E-prostanoid Research Articles

Endogenous prostaglandin E2 amplifies IL-33 production by macrophages through an E prostanoid (EP)2/EP4-cAMP-EPAC-dependent pathway

When activated through toll-like receptors (TLRs), macrophages generate IL-33, an IL-1 family member that induces innate immune responses through ST2 signaling. LPS, a TLR4 ligand, induces macrophages to generate prostaglandin E2 (PGE2) through inducible COX-2 and microsomal PGE2 synthase 1 (mPGES-1) (1). We demonstrate that IL-33 production by bone marrow-derived murine macrophages (bmMFs) requires the generation of endogenous PGE2 and the intrinsic expression of EP2 receptors to amplify NF-κB-dependent, LPS-induced IL-33 expression via exchange protein activated by cAMP (EPAC). Compared with WT cells, bmMFs lacking either mPGES-1 or EP2 receptors displayed reduced LPS-induced IL-33 levels. A selective EP2 agonist and, to a lesser extent, EP4 receptor agonist potentiated LPS-induced IL-33 generation from both mPGES-1-null and WT bmMFs, whereas EP1 and EP3 receptor agonists were inactive. The effects of PGE2 depended on cAMP, were mimicked by an EPAC-selective agonist, and were attenuated by EPAC-selective antagonism and knockdown. LPS-induced p38 MAPK and NF-κB activations were necessary for both IL-33 production and PGE2 generation, and exogenous PGE2 partly reversed the suppression of IL-33 production caused by p38 MAPK and NF-κB inhibition. Mice lacking mPGES-1 showed lower IL-33 levels and attenuated lung inflammation in response to repetitive Alternaria inhalation challenges. Cumulatively, our data demonstrate that endogenous PGE2, EP2 receptors, and EPAC are prerequisites for maximal LPS-induced IL-33 expression and that exogenous PGE2 can amplify IL-33 production via EP2 and EP4 receptors. The ubiquitous induction of mPGES-1-dependent PGE2 may be crucial for innate immune system activation during various IL-33 driven pathologic disorders.

Improvement of metabolic disorders by an EP2 receptor agonist via restoration of the subcutaneous adipose tissue in pulmonary emphysema

Chronic obstructive pulmonary disease (COPD) is often associated with co-morbidities. Metabolic disorders like hyperlipidemia and diabetes occur also in underweight COPD patients, although the mechanism is uncertain. Subcutaneous adipose tissue (SAT) plays an important role in energy homeostasis, since restricted capacity to increase fat cell number with increase in fat cell size occurring instead, is associated with lipotoxicity and metabolic disorders. The aim of this study is to show the protective role of SAT for the metabolic disorders in pulmonary emphysema of a murine model. We found ectopic fat accumulation and impaired glucose homeostasis with wasting of SAT in a murine model of elastase-induced pulmonary emphysema (EIE mice) reared on a high-fat diet. ONO-AE1-259, a selective E-prostanoid (EP) 2 receptor agonist, improved angiogenesis and subsequently adipogenesis, and finally improved ectopic fat accumulation and glucose homeostasis with restoration of the capacity for storage of surplus energy in SAT. These results suggest that metabolic disorders like hyperlipidemia and diabetes occured in underweight COPD is partially due to the less capacity for storage of surplus energy in SAT, though the precise mechanism is uncertained. Our data pave the way for the development of therapeutic interventions for metabolic disorders in emphysema patients, e.g., use of pro-angiogenic agents targeting the capacity for storage of surplus energy in the subcutaneous adipose tissue.

Abstract TP346: Pge2 Ep1-4 Temporospatial Expression Level Changes in the Brain After Intracerebral Hemorrhage in Young, Aged, and Ep1 Deficient Mice Suggests Cross-talk Interactions

