STING controls nociception via type I interferon signalling in sensory neurons - PubMed (original) (raw)

. 2021 Mar;591(7849):275-280.

doi: 10.1038/s41586-020-03151-1. Epub 2021 Jan 13.

Changyu Jiang 2, Amanda S Andriessen 2, Kaiyuan Wang 2, Zilong Wang 2, Huiping Ding 3, Junli Zhao 2, Xin Luo 2, Michael S Lee 2, Yu L Lei 4 5, William Maixner 2, Mei-Chuan Ko 3 6, Ru-Rong Ji 7 8 9

Affiliations

STING controls nociception via type I interferon signalling in sensory neurons

Christopher R Donnelly et al. Nature. 2021 Mar.

Abstract

The innate immune regulator STING is a critical sensor of self- and pathogen-derived DNA. DNA sensing by STING leads to the induction of type-I interferons (IFN-I) and other cytokines, which promote immune-cell-mediated eradication of pathogens and neoplastic cells1,2. STING is also a robust driver of antitumour immunity, which has led to the development of STING activators and small-molecule agonists as adjuvants for cancer immunotherapy3. Pain, transmitted by peripheral nociceptive sensory neurons (nociceptors), also aids in host defence by alerting organisms to the presence of potentially damaging stimuli, including pathogens and cancer cells4,5. Here we demonstrate that STING is a critical regulator of nociception through IFN-I signalling in peripheral nociceptors. We show that mice lacking STING or IFN-I signalling exhibit hypersensitivity to nociceptive stimuli and heightened nociceptor excitability. Conversely, intrathecal activation of STING produces robust antinociception in mice and non-human primates. STING-mediated antinociception is governed by IFN-Is, which rapidly suppress excitability of mouse, monkey and human nociceptors. Our findings establish the STING-IFN-I signalling axis as a critical regulator of physiological nociception and a promising new target for treating chronic pain.

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Figures

Extended Data Figure 1.

Extended Data Figure 1.. STING is expressed in nociceptive sensory neurons in the DRG.

(a) Sting1 (STING) mRNA expression in sensory neuron populations recently profiled and described by Zhang et al. (2019). Peptidergic nociceptive sensory neurons exhibit the highest expression of STING. b-c. In situ hybridization of Sting1 in adult DRG sensory neurons using RNAscope, in conjunction with Nissl staining to label all neurons (b). STING expression is primarily observed in small-diameter sensory neurons. Quantification of DRG neurons expressing STING ( ≥5 puncta) or lacking STING found that approximately 60% of DRG neurons express STING. Scale bar is 100 μm. (c) Quantification of somal diameter in STING+ and STING− neurons indicated that STING-expressing neurons are primarily small-diameter sensory neurons. (d) Schematic indicating the various stimuli which activate STING and its downstream pathway. See Supplementary Table-1 for complete sample sizes, sex, and statistical information.

Extended Data Figure 2.

Extended Data Figure 2.. Antinociceptive effects of STING agonists in naïve mice and in mouse models of neuropathic and cancer pain.

