Resolution, the Grail for Healthy Ocular Inflammation

3.1. Resident Protective Lipid Mediator Circuits

Lipid autacoids are of particular interest as virtually every cell can generate specific and highly conserved bioactive lipid signals such as arachidonic acid (AA, ω-6 C20:4, eicosatetraenoic acid) derived eicosanoids. More importantly, these short-lived lipid signals are some of the earliest cellular responses to injury, infection or stress. It is thus not surprising that primary treatment options for inflammation, such as nonsteroidal anti-inflammatory drugs (NSAIDS) and corticosteroids, target biosynthetic enzymes (cyclooxygenase and phospholipase A₂) for the formation of eicosanoids. However, it is now recognized that several members in the eicosanoid family do not amplify or initiate inflammation but are key counter-regulatory signals that inhibit inflammatory circuits and promote inflammatory resolution (Lawrence et al., 2002; Gilroy et al., 2004; Serhan et al., 2007; Gronert, 2008; Serhan et al., 2008a). In this regard a key enzyme in the formation of protective lipid mediators 15-LOX is the focus of ongoing research efforts. 15-LOX is one of the most inducible genes in macrophages and its expression is induced by interleukin-13 and interleukin-4 (Kuhn & O'Donnell, 2006), cytokines that induce the wound healing macrophages (M2) phenotype (Mosser & Edwards, 2008). Its essential role in maintaining tissue function is emphasized by the fact that 15-LOX is highly expressed in mucosal epithelial cells, corneal epithelium and retinal pigment epithelium. Moreover, it is intriguing that virtually all human epithelial cancers lose expression of 15-LOX indicating that the enzyme has an essential role in regulating epithelial proliferation and apoptosis (Shureiqi et al., 2005; Wu et al., 2008).

Even though, 15-LOX is a resident enzyme the cornea and retina its tissue levels and activity is highly upregulated by recruited 15-LOX expressing macrophages during acute inflammation or normal housekeeping activity of scavenging macrophages. 15-LOX is a key enzyme for the formation of the anti-inflammatory and pro-resolving eicosanoid, lipoxin A₄ (LXA₄), and the recently identified family of ω-3 PUFA derived lipid signals, docosahexaenoic acid (ω-3 C22:6, DHA) derived resolvins (RvD1-4) and protectins (NPD1). 15-LOX can either directly generate protective autacoids like NPD1 or it generates pivotal metabolic intermediates that are further transformed by 5-LOX (Figure 2), a prominent enzyme in macrophages and PMN, to LXA₄ or RvD1-4 (Serhan et al., 2008b). These prominent endogenous lipid autacoids restrain PMN and lymphocyte activation, dendritic cell function, formation of pro-inflammatory chemokines by epithelial cells, promote clearance of apoptotic PMN by macrophages and regulate epithelial wound healing; hence, this lipid circuit is considered critical to the resolution of acute inflammation (Lawrence et al., 2002; Gilroy et al., 2004; Serhan et al., 2007; Gronert, 2008; Serhan et al., 2008b; Kenchgowda & Bazan, 2009). Enzymatic formation and structures of these oxygenated PUFA are highly conserved in most vertebrates including fish and frogs (Gronert et al., 1995; Hong et al., 2005; Serhan et al., 2008a; Serhan et al., 2008b), and it is remarkable that human pathogens such as Pseudomonas areuginosa and Toxoplasma gondii exploit this anti-inflammatory lipid circuit to redirect host immune responses (Aliberti et al., 2002a; Aliberti et al., 2002b; Bannenberg et al., 2004; Vance et al., 2004).