Intracerebral hemorrhage (ICH) is the most severe form of stroke with the highest mortality. Neuroinflammation contributes to ICH-induced brain injury and the upregulation of prostaglandin E 2 (PGE 2 ) has been implicated in modulating these deleterious pathways. PGE 2 acts mainly on four G-protein-coupled E Prostanoid (EP) receptors, EP1-4, each having different downstream pathways, tissue distributions, and expression profiles. Our previous studies demonstrate that EP1 receptor deletion promotes injury following ICH; whereas, deletion of EP2 and EP3 is neuroprotective in an equivalent approach. Here, we aimed to investigate the time course, brain sub-region expression profile, and relative level of EP1-4 mRNA expression in young (5-7mo) and aged (12-13mo) wildtype (WT) and EP1-/- mice. Following ICH or sham surgery, EP1-4 mRNA levels were assessed by RT-qPCR whereby the relative fold change of the gene of interest were determined using the reference gene Tbp and the 2 -ΔΔCT method relative to the control. Minimal EP1-4 expression changes are seen at 24h after ICH; although, EP2 (p=0.036) and EP4 (p=0.066) are 1.6X increased in the cortex of young WT mice. At 72h post-ICH, EP1 is 3.9x elevated in the cortex (p=0.0003) and 4.6x in the striatum (p=0.012). EP3 is also 1.6x elevated in the cortex (p=0.044). In the contralateral hemisphere, a mean 2.4x increase of EP1-4 (p<0.01) expression is seen. In contrast, at 72h after ICH, EP1-/- mice have 0.53x reduced EP3 in the cortex (p=0.030) and 0.68x and 0.71x decreased EP3 (p=0.016) and EP4 (p=0.049), respectively, in the contralateral. Aged EP1 -/- mice show significantly decreased expression levels of EP2-4 in nearly all areas. Due to the contralateral differences, basal expression levels of EP2-4 were investigated in the EP1-/- mice, where EP2 is 2.6x, 3.4x, and 3.8x increased in the cortex, hippocampus, and cerebellum; whereas, EP3 trended oppositely. These data indicate a cross talk between EP1 and the other EP receptors pre- and post-ICH and an association between age and EP receptor expression levels. A better understanding of EP receptor localization and dynamic expression levels after ICH will spark the development of effective pharmacological treatments. Funding: NIH F31NS086441 (JLL) and R01NS046400 (SD)

Altered expression and signalling of EP2 receptor in nasal polyps of AERD patients: role in inflammation and remodelling

Down-regulation of the E-prostanoid (EP)2 receptor has been reported in aspirin exacerbated respiratory disease (AERD). We aimed to evaluate the expression and activation of EP receptors in AERD and their role in prostaglandin (PG) E2 signalling. Samples were obtained from nasal mucosa of control subjects (NM-C, n=7) and from nasal polyps of AERD patients (NP-AERD, n=7). Expression of EP1-4 was assessed at baseline. Fibroblasts were stimulated with receptor agonists to measure cAMP levels, cell proliferation and granulocyte-macrophage colony-stimulating factor (GM-CSF) release. NM-C and NP-AERD samples and fibroblasts expressed EP2, EP3 and EP4 at baseline. Lower expression of EP2 and higher expression of EP4 was observed in NP-AERD compared with NM-C. Stimulation with PGE2 and butaprost caused a higher increase in cAMP in NM-C than in NP-AERD. On the contrary, CAY10598 produced a higher production of cAMP in NP-AERD compared with NM-C. The anti-proliferative effect of PGE2 and butaprost was lower in NP-AERD than in NM-C fibroblasts. Similarly, the capacity of PGE2 and butaprost to inhibit GM-CSF release was lower in NP-AERD than in NM-C. The altered expression of EP2 in AERD may contribute to reduce the capacity of PGE2 to mediate anti-proliferative and anti-inflammatory effects.

Efferocytosis-induced prostaglandin E2 production impairs alveolar macrophage effector functions during Streptococcus pneumoniae infection.

Alveolar macrophages (AMs) are multitasking cells that maintain lung homeostasis by clearing apoptotic cells (efferocytosis) and performing antimicrobial effector functions. Different PRRs have been described to be involved in the binding and capture of non-opsonized Streptococcus pneumoniae, such as TLR-2, mannose receptor (MR) and scavenger receptors (SRs). However, the mechanism by which the ingestion of apoptotic cells negatively influences the clearance of non-opsonized S. pneumoniae remains to be determined. In this study, we evaluated whether the prostaglandin E2 (PGE2) produced during efferocytosis by AMs inhibits the ingestion and killing of non-opsonized S. pneumoniae. Resident AMs were pre-treated with an E prostanoid (EP) receptor antagonist, inhibitors of cyclooxygenase and protein kinase A (PKA), incubated with apoptotic Jurkat T cells, and then challenged with S. pneumoniae. Efferocytosis slightly decreased the phagocytosis of S. pneumoniae but greatly inhibited bacterial killing by AMs in a manner dependent on PGE2 production, activation of the EP2-EP4/cAMP/PKA pathway and inhibition of H2O2 production. Our data suggest that the PGE2 produced by AMs during efferocytosis inhibits H2O2 production and impairs the efficient clearance non-opsonized S. pneumoniae by EP2-EP4/cAMP/PKA pathway.

