CRTH2 Research Articles

Inhibition of the prostaglandin D2\u2013GPR44/DP2 axis improves human islet survival and function

Aims/hypothesisInflammatory signals and increased prostaglandin synthesis play a role during the development of diabetes. The prostaglandin D2 (PGD2) receptor, GPR44/DP2, is highly expressed in human islets and activation of the pathway results in impaired insulin secretion. The role of GPR44 activation on islet function and survival rate during chronic hyperglycaemic conditions is not known. In this study, we investigate GPR44 inhibition by using a selective GPR44 antagonist (AZ8154) in human islets both in vitro and in vivo in diabetic mice transplanted with human islets.MethodsHuman islets were exposed to PGD2 or proinflammatory cytokines in vitro to investigate the effect of GPR44 inhibition on islet survival rate. In addition, the molecular mechanisms of GPR44 inhibition were investigated in human islets exposed to high concentrations of glucose (HG) and to IL-1β. For the in vivo part of the study, human islets were transplanted under the kidney capsule of immunodeficient diabetic mice and treated with 6, 60 or 100 mg/kg per day of a GPR44 antagonist starting from the transplantation day until day 4 (short-term study) or day 17 (long-term study) post transplantation. IVGTT was performed on mice at day 10 and day 15 post transplantation. After termination of the study, metabolic variables, circulating human proinflammatory cytokines, and hepatocyte growth factor (HGF) were analysed in the grafted human islets.ResultsPGD2 or proinflammatory cytokines induced apoptosis in human islets whereas GPR44 inhibition reversed this effect. GPR44 inhibition antagonised the reduction in glucose-stimulated insulin secretion induced by HG and IL-1β in human islets. This was accompanied by activation of the Akt–glycogen synthase kinase 3β signalling pathway together with phosphorylation and inactivation of forkhead box O-1and upregulation of pancreatic and duodenal homeobox-1 and HGF. Administration of the GPR44 antagonist for up to 17 days to diabetic mice transplanted with a marginal number of human islets resulted in reduced fasting blood glucose and lower glucose excursions during IVGTT. Improved glucose regulation was supported by increased human C-peptide levels compared with the vehicle group at day 4 and throughout the treatment period. GPR44 inhibition reduced plasma levels of TNF-α and growth-regulated oncogene-α/chemokine (C-X-C motif) ligand 1 and increased the levels of HGF in human islets.Conclusions/interpretationInhibition of GPR44 in human islets has the potential to improve islet function and survival rate under inflammatory and hyperglycaemic stress. This may have implications for better survival rate of islets following transplantation.

Promoter Genotyping and mRNA Expression - Based Analysis of the PTGDR Gene in Allergy.

Prostaglandin D2 receptors are acquiring a relevant role as potential therapeutic targets in allergy. PTGDR has been described as a candidate gene in allergic disease, although functional studies on this gene are lacking. Objective: The objective of this case-control study was to investigate the potential role of PTGDR in allergy. The study population comprised 195 allergic patients and 112 healthy controls. The PTGDR promoter polymorphisms -1289G>A, -1122T>C, -881C>T, -834C>T, -613C>T, -549T>C, -441C>T, -197T>C, and -95G>T were amplified by polymerase chain reaction (PCR) and sequenced. PTGDR expression levels were analyzed using quantitative PCR and normalized to GAPDH and TBP mRNA levels. All procedures were performed following the Minimum Information for Publication of Quantitative Real-Time PCR Experiment guidelines. PTGDR expression levels were significantly higher in allergic patients than in controls (P<.001). Receiver operating characteristic analysis for expression of PTGDR showed a sensitivity of 81.4% compared with 67% for IgE levels. In addition, differences in the genotypic distribution of the polymorphisms -1289G>A and -1122T>C were found in allergic patients (P=.009). The results indicate that PTGDR overexpression is associated with allergy. The polymorphisms -1289G>A and -1122T>C partly explain the variation in expression we observed. PTGDR expression could have a potential role as a biomarker and pharmacogenetic factor in allergy.

An Agonist Dependent Allosteric Antagonist of Prostaglandin EP2 Receptors.

All reported prostaglandin EP2 receptor antagonists have a purely orthosteric, competitive mode of action. Herein, we report the characterization of compound 1 (pubchem CID 664888) as the first EP2 antagonist that features a reversible, agonist dependent allosteric mode of action. Compound 1 displayed an unsurmountable inhibition of cAMP accumulation stimulated by different EP2 agonists in C6 glioma cells overexpressing human EP2 (C6G-hEP2). The degree of reduction of agonist potency and efficacy depended on the agonist employed. Negative allosteric modulation was not observed in C6G cells overexpressing human EP4, IP, or DP1 receptors. Moreover, in the murine microglial cell line that stably expresses human EP2 receptors (BV2-hEP2), compound 1 reduced the EP2 agonist-induced elevation of interleukin 6 (IL-6), IL-1β, and hEP2 mRNA levels and increased that of tumor necrosis factor (TNF)-α. Compound 1 was docked into a homology model of hEP2. The predicted binding site on the cytoplasmic receptor surface was similar to that of allosteric inhibitors of the β2-adrenergic, CC chemokine receptor 9 (CCR9), and CC chemokine receptor 2 (CCR2) receptors, which supports the notion of a conserved G-protein-coupled receptor (GPCR) binding pocket for allosteric inhibitors. As the first agonist dependent negative allosteric modulator of EP2 receptor, the structure of this compound may provide a basis for developing improved allosteric modulators of EP2 receptors.

