Journal: Nature Communications

Article Title: Post-resolution macrophages shape long-term tissue immunity and integrity in a mouse model of pneumococcal pneumonia

doi: 10.1038/s41467-024-48138-y

Figure Lengend Snippet: A Osmotic pumps loaded with either the pan COX inhibitor naproxen or the EP4 antagonist MF498 were implanted into mice once inflammation resolved (day 4) and their effects of blocking the biological action of post-resolution PGE 2 was established 6 weeks later as determined by measuring ( B ) late effector T cell numbers and ( C ) their effector function as well as ( D ) early effector T cells and their expression of ( E , F ) the α-integrin, CD103. as a marker of T cell residence potential. Linking PGE 2 with this post-resolution T cell phenotype, T cells were incubated PGE 2 and its direct effect on ( G ) CD103 expression or with other cytokines expressed during post-resolution that are known to affect lymphocyte function, ( H ) namely TGFβ, were examined for their collective effects on intracellular ( I ) IL-17 and ( J ) IFNγ. Data were analysed by one-way analysis of variance (ANOVA) and Tukey’s multiple comparisons test. A p value of <0.05 was taken as the threshold of significance with graphical representation as; p < 0.05 = *, p < 0.01 = ** and p < 0.001 = *** and presented as mean ± SEM ( n = 3–6 mice/group).

Article Snippet: The EP4 receptor antagonist MF498 (30 mg/kg, Cayman Chemical) was delivered via osmotic pump infusion over a period of 14 days.

Techniques: Blocking Assay, Expressing, Marker, Incubation

Journal: Nature Communications

Article Title: Post-resolution macrophages shape long-term tissue immunity and integrity in a mouse model of pneumococcal pneumonia

doi: 10.1038/s41467-024-48138-y

Figure Lengend Snippet: A Minipumps loaded with either the pan COX inhibitor naproxen or the EP4 antagonist MF498 were implanted into mice post-resolution four days after intranasal S. pneumoniae . 6 weeks after the initial challenge (or 38 days after this intervention), these sensitised mice or their controls were challenged with S. pneumoniae and impact of this intervention on ( B ) CD4 + /CD44 + /CD62L + and ( C ) CD4 + /CD44 + /CD62L - /CD27 + T cell numbers was determined 24 h later. Indeed, levels of expression of ( D ) CD103 and the T cell activation markers ( E ) CD69 and ( F ) CD44 was also determined on CD4 + /CD44 + /CD62L - /CD27 + T cells. Besides lymphocytes we determined how inhibition of PGE 2 synthesis or EP4 antagonism also impacted on the ability to recruit ( G ) neutrophils and ultimately clear the secondary challenge of ( H ) S. pneumoniae . Data were analysed by one-way analysis of variance (ANOVA) and Tukey’s multiple comparisons test. A p value of <0.05 was taken as the threshold of significance with graphical representation as; p < 0.05 = *, p < 0.01 = ** and p < 0.001 = *** and presented as mean ± SEM ( n = 4–5 mice/group).

Article Snippet: The EP4 receptor antagonist MF498 (30 mg/kg, Cayman Chemical) was delivered via osmotic pump infusion over a period of 14 days.

Techniques: Expressing, Activation Assay, Inhibition

Journal: Nature Communications

Article Title: Post-resolution macrophages shape long-term tissue immunity and integrity in a mouse model of pneumococcal pneumonia

doi: 10.1038/s41467-024-48138-y

Figure Lengend Snippet: The dosing regime in ( A ) revealed the impact of therapeutically antagonising post-resolution EP4 on ( B ) fibrosis along with examples of vascular occlusion (arrows) and ( C ) macrophage infiltration by confocal microscopy as well as ( D – G ) flow cytometry at day 14. These infiltrated macrophages were examined for their ( H ) phagocytic ability. The infiltration of post-resolution macrophages was associated with a second wave of ( I , J ) CCL2 expression with its receptor ( K , L ) CCR2 being negatively controlled by ( M ) EP4. A Alveoli, B bronchiole, BV blood vessel. Students unpaired t -test was used to compare the means of two groups. Differences between multiple groups were analysed using one-way analysis of variance (ANOVA) and Tukey’s multiple comparisons test. A p value of <0.05 was taken as the threshold of significance with graphical representation as; p < 0.05 = *, p < 0.01 = ** and p < 0.001 = *** and presented as mean ± SEM ( n = 3–5 mice/group).

