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Anthrax toxins regulate pain signaling and can deliver molecular cargoes into ANTXR2+ DRG sensory neurons

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Abstract

Bacterial products can act on neurons to alter signaling and function. In the present study, we found that dorsal root ganglion (DRG) sensory neurons are enriched for ANTXR2, the high-affinity receptor for anthrax toxins. Anthrax toxins are composed of protective antigen (PA), which binds to ANTXR2, and the protein cargoes edema factor (EF) and lethal factor (LF). Intrathecal administration of edema toxin (ET (PA + EF)) targeted DRG neurons and induced analgesia in mice. ET inhibited mechanical and thermal sensation, and pain caused by formalin, carrageenan or nerve injury. Analgesia depended on ANTXR2 expressed by Nav1.8+ or Advillin+ neurons. ET modulated protein kinase A signaling in mouse sensory and human induced pluripotent stem cell-derived sensory neurons, and attenuated spinal cord neurotransmission. We further engineered anthrax toxins to introduce exogenous protein cargoes, including botulinum toxin, into DRG neurons to silence pain. Our study highlights interactions between a bacterial toxin and nociceptors, which may lead to the development of new pain therapeutics.

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Fig. 1: Nav1.8+ mouse DRG neurons and human DRG neurons express Antxr2.
Fig. 2: ET intrathecal administration silences mechanical and thermal sensation in mice.
Fig. 3: ANTXR2 expressed by Nav1.8+ or Advillin+ neurons mediates ET-induced analgesia.
Fig. 4: ET induces PKA signaling in DRG neurons but does not affect neuronal viability.
Fig. 5: ET attenuates neurotransmission at nociceptor central terminals.
Fig. 6: Anthrax ET silences pain in mouse models of neuropathic and inflammatory pain.
Fig. 7: Engineered anthrax toxins deliver molecular cargo into DRG sensory neurons and block pain in vivo.

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Data availability

Transcriptional profiling data of the DRG after ET administration is available from the GEO database under no. GSE184619. Microarray data of sorted mouse DRG neuron populations3 is available from the GEO database under accession no. GSE55114. ScRNA-seq data of the mouse nervous system4 are available from the SRA under the accession no. SRP135960 and at http://mousebrain.org. ScRNA-seq data of mouse DRG neurons across development5 are available from the GEO database under accession no. GSE139088. Microarray data from BioGPS.org55 are available at http://biogps.org. ISH data of mouse brain and spinal cord are available from the 2004 Allen Mouse Brain Atlas55 (http://mouse.brain-map.org) and the 2008 Allen Spinal Cord Atlas (http://mousespinal.brain-map.org). Source data are provided with this paper. Datasets generated during the present study are presented in the figures and available from the corresponding author upon reasonable request.

Materials availability

The LFN–LC/AC699S construct is available from Ipsen and requires a material transfer agreement.

Code availability

Customized code or algorithms were not used to generate results in the present study. HCS analysis of DRG neurons was performed using the Cellomics software package as described in Methods. Analysis of DRG sections was performed using Fiji ImageJ plugins as described in Methods. Transcriptional profiling data of the DRG were analyzed using RStudio as described in Methods.

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Acknowledgements

We thank K. Goldstein, S. Sannajust, M. Heyang, N. LoRocco and A. Albakri for excellent technical assistance, and T. Voisin for helpful discussions. We thank M. Dong and S. Tian for helpful discussions and related collaboration. Research was supported by the Burroughs Wellcome Fund, Chan-Zuckerberg Initiative and NIH (DP2AT009499, R01AI130019) to I.M.C.; NIH NIA 5T32AG000222 fellowship to N.J.Y.; NIH NIGMS T32GM007753 fellowship to D.V.N.; the Intramural Program of the NIAID, NIH to S.H.L.; Internal funds from Department of Anesthesiology, Stony Brook Medicine to M.P.; NIH R01NS036855 to B.P.B.; the European Regional Development Fund (NeuRoWeg; nos. EFRE-0800407 and EFRE-0800408) and the Innovative Medicines Initiative 2 Joint Undertaking (116072-NGN-PET) to O.B.; NIH NINDS NS111929 to T.J.P; and the São Paulo Research Foundation (FAPESP) (2013/08216-2 (Center for Research in Inflammatory Diseases)) to T.M.C; Deutsche Forschungsgemeinschaft (nos. 271522021 and 413120531), EFRE-0800384 and Leitmarktagentur.NRW LS-1-1-020d to T.H. The present study was supported in part by a sponsored research grant from Ipsen Pharmaceuticals to I.M.C.