GGA3 interacts with L-type prostaglandin D synthase and regulates the recycling and signaling of the DP1 receptor for prostaglandin D2 in a Rab4-dependent mechanism

Mechanisms controlling the recycling of G protein-coupled receptors (GPCRs) remain largely unclear. We report that GGA3 (Golgi-associated, γ adaptin ear containing, ADP-ribosylation factor-binding protein 3) regulates the recycling and signaling of the PGD2 receptor DP1 through a new mechanism. An endogenous interaction between DP1 and GGA3 was detected by co-immunoprecipitation in HeLa cells. The interaction was promoted by DP1 agonist stimulation, which was supported by increased DP1-GGA3 colocalization in confocal microscopy. Pulldown assays showed that GGA3 interacts with the intracellular loop 2 and C-terminus of DP1, whereas the receptor interacts with the VHS domain of GGA3. The Arf-binding deficient GGA3 N194A mutant had the same effect as wild-type GGA3 on DP1 trafficking, suggesting a new mechanism for GGA3 in recycling. Depletion of Rab4 inhibited the GGA3 effect on DP1 recycling, revealing a Rab4-dependent mechanism. Interestingly, depletion of L-PGDS (L-type prostaglandin synthase, the enzyme that produces the agonist for DP1) impaired the ability of GGA3 to mediate DP1 recycling, while GGA3 knockdown prevented L-PGDS from promoting DP1 recycling, indicating that both proteins function interdependently. A novel interaction was observed between co-immunoprecipitated endogenous L-PGDS and GGA3 proteins in HeLa cells, and in vitro using purified recombinant proteins. Redistribution of L-PGDS towards GGA3- and Rab4-positive vesicles was induced by DP1 activation. Silencing of GGA3 inhibited ERK1/2 activation following DP1 stimulation. Altogether, our data reveal a novel function for GGA3, in a newly described association with L-PGDS, in the recycling and signaling of a GPCR, namely DP1.

A Noncompetitive and Selective Prostaglandin EP2 Antagonist with Anti‐inflammatory Property

The prostaglandin E2 (PGE2) receptor subtype EP2 promotes chronic inflammation, modulates tumorigenesis, and regulates immune function. Inhibition of EP2 receptors reduces inflammation in rodent models of epilepsy, endometriosis, and arthritis, and decreases tumor cell growth. Therefore, inhibiting EP2 receptors could be a promising method for treatment of inflammatory diseases. To date all reported EP2 antagonists have a purely competitive mode of action, which can be overcome by high concentrations of agonist. Herein, we report the characterization of a novel compound belongs to substituted pyrroloquinoxaline scaffold as the first EP2 antagonist that features a noncompetitive mode of action. Cell‐based time‐resolved fluorescence resonance energy transfer (TR‐FRET) assays were developed in 384‐well format to measure the accumulation of cyclic adenosine monophosphate (cAMP) induced by PGE2, on the basis of the fact that activation of the Gas‐coupled EP2 receptor induces cAMP formation. At a low concentration (3 μM) of the pyrroloquinoxaline antagonist, a saturating concentration of PGE2 (3 μM) could reverse the inhibition of EP2 receptor activation in human EP2‐overexpressing C6 glioma (C6G‐EP2) cells. At higher antagonist concentrations (10–30 μM), the inhibition of EP2 receptors could not be fully overcome by the agonist. Meanwhile, the maximal agonist response inhibition by the antagonist increased with increasing concentration. The maximum effect (cAMP induction) by PGE2 was decreased to 81.9 ±1.44%, unlike 100% for a typical competitive antagonist. The antagonist itself had no effect on the TR‐FRET signal since there was no change in the TR‐FRET signal in the absence of C6G‐EP2 cells. The antagonist was reversible as demonstrated by washing out the antagonist three times each at 80% efficiency. After washing out the antagonist, the maximal response of C6G‐EP2 cells to PGE2 increased to 97.6 ± 0.69% from 86.4 ± 0.85% (p &lt; 0.0001, one‐way ANOVA with Bonferroni). Furthermore, the antagonist exhibited little to no effect on the EC50 or maximal response of other Gas‐coupled prostanoid receptors (EP4, IP, and DP1). The agonist EC50s for EP2, EP4, IP and DP1 receptors were shifted 13.2, 2.2, 1.1 and 0.95‐fold to the right, respectively, in 30 μM antagonist. To determine whether the noncompetitive antagonist exerts an anti‐inflammatory action we studied the expression of IL‐6 and IL‐1β in the murine BV2 microglial cell line stably expressing human EP2 receptors (BV2‐hEP2). Treatment of these cells with a &gt;EC90 concentration (30 nM) of the EP2 selective agonist, ONO‐AE1‐259, caused elevated mRNA levels of the two cytokines (measured by qRT‐PCR) that was suppressed by the antagonist (20 μM). Thus, the pyrroloquinoxaline is the first noncompetitive EP2 receptor antagonist. This compound may provide a basis for further development of novel noncompetitive EP2 antagonists.Support or Funding InformationThis work was supported by the National Institutes of Health [Grant Numbers 1R01NS097776 (RD), 1 U01 AG052460 (TG)]. C.J. is a member of the joint training program between Emory University School of Medicine and Xiangya School of Medicine, Central South University, and is supported by the China Scholarship Council. We gratefully acknowledge the gift of ONO‐AE1‐259 by Ono Pharmaceuticals.

