Type I Interferons Act Directly on Nociceptors to Produce Pain Sensitization: Implications for Viral Infection-Induced Pain

Abstract

One of the first signs of viral infection is body-wide aches and pain. Although this type of pain usually subsides, at the extreme, viral infections can induce painful neuropathies that can last for decades. Neither of these types of pain sensitization is well understood. A key part of the response to viral infection is production of interferons (IFNs), which then activate their specific receptors (IFNRs) resulting in downstream activation of cellular signaling and a variety of physiological responses. We sought to understand how type I IFNs (IFN-α and IFN-β) might act directly on nociceptors in the dorsal root ganglion (DRG) to cause pain sensitization. We demonstrate that type I IFNRs are expressed in small/medium DRG neurons and that their activation produces neuronal hyper-excitability and mechanical pain in mice. Type I IFNs stimulate JAK/STAT signaling in DRG neurons but this does not apparently result in PKR-eIF2α activation that normally induces an anti-viral response by limiting mRNA translation. Rather, type I IFNs stimulate MNK-mediated eIF4E phosphorylation in DRG neurons to promote pain hypersensitivity. Endogenous release of type I IFNs with the double-stranded RNA mimetic poly(I:C) likewise produces pain hypersensitivity that is blunted in mice lacking MNK-eIF4E signaling. Our findings reveal mechanisms through which type I IFNs cause nociceptor sensitization with implications for understanding how viral infections promote pain and can lead to neuropathies.SIGNIFICANCE STATEMENT It is increasingly understood that pathogens interact with nociceptors to alert organisms to infection as well as to mount early host defenses. Although specific mechanisms have been discovered for diverse bacterial and fungal pathogens, mechanisms engaged by viruses have remained elusive. Here we show that type I interferons, one of the first mediators produced by viral infection, act directly on nociceptors to produce pain sensitization. Type I interferons act via a specific signaling pathway (MNK-eIF4E signaling), which is known to produce nociceptor sensitization in inflammatory and neuropathic pain conditions. Our work reveals a mechanism through which viral infections cause heightened pain sensitivity.

Keywords: MNK; dorsal root ganglion; eIF4E; nociceptor; type I interferon.

Figure 1.

Type I IFNs induce mechanical nociceptive hypersensitivity responses via a peripheral action in male and female mice. A–D, In male mice, intraplantar (i.pl.) administration of IFN-α (300 U/25 μl) or IFN-β (300 U/25 μl) increased paw mechanical hypersensitivity (g) to von Frey stimulation (A, B) with no significant changes in paw withdrawal latency (s) to thermal stimulation (C, D). n = 9 (vehicle groups) and n = 12 (IFN groups) in A and B. n = 6 (vehicle groups) and n = 12 (IFN groups) in C and D. E–H, In female mice, intraplantar administration of IFN-α (300 U/25 μl) and IFN-β (300 U/25 μl) increased paw mechanical sensitivity to von Frey stimulation (E, F) with no significant changes in paw withdrawal latency (s) to thermal stimulation (G, H). n = 6 per group. I–L, When versus female, no significant sex differences in the development of mechanical hypersensitivity (I, J) or the presence of thermal sensitivity (K, L) were observed between groups following either IFN-α (300 U/25 μl) or IFN-β (300 U/25 μl) intraplantar administration. Data are presented as mean±SEM. Group differences were assessed using two-way ANOVA for A, B, and E, F, followed by Bonferroni's multiple-comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 2.