3.2. The Lipoxin A₄ Circuit

A rapidly evolving field of research now established the endogenous formation and/or protective bioactions of a 15-LOX-initiated circuit in the cornea, retina and uvea (Gronert et al., 2005; Bazan, 2006; Biteman et al., 2007; Qin et al., 2008; Bazan, 2009; Calandria et al., 2009; Kenchgowda & Bazan, 2009; Cortina et al., 2010; Leedom et al., 2010). Direct evidence for a central role of 15-LOX in corneal inflammatory response was obtained with 12/15-LOX knockout mice, which exhibit a phenotype of impaired wound healing and amplified inflammatory responses and inflammatory neovascularization, which correlate with impaired endogenous LXA₄ formation (Gronert et al., 2005; Biteman et al., 2007; Leedom et al., 2010). A loss of function phenotype that is rescued by topical treatment with LXA₄. In addition to LXA₄, other mouse 12/15-LOX-derived metabolites, namely, 12(S)-HETE and 15(S)-HETE, can exert beneficial actions in the cornea. 12(S)-HETE and 15(S)-HETE can function as intracellular second messengers for growth factors and modulate the proliferation of corneal epithelial cells, thereby promoting wound closure (Kenchgowda & Bazan, 2009). 15-LOX in the healthy cornea is predominantly expressed in epithelial cells (Chang et al., 2005; Gronert et al., 2005) as expression and activity of 15-LOX is abrogated by de-epithelialization and restored during epithelial wound healing in mice (Gronert et al., 2005). More importantly, G-protein coupled receptors for LXA₄ (ALX) are expressed in both human and mouse corneas (Gronert et al., 1998; Gronert et al., 2005; Jin et al., 2009; Leedom et al., 2010). ALX has been established as an anti-inflammatory receptor that transduces the counter-regulatory and pro-resolving bioactions of LXA₄ and DHA-derived RvD1 and the receptor is expressed in epithelial cells, PMN, macrophages and peripheral blood T lymphocytes (Chiang et al., 2006; Krishnamoorthy et al., 2010). Its role in inflammatory resolution was established in a gain of function model employing mice that were engineered to express the human ALX receptor in leukocytes (Devchand et al., 2003). These transgenic mice in response to yeast antigen-induced peritonitis demonstrate reduced amplitude of acute inflammation and accelerated PMN resolution. It is thus of interest that 15-LOX and ALX receptor expression and activity are dynamically regulated during inflammatory responses in the cornea, which points towards a fundamental role of the LXA₄ circuit in corneal inflammatory responses (Gronert et al., 2005; Jin et al., 2009; Leedom et al., 2010). In addition to corneal inflammatory responses the LXA₄ circuit may have a role in intraocular inflammatory diseases as recent reports demonstrate that LXA₄ treatment attenuates endotoxin-induced uveitis (EIU) (Medeiros et al., 2008; Karim et al., 2009). EIU is a common animal model that exhibits many of the key features of human uveitis, including dilation of vessels, macromolecule leakage, edema, and infiltration of leukocytes and lymphocytes. Topical treatment with LXA₄ greatly attenuated EIU reducing protein leakage, PMN infiltration and pro-inflammatory circuits (i.e. TNF-α, COX-2, VEGF and NF-κB activation). The role of the 15-LOX/LXA₄ circuit in retina or conjunctiva remains to be explored.

3.3. DHA an Essential ω-3 PUFA: Precursor for Protective Mediators

In addition to AA, cells release other essential PUFAs that are substrates for 15-LOX, especially DHA. Like AA, DHA is found in all human tissues, plasma and milk at concentrations 1-20% of total fatty acids (Arterburn et al., 2006). Moreover, in the cerebral cortex, sperm and retina, DHA is present at higher concentrations than AA. In particular, retinal photoreceptor outer segments have the highest DHA content of any cell and an unusual DHA-retention ability and numerous lines of evidence establish a critical role for DHA in human physiology, namely, neuronal and retinal photoreceptor functions (Bazan, 2006). Indeed, experimental evidence demonstrates decreased blood levels of DHA in various forms of retinitis pigmentosa and in animal models of inherited retinal degeneration (Bazan, 2006). Moreover, current clinical trials (i.e. NEI Age-Related Eye Disease Study) have demonstrated that dietary ω-3 PUFA intake is inversely associated with neovascular age-related macular degeneration (AMD) (SanGiovanni et al., 2000; SanGiovanni & Chew, 2005; SanGiovanni et al., 2007; SanGiovanni et al., 2008; SanGiovanni et al., 2009; Sangiovanni et al., 2009), namely consumption of fish oils (eicosapentaenoic acid (EPA) and DHA) lowers progression of bilateral drusen to central geographic atrophy and neovascularization. Increased dietary intake of ω-3 PUFAs also correlated with a decreased incidence of dry eye syndrome in a well-characterized population of women participating in the Women's Health Study (Miljanovic et al., 2005). These clinical findings are supported by animal studies, which show that topical treatment with α-linolenic acid (ω-3, C18:3), a precursor for essential ω-3 PUFA (EPA and DHA), reversed leukocyte infiltration and expression of TNF-α and IL-1α in a murine model of dry eye (Rashid et al., 2008). Moreover, in animal models of macular degeneration and retinopathy of prematurity diets enriched in DHA and EPA reduced the development of retinal lesions, inflammation and pathological neovascularization (Connor et al., 2007; Tuo et al., 2009). These and numerous other reports have identified dietary ω-3 PUFA, especially DHA, as an important factor for ocular health and inflammatory diseases (Bazan, 2006).