Blockade of thymic stromal lymphopoietin and CRTH2 attenuates airway inflammation in a murine model of allergic asthma.

Background/AimsThymic stromal lymphopoietin (TSLP) is an epithelial cell-derived cytokine that plays a key role in Th2-mediated inflammation, both directly by promoting the proliferation of naïve CD4 Th2 cells, and indirectly by activating dendritic cells (DCs). TSLP-activated DCs induce the expansion of chemoattractant receptor homologous molecule expressed on Th2 (CRTH2)+ CD4+ Th2 memory cells, which undergo a Th2 response and express prostaglandin D2 (PGD2) synthase. CRTH2, a PGD2 receptor, is a selective Th2-cell surface marker. We investigated the effects of an anti-TSLP antibody (Ab) and a CRTH2 antagonist, as well as their mechanisms of action, in a mouse model of acute asthma.MethodsBALB/c mice were sensitized and challenged with ovalbumin. We then evaluated the effects of the administration of an anti-TSLP Ab either alone or together with a CRTH2 antagonist on cell counts, Th2 cytokine levels in bronchoalveolar fluid, and the levels of epithelium-derived cytokines such as TSLP, interleukin (IL) 33, and IL-25 in lung homogenates, as well as airway hyper-responsiveness (AHR).ResultsAnti-TSLP Ab and the CRTH2 antagonist significantly attenuated eosinophilic airway inflammation, AHR, and the expression of Th2 cytokines. The expression of GATA-3 and the levels of IL-33 and IL-25 in lung tissues were affected by the combined anti-TSLP and CRTH2 antagonist treatment.ConclusionsThese results suggest that the dual blockade of TSLP and CRTH2 may serve as an effective treatment target for eosinophilic asthma.

Lipocalin-type prostaglandin D2 synthase deletion induces dyslipidemia and non-alcoholic fatty liver disease

Non-alcoholic fatty liver disease (NAFLD) is an emerging risk factor for type 2 diabetes mellitus, cardiovascular disease, and all-cause mortality. Previously, we demonstrated that lipocalin-type prostaglandin D2 synthase (L-PGDS) knockout mice show increased glucose intolerance and accelerated atherosclerosis. In the present study, we investigated the role of L-PGDS in mediating NAFLD utilizing L-PGDS knockout (KO) and control C57BL/6 mice fed either low fat (LFD) or high fat diet (HFD) for 14 weeks. Our present study demonstrates that L-PGDS KO mice remain slightly lighter in weight compared to control mice, yet develop NAFLD faster and eventually progress to the more severe non-alcoholic steatohepatitis (NASH). We found increased lipid accumulation in the liver of KO mice over time on both diets, as compared to control mice. The L-PGDS KO mice showed elevated fasting glucose and insulin levels and developed insulin resistance on both LFD and HFD. Lipogenesis marker proteins such as SREBP-1c and LXRα were increased in L-PGDS KO mice after 14 weeks on both diets, when compared to control mice. We replicated our in vivo findings in vitro using HepG2 cells treated with a combination of free fatty acids (oleic and palmitic acid) and exposure to a L-PGDS inhibitor and prostaglandin D2 receptor (DP1) antagonists. We conclude that the absence or inhibition of L-PGDS results in dyslipidemia, altered expression of lipogenesis genes and the acceleration of NAFLD to NASH, independent of diet and obesity. We propose L-PGDS KO mice as a useful model to explore the pathogenesis of NAFLD and NASH, and L-PGDS as a potential therapeutic target for treatment.

Effect of levocetirizine hydrochloride on the growth of human dermal papilla cells: a preliminary study.

