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STING controls nociception via type I interferon signalling in sensory neurons

Nature volume 591, pages 275–280 (2021)Cite this article

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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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Fig. 1: STING inhibits nociception in naive and injured mice.
Fig. 2: STING inhibits nociception via type-I interferon signalling in nociceptors.
Fig. 3: STING-mediated IFN-I signalling suppresses pain in monkeys and nociceptor excitability in monkeys and humans.

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Data availability

Raw data are available from the corresponding author upon request. Source data are provided with this paper.

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Acknowledgements

We thank J. P.-Y. Ting and B. D. X. Lascelles for helpful discussions and R. Kuner for providing Nav1.8-cre mice. This study was supported by Duke University Anesthesiology research funds and Duke Microbiome Center fund granted to R.-R.J. C.R.D. was also supported by the International Association for the Study of Pain John J. Bonica Trainee Fellowship, NIH T32 training grant GM008600, and the Department of Anesthesiology, Duke University. M.-C.K. and the monkey study was supported by NIH R21 grant DA044450.

Author information

Authors and Affiliations

  1. Center for Translational Pain Medicine, Department of Anesthesiology, Duke University Medical Center, Durham, NC, USA

    Christopher R. Donnelly, Changyu Jiang, Amanda S. Andriessen, Kaiyuan Wang, Zilong Wang, Junli Zhao, Xin Luo, Michael S. Lee, William Maixner & Ru-Rong Ji

  2. Department of Physiology and Pharmacology, Wake Forest School of Medicine, Winston-Salem, NC, USA

    Huiping Ding & Mei-Chuan Ko

  3. Department of Periodontics and Oral Medicine, University of Michigan, Ann Arbor, MI, USA

    Yu L. Lei

  4. University of Michigan Rogel Cancer Center, Ann Arbor, MI, USA

    Yu L. Lei

  5. W.G. Hefner Veterans Affairs Medical Center, Salisbury, NC, USA

    Mei-Chuan Ko

  6. Department of Neurobiology, Duke University Medical Center, Durham, NC, USA

    Ru-Rong Ji

  7. Department of Cell Biology, Duke University Medical Center, Durham, NC, USA

    Ru-Rong Ji

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  1. Christopher R. Donnelly
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Contributions

C.R.D. and R.-R.J. conceived and shaped the overall direction of the project. C.R.D., C.J., K.W., A.S.A., Z.W., J.Z., X.L. and M.S.L. performed experiments. H.D. performed behavioural testing in monkeys under the guidance of M.-C.K. Y.L.L. and W.M. contributed to the experimental design. Illustrations were created by C.R.D. using BioRender. C.R.D. and R.-R.J. wrote the manuscript with feedback and discussions from all co-authors.

Corresponding authors

Correspondence to Christopher R. Donnelly or Ru-Rong Ji.

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Competing interests

R.-R.J. is a consultant of Boston Scientific and received research support from the company unrelated to the present study. C.R.D. and R.-R.J. have also filed a patent, ‘Treatment of Neuropathic and Cancer Pain Using Agonists of Stimulator of Interferon Response cGAMP Interactor-1 (STING1)’ in association with Duke University. The other authors declare no competing interests.

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Peer review information Nature thanks Bruce Bean, Marzia Malcangio and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data figures and tables

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

a, Sting1 (STING) mRNA expression in sensory neuron populations recently profiled and previously described9. 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 Information for complete sample sizes, sex, and statistical information.

Source data

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

a, Naive 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 4 h following the first (day 1, D1) or second (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. BL, baseline. b, Naive mice were administered vehicle or ADU-S100, a STING agonist with cross-species activity, via i.t. injection (red arrows) and tested as in 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 naive mice for up to 24 h. d, In the CIPN model, i.p. DMXAA and ADU-S100 suppressed mechanical allodynia for up to 48 h. Some toxicity was observed with systemic administration in the CIPN model, as 3 mice in the DMXAA group died 24 h after the second 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, Intrathecal 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, Intrathecal 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 antitumour effects, as tumour burden was unaffected by STING agonist treatment and treatment groups still show significant tumour growth (l). m–o, Effects of naloxone on morphine-, DMXAA-, and ADU-S100-induced antinociception. Naloxone (10 mg/kg, i.p.) reversed morphine (2 nmol i.t.)-induced antinociception (m) 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 naive mice. q–t, Pain and glial reaction after spared nerve injury (SNI). q, Paradigm of experimental design. Pain behaviours and spinal dorsal horn glial cell reaction were assessed following repeated administration with vehicle or DMXAA (red arrows) in mice receiving SNI. r, s, Mechanical allodynia (r) and cold allodynia (s) were significantly reduced in DMXAA-treated mice at later time points (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 q. 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–d, f, g, k), Sidak’s (h, j, l, s), Bonferroni’s (m–p, r, t), or Tukey’s post hoc test (m–o). See Supplementary Information for complete sample sizes, sex, and statistical information.

