa were also impacted, implying that Lipoxin A₄ and analogues, inhibiting both proliferative and inflammatory pathways, might be considered as potential therapeutics.

The actions of LXB₄ and 15-epi-LXB₄ have been far less well defined than those of their LXA₄ analogs. Their mechanism of stimulating target cells (e.g. receptors) is not known. One or both of these analogs have been shown to inhibit the recruitment of neutrophils to sites of inflammation, inhibit the cytotoxicity of NK cells, stimulate the recruitment of monocytes to inflammatory sites, enhance macrophage phagocytosis, and suppress the perception of inflammatory pain in rodents.

Animal model studies

Noninfectious inflammation

One or more of the lipoxins or their analogs have been demonstrated to suppress, limit severity, and/or increase survival in multiple inflammatory and allergic diseases in mouse and rat model studies. These studies include models of experimentally evoked endometriosis, colitis, peritonitis, pancreatitis, kidney inflammation and glomerulonephritis, lung asthma, acid-induced lung injury, cystic fibrosis, pleurisy, brain inflammation and the inflammatory component of Alzheimer's disease, vascular ischemia-reperfusion injuries to various organs including the heart and hind limb, transplant rejection of heart, kidney, and bone marrow, arthritis, dermatitis, periodontitis, cornea inflammation, and inflammation-based pain, hyperalgesia,

Infection-related inflammation

Lipoxins have protective effects in animal models of infection-based inflammation:

• LXA₄ and a LXA₄ analog decreased systemic inflammation and improved survival in rat models of gram-negative bacterial sepsis;
• 15-epi-LXA₄ suppressed the lung injury (i.e., shock lung or acute respiratory distress syndrome) caused by intraperitoneal injection of Escherichia coli in mice;
• transgenic mice made deficient in lipoxin synthesis by deletion of their Alox5 gene were more susceptible to the inflammatory and lethal effects of Toxoplasma gondii and were rescued from these defects by LXA4₄;
• LXA₄ restored macrophage function caused by respiratory syncytial virus in transgenic mice made deficient of lipoxin synthesis by Alox5 gene deletion;

Clinical studies

In a randomized controlled trial, topical application of 15-epi-LXA₄ or a comparatively stable analog of LXB₄, 15R/S-methyl-LXB₄, reduced the severity of eczema in a study of 60 infants.

As of 2015, BLXA₄, a lipoxin analog, was undergoing a phase 1 clinical trial for treating oral gingivitis.

See also

• Epi-lipoxins
• Specialized pro-resolving mediators
• 15-Hydroxyeicosatetraenoic acid

References

External links