To conduct an in vitro investigation into the effect of different concentrations of levocetirizine hydrochloride on the growth of human dermal papilla cells (hDPCs) the underlying mechanisms involved. hDPCs were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing different concentrations of levocetirizine hydrochloride for 48 h. The growth of hDPCs was observed by immunofluorescence staining, and the cell proliferation was detected by MTT assay. After the hDPCs were cultured in DMEM containing 1, 10, 100, 1,000, and 10,000 ng/mL levocetirizine hydrochloride for 48 h, the mRNA expressions of cyclooxygenase 2 (COX-2), prostaglandin D2 synthase (PTGDS), prostaglandin E2 (PGE2), prostaglandin F2α (PGF2α), G protein-coupled receptor 44 (GPR44), protein kinase B (AKT), and glycogen synthase kinase 3β (GSK3β) were determined by real-time fluorescence-based quantitative polymerase chain reaction (PCR), and the protein expressions of PTGDS, phosphorylated protein kinase B (pAKT), and phosphorylated glycogen synthase kinase 3β (pGSK3β) were detected by Western blotting. After the hDPCs were cultured in DMEM containing 1, 10, 100, 1,000, 10,000 ng/mL levocetirizine hydrochloride for 24 h, the secretion levels of prostaglandin D2 (PGD2) and PGD2 receptor (PGD2R) in the culture supernatant were determined by enzyme-linked immunosorbent assay (ELISA). One-way analysis of variance (ANOVA) was performed using SPSS 17.0 software, and the LSD-t test was used for pairwise comparisons. Immunofluorescence staining showed that hDPCs in the 100 ng/mL group grew well, with over 90% confluency. Methyl thiazolyl tetrazolium (MTT) method showed that the proliferation rate of hDPCs significantly differed between different levocetirizine hydrochloride groups and the blank control group (F=42.22, P<0.05), while the proliferation rate was significantly higher in the 100 ng/mL group (115.80%±5.10%) than in the blank control group (100%) (t=28.26, P<0.05). The relative mRNA expressions of COX-2, PGF2a, PTGDS, GPR44, and AKT showed significant differences in different levocetirizine hydrochloride groups (the F values were 1.97, 3.66, 2.17, 2.66, and 7.32, respectively; all P<0.05), whereas the mRNA expressions of PGE2 and GSK3β showed no significant difference (F=0.87, F=1.19, respectively; both P>0.05). The mRNA expressions of COX-2, PTGDS, and GPR44 in the 100 ng/mL group (0.840.08, 0.810.10, and 0.85±0.09, respectively) were significantly lower than those in the blank control group (t=1.97, t=2.17, and t=2.65, respectively; all P<0.05), whereas the mRNA expressions of PGF2α and AKT in the 100 ng/mL group (1.96±0.25 and 1.74±0.32, respectively) were significantly higher than those in the blank control group (t=3.662 and t=7.325, respectively; both P<0.05). There were significant differences in the levels of PTGDS, pAKT, pGSK3β, PGD2, and PGD2R proteins between the different levocetirizine hydrochloride groups (the F values were 11.84, 3.89, 4.07, 66.15, and 44.33, respectively). The protein expressions of PTGDS, PGD2, and PGD2R in the 100 ng/mL group (0.32±0.05, 141.62±5.44, and 215.08±9.55, respectively) were significantly lower than those in the blank control group (0.73±0.06, 180.08±6.15, and 273.24±3.18, respectively) (the t values were 5.66, 45.07, and 92.05, respectively; all P<0.05), whereas the protein expressions of pAKT and pGSK3β in the 100 ng/mL group (0.59±0.05 and 0.46±0.03, respectively) were significantly higher than those in the blank control group (0.46±0.02 and 0.35±0.042, respectively) (t=16.59, t=7.73, respectively; both P<0.05). Levocetirizine hydrochloride may promote the growth and proliferation of hDPC in vitro by inhibiting the PGD2-GPR44 pathway and activating the AKT signaling pathway.

Identification of DNA methylation-driven genes by integrative analysis of DNA methylation and transcriptome data in pancreatic adenocarcinoma.

Pancreatic adenocarcinoma (PAAD) is a painful and fatal disease that undoubtedly remains a health care priority and offers significant therapeutic challenges. The significance of epigenetic modifications, including DNA methylation in tumor development, has gained the attention of researchers. Identifying DNA methylation-driven genes and investigating the mechanisms underlying the tumorigenesis of PAAD are of substantial importance for developing methods of physiological evaluation, treatment planning and prognostic prediction for PAAD. In the present study, a comprehensive analysis of DNA methylation and gene expression data from 188 clinical samples was performed to identify DNA methylation-driven genes in PAAD. In addition, the diagnostic and prognostic value of DNA methylation-driven genes was evaluated using receiver operating characteristic curve, survival and recurrence analyses. A total of 7 DNA methylation-driven genes, namely zinc finger protein 208 (ZNF208), eomesodermin (EOMES), prostaglandin D2 receptor (PTGDR), chromosome 12 open reading frame 42 (C12orf42), integrin subunit α 4 (ITGA4), dedicator of cytokinesis 8 and protein phosphatase 1 regulatory inhibitor subunit 14D (PPP1R14D), were identified. All of them may be used to diagnose PAAD with excellent specificity and sensitivity (area under curve, >0.8). Of the 7 DNA methylation-driven genes, 6 were significantly associated with overall survival (OS) and recurrence-free survival (RFS) P<0.05). Among them, ZNF208, EOMES, PTGDR, C12orf42 and ITGA4 were significantly negatively associated with the OS rate and positively associated with the recurrence rate, while PPP1R14D was significantly positively associated with the OS rate and negatively associated with the recurrence rate. The present study provides novel insight into the epigenetic alterations associated with the occurrence and progression of PAAD, thereby increasing the mechanistic understanding of this disease, offering potential novel molecular biomarkers and contributing to the development of therapeutic targets for PAAD.