The anti-inflammatory, cardioprotective and immunosuppressive actions of dietary ω-3 PUFA are well established based on long standing research efforts; but the molecular mechanism for this striking actions was only recently uncovered (Bazan, 2006; Gronert & Hassan, 2007; Gronert, 2008; Serhan et al., 2008a; Serhan et al., 2008b). Specifically, EPA and DHA are transformed in a fashion analogous to eicosanoid biosynthesis to novel classes of ω-3 PUFA-derived mediators. These mediators have been named resolvins and protectins as they are formed during the resolution phase of inflammation and exhibit potent anti-inflammatory, pro-resolving and neuroprotective properties. Reports from several laboratories provide compelling evidence that these protective ω-3 PUFA-derived mediators provide the elusive molecular mechanism for the remarkable beneficial and essential effects of dietary ω-3 PUFA(Lukiw et al., 2005; Gonzalez-Periz et al., 2006; Connor et al., 2007; Gronert & Hassan, 2007; Levy et al., 2007; Schwab et al., 2007; Serhan et al., 2008a; Bazan, 2009; Gonzalez-Periz et al., 2009; Hassan & Gronert, 2009; Kenchgowda & Bazan, 2009).

In view of the abundance of DHA, especially in the retina and brain, considerable research interest has focused on novel protective DHA-derived autacoids, in particular NPD1, which regulates critical events in the acute inflammatory/reparative response, its resolution and protects neurons and retinal pigment epithelial cells against oxidative stress induced apoptosis (Gronert, 2008; Bazan, 2009; Kenchgowda & Bazan, 2009; Zhang & Bazan, 2010). It is of particular interest that this protective mediator is generated by a single enzyme, namely 15-LOX, which is highly expressed in retinal pigmented and corneal epithelial cells and wound healing macrophages. 15-LOX directly generates NPD1 (10,17-dihydroxy-DHA) and 17-hydroxy-DHA (17-HDHA) (Figure 2); the complete structure of NPD1 has been assigned (Serhan et al., 2006). 17-HDHA is further metabolized by leukocyte/epithelial 5-LOX (Figure 2), in mechanism analogous to lipoxin biosynthesis, to several distinct 17S-resolvins (RvD1-4), which like NPD1 exhibit anti-inflammatory actions (Serhan et al., 2008b). Hence, during acute inflammation interactions of leukocytes with epithelial cells leads to the formation of a temporally defined arrays of protective DHA-derived mediators, which are an integral component of the resolution pathway. Significant endogenous formation of these protective DHA-derived mediators (NPD1, RvD1, 17-HDHA) has been reported by several laboratories, including our group, in both human and mouse tissues (Lukiw et al., 2005; Gonzalez-Periz et al., 2006; Levy et al., 2007; Serhan et al., 2008b; Gonzalez-Periz et al., 2009; Hassan & Gronert, 2009), which includes reports in the retina and cornea (Gronert et al., 2005; Bazan, 2006; Connor et al., 2007; Qin et al., 2008). The protective DHA-derived signals have distinct but overlapping bioactions and both in vitro and in vivo experiments have demonstrated that similar to LXA₄, resolvins and protectins reduce leukocyte infiltration, regulate cytokines/chemokines and reactive oxygen species, and more importantly increase macrophage phagocytosis of apoptotic PMN (Bazan, 2006; Gronert & Hassan, 2007; Schwab et al., 2007; Gronert, 2008; Zhang & Bazan, 2010). Hence, 15-LOX sets in place a resident lipid circuit that counter-regulates inflammatory circuits, contributes to the anti-inflammatory tone of the tissue and during inflammation is amplified by recruited effector cells to promote resolution of PMN.

The important role of DHA derived signals and the resident epithelial 15-LOX circuit is underscored in the retina where retinal pigment epithelial cells are essential to maintain and phagocytose photoreceptors, recycle DHA and form the critical retinal-blood barrier. Failure of RPE cells to function properly leads to photoreceptor damage or death, and consequently, impaired vision or blindness (Bazan, 2006; Zhang & Bazan, 2010). The retina is particularly prone to oxidative stress due to the high oxygen demand and blood flow in the tissue. Oxidative stress, trauma and vascular inflammation induce RPE dysfunction, which contributes to retinopathies, including pathological neovascularization and AMD. Hence, it is striking that the 15-LOX circuit is emerging as an RPE resident pathway that counteract these challenges (Bazan, 2006, 2009; Zhang & Bazan, 2010). Oxidative stress increases production of DHA-derived NPD1 in RPE cells, which inhibits oxidative stress-mediated pro-inflammatory gene induction and apoptosis, and ultimately promotes RPE cell survival. More importantly, human RPE cells deficient in 15-LOX exhibit increased sensitivity to oxidative stress-induced apoptosis, an impaired protective function that is restored selectively by treatment with NPD1. NPD1 has also been shown to inhibit retinal ganglion cell apoptosis following optic nerve transaction, pathological retinal angiogenesis and inflammation, and promote nerve regeneration in the innervated cornea (Bazan, 2006; Connor et al., 2007; Qin et al., 2008; Bazan, 2009; Cortina et al., 2010; Zhang & Bazan, 2010). Taken together, these recent studies demonstrate a role for 15-LOX-triggered DHA signals in maintaining and protecting the neural retina.

