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. 2025 Sep 25;16(1):8262.
doi: 10.1038/s41467-025-63782-8.

Targeting prostaglandin E2 receptor 2 in Schwann cells inhibits inflammatory pain but not inflammation

Affiliations

Targeting prostaglandin E2 receptor 2 in Schwann cells inhibits inflammatory pain but not inflammation

Romina Nassini et al. Nat Commun. .

Abstract

Analgesia by non-steroidal anti-inflammatory drugs (NSAIDs) is ascribed to inhibition of prostaglandin (PG) biosynthesis and ensuing inflammation. However, NSAIDs have life-threatening side effects, and inhibition of inflammation delays pain resolution. Decoupling the mechanisms underlying PG-evoked pain vs. protective inflammation would facilitate pain treatment. Herein, we reveal that selective silencing of the PGE2 receptor 2 (EP2) in Schwann cells via adeno-associated viral vectors abrogates the indomethacin-sensitive component of pain-like responses in mice elicited by inflammatory stimuli without affecting inflammation. In human Schwann cells and in mice, EP2 activation and optogenetic stimulation of adenylyl cyclase evokes a plasma membrane-compartmentalized cyclic adenosine monophosphate (cAMP) signal that, via A-kinase anchor protein-associated protein kinase A, sustains inflammatory pain-like responses, but does not delay their resolution. Thus, an unforeseen and druggable EP2 receptor in Schwann cells, via specific cAMP nanodomains, encodes PGE2-mediated persistent inflammatory pain but not PG-dependent protective inflammation.

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Conflict of interest statement

Competing interests: N.W.B. is a founding scientist of Endosome Therapeutics and pHArm Therapeutics. P.G. is a member of the Scientific Advisory Board of Endosome Therapeutics. R.N., F.D.L. and P.G. are founding scientists of FloNext Srl. G.B. has been fully employed at FloNext Srl, Italy. The remaining authors declare no competing interests.