Steroids Inhibit Eosinophil Accumulation and Downregulate Hematopoietic Chemotaxic Prostaglandin D2 Receptor in Aspirin-Exacerbated Respiratory Disease.

Aspirin-exacerbated respiratory disease (AERD) is characterized by eosinophilic rhinosinusitis, nasal polyposis, aspirin sensitivity, and asthma. Aims/Objectives: This study aims to identify a mechanism to target for the future treatment of AERD via the elucidation of the effect of systemic steroids on the expression of hematopoietic prostaglandin D2 synthase (HPGDS) and chemotaxic prostaglandin D2 (DP2) receptor relative to eosinophil activation in the nasal polyps of patients with AERD. Among 37 patients undergoing endoscopic sinus surgery, 28 received systemic steroids preoperatively. Nasal polyps were harvested from all 37 patients. After routine processing of paraffin sections, immunohistochemistry was performed using specific antibodies for HPGDS, eosinophil peroxidase (EPX), and DP2. Expression of HPGDS, DP2, and EPX by eosinophils was higher and more frequent in patients with non-preoperative steroid therapy. Likewise, HPGDS and DP2 were highly expressed in activated eosinophils in the nasal polyps, but not in normal eosinophils. This study provides clear evidence that systemic steroid therapy inhibits eosinophil activation and decreases HPGDS and DP2 expression in patients with AERD, indicating a reduction in prostaglandin D2 production and hence control hyperplasia of nasal polyps.

The Prostaglandin D2 Receptor CRTH2 Promotes IL-33-Induced ILC2 Accumulation in the Lung.

Group 2 innate lymphoid cells (ILC2s) are rare innate immune cells that accumulate in tissues during allergy and helminth infection, performing critical effector functions that drive type 2 inflammation. ILC2s express ST2, the receptor for the cytokine IL-33, and chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2), a receptor for the bioactive lipid prostaglandin D2 (PGD2). The IL-33-ST2 and the PGD2-CRTH2 pathways have both been implicated in promoting ILC2 accumulation during type 2 inflammation. However, whether these two pathways coordinate to regulate ILC2 population size in the tissue in vivo remains undefined. In this study, we show that ILC2 accumulation in the murine lung in response to systemic IL-33 treatment was partially dependent on CRTH2. This effect was not a result of reduced ILC2 proliferation, increased apoptosis or cell death, or differences in expression of the ST2 receptor in the absence of CRTH2. Rather, data from adoptive transfer studies suggested that defective accumulation of CRTH2-deficient ILC2s in response to IL-33 was due to altered ILC2 migration patterns. Whereas donor wild-type ILC2s preferentially accumulated in the lungs compared with CRTH2-deficient ILC2s following transfer into IL-33-treated recipients, wild-type and CRTH2-deficient ILC2s accumulated equally in the recipient mediastinal lymph node. These data suggest that CRTH2-dependent effects lie downstream of IL-33, directly affecting the migration of ILC2s into inflamed lung tissues. A better understanding of the complex interactions between the IL-33 and PGD2-CRTH2 pathways that regulate ILC2 population size will be useful in understanding how these pathways could be targeted to treat diseases associated with type 2 inflammation.

Screening for autoantibody targets in post-vaccination narcolepsy using proteome arrays.

Narcolepsy type 1 (NT1) is a chronic sleep disorder caused by a specific loss of hypocretin-producing neurons. The incidence of NT1 increased in Sweden, Finland and Norway following Pandemrix®-vaccination, initiated to prevent the 2009 influenza pandemic. The pathogenesis of NT1 is poorly understood, and causal links to vaccination are yet to be clarified. The strong association with Human leukocyte antigen (HLA) DQB1*06:02 suggests an autoimmune pathogenesis, but proposed autoantigens remain controversial. We used a two-step approach to identify autoantigens in patients that acquired NT1 after Pandemrix®-vaccination. Using arrays of more than 9000 full-length human proteins, we screened the sera of 10 patients and 24 healthy subjects for autoantibodies. Identified candidate antigens were expressed in vitro to enable validation studies with radiobinding assays (RBA). The validation cohort included NT1 patients (n=39), their first-degree relatives (FDR) (n=66), population controls (n=188), and disease controls representing multiple sclerosis (n=100) and FDR to type 1 diabetes patients (n=41). Reactivity towards previously suggested NT1 autoantigen candidates including Tribbles homolog 2, Prostaglandin D2 receptor, Hypocretin receptor 2 and α-MSH/proopiomelanocortin was not replicated in the protein array screen. By comparing case to control signals, three novel candidate autoantigens were identified in the protein array screen; LOC401464, PARP3 and FAM63B. However, the RBA did not confirm elevated reactivity towards either of these proteins. In summary, three putative autoantigens in NT1 were identified by protein array screening. Autoantibodies against these candidates could not be verified with independent methods. Further studies are warranted to identify hypothetical autoantigens related to the pathogenesis of Pandemrix®-induced NT1.