(a) Naïve mice were administered vehicle or the STING agonist DMXAA via i.t. injection (red arrows), followed by Von Frey testing to determine mechanical thresholds at 4h following the 1st (day 1, D1) or 2nd (D2) injection. STING agonists induced a dose-dependent increase in paw withdrawal thresholds, which was further amplified by multiple injections. 10 μg (35 nmol) exhibited the largest effect, and therefore, was used throughout the rest of the study. (b) Naïve mice were administered vehicle or ADU-S100, a STING agonist with cross-species activity, via i.t. injection (red arrows) and tested as in panel a. 25 μg (35 nmol) exhibited the largest increase in paw withdrawal thresholds and this dose was used throughout the rest of the study. (c) Systemic administration of DMXAA and ADU-S100 increased paw withdrawal threshold in naïve mice for up to 24h. (d) In the CIPN model, i.p. DMXAA and ADU-S100 suppressed mechanical allodynia for up to 48h. Some toxicity was observed with systemic administration in the CIPN model, as 3 mice in the DMXAA group died 24h after the 2nd injection. No mice died in the vehicle or ADU-S100 groups. (e) A chemotherapy-induced peripheral neuropathy (CIPN) model of neuropathic pain was established with paclitaxel. f-g. In the CIPN model, i.t. DMXAA and ADU-S100 suppressed mechanical allodynia (f) and cold allodynia (g), as determined by response duration (in seconds) to acetone. (h) i.t. administration with the natural STING ligand 3’3’-cGAMP also reduced CIPN-induced mechanical allodynia. (i) STING agonists were also tested in the sciatic nerve chronic constriction injury (CCI) model of neuropathic pain. (j) i.t. treatment with DMXAA and ADU-S100 led to prolonged inhibition of mechanical allodynia (as determined by withdrawal threshold). k-l. Administration of DMXAA and ADU-S100 also suppressed cold allodynia (k) in the BCP model. These effects were not secondary to direct antitumor effects, as tumor burden was unaffected by STING agonist treatment (l). m-o. Effects of naloxone on morphine-, DMXAA-, and ADU-S100-induced antinociception. (m) Naloxone (10 mg/kg, i.p.) reversed morphine (2 nmol i.t.)-induced antinociception but had no effect on the antinociceptive effects of DMXAA (35 nmol, i.t.) (n) or ADU-S100 (35 nmol, i.t.) (o). (p) Repeated administration of DMXAA (35 nmol, i.t.) did not induce tolerance in naïve mice. q-t. Pain and glial reaction after spared nerve injury (SNI). (q) Paradigm of experimental design. Pain behaviors and spinal dorsal horn glial cell activation were assessed following repeated administration with vehicle or DMXAA (red arrows) in mice receiving SNI . Mechanical allodynia (r) and cold allodynia (s) were significantly reduced in DMXAA-treated mice at later timepoints (starting at D12). (t) Quantification of GFAP+ (astrocytes), Iba1+ (microglia), or DAPI (all cells) in the spinal dorsal horn (SDH) of mice at D21 after sustained vehicle or DMXAA treatment as indicated in panel o. SNI increased astrocyte activation (GFAP) in vehicle- but not DMXAA-treated mice. Values represent the ipsilateral (injured) SDH normalized to the contralateral (uninjured) SDH. All data are expressed as the mean ± s.e.m. * P < 0.05; ** P < 0.01; *** P < 0.001, **** P < 0.0001. Statistical comparisons were conducted with two-way ANOVA with Dunnett’s (a, b, c, d, f, g, k), Sidak’s (h, j, l, s), Bonferroni’s (m, n, o, p, r, t), or Tukey’s post-hoc test (m, n, o) See Supplementary Table-1 for complete sample sizes, sex, and statistical information.

Extended Data Figure 3.

Extended Data Figure 3.. STING agonists induce conditioned place preference in mice with neuropathic pain but not in naïve mice.

(a) Schematic indicating the experimental setup for the conditioned place preference (CPP) assay used to measure ongoing pain in mice. (b) Schematic indicating CPP protocol for naïve mice. CPP in naïve mice was performed using repeated pairings (3 trials). (c) Repeated pairing with i.t. morphine (2 nmol), but not DMXAA or ADU-S100 (35 nmol), induces CPP in naïve mice. (d) Schematic indicating CPP protocol for mice in the chemotherapy-induced peripheral neuropathy (CIPN) model of neuropathic pain. (e) A single pairing with i.t. DMXAA and ADU-S100 (35 nmol) induced comparable CPP as clonidine (35 nmol), a strong analgesic when administered via i.t. injection. f. Schematic depicting patch clamp recordings in small diameter dissociated DRG neurons from STING+/+ (WT) or STING_gt/gt_ (gKO) mice. (g-h) STING_gt/gt_ mice exhibit increased excitability, as determined by number of action potentials evoked per current step (in 10 pA increments). (i-j) Nociceptors from STING_gt/gt_ mice also exhibit decreased rheobase at baseline. All data are expressed as the mean ± s.e.m. * P < 0.05; ** P < 0.01; *** P < 0.001, **** P < 0.0001. Statistical comparisons were conducted with one-way ANOVA with Fisher’s LSD test (c, e), two-way ANOVA with Bonferroni’s post-hoc test (h), or two-tailed t-test (j). See Supplementary Table-1 for complete sample sizes, sex, and statistical information.

Extended Data Figure 4.