Article Snippet: The EP4 receptor antagonist MF498 (30 mg/kg, Cayman Chemical) was delivered via osmotic pump infusion over a period of 14 days.

Techniques: Confocal Microscopy, Flow Cytometry, Expressing

Journal: The Journal of Allergy and Clinical Immunology

Article Title: Prostaglandin E 2 suppresses human group 2 innate lymphoid cell function

doi: 10.1016/j.jaci.2017.09.050

Figure Lengend Snippet: Human tonsillar ILC2s selectively express EP2 and EP4 receptors. A, EP receptor transcripts of freshly sorted human tonsillar ILC2s were detected by using single-cell RNA-seq. Expression distribution (violin plots) are shown for genes of PGE 2 receptors ( PTGER1 to PTGER4 ) in each ILC subset (group 1 to group 3 ILCs and NK cells), where expression patterns were obtained as reads per kilobase gene model and million mappable reads. B, mRNA expression of EP receptors ( PTGER1 to PTGER4 ) in sorted and expanded tonsillar ILC2s was determined by using RT-qPCR (n = 5). C, mRNA expression of EP2 and EP4 receptors after 24 hours of stimulation normalized to nonstimulated cells. Graphs show mean + SEM expression (n = 5).

Article Snippet: The EP2 receptor antagonist PF-04418948 and the EP4 receptor antagonist ONO AE3-208 (both from Cayman Chemicals) were added 20 minutes before PGE 2 in 1 μmol/L concentration.

Techniques: RNA Sequencing, Expressing, Quantitative RT-PCR

Journal: The Journal of Allergy and Clinical Immunology

Article Title: Prostaglandin E 2 suppresses human group 2 innate lymphoid cell function

doi: 10.1016/j.jaci.2017.09.050

Figure Lengend Snippet: PGE 2 reduces IL-5 and IL-13 production in ILC2s through activation of EP2 and EP4 receptors. Sorted and expanded tonsillar ILC2s were stimulated as indicated for 24 hours. The EP2 receptor antagonist PF-04418948 ( PF ; 1 μmol/L) and the EP4 receptor antagonist ONO AE3-208 ( ONO ; 1 μmol/L) were added separately or together 20 minutes before PGE 2 . A and B, Concentrations of released IL-5 (Fig 6, A ) and IL-13 (Fig 6, B ) in ILC2 supernatants were determined by means of ELISA. Bar graphs show means + SEMs. (n = 6-7). * P < .05 and ** P < .01. C, Representative dot plots show intracellular expression of IL-5 and IL-13 assessed by using flow cytometry. D and E, Graphs show mean + SEM percentages of IL-5 + (Fig 6, D ) and IL-13 + (Fig 6, E ) ILC2s (n = 6). * P < .05 and ** P < .01. Expression of GATA-3 was analyzed by using flow cytometry. F and G, Graphs show geometric mean + SEM fluorescence intensity (Fig 6, F ) and geometric mean fluorescence intensity normalized to nonstimulated cells + SEM (Fig 6, G ) of GATA-3 expression (n = 5). * P < .05. H and I, Released IL-5 (Fig 6, H ) and IL-13 (Fig 6, I ) are shown after 10 minutes of pretreatment with the EP2 receptor agonist butaprost (But) and the EP4 receptor agonist L-902,688 (L9) separately or together in 100 nmol/L concentrations. Concentrations were determined by means of ELISA and are shown as means ± SEMs (n = 4; compared to interleukin treatment). * P < .05 and ** P < .01.

Article Snippet: The EP2 receptor antagonist PF-04418948 and the EP4 receptor antagonist ONO AE3-208 (both from Cayman Chemicals) were added 20 minutes before PGE 2 in 1 μmol/L concentration.

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Expressing, Flow Cytometry, Fluorescence