Are Aspirin and Apixaban Sufficient to Prevent Immunothrombosis in COVID-19?

Severe COVID-19 disease is associated with respiratory and vascular injury and microvascular immunothrombosis mediated by complement activation, inflammatory lipid storm, platelet activation, platelet-leukocyte adhesion and activation of coagulation cascade. Amongst the inflammatory lipids, thromboxane A2 (TxA2) is a known key mediator of microvascular thrombosis. The levels of TxB2, a stable metabolite of TxA2 are markedly increased in the bronchoalveolar lavage fluid and plasma in severe COVID-19 patients. Low-dose aspirin mitigates the generation of prostanoids including TxA2 by irreversible inactivation of the constitutive enzyme cyclooxygenase (COX)-1. The anti-thrombotic action of aspirin is currently being investigated in outpatient and hospitalized COVID-19 patients in the global RECOVERY and ACTIV-4 clinical trials. Several lines of investigations suggest that COX-2 plays an important and critical role in the immunothrombotic effects mediated by COVID-19. Pharmacologic inhibition of either COX-1 or COX-2 can prevent a plethora of lipid mediators of inflammation that are both pro- and anti-inflammatory in function. Thus, a more definitive approach to prevent immunothrombotic events in COVID-19 will be to directly block the prothrombotic effects of TxA2. Although thromboxane synthase (TS) inhibitors suppress TxA2 formation, accumulation of the substrate PGH2 is known to interact with the platelet and vessel wall TxA2 prostanoid receptor (TPR), thus reducing the antiplatelet effects of TS inhibitors. TPR antagonists block the activity of both TxA2 and PGH2 on platelets and vessels but do not block TxA2 production, which leads to increased generation of 11-dehydro-thromboxane B2, a stable metabolite of TxA2, and a potent agonist of the DP2 (CRTH2) receptor for prostaglandin D2 (PGD2). PGD2/DP2 receptor signaling has been implicated in immune dysregulation in viral infections including COVID-19. Ramatroban, an orally bioavailable, potent, dual TxA2/TP and PGD2/DP2 receptor antagonist, has demonstrated efficacy in a variety of animal models of atherosclerosis, thrombosis and sepsis. Ramatroban has a proven safety profile, having been used in Japan over the past 20 years as a treatment of allergic rhinitis and therefore, merits investigation as a promising antithrombotic and immunomodulator agent for chemoprophylaxis and treatment in COVID-19 patients.

Ramatroban as a Novel Immunotherapy for COVID-19.

SARS-CoV-2 virus suppresses host innate and adaptive immune responses, thereby allowing the virus to proliferate, and cause multiorgan failure, especially in the elderly. Respiratory viruses stimulate cyclooxygenase-2 (COX-2) to generate prostanoids including Prostaglandin D2 (PGD2) and thromboxane A2. Furthermore, PGD2 concentrations in the airways increase with aging. PGD2 action mediated via DP2 receptors suppresses both innate and adaptive immune responses, by inhibiting interferon-λ and stimulation of myeloid monocyte-derived suppressor cells respectively. PGD2 and thromboxane A2 actions via the TP receptors activate platelets leading to a prothrombotic state. Ramatroban, a small-molecule antagonist of DP2 and TP receptors, reverses viremia-associated proinflammatory, immunosuppressive5 and prothrombotic processes which are similar to those induced by SARS-Cov-2. Ramatroban, used for the treatment of allergic rhinitis in Japan for the past 20 years has an excellent safety profile. Therefore, Ramatroban merits investigation as a novel immunotherapy for the treatment of COVID-19 disease.

Expression of Th1/Th2 cell-related chemokine receptors on CD4+ lymphocytes under physiological conditions.

Chemokine receptors (CRs) and the prostaglandin D2 receptor, CRTH2, have been used as surrogate markers for cytoplasmic Th1 and Th2 cytokines. The presence of regulatory T (Treg) and Th17 cells may affect the analysis of such surrogate markers, as they share several CRs with Th1 and Th2 cells. This study aimed to determine the optimal surrogate markers of Th1 and Th2 cells under physiological conditions. Surface and cytoplasmic markers of CD4+ peripheral lymphocytes were analyzed in healthy volunteers by flow cytometry. Th1, Th2, Th17, and Treg cells were identified as IFN-γ+ , IL-4+ IL-13+ , IL-17+ , and CD25+ FoxP3+ CD4+ lymphocytes, respectively. The percentages of CXCR3+ and CCR5+ CD4+ lymphocytes clearly correlated with those of Th1 cells. The percentage of CRTH2+ CD4+ lymphocytes showed the closest correlation with that of Th2 cells. The percentages of Th2 cells correlated with those of CCR3+ or CCR8+ CD4+ lymphocytes, with the majority of CCR3+ and CCR8+ cells unlikely to be Th2 cells, themselves. The proportions of CCR4+ or CCR7+ CD4+ lymphocytes did not correlate with those of Th2 cells, possibly due to their expression on the surface of Treg and Th17 cells. Th2-related receptors were classified into three different groups for better understanding. CXCR3 and CCR5 are useful markers of Th1 cells. With the exception of CCR4 and CCR7 expressed at measurable levels on Treg and Th17 cells, CRTH2 and CRs, CCR3, and CCR8 may be employed as surrogate markers of Th2 cells. The proposed surrogate markers may help physicians in interpreting the disease state.