3.4. The Cytoprotective Heme-Oxygenase System

Heme-oxygenases (HO-1 and HO-2) constitute an essential cytoprotective system that is the rate-limiting enzyme for metabolizing essential heme and recycling iron. In the process, HO-1 generates the antioxidant biliverdin, which is converted to bilirubin, and the bioactive gas carbon monoxide. The heme-oxygenase system contains two principle enzymes: 1) HO-2, which is expressed at basal level in many healthy uninjured tissues and 2) HO-1, which is a highly inducible cytoprotective and early response gene. HO-1 amplification by genetic or pharmacological approaches causes striking anti-proliferative, anti-inflammatory and anti-apoptotic actions in many disease models (Otterbein et al., 2003; Ryter et al., 2006; Abraham & Kappas, 2008). HO-1 essential role in health and inflammation are underscored by the fact that human deficiencies in HO-1 and genetic deletion of HO-1 in mice leads to spontaneous chronic and non-resolving inflammation without injury or infection. Moreover, HO-1 expression is induced in macrophages specifically during the resolution phase and HO-1 mediates the anti-inflammatory actions of the key anti-inflammatory and immune regulatory cytokine IL-10 (Willis et al., 2000; Willoughby et al., 2000; Bach, 2005).

The HO system is constitutively expressed in the healthy cornea (HO-2) and is highly upregulated during acute and self-resolving injury responses (HO-1). Deletion of HO-2 in mice results not only in a defect of basal HO activity but also failed upregulation of HO-1 in acute inflammatory/reparative response of the cornea. In these HO deficient mice normal self-resolving inflammation and epithelial injury shifts to sustained and amplified leukocyte infiltration, failed wound healing and pronounced pathological neovascularization, hallmarks of chronic inflammation (Abraham et al., 1995; Seta et al., 2006; Biteman et al., 2007; Patil et al., 2008; Bellner et al., 2009). Strikingly, the phenotype of chronic inflammation correlates with impaired formation of LXA₄ and in 12/15-LOX deficient mice (Alox15 KO) induction of HO-1 is impaired and correlates with amplified PMN infiltration, chemokine formation and delayed wound healing. The 15-LOX KO phenotype of dysregulated inflammation is rescued by adding back LXA₄, which restores normal HO-1 expression, wound healing and inflammation. Experiments with human corneal epithelial cells have confirmed that specific 15-LOX derived mediators, LXA₄, NPD1 and 17-HDHA, amplify expression of cytoprotective HO-1 (Biteman et al., 2007). In addition, systemic pharmacological upregulation of HO-1 in endotoxin-induced uveitis affords effective protection against acute inflammation by reducing leukocytes in the aqueous humor and down-regulating pro-inflammatory cytokines and inducible nitric oxide (iNOS) (Ohta et al., 2003). The role of the HO system in the retina and conjunctiva remains to be defined.

Taken together, these findings suggest an essential role of the HO system in ocular inflammation, which is consistent with an impressive body of work that has demonstrated that HO is a fundamental protective response to inflammation and oxidative stress as well as a key feature of inflammatory resolution (Willoughby et al., 2000; Ohta et al., 2003; Otterbein et al., 2003; Bach, 2005; Abraham & Kappas, 2008; Gronert, 2008). Moreover, these studies as well as reports from other inflammatory models (Willis et al., 2000; Willoughby et al., 2000; Nascimento-Silva et al., 2005) suggest an interdependence of the HO system with lipid mediators circuits that have been identified as key component of the resolution program (Lawrence et al., 2002; Gilroy et al., 2004; Serhan et al., 2007), such as the 15-LOX/LXA₄ circuit. Dysregulation of these ocular resolution circuits may provide insight and therapeutic targets for inflammatory diseases in the visual axis.