Host knockout of E-prostanoid 2 receptors reduces tumor growth and causes major alterations of gene expression in prostaglandin E2-producing tumors

Prostaglandin E2 (PGE2) is elevated in a variety of malignant tumors and has been shown to affect several hallmarks of cancer. Accordingly, the PGE2 receptor, E-prostanoid 2 (EP2), has been reported to be associated with patient survival and reduced tumor growth in EP2-knockout mice. Thus, the aim of the present study was to screen for major gene expression alterations in tumor tissue growing in EP2-knockout mice. EP2-knockout mice were bred and implanted with EP2 receptor-expressing and PGE2-producing epithelial-like tumors. Tumor tissue and plasma were collected and used for analyses with gene expression microarrays and multiplex enzyme-linked immunosorbent assays. Tumor growth, acute phase reactions/systemic inflammation and the expression of interleukin-6 were reduced in EP2-knockout tumor-bearing mice. Several hundreds of genes displayed major changes of expression in the tumor tissue when grown in EP2-knockout mice. Such gene alterations involved several different cellular functions, including stemness, migration and cell signaling. Besides gene expression, several long non-coding RNAs were downregulated in the tumors from the EP2-knockout mice. Overall, PGE2 signaling via host EP2 receptors affected a large number of different genes involved in tumor progression based on signaling between host stroma and tumor cells, which caused reduced tumor growth.

Inhibition of STAT3- and MAPK-dependent PGE2 synthesis ameliorates phagocytosis of fibrillar β-amyloid peptide (1-42) via EP2 receptor in EMF-stimulated N9 microglial cells.

BackgroundProstaglandin E2 (PGE2)-involved neuroinflammatory processes are prevalent in several neurological conditions and diseases. Amyloid burden is correlated with the activation of E-prostanoid (EP) 2 receptors by PGE2 in Alzheimer’s disease. We previously demonstrated that electromagnetic field (EMF) exposure can induce pro-inflammatory responses and the depression of phagocytosis in microglial cells, but the signaling pathways involved in phagocytosis of fibrillar β-amyloid (fAβ) in microglial cells exposed to EMF are poorly understood. Given the important role of PGE2 in neural physiopathological processes, we investigated the PGE2-related signaling mechanism in the immunomodulatory phagocytosis of EMF-stimulated N9 microglial cells (N9 cells).MethodsN9 cells were exposed to EMF with or without pretreatment with the selective inhibitors of cyclooxygenase-2 (COX-2), Janus kinase 2 (JAK2), signal transducer and activator of transcription 3 (STAT3), and mitogen-activated protein kinases (MAPKs) and antagonists of PG receptors EP1-4. The production of endogenous PGE2 was quantified by enzyme immunoassays. The phagocytic ability of N9 cells was evaluated based on the fluorescence intensity of the engulfed fluorescent-labeled fibrillar β-amyloid peptide (1-42) (fAβ42) measured using a flow cytometer and a fluorescence microscope. The effects of pharmacological agents on EMF-activated microglia were investigated based on the expressions of JAK2, STAT3, p38/ERK/JNK MAPKs, COX-2, microsomal prostaglandin E synthase-1 (mPGES-1), and EP2 using real-time PCR and/or western blotting.ResultsEMF exposure significantly increased the production of PGE2 and decreased the phagocytosis of fluorescent-labeled fAβ42 by N9 cells. The selective inhibitors of COX-2, JAK2, STAT3, and MAPKs clearly depressed PGE2 release and ameliorated microglial phagocytosis after EMF exposure. Pharmacological agents suppressed the phosphorylation of JAK2-STAT3 and MAPKs, leading to the amelioration of the phagocytic ability of EMF-stimulated N9 cells. Antagonist studies of EP1-4 receptors showed that EMF depressed the phagocytosis of fAβ42 through the PGE2 system, which is linked to EP2 receptors.ConclusionsThis study indicates that EMF exposure could induce phagocytic depression via JAK2-STAT3- and MAPK-dependent PGE2-EP2 receptor signaling pathways in microglia. Therefore, pharmacological inhibition of PGE2 synthesis and EP2 receptors may be a potential therapeutic strategy to combat the neurobiological deterioration that follows EMF exposure.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-016-0762-9) contains supplementary material, which is available to authorized users.