Journal: The Journal of Allergy and Clinical Immunology

Article Title: Prostaglandin E 2 suppresses human group 2 innate lymphoid cell function

doi: 10.1016/j.jaci.2017.09.050

Figure Lengend Snippet: Dual engagement of EP2 and EP4 receptors is essential for the PGE 2 -induced reduction of ILC2 cytokine production. Sorted and expanded tonsillar ILC2s were stimulated as indicated for 24 hours. Additionally, the EP2 receptor antagonist PF-04418948 (PF) and the EP4 receptor antagonist ONO AE3-208 (ONO) were added separately or together in different concentrations (100 nmol/L, 300 nmol/L, 1 μmol/L, and 3 μmol/L) 20 minutes before PGE 2 . A and B, Concentrations of released IL-5 ( , A ) and IL-13 ( , B ) in ILC2 supernatants were determined by using ELISA and shown as means ± SEM (n = 4). C and D, Sorted and expanded tonsillar ILC2s were treated with the EP2 receptor agonist butaprost (But) and the EP4 receptor agonist L-902,688 (L9) separately or together in different concentrations (10 nmol/L, 30 nmol/L, 100 nmol/L, 300 nmol/L, and 1 μmol/L) 10 minutes before the stimulatory cytokines. Concentrations of released IL-5 ( , C ) and IL-13 ( , D ) in ILC2 supernatants were determined by using ELISA and are shown as means ± SEMs (n = 4; compared with IL treatment). * P < .05 and ** P < .01. ns , Nonstimulated.

Article Snippet: The EP2 receptor antagonist PF-04418948 and the EP4 receptor antagonist ONO AE3-208 (both from Cayman Chemicals) were added 20 minutes before PGE 2 in 1 μmol/L concentration.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: The Journal of Allergy and Clinical Immunology

Article Title: Prostaglandin E 2 suppresses human group 2 innate lymphoid cell function

doi: 10.1016/j.jaci.2017.09.050

Figure Lengend Snippet: Activation of EP2 and EP4 receptors inhibits ILC2 cytokine release, likely through preventing ILC2 proliferation. Sorted and expanded tonsillar ILC2s were stimulated as indicated for 72 hours. Additionally, the EP2 receptor antagonist PF-04418948 ( PF ; 1 μmol/L) and the EP4 receptor antagonist ONO AE3-208 ( ONO ; 1 μmol/L) were added separately or together 20 minutes before PGE 2 . A, Proliferation of CellTrace violet dye–loaded ILC2s after 72 hours of incubation was analyzed by using flow cytometry. B, Data are shown as percentages of ILC2s in different division phases (D0-D4; n = 4). * P < .05 and *** P < .001. C and D, Concentrations of released IL-5 (Fig 7, C ) and IL-13 (Fig 7, D ) in ILC2 supernatants were detected by means of ELISA, and bars show means + SEMs (n = 7). * P < .05 and ** P < .01. Expression of GATA-3 was analyzed by using flow cytometry. E and F, Graphs show geometric mean fluorescence intensity + SEM (Fig 7, E ) and geometric mean fluorescence intensity normalized to nonstimulated cells + SEM (Fig 7, F ) of GATA-3 expression (n = 7). * P < .05 and ** P < .01.

Article Snippet: The EP2 receptor antagonist PF-04418948 and the EP4 receptor antagonist ONO AE3-208 (both from Cayman Chemicals) were added 20 minutes before PGE 2 in 1 μmol/L concentration.

Techniques: Activation Assay, Incubation, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Expressing, Fluorescence

Journal:

Article Title: Chemotactic action of prostaglandin E 2 on mouse mast cells acting via the PGE 2 receptor 3

doi: 10.1073/pnas.0701700104

Figure Lengend Snippet: The chemoattractant activity of PGE2 acts via the EP3 receptor. (a) Chemotaxis of 2-wk BMMC to PGE2 in the absence (■) or presence (□) of 1 μg/ml pertussis toxin. Data are mean ± SEM for n = 4. (b) Selective agonists; PGE1 alcohol (■, EP2, and EP4), 17pt PGE2 (▴, EP1), Sulprostone (▾, EP1 and EP3), Misoprostol (♦, EP2, EP3, and EP4) were added to the bottom wells of a chemotaxis plate, and 2-wk BMMC were added to the top. Data are mean ± SEM for n = 3 cell cultures. No cells migrated to assay buffer alone. (c) Real-time PCR analysis of EP1, EP2, EP3, and EP4 receptor mRNA expression in 2- and 10-wk BMMC. Data are mean ± SEM for n = 3 using RNA isolated from separate cell cultures.

Article Snippet: We thank Ms. Gill Martin for help with processing BMMC and Merck Frosst Canada Ltd., Pointe Claire–Dorval, Quebec, PQ, Canada, for the kind gift of the EP3 and EP4 receptor antagonists.