Expression of IFNRs in DRG sensory neurons. A, Single DRG neuron-sequencing tSNE clusters showing expression of both Ifnar1 (IFNR1) and Ifnar2 (IFNR2) mRNAs with the neuronal marker rbfox3 (NeuN). B, Ifnar1 and Ifnar2 mRNAs expression overlaps with the small/medium-sized neuron subpopulation expressing Prph (peripherin) and Scn10a (Nav1.8). C, Ifnar1 and Ifnar2 mRNAs distribution across DRG sensory neurons of peptidergic [Trpv1 (TRPV1), Calca (CGRP)] and nonpeptidergic [P2rx3 (P2X3)] sub-clusters. D, Ifnar1 and Ifnar2 mRNAs expression differences in a subpopulation that express F2rl1 (PAR2) and Nppb (NPPB). E, In situ hybridization of Ifnr1 and Ifnr2 (red) in neurons expressing Calca (green), P2rx3 (blue) and NF200 (cyan). F, G, Coexpression analysis of Ifnr1 and Ifnr2 across the nociceptive population in the DRG. H, Quantification of percentage Ifnr1 and Ifnr2 mRNA subunits based on total DRG neuronal population. n = 3. I, J, Frequency analysis of Ifnr1 and Ifnr2 in the DRG based on neuronal size.

Figure 3.

Downstream signaling events associated with IFNRs activation. A, Direct stimulation of IFNRs with IFN-α (300 U/ml) and IFN-β (300 U/ml) activated downstream JAK/STAT signaling pathways in cultured DRG neurons. B, Neither IFN-α (300 U/ml) nor IFN-β (300 U/ml) induced mTOR or RS6 phosphorylation in DRG cultures over a time course of 6 h. C, Time course of the effects produced by IFN-α (300 U/ml) and IFN-β (300 U/ml) on ERK, eIF4E and AKT phosphorylation. Data are presented as mean±SEM; n = 3–6 per group for WB analysis. Group differences (treated vs vehicle) in A–C were assessed using one-way ANOVA followed by Dunnett's multiple-comparisons test. *p < 0.05, **p < 0.01, ****p < 0.0001.

Figure 4.

Type I IFNRs activity in cultured DRG neurons is neither associated with BiP-PKR-eIF2α stimulation nor suppressed by the small-molecule ISR inhibitor ISRIB. A, B, Application of either IFN-α (300 U/ml; A) or IFN-β (300 U/ml; B) for 1–6 h did not modify BiP expression or signaling via PKR and downstream p-eIF2α in cultured DRG neurons. C, Long exposure (24 h) to IFN-α (300 U/ml) or IFN-β (300 U/ml) did not modify PKR phosphorylation in cultured DRG neurons. Likewise, no changes on PKR phosphorylation after a 24 h IFN-α (300 U/ml) treatment were observed in the presence of the integrated stress response inhibitor ISRIB (200 nm). D, E, The ISR inhibitor ISRIB (200 nm) did not modulate components of the signaling pathways that are activated after either IFN-α (300 U/ml; D) or IFN-β (300 U/ml; E) application. n = 3 per group. Data are presented as mean ± SEM. n.s., not significant.

Figure 5.

IFN-α treatment causes DRG neuron hyperexcitability. A, Small and medium sized DRG neurons were sampled for patch-clamp electrophysiology experiments. The resting membrane potential was similar across the groups, with no significant effect of IFN-α treatment. B, Representative traces of action potential firing in the control (n = 8 cells) and IFN-α (n = 6 cells) groups. Action potentials were elicited by slowly depolarizing ramp currents of varying intensities. C, Mean number of action potentials were higher in the IFN-α group at each ramp intensity. D, IFN-α treatment significantly shortened the latency to the first spike. Group differences were assessed using two-way ANOVA followed by Fisher's LSD test. *p < 0.05, **p < 0.01.

Figure 6.