Abstract 067: Unexpected Actions of the Endothelial-EP 4 Receptor for PGE 2 to Promote Hypertension

Prostaglandin E2 (PGE 2 ) is a major prostanoid produced by the kidney with vasodilator and natriuretic actions and its actions are mediated by four distinct E-prostanoid (EP) receptor isoforms: EP 1 -EP 4 . The EP 4 receptor (EP 4 R) has multiple actions that could impact blood pressure (BP) by triggering macula densa stimulation of renin, inducing vasodilation, and inhibiting epithelial sodium transport. Accordingly, we examined the role of EP 4 R on BP regulation by generating EP 4 R-deficient mice. Because deletion of EP 4 R in utero causes peri-natal mortality due to persistent patent ductus arteriosus, we carried out conditional deletion using an EP 4 flox/flox mouse line. We first generated mice completely lacking EP 4 R in all tissues (TBKO) using a tamoxifen-inducible transgene driving Cre expression in all tissues. Resting mean arterial pressure (MAP) measured by radiotelemetry tended to be elevated in TBKOs compared to controls (106±2 vs 111±2 mmHg; p=0.06). In addition TBKOs showed exaggerated salt sensitivity and enhanced hypertensive response to chronic Ang II infusion compared to controls (MAP increase: 25±3 vs. 37±2 mmHg; p<0.05). To determine whether altered BP responses in the TBKOs were due to elimination of EP 4 R-depedent actions in vascular smooth muscle cells (VSMCs) or in endothelial cells (ECs), we generated mice lacking EP 4 R in VSMCs (SMKO) or ECs (ECKO) using EP 4 flox/flox and transgenic mice with tamoxifen-inducible expression of Cre limited to VSMCs or ECs. Resting MAP in SMKO mice was significantly reduced compared to controls (109±1 vs. 104±2 mmHg; p<0.05), but salt sensitivity and Ang II-dependent hypertension were unaffected. Although no statistically significant differences in baseline MAP or salt sensitivity were observed between ECKOs and controls, the hypertensive response to AngII infusion was significantly reduced in ECKOs (MAP increase: 31±3 vs 24±2 mmHg; p<0.05). In summary, our work suggests a complex role for PGE 2 acting via its EP 4 R in BP regulation, with a major effect to promote resistance to hypertension, apparent in the TBKOs. However, we have also uncovered an unexpected and opposing effect of EP 4 R in endothelium to promote hypertension.

Altered expression and signalling of EP2 receptor in nasal polyps of AERD patients: role in inflammation and remodelling.

Down-regulation of the E-prostanoid (EP)2 receptor has been reported in aspirin exacerbated respiratory disease (AERD). We aimed to evaluate the expression and activation of EP receptors in AERD and their role in prostaglandin (PG) E2 signalling. Samples were obtained from nasal mucosa of control subjects (NM-C, n=7) and from nasal polyps of AERD patients (NP-AERD, n=7). Expression of EP1-4 was assessed at baseline. Fibroblasts were stimulated with receptor agonists to measure cAMP levels, cell proliferation and granulocyte-macrophage colony-stimulating factor (GM-CSF) release. NM-C and NP-AERD samples and fibroblasts expressed EP2, EP3 and EP4 at baseline. Lower expression of EP2 and higher expression of EP4 was observed in NP-AERD compared with NM-C. Stimulation with PGE2 and butaprost caused a higher increase in cAMP in NM-C than in NP-AERD. On the contrary, CAY10598 produced a higher production of cAMP in NP-AERD compared with NM-C. The anti-proliferative effect of PGE2 and butaprost was lower in NP-AERD than in NM-C fibroblasts. Similarly, the capacity of PGE2 and butaprost to inhibit GM-CSF release was lower in NP-AERD than in NM-C. The altered expression of EP2 in AERD may contribute to reduce the capacity of PGE2 to mediate anti-proliferative and anti-inflammatory effects.

NLRP3 inflammasome inhibition is disrupted in a group of auto-inflammatory disease CAPS mutations.

Inflammasomes are positioned to rapidly escalate the intensity of inflammation by activating interleukin (IL)-1β, IL-18 and cell death by pyroptosis. However, negative regulation of inflammasomes remains poorly understood, as is the signaling cascade that dampens inflammasome activity. We found that rapid NLRP3 inflammasome activation was directly inhibited by protein kinase A (PKA), which was induced by prostaglandin E2 (PGE2) signaling via the PGE2 receptor E-prostanoid 4 (EP4). PKA directly phosphorylated the cytoplasmic receptor NLRP3 and attenuated its ATPase function. We found that Ser295 in human NLRP3 was critical for rapid inhibition and PKA phosphorylation. Mutations in NLRP3-encoding residues adjacent to Ser295 have been linked to the inflammatory disease CAPS (cryopyrin-associated periodic syndromes). NLRP3-S295A phenocopied the human CAPS mutants. These data suggest that negative regulation at Ser295 is critical for restraining the NLRP3 inflammasome and identify a molecular basis for CAPS-associated NLRP3 mutations.

