PGD Synthase and PGD2 in Immune Resposne

PGD2 is formed from arachidonic acid by successive enzyme reactions: oxygenation of arachidonic acid to PGH2, a common precursor of various prostanoids, catalyzed by cyclooxygenase, and isomerization of PGH2 to PGD2 by PGD synthases (PGDSs). PGD2 can be either pro- or anti-inflammatory depending on disease process and etiology. The anti-inflammatory and immunomodulatory attributes of PGDS/PGD2 provide opportunities for development of novel therapeutic approaches for resistant infections and refractory inflammatory diseases. This paper highlights the role of PGD synthases and PGD2 in immune inflammatory response.


Introduction
Prostaglandins (PG) are a family of structurally related eicosanoids that have regulatory roles in normal physiological as well as pathological contexts [1]. Cyclooxygenase enzymes catalyze the conversion of arachidonic acid to PGH 2 , which is converted to other prostanoid species including PGD 2 , PGE 2 , PGF 2α , prostacyclin (PGI 2 ), and thromboxane (TX) A 2 by the action of specific synthases [1][2][3].
The importance of the role of PGD 2 in the pathogenesis and resolution of inflammation and innate immune response is increasingly recognized [6,7]. However, the effect of PGD 2 on inflammation is complex because PGD 2 either promotes or suppresses inflammation depending on the inflammatory milieu. This is further complicated by the fact that PGD 2 undergoes nonenzymatic processes to generate 15d-PGJ 2 , an anti-inflammatory lipid. Therefore, the net effect may depend on the rate of production of distal products of PGD 2 depending upon the disease process. Here, we review the biology and role of PGD synthases and PGD 2 in inflammation and host immune response.

PGD Synthases
The arachidonate cyclooxygenase pathway can generate PGD 2 by the functional linkage of a series of isoformic enzymes corresponding to phospholipase A 2 , cyclooxygenase and PGDS. Prostanoid formation occurs when cyclooxygenase oxygenates arachidonate converting it to PGG 2 , which is then reduced to PGH 2 . PGH 2 , in turn, is converted to five primary active metabolites, PGD 2 , PGE 2 , PGF 2α , PGI 2 , or thromboxane A 2 via distinct synthases such as PGD synthase and PGE synthase [1,2]. Two PGD synthases have been identified, lipocalin (L-PGDS) and hematopoietic (H-PGDS) 2 Mediators of Inflammation [4,[8][9][10]. L-PGDS and H-PGDS are quite different from each other biochemically in terms of their amino acid sequence, tertiary structure, evolutional origin, chromosomal and cellular localization, and tissue distribution and immunologically in terms of their functional relevance [10,11].