G Protein-Coupled Receptors in Asthma Therapy: Pharmacology and Drug Action.

Asthma is a heterogeneous inflammatory disease of the airways that is associated with airway hyperresponsiveness and airflow limitation. Although asthma was once simply categorized as atopic or nonatopic, emerging analyses over the last few decades have revealed a variety of asthma endotypes that are attributed to numerous pathophysiological mechanisms. The classification of asthma by endotype is primarily routed in different profiles of airway inflammation that contribute to bronchoconstriction. Many asthma therapeutics target G protein–coupled receptors (GPCRs), which either enhance bronchodilation or prevent bronchoconstriction. Short-acting and long-acting β2-agonists are widely used bronchodilators that signal through the activation of the β2-adrenergic receptor. Short-acting and long-acting antagonists of muscarinic acetylcholine receptors are used to reduce bronchoconstriction by blocking the action of acetylcholine. Leukotriene antagonists that block the signaling of cysteinyl leukotriene receptor 1 are used as an add-on therapy to reduce bronchoconstriction and inflammation induced by cysteinyl leukotrienes. A number of GPCR-targeting asthma drug candidates are also in different stages of development. Among them, antagonists of prostaglandin D2 receptor 2 have advanced into phase III clinical trials. Others, including antagonists of the adenosine A2B receptor and the histamine H4 receptor, are in early stages of clinical investigation. In the past decade, significant research advancements in pharmacology, cell biology, structural biology, and molecular physiology have greatly deepened our understanding of the therapeutic roles of GPCRs in asthma and drug action on these GPCRs. This review summarizes our current understanding of GPCR signaling and pharmacology in the context of asthma treatment.SIGNIFICANCE STATEMENTAlthough current treatment methods for asthma are effective for a majority of asthma patients, there are still a large number of patients with poorly controlled asthma who may experience asthma exacerbations. This review summarizes current asthma treatment methods and our understanding of signaling and pharmacology of G protein–coupled receptors (GPCRs) in asthma therapy, and discusses controversies regarding the use of GPCR drugs and new opportunities in developing GPCR-targeting therapeutics for the treatment of asthma.

CD8+ Tc2 cells: underappreciated contributors to severe asthma.

The complexity of asthma is underscored by the number of cell types and mediators implicated in the pathogenesis of this heterogeneous syndrome. Type 2 CD4+ T-cells (Th2) and more recently, type 2 innate lymphoid cells dominate current descriptions of asthma pathogenesis. However, another important source of these type 2 cytokines, especially interleukin (IL)-5 and IL-13, are CD8+ T-cells, which are increasingly proposed to play an important role in asthma pathogenesis, because they are abundant and are comparatively insensitive to corticosteroids. Many common triggers of asthma exacerbations are mediated via corticosteroid-resistant pathways involving neutrophils and CD8+ T-cells. Extensive murine data reveal the plasticity of CD8+ T-cells and their capacity to enhance airway inflammation and airway dysfunction. In humans, Tc2 cells are predominant in fatal asthma, while in stable state, severe eosinophilic asthma is associated with greater numbers of Tc2 than Th2 cells in blood, bronchoalveolar lavage fluid and bronchial biopsies. Tc2 cells strongly express CRTH2, the receptor for prostaglandin D2, the cysteinyl leukotriene receptor 1 and the leukotriene B4 receptor. When activated, these elicit Tc2 cell chemotaxis and production of chemokines and type 2 and other cytokines, resulting directly or indirectly in eosinophil recruitment and survival. These factors position CD8+ Tc2 cells as important and underappreciated effector cells contributing to asthma pathogenesis. Here, we review recent advances and new insights in understanding the pro-asthmatic functions of CD8+ T-cells in eosinophilic asthma, especially corticosteroid-resistant asthma, and the molecular mechanisms underlying their pathologic effector function.

Mesenchymal COX2-Derived PGD2 Activates an ILC2-Treg Axis to Promote Proliferation of Normal and Malignant HSPCs

