ephrin-B2 promotes nociceptive plasticity and hyperalgesic priming through EphB2-MNK-eIF4E signaling in both mice and humans

Abstract

Ephrin-B-EphB signaling can promote pain through ligand-receptor interactions between peripheral cells, like immune cells expressing ephrin-Bs, and EphB receptors expressed by DRG neurons. Previous studies have shown increased ephrin-B2 expression in peripheral tissues like synovium of rheumatoid and osteoarthritis patients, indicating the clinical significance of this signaling. The primary goal of this study was to understand how ephrin-B2 acts on mouse and human DRG neurons, which express EphB receptors, to promote pain and nociceptor plasticity. We hypothesized that ephrin-B2 would promote nociceptor plasticity and hyperalgesic priming through MNK-eIF4E signaling, a critical mechanism for nociceptive plasticity induced by growth factors, cytokines and nerve injury. Both male and female mice developed dose-dependent mechanical hypersensitivity in response to ephrin-B2, and both sexes showed hyperalgesic priming when challenged with PGE₂ injection either to the paw or the cranial dura. Acute nociceptive behaviors and hyperalgesic priming were blocked in mice lacking MNK1 (Mknk1 knockout mice) and by eFT508, a specific MNK inhibitor. Sensory neuron-specific knockout of EphB2 using Pirt-Cre demonstrated that ephrin-B2 actions require this receptor. In Ca²⁺-imaging experiments on cultured DRG neurons, ephrin-B2 treatment enhanced Ca²⁺ transients in response to PGE₂ and these effects were absent in DRG neurons from MNK1^-/- and EphB2-Pirt^Cre mice. In experiments on human DRG neurons, ephrin-B2 increased eIF4E phosphorylation and enhanced Ca²⁺ responses to PGE₂ treatment, both blocked by eFT508. We conclude that ephrin-B2 acts directly on mouse and human sensory neurons to induce nociceptor plasticity via MNK-eIF4E signaling, offering new insight into how ephrin-B signaling promotes pain.

Keywords: EphB; Ephrin; MNK1; MNK2; acute to chronic pain; hyperalgesic priming; nociceptor.

Figure 1.. Response to intraplantar injection of EphrinB2-Fc or vehicle in both male and female mice.

A, Overview of behavioral testing procedure. B, Mechanical thresholds after EphrinB2-Fc intraplantar injection in male mice. Mice received an intraplantar injection of 100 ng of PGE₂ after initial mechanical hypersensitivity had resolved to assess the presence of hyperalgesic priming. C, Mechanical thresholds after EphrinB2-Fc intraplantar injection in female mice. Hyperalgesic priming was assessed over the same time course as in male mice. D, Male mice were injected with EphrinB2-Fc or vehicle and tested for thermal hyperalgesia using the Hargreaves method. E, Female mice were injected with EphrinB2-Fc or vehicle and tested for thermal hyperalgesia using the Hargreaves method. Males and females n = 6/group, stars represent significant differences between groups. EB2 = EphrinB2-Fc. Repeated measures two-way ANOVA with Bonferroni’s post hoc test: *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 2.. MNK1 is necessary for EphrinB2-Fc induced mechanical hypersensitivity, thermal hyperalgesia, and hyperalgesic priming.

A, B, Wild-type or MNK1^−/− male mice were given an intraplantar injection of 100ng EphrinB2-Fc or vehicle followed by an injection of 100ng PGE₂ after initial hypersensitivity had resolved. C, D, Wild-type or MNK1^−/− mice were given intraplantar injections of either EphrinB2-Fc or vehicle and tested for thermal hyperalgesia using the Hargreaves method. Stars represent statistical significance. Males and females n = 6/group. EB2 = EphrinB2-Fc. Repeated measures two-way ANOVA with Bonferroni’s post hoc test: *p < 0.05, **p < 0.01, ***p < 0.001

Fig 3:. MNK1/2 inhibition with eFT508 blocks EphrinB2-driven nociceptive behaviors.