Hematopoietic PGD Synthase
Hematopoietic PGD synthase (H-PGDS) was previously known as the spleen-type PGDS [9,12] or glutathione-(GSH-) requiring enzyme for the production of PGD 2 in the peripheral tissues [12,13]. H-PGDS is characterized as a member of Sigma class of glutathione S-transferase (GST) gene family [14] that catalyze the conjugation of GSH to an electrophilic substrate. The enzyme is a homodimer and folds like other glutathione transferases. H-PGDS is localized in antigen-presenting cells and mast cells of a variety of tissues and is involved in the activation and differentiation of mast cells. It is also expressed in dendritic cells, Langerhans, and megakaryoblasts [15]. H-PGDS isomerizes PGH 2 to PGD 2 selectively and effectively, whereas other GST isozymes catalyze the conversion of PGH 2 nonselectively to produce PGD 2 , PGE 2 , and PGF 2α . The high specificity of H-PGDS for the production of PGD 2 is attributed to the unique architecture of the catalytic pocket. The deep and wide catalytic cavity of H-PGDS is striking in comparison with the narrow shallow cavities of other GSTs. The unique 3 D architecture of the cleft leads to the putative substrate binding mode and its catalytic mechanism, responsible for the specific isomerization from PGH 2 to PGD 2 [14].
H-PGDS contributes to the production of the D and J series of prostanoids in the immune system and is involved in allergic inflammatory response. Since H-PGDS is present in mast cells, Th2 cells, and other leukocytes, it is thought to be responsible for the bulk of PGD 2 production during allergic responses [16,17]. In mouse models of asthma and allergic disease, H-PGDS has a substantial proinflammatory effect, regulating many hallmark characteristics including eosinophilia, airway hyperreactivity, mucus production, and Th2 cytokine levels. Inhibitors of H-PGDS have shown to be protective in mouse models of allergic airway inflammation [18]. The compound, HQL-79, is characterized as a specific inhibitor of human H-PGDS and has shown to exhibit a therapeutic effect when used in animal models of allergic disease and neuroinflammation [19]. Thus, selective inhibitors of H-PGDS may prove to be more useful to suppress allergic and inflammatory reactions rather than COX-1 or COX-2 inhibitors, such as aspirin, indomethacin, and coxibs because these COX inhibitors suppress the production of all prostaglandins in comparison to H-PGDS inhibitors [20][21][22][23].
While H-PGDS is proinflammatory in allergic airway diseases, H-PGDS has shown to be protective in other models of inflammation. Trivedi et al. showed that H-PGDS negatively regulates the severity and duration of delayed type hypersensitivity responses. Their data suggests that contrary to the role of H-PGDS in driving Th2-like responses in models of asthma, HPGDS may act as an internal braking signal essential for bringing about the resolution of Th1-driven delayed type hypersensitivity reactions [24]. Rajakariar et al. using H-PGDS knockout mice showed that H-PGDS synthesizes 15d-PGJ 2 during mammalian defense responses and together with PGD 2 , acting through the DP1 receptor, plays a central role in controlling the onset of acute inflammation and its resolution by balancing proversus anti-inflammatory cytokines. These data highlight the anti-inflammatory and proresolution properties of cyclopentanone prostaglandins, PGD2 and DP1 receptors [25].

Lipocalin-Type PGD Synthase
Lipocalin-type PGDS is GSH independent and is identical to beta trace protein, which was discovered in 1961 as a major protein of human cerebrospinal fluid [26][27][28]. Because it resembles lipophilic ligand carrier proteins it was named lipocalin-type PGD synthase. L-PGDS is a bifunctional protein, acting as a PGD 2 -producing enzyme as well as an intercellular transporter of retinoids or other lipophilic molecules [29]. It is the only enzyme among the members of the lipocalin gene family that binds small lipophilic substances like retinoic acid, bilirubin, and ganglioside. Structurally it is a monomer with a β-barrel structure and a hydrophobic pocket and was initially identified as responsible for PGD 2 production in the brain [10,19,30]. Since then it has been shown that L-PGDS is also expressed in other tissues including the heart, kidneys [31,32], and lungs [33,34].
L-PGDS is secreted into various body fluids, such as CSF, plasma, seminal plasma, and urine, and functions as both a PGD 2 -producing enzyme and an extracellular transporter of various lipophilic substances. The L-PGDS/β-trace concentration in human serum fluctuates with circadian rhythmicity and exhibits a nocturnal increase and is best known because of its ability to induce sleep. The role of L-PGDS in several metabolic functions has since been identified. Deletion of L-PGDS leads to accelerated glucose intolerance and induces obesity [35], nephropathy, and aortic thickening [36,37]. In animal models of ischemia lack of L-PGDS confers susceptibility to injury in brain and heart [31,32,38]. L-PGDS also has an inhibitory effect on progression of lung, ovarian, and colorectal cancer and some forms of leukemia [39]. Thus, it is evident that L-PGDS has several key regulatory roles that extend beyond its function in the brain.
Similar to H-PGDS in models of allergic inflammation L-PGDS has shown to be proinflammatory. Fujitani et al. reported that L-PGDS transgenic mice exhibit strong allergic lung responses and eosinophilia [40] with enhanced allergic airway inflammation. In a model of chronic allergic dermatitis blockade of L-PGDS with an inhibitor led to significant attenuation of inflammatory response [41], which was also confirmed in CRTH2 knockout mice. The proinflammatory role of L-PGDS has also been suggested in human ulcerative colitis. Hokari et al. showed that the level of L-PGDS mRNA expression is increased in UC patients in parallel with disease activity [42]. In a diabetic rat model Ogawa et al. showed that urinary excretion of L-PGDS increased preceding diabetic nephropathy [43] and the levels of L-PGDS could predict the progression of renal injury [44]. These findings have been independently confirmed by other investigators [45]. L-PGDS in the urine is being investigated as a diagnostic biomarker of acute kidney injury and inflammation associated with diabetes, hypertension, and drug-induced nephropathy [46]. Because L-PGDS has a smaller molecular weight than serum albumin it may be expected to appear in the urine even before albuminuria and hence prove as a more sensitive marker for early detection of renal injury.
We have studied the role of L-PGDS in LPS-induced inflammation and shown that L-PGDS is induced in vitro in macrophages [33,34] and in vivo in the lung in response to LPS and P. aeruginosa [33]. Our study showed that H-PGDS was constitutively expressed in vitro in macrophages whereas L-PGDS is induced in a time-dependent manner in response to LPS or PA103. Similarly in vivo studies in mice showed that the expression of L-PGD synthase was induced in response to LPS and PA103 [33]. However, the immunomodulatory effects of L-PGDS are less well studied. In a mouse model of P. aeruginosa infection we have shown that L-PGDS −/− mice have impaired host defenses whereas overexpression of L-PGDS is protective in P. aeruginosa-induced pneumonia suggesting a pivotal role for L-PGDS in innate immune response [33]. These studies suggest an important role of L-PGDS in immunomodulation.