Extended Data Figure 4.. Mice lacking STING exhibit normal innervation patterns, sensory neuron numbers, and sensorimotor behaviors.

a-f. Hindpaw skin immunostaining for nerve fibers (a, c, e) and their quantification (b, d, f) from STING+/+ or STING_gt/gt_ mice. (a) TuJ1 to label all nerve fibers, (c) CGRP to label fibers from peptidergic nociceptors, or (e) IB4 to label fibers from nonpeptidergic nociceptors. In each case, sections were counterstained with DAPI to identify the dermis-epidermis junction. Quantification of (b) TuJ1+, (d) CGRP+, and (f) IB4+ nerve fibers indicate that STING-WT and -gKO mice have similar peripheral innervation densities. Scale bar for (a-f) is 50 μm. g-i. L3-L5 spinal cord segments were collected from STING+/+ and STING_gt/gt_ mice and immunostained for CGRP (red) and IB4 (green) to label central nociceptive terminals. (g) Representative images, scale bar is 100 μm. Image J quantification of (h) CGRP and (i) IB4 pixel density in the spinal dorsal horn (displayed in arbitrary units, A.U.) indicates that there are no changes in central innervation density in STING_gt/gt_ mice. j-k. Analysis of total DRG neuron numbers in STING+/+ and STING_gt/gt_ mice. (j) L5 DRGs were collected from STING+/+ and STING_gt/gt_ mice, serially sectioned at 14 μm, and every 3rd section was immunostained for the pan-neuronal marker TuJ1 (purple), counterstained with DAPI (blue). Scale bar is 200 μm. (k) Quantification of total DRG neuron numbers indicated that STING+/+ and STING_gt/gt_ have similar sensory neuron numbers. l-n. No differences were observed between STING+/+ and STING_gt/gt_ mice in the (l) tape test, a measure of sensorimotor function, (m) in an accelerating rotarod test to measure motor function, or (n) in locomotor activity in the open field test (30 min test duration). All data are expressed as the mean ± s.e.m. * P < 0.05; ** P < 0.01; *** P < 0.001, **** P < 0.0001. Statistical comparisons were conducted with two-tailed t-test. See Supplementary Table-1 for complete sample sizes, sex, and statistical information.

Extended Data Figure 5.

Extended Data Figure 5.. STING signaling in nociceptors is required for the antinociceptive effects of STING agonists.

(a) Schematic showing mechanism of action of the small molecule STING inhibitors H-151 and C-176. (b-d) i.t. administration of H-151 and C-176 induced transient mechanical (b-c) and cold hypersensitivity (d) in naïve mice. (e) DMXAA and ADU-S100 (35 nmol each, i.t.) increased paw withdrawal thresholds in STING+/+ but not in STING_gt/gt_ mice. (f). Administration of ADU-S100 (35 nmol, i.t.) did not alter paw withdrawal frequency to a low-threshold 0.16g Von Frey filament in STING_gt/gt_ mice. (g) DMXAA and ADU-S100 increased paw withdrawal threshold in STING_fx/fx_ (WT) mice, but no significant effects were observed in STING_fx/fx_; Nav1.8-Cre mice. A non-statistically significant increase in withdrawal thresholds was observed at later timepoints (24h, 48h) in STING_fx/fx_; Nav1.8-Cre mice. (h) DMXAA and ADU-S100 (35 nmol each, i.t.) reduced mechanical hypersensitivity (determined by withdrawal frequency to a 0.16g Von Frey filament) in STING_fx/fx_; Nav1.8-Cre mice at 24h and 48h. (i) Schematic indicating method of resiniferatoxin (RTX) treatment and the effects of STING agonists in RTX-treated mice. (j) RTX increased withdrawal latency in the hotplate test (cutoff time = 40s). (k) The early (1h, 4h, 24h) antinociceptive effects of DMXAA and ADU-S100 (35 nmol, i.t.) were abolished by RTX. (l) The antinociceptive effects of DMXAA and ADU-S100 (35 nmol, i.t.) showed a normal time course of effects (relative to Fig. 1b) in Prkdcscid mice, which lack mature B and T cells. (m) Schematic indicating possible upstream activators of STING in sensory neurons, and proposed mechanism by which STING+ neurons regulate nociception through induction of cytokines and chemokines. While our data support that STING signaling in nociceptors contributes to the early antinociceptive effects of STING agonists, additional cell types including other sensory neuron populations and peripheral immune cells may also contribute to the prolonged antinociceptive effects at later timepoints. All data are expressed as the mean ± s.e.m. * P < 0.05; ** P < 0.01; *** P < 0.001, **** P < 0.0001. Statistical comparisons were conducted with two-way ANOVA with Dunnett’s (vs. BL; b, c, d, e, f, g, h) or Tukey’s (k, l) post-hoc tests. Comparisons between two groups (j) were conducted with two-tailed t-test. See Supplementary Table-1 for complete sample sizes, sex, and statistical information.