Vascular Smooth Muscle-Specific EP4 Receptor Deletion in Mice Exacerbates Angiotensin II-Induced Renal Injury.

Cyclooxygenase inhibition by non-steroidal anti-inflammatory drugs is contraindicated in hypertension, as it may reduce glomerular filtration rate (GFR) and renal blood flow. However, the identity of the specific eicosanoid and receptor underlying these effects is not known. We hypothesized that vascular smooth muscle prostaglandin E2 (PGE2) E-prostanoid 4 (EP4) receptor deletion predisposes to renal injury via unchecked vasoconstrictive actions of angiotensin II (AngII) in a hypertension model. Mice with inducible vascular smooth muscle cell (VSMC)-specific EP4 receptor deletion were generated and subjected to AngII-induced hypertension. EP4 deletion was verified by PCR of aorta and renal vessels, as well as functionally by loss of PGE2-mediated mesenteric artery relaxation. Both AngII-treated groups became similarly hypertensive, whereas albuminuria, foot process effacement, and renal hypertrophy were exacerbated in AngII-treated EP4VSMC-/- but not in EP4VSMC+/+ mice and were associated with glomerular scarring, tubulointerstitial injury, and reduced GFR. AngII-treated EP4VSMC-/- mice exhibited capillary damage and reduced renal perfusion as measured by fluorescent bead microangiography and magnetic resonance imaging, respectively. NADPH oxidase 2 (Nox2) expression was significantly elevated in AngII-treated EP4-/- mice. EP4-receptor silencing in primary VSMCs abolished PGE2 inhibition of AngII-induced Nox2 mRNA and superoxide production. These data suggest that vascular EP4 receptors buffer the actions of AngII on renal hemodynamics and oxidative injury. EP4 agonists may, therefore, protect against hypertension-associated kidney damage. Antioxid. Redox Signal. 25, 642-656.

Human Mesenchymal Stem Cell-Derived Microvesicles Prevent the Rupture of Intracranial Aneurysm in Part by Suppression of Mast Cell Activation via a PGE2-Dependent Mechanism.

Activation of mast cells participates in the chronic inflammation associated with cerebral arteries in intracranial aneurysm formation and rupture. Several studies have shown that the anti-inflammatory effect of mesenchymal stem cells (MSCs) is beneficial for the treatment of aneurysms. However, some long-term safety concerns exist regarding stem cell-based therapy for clinical use. We investigated the therapeutic potential of microvesicles (MVs) derived from human MSCs, anuclear membrane bound fragments with reparative properties, in preventing the rupture of intracranial aneurysm in mice, particularly in the effect of MVs on mast cell activation. Intracranial aneurysm was induced in C57BL/6 mice by the combination of systemic hypertension and intrathecal elastase injection. Intravenous administration of MSC-derived MVs on day 6 and day 9 after aneurysm induction significantly reduced the aneurysmal rupture rate, which was associated with reduced number of activated mast cells in the brain. A23187-induced activation of both primary cultures of murine mast cells and a human mast cell line, LAD2, was suppressed by MVs treatment, leading to a decrease in cytokine release and tryptase and chymase activities. Upregulation of prostaglandin E2 (PGE2) production and E-prostanoid 4 (EP4) receptor expression were also observed on mast cells with MVs treatment. Administration of an EP4 antagonist with the MVs eliminated the protective effect of MVs against the aneurysmal rupture in vivo. Human MSC-derived MVs prevented the rupture of intracranial aneurysm, in part due to their anti-inflammatory effect on mast cells, which was mediated by PGE2 production and EP4 activation. Stem Cells 2016;34:2943-2955.

Prostaglandin E2 inhibits collagen synthesis in dermal fibroblasts and prevents hypertrophic scar formation in vivo.