Source data

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

a, Schematic indicating the experimental setup for the conditioned place preference (CPP) assay used to measure ongoing pain in mice. b, Schematic and timeline indicating CPP protocol for naive mice. CPP in naive 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 naive mice. d, Schematic and timeline 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+/+ (gWT) or STINGgt/gt (gKO) mice. g, h, STINGgt/gt mice exhibit increased excitability, as determined by number of action potentials evoked per current step (in 10 pA increments), as indicated by traces (g) and quantification (h). i, j, Nociceptors from STINGgt/gt mice also exhibit decreased rheobase at baseline, as indicated by traces (i) and quantification (j). 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 Information for complete sample sizes, sex, and statistical information.

Source data

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

a–f, Hindpaw skin immunostaining for nerve fibres (a, c, e) and their quantification (b, d, f) from STING+/+ (gWT) or STINGgt/gt (gKO) mice. TuJ1 to label all nerve fibres (a), CGRP to label fibres from peptidergic nociceptors (c), or IB4 to label fibres from nonpeptidergic nociceptors (e). In each case, sections were counterstained with DAPI to identify the dermis-epidermis junction. Quantification of TuJ1+ (b), CGRP+ (d) and IB4+ nerve fibres (f) 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 STINGgt/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 CGRP (h) and IB4 (i) pixel density in the spinal dorsal horn (displayed in arbitrary units, A.U.) indicates that there are no changes in central innervation density in STINGgt/gt mice. j, k, Analysis of total DRG neuron numbers in STING+/+ and STINGgt/gt mice. j, L5 DRGs were collected from STING+/+ and STINGgt/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 STINGgt/gt have similar sensory neuron numbers. l–n, No differences were observed between STING+/+ and STINGgt/gt mice in the tape test (l), a measure of sensorimotor function, in an accelerating rotarod test to measure motor function (m), or in locomotor activity in the open field test (30 min test duration) (n). 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 Information for complete sample sizes, sex, and statistical information.

Source data

Extended Data Fig. 5 STING signalling 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 naive mice. e, DMXAA and ADU-S100 (35 nmol each, i.t.) increased paw withdrawal thresholds in STING+/+ (gWT) but not in STINGgt/gt (gKO) 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 STINGgt/gt mice. g, DMXAA and ADU-S100 increased paw withdrawal threshold in Stingfl/fl (cWT) mice, but no significant effects were observed in Stingfl/fl;Nav1.8-Cre (cKO) mice. A non-statistically significant increase in withdrawal thresholds was observed at later time points (24 h, 48 h) in Stingfl/fl;Nav1.8-Cre mice. h, DMXAA and ADU-S100 (35 nmol each, i.t.) reduced mechanical hypersensitivity (determined by withdrawal frequency to a 0.16 g Von Frey filament) in Stingfl/fl;Nav1.8-Cre mice at 24 h and 48 h. 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 = 40 s). k, The early (1 h, 4 h, 24 h) 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 signalling 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 time points. 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–h) or Tukey’s (k, l) post hoc tests. Comparisons between two groups (j) were conducted with two-tailed t-test. See Supplementary Information for complete sample sizes, sex, and statistical information.

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Extended Data Fig. 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 24 h following injection in WT, but not STINGgt/gt mice. b, While basal IFN-β could be detected in DRG tissue from all genotypes, STINGgt/gt mice exhibited significantly lower IFN-β levels. c, d, ADU-S100 treatment of high density DRG neuron cultures from STING+/+ (gWT) or STINGgt/gt (gKO) 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 slightly reduced by ADU-S100 treatment. i–k, Mice lacking Ifnar1 globally (Ifnar1−/−) or selectively in sensory neurons (Ifnar1fl/fl;Nav1.8-Cre) exhibit significantly increased sensitivity to mechanical stimuli determined by paw withdrawal threshold (i) or paw withdrawal frequency (j) to a low stimulus 0.16 g 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 0.04 g 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 Fig. 2h–k. 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 STINGgt/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–k), two-way ANOVA with Dunnett’s post hoc test (a, c, d, o), or two-tailed t-test (h, m, q–s). See Supplementary Information for complete sample sizes, sex, and statistical information.