Prostaglandin D 2
PGs are a group of 20-carbon polyunsaturated fatty acids containing a unique 5-carbon ring structure. Prostaglandins are all produced from arachidonic acid (C20:4 fatty acid) via their common intermediate, PGH 2 , and are a family of structurally related eicosanoids that not only have an important role in homeostasis but also contribute to the pathology of numerous inflammatory diseases. Each prostanoid is then produced from PGH 2 by its specific terminal PG synthase such as PGD synthase in the case of PGD 2 [13]. PGD 2 is an acidic lipid mediator derived from arachidonic acid by sequential action of cyclooxygenase and PGD 2 synthases. Both H-and L-PGD synthase enzymes may form PGD 2 in vitro, but it is not fully understood which PGDS enzyme predominates under varied conditions in vivo. PGD 2 for a long time was considered as a minor and biologically inactive prostaglandin. In the 1980s, however, PGD 2 was found to be the most abundant prostaglandin in the brains of rats [52] and other mammals including humans [53], thus suggesting that it may have an important function in the central nervous system (CNS). The physiological functions of PGD 2 have now been extensively defined and include regulation of sleep and body temperature, olfactory function, hormone release, and nociception in the central nervous system. PGD 2 also prevents platelet aggregation and induces vasodilation and bronchoconstriction. It is released from mast cells as an allergic and inflammatory mediator [54] and is responsible for the symptoms in mastocytosis patients, such as flushing, diarrhea, tachycardia, dyspnea, and deep sleep [55]. PGD 2 is further converted to 9α, 11β-PGF 2 , a stereoisomer of PGF 2α , which exerts various pharmacological actions different from those induced by PGF 2α . PGD 2 is also readily dehydrated in vitro and in vivo [56] to produce prostaglandins of the J series, such as PGJ 2 , Δ 12 -PGJ 2 , and 15deoxy-Δ 12,14 -PGJ 2 . 15-Deoxy-Δ 12,14 -PGJ 2 has been identified as an endogenous ligand for a nuclear receptor (peroxisome proliferator-activated receptor γ), and it promotes adipocyte differentiation [5,6].
Prostaglandin D 2 may exert proinflammatory or antiinflammatory effects in different biologic systems. PGD 2 has been implicated in the initiation and progression of inflammation. In mouse models of asthma and allergic disease, PGD 2 has a substantial proinflammatory effect, regulating many hallmark characteristics including eosinophilia, airway hyperreactivity, mucus production, and Th2 cytokine levels [40,47]. Moreover, inhibition of PGD 2 synthesis and PGD 2 signaling blockade has a suppressive effect on neuroinflammation in mouse models of Krabbe's disease [48]. The injection of PGD 2 into the skin has been shown to result in erythema, edema, induration, and leukocyte infiltration [49]. PGD 2 and other vasodilator prostaglandins may also contribute to inflammation by increasing local blood flow.
In contrast to these proinflammatory effects, PGD 2 and its cyclopentenone prostaglandin derivatives also have antiinflammatory properties, with functions in resolution of inflammation. There is considerable interest in the importance of PGD 2 and its distal products in the mediation and resolution of inflammation [3,57]. Gilroy et al. showed that in a model of experimental pleuritis PGD 2 significantly attenuated inflammation [3]. Similarly in a model of experimental colitis COX-2-derived PGD 2 , acting via the DP receptor, was shown to attenuate neutrophilic infiltration into colonic mucosa [50]. In a human model of an acute inflammatory response induced by administration of LPS, which evokes transient flu-like symptoms with pyrexia and a hemodynamic response, Song et al. have shown that tetranor PGDM increases markedly during this response and that PGJ 2 has antipyretic effects [58]. These data strongly support the anti-inflammatory effects of PGD 2 .
Although several studies have investigated the role of PGD 2 in inflammation, the role of PGD 2 in host immune response has been scantly studied. An earlier study showed that PGD 2 concentration, but not the PGE 2 or IL-1β concentrations, is elevated in a time-dependent manner in the CSF of patients with African sleeping sickness, caused by Trypanosoma brucei [59]. These investigators have also shown that mouse astrocytes and fibroblasts in culture induce the production of PGD 2 in response to T. brucei [60]. Although the production of PGD2 was increased in vitro, the functional effects of PGD2 in this setting remain unclear. In a recent investigation Zhao et al. showed that an age-related increase in PGD 2 in mice led to diminished respiratory dendritic cell migration resulting in defects in virus-specific T-cell responses in vivo. They further showed that administration of PGD 2 antagonist reversed this defect resulting in migration of dendritic cells with enhancement of T-cell antivirus response with increased clearance and survival [51]. These data suggest that similar to allergic airway disease PGD 2 may have immunosuppressive effects in viral infections.
In a mouse model of P. aeruginosa lung infection we have shown that inhibition of COX-2 improves survival in a lethal Table 1: Summary of PGDS and PGD 2 effects in models of inflammation.