Hypertrophic scarring is a common dermal fibroproliferative disorder characterized by excessive collagen deposition. Prostaglandin E2 (PGE2 ), an important inflammatory product synthesized via the arachidonic acid cascade, has been shown to act as a fibroblast modulator and to possess antifibroblastic activity. However, the mechanism underlying the antifibrotic effect of PGE2 remains unclear. In this study, we explored the effects of PGE2 on TGF-β1-treated dermal fibroblasts in terms of collagen production and to determine the regulatory pathways involved, as well as understand the antiscarring function of PGE2 in vivo. We found that PGE2 inhibited TGF-β1-induced collagen synthesis by regulating the balance of matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinase (TIMP). It did so by upregulating cAMP through the E prostanoid (EP)2 receptor. We determined that inhibition of the TGF-β1/Smad pathway by PGE2 is associated with its ability to inhibit collagen synthesis. An in vivo study further confirmed that PGE2 inhibits hypertrophic scar formation by decreasing collagen production. Our results demonstrate that the novel anti-scarring function of PGE2 is achieved by balancing MMPs/TIMP expression and decreasing collagen production.

E-Prostanoid 2 Receptor Overexpression Promotes Mesenchymal Stem Cell Attenuated Lung Injury.

Mesenchymal stem cells (MSCs) represent a promising approach for the treatment of acute respiratory distress syndrome (ARDS). However, their low efficiency in homing to injured lung tissue limits their therapeutic effect. Prostaglandin E2 (PGE2) biosynthesis substantially enhances the inflammatory response of the tissue. Moreover, it also facilitates the migration of MSCs by activating the E-prostanoid 2 (EP2) receptor in vitro. Given these observations, it would seem reasonable that PGE2 might act as a chemokine to promote the migration of MSCs through activation of the EP2 receptor. Herein, we confirmed that PGE2 was significantly increased in lung tissue as a result of stimulation by LPS. In addition, we constructed a lentiviral vector carrying the EP2 gene, which was successfully transduced into MSCs (MSCs-EP2). Near-infrared imaging and immunofluorescence showed that compared with MSCs-GFP, MSCs-EP2 significantly enhanced MSC homing to injured lung tissue. Moreover, the diminished amounts of Evans blue in homogeneous lung parenchyma in vivo indicated, in comparison with MSCs-GFP, that MSCs-EP2 significantly decreased LPS-induced pulmonary vascular permeability. In addition, administration of MSCs-EP2 largely decreased the levels of interleukin-1β and tumor necrosis factor-α compared with that observed after administration of MSCs-GFP at both 24 and 72 hr. Our results suggested that treatment with MSCs-EP2 markedly enhanced MSC homing to damaged lung tissue and, in addition, improved both lung inflammation and permeability. Thus, MSCs and EP2 combination gene therapy could markedly facilitate MSC homing to areas of inflammation, representing a novel strategy for MSC-based gene therapy in inflammatory diseases.

PGE2 maintains self-renewal of human adult stem cells via EP2-mediated autocrine signaling and its production is regulated by cell-to-cell contact.

Mesenchymal stem cells (MSCs) possess unique immunomodulatory abilities. Many studies have elucidated the clinical efficacy and underlying mechanisms of MSCs in immune disorders. Although immunoregulatory factors, such as Prostaglandin E2 (PGE2), and their mechanisms of action on immune cells have been revealed, their effects on MSCs and regulation of their production by the culture environment are less clear. Therefore, we investigated the autocrine effect of PGE2 on human adult stem cells from cord blood or adipose tissue, and the regulation of its production by cell-to-cell contact, followed by the determination of its immunomodulatory properties. MSCs were treated with specific inhibitors to suppress PGE2 secretion, and proliferation was assessed. PGE2 exerted an autocrine regulatory function in MSCs by triggering E-Prostanoid (EP) 2 receptor. Inhibiting PGE2 production led to growth arrest, whereas addition of MSC-derived PGE2 restored proliferation. The level of PGE2 production from an equivalent number of MSCs was down-regulated via gap junctional intercellular communication. This cell contact-mediated decrease in PGE2 secretion down-regulated the suppressive effect of MSCs on immune cells. In conclusion, PGE2 produced by MSCs contributes to maintenance of self-renewal capacity through EP2 in an autocrine manner, and PGE2 secretion is down-regulated by cell-to-cell contact, attenuating its immunomodulatory potency.

PGE2-EP3 signaling exacerbates intracerebral hemorrhage outcomes in 24-mo-old mice.