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

a–f, Hindpaw skin was collected from Ifnar1+/+ or Ifnar1−/− mice and immunostained for TuJ1 (a), CGRP (c) or IB4 (e) with DAPI as in Extended Data Fig. 4. Quantification of epidermal TuJ1+ (b), CGRP+ (d) and IB4+ (f) nerve fibres 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 CGRP (h) and IB4 (i) pixel density (displayed in arbitrary units, A.U.) indicates similar densities of central innervation. j, k, 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) (j). 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 tape test (l), in the accelerating rotarod test (m), or in locomotor activity in the open field test (n) (30 min 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 Information for complete sample sizes, sex, and statistical information.

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

a, b, Inhibition of endogenous IFN-I signalling via i.t. administration of an anti-IFN-α neutralizing antibody (a) or an anti-IFN-β neutralizing antibody (b) (vs. IgG control, 300 ng) induced transient mechanical allodynia in naive mice. c, Inhibition of the IFN-I signalling adaptor Tyk2 with PF-06700841 (i.t., 1 μg) induced transient, dose-dependent mechanical allodynia in naive mice. d–f, i.t. injection of recombinant murine IFN-α (d), murine IFN-β (produced in mammalian cells) (e), or universal IFN-I (f) increased paw withdrawal thresholds in naive 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 signalling pathways and the pharmacologic inhibitors used to target Tyk2, PI3K, or MAPKK. h, Pretreatment of naive mice with the Tyk2 inhibitor PF-06700841 (i.t., 1 μg) abolished IFN-β-induced antinociception. i, j, Pre-treatment with 1 μg i.t. U0126, a MAPKK (MEK) inhibitor (i) or 1 μg LY294002, a PI3K inhibitor (j), failed to affect IFN-β-induced increased paw withdrawal threshold. k, Intraplantar (i.pl., for example, 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 (0.04 g, 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–f, h–l). See Supplementary Information for complete sample sizes, sex, and statistical information.

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

a, L1-L5 DRGs were isolated from STINGfl/fl (cWT), STINGfl/fl;Nav1.8-Cre (cKO), and STINGgt/gt (gKO) 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, c, IFN-α (b) and IFN-β (c) 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 STINGfl/fl;Nav1.8-Cre DRG lysate, but this increase was significantly lower than that seen in WT mice. d, e, DRGs from naive mice were incubated ex vivo in control IgG or anti-IFNβ for 2h followed by whole-mount patch clamp recordings from DRG nociceptors. Representative traces (d) and quantification (e) 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. Representative traces (f) and quantification of mEPSC frequency (g) and amplitude (h). Ifnar1−/− mice exhibit increased frequency and amplitude compared to WT littermates. No significant increase was observed in Ifnar1fl/fl;Nav1.8-Cre mice relative to Ifnar1fl/fl 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, l, IFN-α (k) and IFN-β (l) levels in SDH lysate in mice of the indicated genotypes 24 h after STING agonist administration showing induction of IFN-α but not IFN-β. Basal IFN-β production was significantly lower in STINGgt/gt mice. m–o. Ex vivo incubation of spinal cord slices from naive mice with ADU-S100. Representative traces (m) and quantification of frequency (n) and amplitude (o) 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 frequency (q) and amplitude (r) 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 Information for complete sample sizes, sex, and statistical information.

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Extended Data Fig. 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 STINGgt/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 time points. c, Intrathecal poly(I:C) elevated mechanical thresholds in Ifnar1fl/fl (WT), STINGgt/gt, and cGAS−/− mice, but these effects were abolished in Ifnar1fl/fl;Nav1.8-Cre and RIG-I−/− mice. d, Intrathecal poly(dA:dT) elevated mechanical thresholds in WT and RIG-I−/− mice for up to 24 h, but these effects were abolished in Ifnar1fl/fl;Nav1.8-Cre, STINGgt/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 min as indicated. Representative traces (e) and quantification (f) 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. Representative traces (h) and Timecourse (i) 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 Information for complete sample sizes, sex, and statistical information.

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Donnelly, C.R., Jiang, C., Andriessen, A.S. et al. STING controls nociception via type I interferon signalling in sensory neurons. Nature 591, 275–280 (2021). https://doi.org/10.1038/s41586-020-03151-1

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