A, B, Mice given an oral dose of eFT508 (10 mg/kg) compared to vehicle 30 minutes prior to receiving an intraplantar injection of 100 ng of EphrinB2-Fc show attenuated mechanical hypersensitivity. After initial hypersensitivity was resolved, mice received an intraplantar injection of 100 ng of PGE₂ to assess the presence of hyperalgesic priming. C, D, Mice were given an oral dose of eFT508 or vehicle 30 minutes prior to receiving an intraplantar injection of EphrinB2-Fc and tested for thermal hyperalgesia. Stars represent statistical significance. Males and females n = 6/group. EB2, EphrinB2-Fc. Repeated measures two-way ANOVA with Bonferroni’s post hoc test: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig 4:. EphB2-Pirt^+/− mice show significantly reduced nociceptive behaviors in response to EphrinB2 treatment.

A, B, EphB2-Pirt^+/+ and EphB2-Pirt^Cre mice were given an intraplantar injection of 100 ng of EphrinB2-Fc and tested for mechanical hypersensitivity. After resolution of initial hypersensitivity, mice were given an injection of PGE₂ to assess the presence of hyperalgesic priming. C, D, EphB2-Pirt^+/+ and EphB2-Pirt^Cre mice were given a 100 ng injection of EphrinB2-Fc intraplantarly and tested for thermal hyperalgesia. Males and females n = 6/group. EB2 = EphrinB2-Fc. Repeated measures two-way ANOVA with Bonferroni’s post hoc test: *p < 0.05, **p < 0.01.

Fig 5:. Supradural injection of EphrinB2-Fc promotes peri-orbital mechanical hypersensitivity and priming that is MNK1-dependent.

A, B, Naïve male (A) and female (B) mice were given a supradural injection of 100ng EphrinB2-Fc or vehicle and periorbital mechanical hypersensitivity was assessed. After initial hypersensitivity resolved, mice were given a supradural injection of 100ng PGE₂ or vehicle to assess the presence of hyperalgesic priming. Stars represent statistical significance of EphrinB2-Fc/PGE₂ group versus vehicle/vehicle. Pound represents statistical significance of EphrinB2-Fc/vehicle versus vehicle/vehicle. EphrinB2/PGE₂ (n = 8). EphrinB2-Fc/Vehicle and vehicle/vehicle (n = 6). Vehicle/PGE2 (n = 7). C, D, Wild-type and MNK1^−/− mice received a supradural injection of 100ng EphrinB2-Fc or vehicle. Priming was determined through a dural injection of 100ng PGE₂. Stars represent statistical significance of MNK1^−/− EphrinB2-Fc versus Wild-type vehicle. Pound represents statistical significance of Wild-type EphrinB2-Fc group versus Wild-type vehicle. Wild-type EphrinB2-Fc (n = 7). All other groups (n = 6). EB2 = EphrinB2-Fc. Repeated measures two-way ANOVA with Bonferroni’s post hoc test: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig 6:. EphrinB2-FC treatment sensitizes mouse DRG neurons in an EphB2 and MNK-dependent fashion.