With the population aging at an accelerated rate, the prevalence of stroke and financial burden of stroke-related health care costs are expected to continue to increase. Intracerebral hemorrhage (ICH) is a devastating stroke subtype more commonly affecting the elderly population, who display increased mortality and worse functional outcomes compared with younger patients. This study aimed to investigate the contribution of the prostaglandin E2 (PGE2) E prostanoid (EP) receptor subtype 3 in modulating anatomical outcomes and functional recovery following ICH in 24-mo-old mice. EP3 is the most abundant EP receptor in the brain and we have previously shown that signaling through the PGE2-EP3 axis exacerbates ICH outcomes in young mice. Here, we show that EP3 receptor deletion results in 17.9 ± 6.1% less ICH-induced brain injury (P < 0.05) and improves neurological functional recovery (P < 0.01), as identified by lower neurological deficit scores, decreased resting time, and more gross and fine motor movements. Immunohistological staining was performed to investigate possible mechanisms of EP3-mediated neurotoxicity. Identified mechanisms include reduced blood accumulation and modulation of angiogenic and astroglial responses. Using this aged cohort of mice, we have confirmed and extended our previous results in young mice demonstrating the deleterious role of the PGE2-EP3 signaling axis in modulating brain injury and functional recovery after ICH, further supporting the notion of the EP3 receptor as a putative therapeutic avenue for the treatment of ICH.

Abstract TP100: PGE2 EP1-4 Receptor Expression Levels After Intracerebral Hemorrhage in Young, Aged, and EP1 Deficient Mice

Intracerebral hemorrhage (ICH) is the most severe form of stroke and is associated with high mortality and morbidity. Neuroinflammation significantly contributes to ICH-induced brain injury and upregulation of prostaglandin E 2 (PGE 2 ) has been implicated in modulating these deleterious neuroinflammatory pathways. PGE 2 acts on mainly the four G-protein-coupled E prostanoid (EP) receptors, EP1-4, which each have different downstream signaling pathways, tissue distributions, and expression profiles. We have previously demonstrated that EP1 receptor deletion promotes injury following ICH, whereas deletion of EP2 and EP3 is neuroprotective in an equivalent experimental approach. Here, we aimed to investigate the time course, brain sub-region expression profile, and relative level of EP1-4 mRNA expression in young (5-7mo) and aged (12-13mo) wildtype (WT) and EP1 receptor knockout (EP1 -/- ) mice. Following ICH or sham surgery, EP1-4 mRNA levels were assessed by RT-qPCR. Minimal EP1-4 expression changes are seen at 24h after ICH; although, EP2 (p=0.0.36) and EP4 (p=0.066) are 1.6X increased in the cortex of young WT mice. At 72h post-ICH, EP1 is 3.9x elevated in the cortex (p=0.0003) and 4.6x in the striatum (p=0.012). EP3 is also 1.6x elevated in the cortex (p=0.044). In the contralateral hemisphere, a mean 2.4x increase of EP1-4 (p&lt;0.01) expression is seen. In contrast, at 72h after ICH, EP1 -/- mice have 0.53x reduced EP3 in the cortex (p=0.030) and 0.68x and 0.71x decreased EP3 (p=0.016) and EP4 (p=0.049), respectively, in the contralateral hemisphere. Aged EP1 -/- mice show significantly decreased expression levels of EP2-4 in nearly all areas (cortex, striatum, and contralateral hemisphere). Due to the contralateral differences, basal expression levels of EP2-4 were investigated in the EP1 -/- mice, where EP2 is 2.6x, 3.4x, and 3.8x increased in the cortex, hippocampus, and cerebellum; whereas, EP3 trended oppositely. These data indicate a cross-talk interaction between EP1 and the other EP receptors pre- and post-ICH and an association between age and EP receptor expression levels. A better understanding of EP receptor localization and dynamic expression levels after ICH will facilitate the development of effective pharmacological treatments.

Abstract TP114: Modulation of Blood Accumulation and Angiogenic Responses by PGE2-EP3 Signaling After ICH in Aged Mice