Genetic and pharmacological targeting of the MNK-eIF4E signaling axis attenuates type I IFN-induced pain hypersensitivity. A, B, One hour stimulation with either IFN-α (300 U/ml) or IFN-β (300 U/ml) increased the phosphorylation of the translation initiation factor eIF4E at serine S209 (eIF4E^S209; red) in cultured DRG neurons expressing NeuN (blue) and peripherin (green). Scale bar, 50 μm. Data are presented as mean±SEM. Group differences were assessed using one-way ANOVA followed by Dunnett's multiple-comparisons test. C, MNK1−/− mouse genotyping. D, E, Mechanical hypersensitivity produced by intraplantar (i.pl.) administration of either IFN-α (300 U/25 μl; D) or IFN-β (300 U/25 μl; E) was attenuated in mice lacking MNK1 (Mknk1−/−), the specific kinase that phosphorylates eIF4E. Data are presented as mean±SEM; n = 6 per group. Group differences were assessed using two-way ANOVA followed by Bonferroni's multiple-comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. F, eIF4E^S209A mouse genotyping. G, Mice lacking the phosphorylation site at Serine 209 (eIF4E^S209A) showed absence of eIF4E phosphorylation in L5 DRGs demonstrating antibody specificity. Scale bar, 50 μm. H, I, Mechanical hypersensitivity was attenuated in eIF4E^S209A mice compared with WT mice following an intraplantar injection of either IFN-α (300 U/25 μl; H) or IFN-β (300 U/25 μl; I). Data are presented as mean±SEM; n = 6 per group. Group differences were assessed using two-way ANOVA followed by Bonferroni's multiple-comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 7.

Endogenous type I IFN induction with poly (I:C) produces pain sensitization in mice via MNK1-eIF4E signaling. A, B, The synthetic analog of a double-stranded RNA (dsRNA), poly (I:C) (1 mg/kg, i.p.), injected for 2 consecutive days, produced mechanical (A) and thermal hypersensitivity (B) in mice. n = 6 per group. *p < 0.05, **p < 0.01, compare to vehicle. ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, ^####p < 0.0001 compare to baseline. Group differences were assessed using two-way ANOVA followed by Dunnett's (#) or Bonferroni's (*) multiple-comparisons tests. C, Changes in body temperature produced by intraperitoneal poly (I:C) (1 mg/kg) administration. n = 6 per group. **p < 0.01, ****p < 0.0001 compared with vehicle. Group differences were assessed using unpaired t test. D, Intraperitoneal poly (I:C) administration increased phospho, but not total, eIF4E in L5 DRGs of WT mice at Day 2 (3 h post-second poly I:C injection). Scale bar, 50 μm. n = 3 per group. *p < 0.05 compared with vehicle. Group differences were assessed using unpaired t test. Mechanical (E) and thermal (F) hypersensitivity produced by intraplantar administration of poly (I:C) (1 mg/kg) were partially attenuated in MNK1−/− mice compared with WT mice. ***p < 0.001, compare to WT mice. ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, ^####p < 0.001 compared with baseline. n = 12 (WT) and n = 6 (MNK1−/−) per group. Group differences were assessed using two-way ANOVA followed by Dunnett's (#) or Bonferroni's (*) multiple-comparisons tests. Mechanical (G) and thermal (H) hypersensitivity produced by intraplantar administration of poly (I:C) (1 mg/kg) were attenuated in eIF4E^S209A mice compared with WT mice. n = 12 (WT) and n = 6 (eIF4E^S209A) per group. *p < 0.05, ***p < 0.001 compared with WT mice, ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 compared with baseline. Group differences were assessed using two-way ANOVA followed by Dunnett's (#) or Bonferroni's (*) multiple-comparisons tests. I, Lumbar DRGs (L4-L5), lumbar SDH and sciatic nerve from MNK−/− and eIF4E^S209A mice showed decrease and absence, respectively, on eIF4E phosphorylation compared with WT mice following intraperitoneal poly I:C administration (Day 2, 3 h post-second poly (I:C) injection). n = 3 per group. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.001 compared with WT. Differences were assessed using one-way ANOVA followed by Dunnett's multiple-comparisons test. J, Application of poly (I:C) (10 μg/ml) did not increase p-ERK, p-eIF4E, p-PKR or p-eIF2α in cultured DRG neurons. n = 3 per group.

Figure 8.

Schematic model of downstream signaling events associated with type I interferon actions in nociceptors. Our work demonstrates that type I IFN production rapidly induced nociceptor hyperexcitability and mechanical pain sensitization via IFNR activation, likely on sensory neurons. The signaling mechanisms are dependent on MNK-eIF4E with no apparent ISR induction. Therefore, MNK-eIF4E signaling is a key translation regulation pathway that modulates viral infection-induced pain.