A, Diagram of the experimental timeline. B, Representative Ca²⁺ traces in response to PGE₂ (50 nM) and K⁺ (50 mM). C, D, Total percent neuronal response rate to 50nM PGE₂ treatment. WT vehicle treated (n = 188). WT EphrinB2-FC treated (n = 224). MNK1^−/− vehicle (n = 75). MNK1^−/− EphrinB2-Fc (n = 76). EphB2-Pirt^+/+ vehicle (n = 32). EphB2-Pirt^+/+ EphrinB2-Fc (n = 28). EphB2-Pirt^Cre vehicle (n = 33). EphB2-Pirt^Cre EphrinB2-FC (n = 39). E, F, latency to peak was determined in EphrinB2-Fc or vehicle treated cultures for both PGE₂ (WT vehicle, WT EphrinB2-FC, MNK1^−/− vehicle, MNK1^−/− EphrinB2-Fc, out of n = 17, 62, 6, and 9 responding neurons, respectively) and KCl positive neurons (WT vehicle, WT EphrinB2-FC, MNK1^−/− vehicle, MNK1^−/− EphrinB2-Fc, out of n = 188, 224, 75, and 76 responding neurons, respectively). G, H, latency to peak was determined in EphrinB2-Fc or vehicle treated cultures for both PGE₂ (EphB2-Pirt^+/+ vehicle, EphB2-Pirt^+/+ EphrinB2-Fc, EphB2-Pirt^Cre vehicle, EphB2-Pirt^Cre EphrinB2-Fc, out of n = 5, 18, 5 and 3 responding neurons, respectively) and KCl positive neurons (EphB2-Pirt^+/+ vehicle, EphB2-Pirt^+/+ EphrinB2-Fc, EphB2-Pirt^Cre vehicle, EphB2-Pirt^Cre EphrinB2-Fc, out of n = 32, 28, 33, and 39 responding neurons, respectively). Stars represent statistical significance between groups. WT = Wild-type; ns = not significant; EB2 = EphrinB2-Fc. Bars represent standard error of mean. PGE₂ % response, chi-squared analysis: ***p < 0.001, ****p < 0.0001. Ordinary two-way ANOVA with Bonferroni’s post hoc test: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig 7:. EphrinB2 treatment sensitizes human DRG neurons in a MNK-dependent fashion.

Cultures of human dissociated DRG neurons were pre-treated with either EphrinB2 (0.35μg), IgG control, or EphrinB2 and eFT508 (25 nM) prior to delivery of 50nM PGE₂. A, Representative traces of pre-treated neurons to 50nM PGE₂. B, Total response rate to 50nM PGE₂ in dissociated cultures treated with IgG control, EphrinB2, or EphrinB2 and eFT508, n = 4 (out of 134 total), 61 (out of 155 total), and 0 (out of 65 total) responding neurons, respectively. A total of 7 donors were cultured. C, latency to peak was determined in EphrinB2-FC or vehicle treated cultures for both PGE₂ and K⁺ (50 mM) responsive neurons. Only neurons responsive to both PGE₂ and K⁺ were assessed (IgG control and EphrinB2, n = 4 and 61 responding neurons, respectively). D, Latency to peak in all K⁺-responsive neurons (IgG control, EphrinB2, or EphrinB2 and eFT508, n = 134, 155, and 65 responding neurons, respectively). Stars represent statistical significance between groups. Bars represent standard error of mean. Male (n = 3 independent cultures) and female (n = 4 independent cultures) groups were combined for analysis. PGE₂ % response, Chi-squared analysis: ****p < 0.0001 PGE₂ latency peak, Unpaired t test: **p < 0.01. K⁺ latency to peak, Ordinary one-way ANOVA with Bonferroni’s post hoc test: **p < 0.01, ****p < 0.0001. ns = not significant.

Fig 8:. EphrinB2 causes increased eIF4E phosphorylation in human DRG neurons.

Immunocytochemistry of dissociated human DRG cultures treated with either IgG control, EphrinB2, or EphrinB2 and eFT508 for 1 hour. A, Representative panel stained for Peripherin (green) or p-eIF4E (red). IgG control or EphrinB2 treated at 0.35μg/mL or EphrinB2 and eFT508 (25 nM) treated neurons. B, Quantification of p-eIF4E expression. IgG control, EphrinB2, EphrinB2 and eFT508 treated neurons n = 55, 95, and 50, respectively. C, Quantification of large diameter neurons (≥40μm) p-eIF4E expression. IgG control, EphrinB2, EphrinB2 and eFT508 treated neurons n = 37, 57, and 40, respectively). D, Quantification of small diameter neurons (<40μm) p-eIF4E expression. IgG control, EphrinB2, EphrinB2 and eFT508 treated neurons n = 18, 77, 10, respectively). Stars represent statistical significance between groups. Bars represent standard error of mean. ns, not significant. Male (n = 3 independent cultures) and female (n = 2 independent cultures) groups were combined for analysis. Ordinary one-way ANOVA with Bonferroni’s post hoc test: **p < 0.01, ****p < 0.0001.