With the population aging at an accelerated rate, the prevalence of stroke and financial burden of stroke-related health care costs is expected to continue to increase. Intracerebral hemorrhage (ICH) is a devastating stroke subtype more commonly affecting the elderly population, whom display increased mortality and worse functional outcomes compared to younger patients. This study aimed to investigate the contribution of the prostaglandin E 2 (PGE 2 ) E prostanoid (EP) receptor subtype 3 in modulating anatomical outcomes and functional recovery following ICH in 24-mo-old mice. PGE 2 levels are dramatically upregulated following brain injury and have been shown to modulate the deleterious excitotoxic and neuroinflammatory processes resulting from activation of glial cells and infiltration of blood cells and proinflammatory molecules through its four EP receptors, EP1-4. EP3 is the most abundant EP receptor in the brain and we have previously shown that signaling through the PGE 2 -EP3 axis exacerbates ICH outcomes in young mice. Here, we show that EP3 receptor deletion results in 17.9±6.1% less ICH-induced brain injury (p=0.0336) and improves neurological functional recovery, as identified by lower neurological deficit scores at 48h (p=0.0013) and 72h (p=0.0063), and increased gross (p=0.0038) and fine motor (p=0.0556) movements and decreased resting time (p = 0.0280) at 72h. Immunohistological staining was performed to investigate possible mechanisms of EP3-mediated neurotoxicity. Identified mechanisms include reduced blood accumulation (p=0.0277) and modulation of angiogenic and astroglial responses, where EP3 -/- mice have increased striatal astrogliosis (p=0.0498) and VEGF immunoreactivity (p=0.0115), and tended to have increased cortical astrogliosis (p=0.1874). Using this aged cohort of mice, we have confirmed and extended our previous results in young mice demonstrating the deleterious role of the PGE 2 -EP3 signaling axis in modulating brain injury and functional recovery after ICH, further supporting the notion of the EP3 receptor as a putative therapeutic avenue for the treatment of ICH.

The influence of prostaglandin E2 on the production of IFN-γ by bovine CD4+, CD8+ and WC1+ T cells

The aim of these studies was to assess the influence of prostaglandin E2 (PGE2) on the production of interferon-gamma (IFN-γ) by bovine CD4+, CD8+, and WC1+ T cells and furthermore, should this effect exist, to identify the E-prostanoid (EP) receptor subtype(s) responsible for this influence. We here report that exposure of bovine peripheral blood mononuclear cells (PBMCs) to PGE2 significantly and dose-dependently decreased the percentage of IFN-γ-producing CD4+ and CD8+ T cells. It was also shown that PGE2 reduced IFN-γ production by WC1+ T cells, but this effect was not dose dependent. The impairment of IFN-γ production should be recognized as an anti-inflammatory and immunosuppressive action, thus the obtained results confirm the paradoxical status of PGE2 as a proinflammatory factor with immunosuppressive activity. The blockade of EP1, EP2, EP3, and EP4 receptors did not prevent PGE2-induced reduction of IFN-γ production by CD4+ and CD8+ T cells, indicating that this effect of PGE2 is not mediated through EP receptors. On the contrary, the blockade of either EP2 or EP4 receptors, but not EP1 or EP3 receptors, prevented the PGE2-induced reduction of percentage of IFN-γ-producing WC1+ T cells. These findings indicate that the ability of PGE2 to impair IFN-γ production by WC1+ T cells is mediated via EP2 and EP4 receptors. These results suggest the possibility of pharmacological manipulation of IFN-γ production by WC1+ T cells via selective antagonists and agonists of EP2 and EP4 receptors.

Impaired E Prostanoid2 Expression and Resistance to Prostaglandin E2 in Nasal Polyp Fibroblasts from Subjects with Aspirin-Exacerbated Respiratory Disease.

Recurrent, rapidly growing nasal polyps are hallmarks of aspirin-exacerbated respiratory disease (AERD), although the mechanisms of polyp growth have not been identified. Fibroblasts are intimately involved in tissue remodeling, and the growth of fibroblasts is suppressed by prostaglandin E2 (PGE2), which elicits antiproliferative effects mediated through the E prostanoid (EP)2 receptor. We now report that cultured fibroblasts from the nasal polyps of subjects with AERD resist this antiproliferative effect. Fibroblasts from polyps of subjects with AERD resisted the antiproliferative actions of PGE2 and a selective EP2 agonist (P < 0.0001 at 1 μM) compared with nasal fibroblasts from aspirin-tolerant control subjects undergoing polypectomy or from healthy control subjects undergoing concha bullosa resections. Cell surface expression of the EP2 receptor protein was lower in fibroblasts from subjects with AERD than in fibroblasts from healthy control subjects and aspirin-tolerant subjects (P < 0.01 for both). Treatment of the fibroblasts with trichostatin A, a histone deacetylase inhibitor, significantly increased EP2 receptor mRNA in fibroblasts from AERD and aspirin-tolerant subjects but had no effect on cyclooxygenase-2, EP4, and microsomal PGE synthase 1 (mPGES-1) mRNA levels. Histone acetylation (H3K27ac) at the EP2 promoter correlated strongly with baseline EP2 mRNA (r = 0.80; P < 0.01). These studies suggest that the EP2 promotor is under epigenetic control, and one explanation for PGE2 resistance in AERD is an epigenetically mediated reduction of EP2 receptor expression, which could contribute to the refractory nasal polyposis typically observed in this syndrome.