Extended Data Figure 6.

Extended Data Figure 6.. STING agonists induce IFN-I production in sensory neurons in vitro and in vivo.

(a) Intrathecal administration of STING agonists increases IFN-α in serum 24h following injection in WT, but not STING_gt/gt_ mice. (b) While basal IFN-β could be detected in DRG tissue from all genotypes, STING_gt/gt_ mice exhibited significantly lower IFN-β levels. c-d. ADU-S100 treatment of high density DRG neuron cultures from STING+/+ or STING_gt/gt_ mice. In WT DRG neurons, ADU-S100 induced release of IFN-α (c) and IFN-β (d) into the culture medium, determined by ELISA. e-h. IFN-β expression in mouse DRG neurons. (e) Schematic showing experimental design to determine which cell types within the DRG produce IFN-β in response to i.t. STING activation with ADU-S100 using an Ifnb1YFP reporter mouse. (f-h) Administration of ADU-S100 induced an increase in YFP expression (indicating IFN-β expressing cells) within the DRG. (f) Representative images showing YFP (purple), TuJ1 (green), and DAPI (blue). Scale bar is 50 μm. (g) ADU-S100 increased the proportion of total neurons expressing YFP+ in Ifnb1YFP/YFP mice; virtually no YFP+ cells detected in WT Ifnb1+/+ mice. (h) YFP expression was primarily detected in TuJ1+ neurons rather than TuJ1− non-neuronal cell types, although this neuronal expression bias was reduced by ADU-S100 treatment. i-k Mice lacking Ifnar1 globally (Ifnar1−/−) or selectively in sensory neurons (Ifnar1fx/fx; Nav1.8-Cre) exhibit significantly increased sensitivity to mechanical stimuli determined by (i) paw withdrawal threshold or (j) paw withdrawal frequency to a low stimulus 0.16g Von Frey filament, compared to their littermate controls. (k) These mice also exhibit increased sensitivity to cold stimulation. l-m. CPA testing was performed as in Fig. 1l. Pairing in the preferred chamber with 0.04g filament induced CPA in Ifnar1−/− mice, but not Ifnar1+/+ littermate control mice. (l) Representative trackplots of mouse movement pre- and post-pairing. (m) Quantification of CPA score (Pre – post, in seconds). n-q. Action potentials (n-o) and rheobases (p-q) in DRG nociceptors as in Figure 2g-j. Ifnar1−/− mice exhibit increased excitability, as determined by (n-o) number of action potentials evoked per current step (in 10 pA increments) or (p-q) basal rheobase. r-s. Input resistance was calculated from patch clamp recordings from DRG nociceptors from mice of the indicated genotypes. Increased input resistance was observed in neurons from both Ifnar1−/− (r) and STING_gt/gt_ (s) mice compared to their WT littermates. All data are expressed as the mean ± s.e.m. * P < 0.05; ** P < 0.01; *** P < 0.001, **** P < 0.0001. Statistical comparisons were conducted with one-way ANOVA with Tukey’s post-hoc test (b, g, i, j, k) two-way ANOVA with Dunnett’s post-hoc test (a, c, d, o), or two-tailed t-test (h, m, q, r, s). See Supplementary Table-1 for complete sample sizes, sex, and statistical information.

Extended Data Figure 7.