Figures

Fig. 1
Fig. 1. EP2 receptor activation in Schwann cells mediates PGE2-evoked sustained hindpaw mechanical allodynia (allodynia).
a,d, f Dose-dependent non-evoked nociception and b, e, g dose- and time-dependent allodynia after intraplantar (i.pl.) injection of PGE2, L-902,688 (L-902), butaprost (Buta) or vehicle (Veh) in C57BL/6 J mice (B6) (n = 8 mice/group). c Schematic representation of agonists/antagonists targeting EP2 and EP4 receptors. h Non-evoked nociception and i allodynia after i.pl. PGE2 (1.5 nmol) or Veh in B6 pretreated with PF-04448948 (PF, 5nmol), BGC 20-1531 (BGC, 5 nmol) or Veh (n = 8 mice/group). j RT-qPCR for Ptger2, Ptger4 Avil and S100 mRNA in mouse dorsal root ganglia (DRG) sciatic and cutaneous Schwann cells (SCs) (DRG and sciatic SCs n = 4, cutaneous n = 3 independent experiments). k, l Representative images of EP2, EP4, NeuN and S100B expression in mouse DRG and sciatic nerve tissue (scale bar: 20 μm) (n = 3 subjects). m Representative images of EP2, EP4 and SOX10 expression in mouse sciatic and cutaneous SCs (n = 3 independent experiments). n–r Non-evoked nociception (left panel) and allodynia (right panel) after i.pl. PGE2 or Veh in Plp-Cre, Adv-Cre or Control mice infected with AAV for selective silencing of EP4 (-Ptger4) (P1p-Ptger4 or Adv-Ptger4) (n,o) or EP2 (-Ptger2) (P1p-Ptger2 or Adv-Ptger2) (p,q) or with AAV for a scrambled shRNA (Plp-scrambled) (r) (n = 8 mice/group). Data are mean ±  s.e.m. a, d, f, h, n, o, p, q, r 1-way or b, e, g, i, n, o, p, q, r 2-way ANOVA, Bonferroni correction. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. Veh P < 0.05, ††P < 0.01, †††P < 0.001, ††††P < 0.0001 vs. PGE2/Veh, Control/ PGE2. Source data are provided as a Source Data file.
Fig. 2
Fig. 2. EP2 in Schwann cells mediates carrageenan and CFA hindpaw mechanical allodynia (allodynia) but not inflammation.
a, c Dose-dependent non-evoked nociception and b, d dose- and time-dependent allodynia after intraplantar (i.pl.) phospholipase A2 activating protein (PLAA), arachidonic acid (AA) or vehicle (Veh) in C57BL/6 J mice (B6) (n = 8 mice/group). e Time dependent PGE2 assay in B6 paw tissue homogenates after i.pl. PGE2 (1.5 nmol), PLAA (2 nmol), AA (10 nmol) or Veh (n = 6 mice/group) and f pretreated with indomethacin (Indo, 280 nmol) (n = 8 mice/group). g, h Allodynia after i.pl. PLAA, AA or Veh in P1p-Cre or Control mice infected with AAV for selective silencing of EP2 (P1p-Ptger2) and pretreated with Indo or Veh (n = 8 mice/group). i, j Allodynia, paw thickness and myeloperoxydase (MPO) activity assay after i.pl. carrageenan (Cg, 300 μg), complete Freund’s adjuvant (CFA) or Veh in Plp-Ptger2 or Control mice and pretreated with Indo or Veh (n = 8 mice/group). Data are mean ± s.e.m. a, c, e, f and MPO activity in i, j, 1-way or b, d, g, h, allodynia and paw thickness in i, j 2-way ANOVA, Bonferroni correction. ***P < 0.001, ****P < 0.0001 vs. Veh or Control/Veh/Veh ††P < 0.01, †††P < 0.001, ††††P < 0.0001 vs. Control/Veh/PLAA, AA, Cg, CFA. Source data are provided as a Source Data file.
Fig. 3
Fig. 3. Schwann cells cAMP nanodomains promote hindpaw mechanical allodynia (allodynia) by PGE2.
a Global cAMP formation in human Schwann cells (hSCs), after blue light stimulation (450 nm, 10 min) (n = 121 cells, n = 3 independent experiments) or forskolin (FSK 100 μM) (n = 78 cells, n = 3 independent experiments). b Schematic representation of Cre recombinase dependent expression of Beggiatoa photo-activable AC (bPAC) and mCherry separated by T2A self-cleaving peptide sequence. c Representative images of mCherry expression in S100B+ cells in mouse paw tissue after intraplantar (i.pl.) infection with AAV for selective expression of bPAC in SCs (scale bar: 20 μm) (n = 4 subjects). d Illustration of bPAC activation after blue light stimulation and allodynia induced by blue light stimulation (pulsed 1 s/5 s for 10 min) in Plp-Cre or Control mice infected with AAV-bPAC (Plp-bPAC) (n = 8 mice/group). e Representative images and cumulative data of membrane and intracellular localization of EP2 and EP4 in hSCs cells, with or without pre-incubation with unlabeled PGE2 (10 µM, 30 min). Scale bar, 20 µm. (n = 4 independent experiments). f Concentration-dependent global cAMP formation in hSCs induced by PGE2 (n = 8 replicates). g Membrane confined cAMP formation induced by PGE2 (10 nM) in hSCs in the presence of PF-04418948 (PF,1 μM), BGC 20-1531 (BGC, 1 μM) or vehicle (Veh) (cells number: PGE2 = 80, PF = 90, BGC = 88, n = 3 independent experiments). h Illustration of membrane tagged Lyn11-bPAC membrane confined cAMP formation and cAMP formation in hSCs after blue light stimulation (n = 47 cells, n = 3 independent experiments) or FSK (100 μM) (n = 46 cells, n = 3 independent experiments). i Allodynia induced by blue light stimulation in Plp-Cre or Control mice infected with AAV-Lyn11-bPAC (Plp-Lyn11-bPAC) (n = 8 mice/group). j Concentration-dependent PKA activation induced by PGE2 in hSCs (n = 8 replicates). k Membrane confined PKA activation in hSCs induced by PGE2 (10 nM) in the presence of PF (1 μM), BGC (1 μM) or Veh (cells number: PF = 64, BGC = 63, PGE2 = 64, n = 3 independent experiments). Data are mean ±  s.e.m.a,e,h,2-tailed Student’s ttest, g, k 1-way or d, i 2-way ANOVA, Bonferroni correction. AUC area under curve. ****P < 0.0001 vs. Control/blue light. Source data are provided as a Source Data file.