Model
Reference H-PGDS

Pro-inflammatory
Asthma and allergic airway inflammation (mouse model) [40,47] Neuroinflammation/Krabbe's disease (mouse Model) [48] Skin inflammation (mouse model) [49] Anti-inflammatory Mouse model of pleuritis [3] Mouse model of colitis [50] Immunosuppressive Viral infection (mouse Model) [51] model of P. aeruginosa infection [61]. The bacterial clearance of P. aeruginosa was enhanced in COX-2 knockout mice whereas transgenic mice that overexpress COX-2 have an impaired bacterial clearance from the lungs [62]. Our study showed that the immunomodulatory effects of inhibition of COX-2 are related to inhibition of PGE 2 . We also examined the effects of administration of PGD 2 in a model of P. aeruginosa lung infection. Mice that were given intratracheal PGD 2 showed an enhanced clearance of P. aeruginosa from the lungs [33]. These results were in agreement with our studies from L-PGDS knockout and L-PGDS overexpressing mice [33]. Recently we have investigated the mechanisms by which PGD 2 may exhibit immunomodulatory effects.
We have shown that PGD 2 inhibits a key proinflammatory immunoglobulin cell surface receptor TREM-1 in vitro in macrophages [63]. Furthermore, we have shown that PGD 2 induces the expression of Nrf2 in a DP1 receptor-dependent manner [63]. These studies provide a new paradigm and highlight a key regulatory role of PGD 2 in innate immune response to bacterial infections.

Conclusions
The role of PGDS/PGD 2 in regulating inflammation in a variety of organ systems and disease process is burgeoning. The inflammatory response protects the body against infection and injury but can itself become dysregulated with deleterious consequences to the host. It is now evident that endogenous biochemical pathways such as PGDS/PGD 2 get activated during defense reactions. The effect of PGDS/PGD 2 on inflammation is complex because they can either promote or suppresses inflammation depending on the inflammatory milieu. Table 1 provides a summary of the models of different effects of PGDS/PGD 2 . Interdiction of L-PGDS, PGD 2 , and DP receptors provides novel therapeutic approaches to modulate inflammation and innate immune responses.