Extended Data Figure 7.. Mice lacking Ifnar1 exhibit normal innervation patterns, sensory neuron numbers, and sensorimotor behaviors.

a-f. Hindpaw skin was collected from Ifnar1+/+ or Ifnar1−/− mice and immunostained for (a) TuJ1, (c) CGRP, or (e) IB4 with DAPI as in Extended Data Fig. 4. Quantification of epidermal (b) TuJ1+, (d) CGRP+, and (f) IB4+ nerve fibers indicate that similar skin innervation densities for each marker in WT and KO mice. Scale bar for (a-f) is 50 μm. g-i. L3-L5 spinal cord segments were collected from Ifnar1+/+ or Ifnar1−/− mice and immunostained for CGRP (red) and IB4 (green) to label central nociceptive terminals in the dorsal horn. (g) Representative images, scale bar is 100 μm. Image J quantification of (h) CGRP and (i) IB4 pixel density (displayed in arbitrary units, A.U.) indicates similar densities of central innervation. j-k. (j) L5 DRGs were collected from Ifnar1+/+ and Ifnar1−/− mice and total cell counts were performed as in Extended Data Fig. 4 using TuJ1 (purple) and DAPI (blue). Scale bar is 200 μm. (k) Quantification of total DRG neuron numbers indicated that Ifnar1+/+ or Ifnar1−/− mice have similar numbers of L5 DRG neurons. l-n. No differences were observed between Ifnar1+/+ and Ifnar1−/− mice in the (l) tape test, (m) in the accelerating rotarod test, or (n) in locomotor activity in the open field test (30 minute duration). All data are expressed as the mean ± s.e.m. * P < 0.05; ** P < 0.01; *** P < 0.001, **** P < 0.0001. Statistical comparisons were conducted with two-tailed t-test. See Supplementary Table-1 for complete sample sizes, sex, and statistical information.

Extended Data Figure 8.

Extended Data Figure 8.. Type-I interferons regulate nociception in mice via Tyk2.

a-b. Inhibition of endogenous IFN-I signaling via i.t. administration of (a) an anti-IFN-α neutralizing antibody or (b) an anti-IFN-β neutralizing antibody (vs. IgG control, 300 ng) induced transient mechanical allodynia in naïve mice. (c) Inhibition of the IFN-I signaling adapter Tyk2 with PF-06700841 (i.t., 1 μ) induced transient, dose-dependent mechanical allodynia in naïve mice. d-f. i.t. injection of recombinant (d) murine IFN-α, (e) murine IFN-β (produced in mammalian cells), or (f) universal IFN-I increased paw withdrawal thresholds in naïve mice. Notably, 100 U exhibited the greatest effects for each recombinant ligand. At higher concentrations some mice exhibited mechanical hypersensitivity. (g) Schematic showing downstream IFN-I signaling pathways and the pharmacologic inhibitors used to target Tyk2, PI3K, or MAPKK. h. Pretreatment of naïve mice with the Tyk2 inhibitor PF-06700841 (i.t., 1μg) abolished IFN-β-induced antinociception. i-j. Pre-treatment with (i) 1 μg i.t. U0126, a MAPKK (MEK) inhibitor or (j) 1 μg LY294002, a PI3K inhibitor, failed to affect IFN-β-induced increased paw withdrawal threshold. (k) Intraplantar (i.pl., e.g. hindpaw) administration of IFN-α at high concentrations induced prolonged mechanical hypersensitivity. (l), i.pl. injection of 300U IFN-α evokes mechanical hypersensitivity in both WT and Ifnar1−/− mice, as determined by paw withdrawal frequency to repeated stimulation with a very low threshold Von Frey filament (0.04g, selected due to the baseline hypersensitivity of Ifnar1−/− mice). (m) Mechanical hypersensitivity induced by i.pl. injection of 300U IFN-α is attenuated by i.t. IFN-α administration, as determined by paw withdrawal threshold (left panel) or withdrawal frequency (right panel) to repeated stimulation with 0.16g Von Frey filament. All data are expressed as the mean ± s.e.m. * P < 0.05; ** P < 0.01; *** P < 0.001, **** P < 0.0001. Statistical comparisons were conducted with two-way ANOVA with Bonferroni’s (a, b, m) or Dunnett’s post-hoc test (c, d, e, f, h, i, j, k, l). See Supplementary Table-1 for complete sample sizes, sex, and statistical information.

Extended Data Figure 9.

Extended Data Figure 9.. Peripheral and central actions of STING-mediated IFN-I signaling in DRG and spinal cord.