Techniques: Activity Assay, Chemotaxis Assay, Real-time Polymerase Chain Reaction, Expressing, Isolation

Journal:

Article Title: Chemotactic action of prostaglandin E 2 on mouse mast cells acting via the PGE 2 receptor 3

doi: 10.1073/pnas.0701700104

Figure Lengend Snippet: Confirmation that the EP3 receptor mediates mast cell migration to PGE2. Chemotaxis of 2-wk BMMC to either PGE2 (100 nM, ■) or SCF (1 nM, ▴) in the presence of either an EP3 antagonist (L826,266) (a) or an EP4 antagonist (L161,982) (b). Data are mean ± SEM for n = 4. (c) mRNA expression of the EP3 receptor α, β, and γ isoforms in 2-wk BMMC were analyzed by RT-PCR and compared with the housekeeping gene GAPDH. The 400-bp PCR product for the β isoform was sequenced and confirmed as EP3 receptor β, and the additional 500-bp PCR product was sequenced and determined to be EP3 receptor α product. The 500-bp PCR product was probably synthesized because of the close proximity of the EP3 receptor β reverse primer to the splicing site between the α and β isoforms, producing the extra PCR product of 500 bp. Data are mean ± SEM for n = 3 using RNA isolated from Applied Biosystems separate cell cultures. (d) Chemotaxis of 2-wk BMMC to 100 nM PGE2 in the presence of 10 μM LY294002, a phosphatidylinositol 3-kinase inhibitor (LY); SB202190, a p38MAPK inhibitor (SB); or U0126, a MAPKK inhibitor (U). Data are mean ± SEM for n = 6, ∗, P < 0.05.

Article Snippet: We thank Ms. Gill Martin for help with processing BMMC and Merck Frosst Canada Ltd., Pointe Claire–Dorval, Quebec, PQ, Canada, for the kind gift of the EP3 and EP4 receptor antagonists.

Techniques: Migration, Chemotaxis Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Synthesized, Isolation

Journal: Journal of Bone and Mineral Research

Article Title: Mechanical Induction of PGE 2 in Osteocytes Blocks Glucocorticoid-Induced Apoptosis Through Both the β-Catenin and PKA Pathways

doi: 10.1002/jbmr.168

Figure Lengend Snippet: Dexamethasone Inhibits the Expression of COX-2 and the EP2 Receptor Genes and Downstream Targets of the β-Catenin Pathway

Article Snippet: If necessary, cells were pretreated with 5 μM of EP2 antagonist AH6809 (6-isopropoxy-9-oxoxanthene-2-carboxylic acid; Cayman Chemical), 5 μM of EP4 antagonist CP-147499 (kindly provided by Dr Lydia Pan, Pfizer, Inc., Groton, CT, USA), 5 μM of H89 (isobutylmethylxanthine; Sigma-Aldrich), or 1 μM of wortmannin (Sigma-Aldrich) for 0.5 to 1 hour prior to addition of PGE 2 .

Techniques: Expressing

Journal: Journal of Bone and Mineral Research

Article Title: Mechanical Induction of PGE 2 in Osteocytes Blocks Glucocorticoid-Induced Apoptosis Through Both the β-Catenin and PKA Pathways

doi: 10.1002/jbmr.168

Figure Lengend Snippet: A diagram showing the means whereby mechanical loading (ɛ) prevents apoptosis. Mechanical loading, in the form of FFSS, induces the release of prostaglandin (PGE 2 ) through connexin 43 hemichannels (Cx43 HCs), as shown previously by Cherian and colleagues to have both autocrine and paracrine effects through EP2 and EP4 receptors. These receptors not only signal through the traditional cAMP/PKA pathway to reduce apoptosis but also signal through the PI3k/Akt/GSK-3/β-catenin pathway. Lithium chloride (LiCl) also blocks apoptosis. The same mechanism and pathways were shown to be responsible for the transcription of Cx43 and increased gap junction function. For additional information on the potential for crosstalk between these two pathways and role in bone cell function, see the review by Bonewald and Johnson.

Article Snippet: If necessary, cells were pretreated with 5 μM of EP2 antagonist AH6809 (6-isopropoxy-9-oxoxanthene-2-carboxylic acid; Cayman Chemical), 5 μM of EP4 antagonist CP-147499 (kindly provided by Dr Lydia Pan, Pfizer, Inc., Groton, CT, USA), 5 μM of H89 (isobutylmethylxanthine; Sigma-Aldrich), or 1 μM of wortmannin (Sigma-Aldrich) for 0.5 to 1 hour prior to addition of PGE 2 .

Techniques: Cell Function Assay