Cyclooxygenase (COX)-dependent production of prostaglandins (PGs) is known to play important roles in tumorigenesis. PGD2 has recently emerged as a key regulator of tumor- and inflammation-associated functions. We previously reported that mesenchymal stromal cells (MSCs) from patients with acute myeloid leukemia (AML) overexpressed COX-2 and secreted high levels of PGs including PGD2. Since little is known about the role of PGD2 in normal and malignant hematopoiesis, we prioritized this mesenchymal source of PG for further investigation. We observed that AML MSCs or normal MSCs overexpressing COX-2 promotes proliferation of co-cultured hematopoietic stem and progenitor cells (HSPCs), which can be prevented by treatment with COX-2 knockdown or TM30089, a specific antagonist of the PGD2 receptor CRTH2. Mechanistically, we demonstrate that PGD2-CRTH2 signaling acts directly on type 2 innate lymphoid cells (ILC2s), potentiating their expansion and driving them to produce Interleukin-5 (IL-5) and IL-13. We further show that IL-5 but not IL-13 expands CD25+Foxp3+ IL5Ra+ T regulatory cells (Tregs) and promotes HSCP proliferation. Disruption of the PGD2-activated ILC2-Treg axis by specifically blocking the PGD2 receptor CRTH2 or IL-5 impedes proliferation of normal and malignant HSPCs. Conversely, co-transfer of Lin-CD127+CRTH2+ ILC2s and CD4+CD25+IL5Ra+ Tregs promotes malignant HSCP proliferation and accelerates leukemia development in xenotransplanted mice. Collectively, these results indicate that the mesenchymal source of PGD2 promotes proliferation of normal and malignant HSPCs through activation of the ILC2-Treg axis. These findings also suggest that this PGD2-activated ILC2-Treg axis may be a valuable therapeutic target for cancer and inflammation-associated diseases. Disclosures No relevant conflicts of interest to declare.

L-type prostaglandin D synthase regulates the trafficking of the PGD2 DP1 receptor by interacting with the GTPase Rab4

Accumulating evidence indicates that G protein-coupled receptors (GPCRs) interact with Rab GTPases during their intracellular trafficking. How GPCRs recruit and activate the Rabs is unclear. Here, we report that depletion of endogenous L-type prostaglandin D synthase (L-PGDS) in HeLa cells inhibited recycling of the prostaglandin D2 (PGD2) DP1 receptor (DP1) to the cell surface after agonist-induced internalization and that L-PGDS overexpression had the opposite effect. Depletion of endogenous Rab4 prevented l-PGDS-mediated recycling of DP1, and l-PGDS depletion inhibited Rab4-dependent recycling of DP1, indicating that both proteins are mutually involved in this pathway. DP1 stimulation promoted its interaction through its intracellular C terminus with Rab4, which was increased by l-PGDS. Confocal microscopy revealed that DP1 activation induces l-PGDS/Rab4 co-localization. l-PGDS/Rab4 and DP1/Rab4 co-immunoprecipitation levels were increased by DP1 agonist treatment. Pulldown assays with purified GST-l-PGDS and His6-Rab4 indicated that both proteins interact directly. l-PGDS interacted preferentially with the inactive, GDP-locked Rab4S22N variant rather than with WT Rab4 or with constitutively active Rab4Q67L proteins. Overexpression and depletion experiments disclosed that l-PGDS partakes in Rab4 activation following DP1 stimulation. Experiments with deletion mutants and synthetic peptides revealed that amino acids 85-92 in l-PGDS are involved in its interaction with Rab4 and in its effect on DP1 recycling. Of note, GTPγS loading and time-resolved FRET assays with purified proteins suggested that l-PGDS enhances GDP-GTP exchange on Rab4. Our results reveal how l-PGDS, which produces the agonist for DP1, regulates DP1 recycling by participating in Rab4 recruitment and activation.

Prostaglandin D2 stimulates phenotypic changes in vascular smooth muscle cells

Since chronic inflammation is associated with the pathogenesis of atherosclerosis, inflammatory cytokines might contribute to the phenotypic modulation of vascular smooth muscle cells (VSMCs). Tumor necrosis factor α (TNFα) facilitated the transformation of contractile VSMCs to the synthetic phenotype, as determined by the expression of marker proteins and a collagen gel contraction assay. Western blot analysis and a cyclooxygenase-2 (COX2) promoter assay revealed that TNFα stimulation resulted in the induction of COX2. The overexpression, silencing, or pharmacological inhibition of COX2 significantly affected TNFα-induced phenotypic conversion, and of the tested prostaglandins, only PGD2 significantly induced phenotypic conversion. ERK was significantly activated by PGD2 stimulation, and the pharmacological inhibition of ERK blocked the PGD2-induced phenotypic conversion of VSMCs. However, antagonists or agonists of PGD2 receptors did not affect VSMC conversion. In contrast, spontaneously dehydrated forms of PGD2, such as PGJ2, Δ12-PGJ2, and 15-d-PGJ2, strongly induced phenotypic conversion. A reporter gene assay showed that TNFα, PGD2, and 15-d-PGJ2 significantly activated the peroxisome proliferator-responsive element (PPRE) promoter. In addition, the overexpression or silencing of peroxisome proliferator-activated receptor δ (PPARδ) significantly influenced 15-d-PGJ2-induced phenotypic conversion. Finally, atherosclerotic neointima formation was significantly suppressed in mice lacking TNFα. In addition, mice fed celecoxib exhibited complete inhibition of carotid artery ligation-induced neointima formation. This study shows that PGD2 regulates the phenotypic conversion of VSMCs by generating an endogenous ligand of PPAR, and that this leads to neointima formation in occlusive arterial disease.