(a) L1-L5 DRGs were isolated from STING_fx/fx_, STING_fx/fx_; Nav1.8-Cre, and STING_gt/gt_ mice and incubated ex vivo with vehicle (left DRGs) or 30 μM ADU-S100 (right DRGs) for 2h, followed by lysis and IFN-α and IFN-β ELISA. (b) IFN-α and (c) IFN-β levels in DRG lysate were increased by ex vivo incubation with ADU-S100 in WT mice. ADU-S100 induced a significant elevation of IFN-β in STING_fx/fx_; Nav1.8-Cre DRG lysate, but this increase was significantly lower than that seen in WT mice. d-e. DRGs from naïve mice were incubated ex vivo in control IgG or anti-IFNβ for 2h followed by whole-mount patch clamp recordings from DRG nociceptors. (d) Representative traces and (e) quantification of current-evoked action potentials. Anti-IFNβ treatment significantly increased action potential firing. f-h. Recordings of miniature EPSCs (mEPSCs) from outer lamina II spinal dorsal horn neurons in spinal cord slices from mice of the indicated genotypes. (f) Representative traces and quantification of mEPSC (g) frequency and (h) amplitude. Ifnar1−/− mice exhibit increased frequency and amplitude compared to WT littermates. No significant increase was observed in Ifnar1fx/fx; Nav1.8-Cre mice relative to Ifnar1fx/fx controls. (i) Representative images of RNAscope showing Sting1 (STING) mRNA (red) in conjunction with Iba1 immunostaining to label microglia (green) in the spinal cord dorsal horn (SDH). Yellow arrows indicate STING+/Iba1+ cells. Scale bar represents 20 μm. (j) Quantification indicates that STING is predominantly expressed by microglia in the SDH. (k) IFN-α and (l) IFN-β levels in SDH lysate in mice of the indicated genotypes 24h after STING agonist administration showing induction of IFN-α but not IFN-β. Basal IFN-β production was significantly lower in STING_gt/gt_ mice. m-o. Ex vivo incubation of spinal cord slices from naïve mice with ADU-S100. (m) Representative traces and quantification of (n) frequency and (o) amplitude of spontaneous EPSCs (sEPSCs) from outer lamina II spinal dorsal horn neurons. ADU-S100 significantly reduced the frequency and amplitude of sEPSCs. p-r. Acute perfusion of spinal cord slices with IFN-I and recording of TTX-resistant mEPSCs from outer lamina II spinal dorsal horn neurons. (p) Representative traces from Ifnar1+/+ and Ifnar1−/− mice, with IFN-I perfused as indicated and the areas indicated by the black bars magnified for enhanced resolution. Quantification of (q) frequency and (r) amplitude of mEPSC in neurons from WT and KO mice pre- and 1 min post- perfusion with IFN-I. Acute IFN-I treatment significantly reduced the frequency and amplitude of mEPSCs in WT mice. All data are expressed as the mean ± s.e.m. * P < 0.05; ** P < 0.01; *** P < 0.001, **** P < 0.0001. Statistical comparisons were conducted with one-way or two-way ANOVA with Sidak’s (b, c, g, h) or Bonferroni post-hoc test (e, k, l, q, r) or two-tailed t-test (n, o). See Supplementary Table-1 for complete sample sizes, sex, and statistical information.

Extended Data Figure 10.

Extended Data Figure 10.. dsDNA induces antinociception via the cGAS/STING/IFN-I pathway and proposed mechanism of STING/IFN-I regulation of nociception.