Fig. 4
Fig. 4. Schwann cells membrane-bound PKA/AKAP drives hindpaw mechanical allodynia (allodynia) by PGE2 and inflammation.
a Membrane cAMP in hSCs after red light (650 nm/10 min) activation of membrane-tagged light-activated phosphodiesterase (Lyn11-LAPD) and PGE2 (10 nM) (cells: light = 61, no light = 99). b, c Allodynia by carrageenan (Cg, 300 μg) or CFA in Plp-Cre or Control mice after AAV-Lyn11-LAPD (Plp-Lyn11-LAPD, i.pl.) and red light stimulation (n = 8 mice/group). d A-kinase anchor protein 79 (AKAP79) associated cAMP formation and e PKA activation by PGE2 (10 nM) in the presence of PF-04418948 (PF, 1 μM), BGC 20-1531 (BGC, 1 μM) or vehicle (Veh) in hSCs (d, cells: PGE2 = 62, PF = 98, BGC = 57, e, PF = 95, BGC = 47, PGE2 = 52, n = 3 independent experiments). f AKAP79 associated cAMP formation in hSCs after blue light stimulation (n = 43 cells, n = 3 independent experiments) or forskolin (FSK, 100 μM) (n = 46 cells, n = 3 independent experiments). Allodynia by (g) blue light stimulation (1 s/5 s) in Plp-Cre or Control mice after AAV-AKAP150-bPAC (Plp-AKAP150-bPAC, i.pl.) (n = 8 mice/group);h,i,Cg, CFA or Veh in C57BL/6 J mice pretreated with st-Ht31 (17 nmol) or Veh (n = 8 mice/group). j AKAP79 associated cAMP formation in hSCs after red light activation of AKAP79-LAPD and PGE2 (10 nM) (cells: light = 102, No light = 100, n = 3 independent experiments). Allodynia induced by Cg, CFA or Veh in (k, l) Plp-Cre or Control mice infected with AAV for AKAP150 silencing (Plp-AKAP150) (n = 8 mice/group); m, n, Plp-Cre or Control mice after AAV-AKAP150-LAPD (Plp-AKAP150-LAPD, i.pl.) and stimulated with red light (n = 8 mice/group); o, p, Plp-Trpa1 or Control mice (n = 8 mice/group). q Illustration of BRET assay. r Catalytic subunit of PKA (CA) and TRPA1 interaction after PGE2 (100 nM) in the presence of BGC (100 nM) PF(100 nM), st-Ht31 (10 μM) or CGRP (10 μM) (replicates: PGE2 = 8, BGC = 8, PF = 8, st-Ht31 = 8, CGRP = 8). Data are mean ± s.e.m. a, f, j 2-tailed Student’s t test d, e, r, 1-way or b, c, g, h, i, k–p, 2-way ANOVA, Bonferroni correction. AUC area under the curve. **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. Veh, Control/Veh, blue light, red light P < 0.05, ††P < 0.01, †††P < 0.001, ††††P < 0.0001 vs. Cg, CFA, PGE2/Veh, Control/Cg, CFA, Plp-AKAP150-LAPD/no light/Cg, CFA. Source data are provided as a Source Data file.
Fig. 5
Fig. 5. EP2 in Schwann cells blockade abates hindpaw mechanical allodynia (allodynia) but does not delay pain resolution.
a–c Allodynia, paw thickness and myeloperoxidase (MPO) activity assay after complete Freund’s adjuvant (CFA) or vehicle (Veh) in C57BL/6 J mice treated for 6 consecutive days with diclofenac (25 mg/kg, i.p.), PF-04448948 (PF, 10 mg/kg, i.g.) or Veh (n = 8 mice/group). d–i allodynia, paw thickness and MPO activity assay after CFA or Veh in P1p-Cre or Control mice infected with AAV for selective silencing of d–f, EP2 (-Ptger2) (Plp-Ptger2) or g–i, AKAP150 (Plp-Akap150) (n = 8 mice/group). Data are mean ± s.e.m. a, b, d, e, g, h, 2-way or c, f, i, 1-way ANOVA, Bonferroni correction. *P < 0.05, ***P < 0.001, ****P < 0.0001 vs. Veh, Control/Veh P < 0.05, ††P < 0.01, ††††P < 0.0001 vs. CFA/Veh, Control/CFA, §§P < 0.01, §§§§P < 0.0001 vs. CFA/diclofenac. Source data are provided as a Source Data file.
Fig. 6
Fig. 6. Hypothesized mechanism of action of prostaglandins.
Prostaglandins (PGs) released during inflammation promote inflammation and pain by distinct mechanisms. a PGs cause inflammation by an EP2-independent inflammatory process, which is characterized by edema, leukocyte infiltration and release of MPO. b PGs cause pain by activating EP2 on SCs. EP2 generates cAMP, leading to AKAP79-associated activation of PKA in plasma membrane-delimited nanodomains. PKA phosphorylates and activates TRPA1 in SCs, which elicits a calcium-dependent release of reactive oxygen species (H2O2). H2O2 targets TRPA1 on adjacent nociceptors, which results in sustained hypersensitivity to mechanical stimuli.

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