(a) i.t. IFN-α and IFN-β elevated mechanical thresholds in WT mice and could transiently rescue the mechanical hypersensitivity phenotype of STING_gt/gt_ mice back to levels comparable to WT baseline levels. (b) Schematic illustrating intracellular poly(dA:dT) induces cGAS/STING-dependent IFN-I upregulation whereas intracellular poly(I:C) induces IFN-I response through a STING-independent mechanism. c-d. Mice of the indicated genotypes were administered 1 μg poly(I:C) or 1 μg poly(dA:dT) via i.t. injection followed by Von Frey testing to determine mechanical thresholds at the indicated timepoints. (c) i.t. poly(I:C) elevated mechanical thresholds in Ifnar1fx/fx (WT), STING_gt/gt_, and cGAS−/− mice, but these effects were abolished in Ifnar1fx/fx;Nav1.8-Cre and RIG-I−/− mice. (d) i.t. poly(dA:dT) elevated mechanical thresholds in WT and RIG-I−/− mice, but these effects were abolished in Ifnar1fx/fx;Nav1.8-Cre, STING_gt/gt_, and cGAS−/− mice. e-g. Sodium currents were recorded from DRG nociceptors cultured from Ifnar1+/+ or Ifnar1−/− mice, perfused with vehicle or IFN-I for 2 minutes as indicated. (e) Representative traces and (f) quantification of Na+ currents demonstrating that rIFN-I treatment led to a reduction in sodium currents. (g) Nociceptors from _Ifnar1_-gKO mice exhibited increased Na+ current amplitude at baseline. h-i. HEK293 cells stably expressing the voltage-gated sodium channel Nav1.7 were perfused as in panel f and Na+ currents were recorded. (h) Representative traces and (i) Timecourse of Nav1.7-mediated currents. IFN-I perfusion reduced Nav1.7-mediated currents. All data are expressed as the mean ± s.e.m. * P < 0.05; ** P < 0.01; *** P < 0.001, **** P < 0.0001. Statistical comparisons were conducted with two-way ANOVA with Dunnett’s post-hoc test (a, c, d), two-way ANOVA with Bonferroni’s post-hoc test (f, i), or two-tailed t-test (g). See Supplementary Table-1 for complete sample sizes, sex, and statistical information.

Figure 1.

Figure 1.. STING inhibits nociception in naïve and injured mice

a-c. Naïve mice were administered vehicle or STING agonists via intrathecal (i.t., a) injection for two successive days, followed by von Frey testing at the indicated timepoints. (b) STING agonists elevated mechanical thresholds for up to 48h. (c) STING agonists did not affect motor function in rotarod test. (d) i.t. injection of the natural STING ligands 2’3’-cGAMP and 3’3’-cGAMP elevated mechanical thresholds in naïve mice. e-f. A syngeneic bone cancer pain model (BCP) was established, followed by i.t. vehicle or STING agonist 10d post-inoculation. STING agonists suppressed BCP-induced mechanical allodynia. (g) Experimental layout to test whether i.t. STING agonists can suppress ongoing pain in the BCP model using a conditioned place preference (CPP) assay. (h) CPP was observed in STING agonist-paired mice compared to vehicle-paired mice. i-k. Compared to their corresponding wildtype (WT) littermates, STING_gt/gt_ and STING_fx/fx_; Nav1.8-Cre mice exhibit increased sensitivity to (i, j) mechanical stimuli, and (k) cold stimuli. (l) A conditioned place aversion (CPA) assay was used to assess hypersensitivity to mechanical stimulation using repeated stimulation with an innocuous 0.04g Von Frey filament. (m) Representative track plots in WT and STING_gt/gt_ mice. (n) Quantification of CPA score. All data are expressed as the mean ± s.e.m. * P < 0.05; ** P < 0.01; *** P < 0.001, **** P < 0.0001. Statistical comparisons were conducted with two-way ANOVA with Dunnett’s post-hoc test (vehicle vs. agonist; b-f) or Tukey’s post-hoc test (i-k), one-way ANOVA with Fisher’s LSD post-hoc test (vehicle-paired vs. drug-paired; h), or two-tailed t-test (n). See Supplementary Table-1 for complete sample sizes, sex, and statistical information.

Figure 2.

Figure 2.. STING inhibits nociception via type-I interferon signaling in nociceptors.

(a) Intrathecal administration of STING agonists increased IFN-α in DRG lysate 24h following injection in WT, but not STING_gt/gt_ mice. (b) Ifnar1 in situ hybridization (red) in conjunction with immunostaining for the pan-neuronal marker TuJ1 (white) indicates that Ifnar1 is expressed by virtually all DRG neurons in naïve mice. (c) STING agonists elevated mechanical thresholds in Ifnar1fx/fx (WT) but not Ifnar1fx/fx; Nav1.8-Cre (cKO) mice. (d) Schematic of experimental design for whole-mount patch clamp experiments in DRGs. (e) Current-evoked action potentials were recorded, and the percent change by ADU-S100 (relative to vehicle) in Ifnar1+/+ (WT; black bars) or Ifnar1−/− DRGs (gKO; white bars) is displayed (red line indicates no change). ADU-S100 led to a sharp reduction in DRG excitability in WT but not gKO DRGs. (f) IFN-α and (g) IFN-β each induced transient antinociception in WT mice which was abolished in _Ifnar1_-gKO/cKO mice. h-i. Patch clamp recordings in dissociated DRG neurons from Ifnar1+/+ or Ifnar1−/− mice after acute perfusion with vehicle or rIFN-I. (h) Representative traces and (i) quantification of current evoked action potentials. IFN-I inhibited action potential firing in DRG neurons from WT, but not gKO mice. (j-k) rIFN-I treatment also increased rheobase in WT neurons, with representative traces (j) and quantification (k) indicated. (l) Representative traces and (m) quantification of Ca2+ currents. rIFN-I perfusion decreased Ca2+ currents in nociceptors from WT but not _Ifnar1_-gKO mice. (n) Amplitude of Ca2+ currents at baseline in nociceptors from WT and _Ifnar1_-gKO mice. All data are expressed as the mean ± s.e.m. * P < 0.05; ** P < 0.01; *** P < 0.001, **** P < 0.0001. Statistical comparisons were conducted with two-way ANOVA with Bonferroni’s (e, i, m) or Dunnett’s post-hoc test (a, c, f, g, relative to BL within each group), or two-tailed t-test (k, n). See Supplementary Table-1 for complete sample sizes, sex, and statistical information.

Figure 3.

Figure 3.. STING-mediated IFN-I signaling suppresses pain and nociceptor excitability in non-human primates and humans.

a-c Administration of ADU-S100 via i.t. catheter in non-human primates (Macaca mulatta, a) (b) attenuated 2% menthol gel-induced cold allodynia in a dose-dependent manner, and (c) increased IFN-β in cerebrospinal fluid (CSF) in NHPs treated with 3 nmol ADU-S100. d-f. Patch clamp recordings on DRG nociceptors from NHPs acutely treated with vehicle or rIFN-I as in Fig. 2h. (d) Representative traces of current evoked action potentials (left) and rheobase (right). (e) quantification of action potentials and (f) rheobase. rIFN-I perfusion inhibited NHP nociceptor excitability, as evidenced by reduced action potential firing and increased rheobase. g-i. Patch clamp recordings on small-diameter (< 55 μm) human DRG neurons (hDRGs). (g) hDRG neuron with pipette attached for patch clamp recordings. (h) Representative recording following acute application of rIFN-I, which led to hyperpolarization of the membrane potential, quantified in (i). (j) Mechanisms by which autocrine and/or paracrine STING-mediated IFN-I signaling suppresses nociception. All data are expressed as the mean ± s.e.m. * P < 0.05; ** P < 0.01; *** P < 0.001, **** P < 0.0001. Statistical comparisons were conducted with two-way ANOVA with Dunnett’s post-hoc test (b, e), one-way ANOVA with Dunnett’s post-hoc test (c), two-tailed t-test (f), or one-sample t-test (vs. hypothetical value of 0). See Supplementary Table-1 for complete sample sizes, sex, and statistical information.

References

    1. Ishikawa H & Barber GN STING is an endoplasmic reticulum adaptor that facilitates innate immune signalling. Nature 455, 674–678, doi: 10.1038/nature07317 (2008). -DOI -PMC -PubMed
    1. Woo SR et al. STING-dependent cytosolic DNA sensing mediates innate immune recognition of immunogenic tumors. Immunity 41, 830–842, doi: 10.1016/j.immuni.2014.10.017 (2014). -DOI -PMC -PubMed
    1. Kwon J & Bakhoum SF The Cytosolic DNA-Sensing cGAS-STING Pathway in Cancer. Cancer Discov 10, 26–39, doi: 10.1158/2159-8290.Cd-19-0761 (2020). -DOI -PMC -PubMed
    1. Julius D & Basbaum AI Molecular mechanisms of nociception. Nature 413, 203–210 (2001). -PubMed
    1. Donnelly CR, Chen O & Ji RR How Do Sensory Neurons Sense Danger Signals? Trends Neurosci, doi: 10.1016/j.tins.2020.07.008 (2020). -DOI -PMC -PubMed

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