Author information

Authors and Affiliations

Authors

Contributions

N.J.Y., R.J.C. and I.M.C. conceived the project. N.J.Y., D.N., A.K-C. and V.S.T. performed experiments and data analysis. J.I., A.B. and T.H. performed HCS microscopy analysis on DRG neurons, DRG sections and human iPSC-derived sensory neurons. A.U.Q. and T.M.C. performed spinal cord immunostaining and biotelemetry analyses. H.X.B.Z. and B.P.B. performed electrophysiological analysis in DRG neurons. J.L. and M.P. performed spinal cord electrophysiology analysis. S.S. and T.P. performed ISH analysis of human DRG neurons. P.R., A.N. and O.B. provided human iPSC-derived sensory neurons. M.L. and B.L.P. provided recombinant anthrax toxin. M.M. and S.H.L. provided native and engineered anthrax toxins and Antxr2 CKO mice. S.M.L., S.P., V.T. and K.A.F. purified and characterized chimeric anthrax botulinum toxin. S.M. and J.M. performed the mPNHD assay. N.J.Y. and I.M.C. wrote the manuscript with input from all authors.

Corresponding author

Correspondence to Isaac M. Chiu.

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Competing interests

S.M.L., S.P., S.M., J.M., V.T. and K.A.F. are employees of Ipsen. I.M.C. has received sponsored research support from Ipsen, GSK and Allergan Pharmaceuticals, and is a member of scientific advisory boards for GSK and Kintai Pharmaceuticals. This work is related to patent applications PCT/US16/49099 and PCT/US16/49106, ‘Compositions and methods for treatment of pain’, of which R.J.C., I.M.C., B.L.P., K.A.F., S.P. and S.M.L. are co-inventors. O.B. is a co-founder and shareholder of LIFE & BRAIN GmbH. The remaining authors declare no competing interests.

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Nature Neuroscience thanks Stephen McMahon and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Antxr2 but not Antxr1 is enriched in DRG sensory neurons.

(a, b) Transcriptomes of adult DRG sensory neurons plotted as a force-directed layout from public single cell RNA-seq data5, overlaid with expression levels of Antxr2 (a) and Antxr1 (b). Subgroups of neurons are labeled based on the published database. SST, somatostatin. (c) Expression of Antxr2 and Antxr1 across the mouse nervous system from public single cell RNA-seq data4.

Extended Data Fig. 2 Anthrax toxins modulate MAPK and cAMP signaling in DRG sensory neurons.

(a, b) DRG cultures were treated with combinations of PA, LF and EF for 24 h. Levels of MEK3 expression and p38 phosphorylation were quantified by western blot. (n=3 experiments in (a); n=4 experiments for untreated and LT, n=3 experiments for PA, LF, EF and ET in (b).) (c) cAMP levels in DRG cultures treated with combinations of PA, LF and EF for 2 h. (n=4 experiments for untreated, PA, LT, EF and ET; n=3 experiments for LF.) (d) cAMP levels in DRG cultures treated for 2 h with varying concentrations of EF in the presence or absence of 10 nM PA (n=3 experiments). Statistical significance was assessed by one-way RM ANOVA with Dunnett’s post hoc test (a), mixed-effect model with Dunnett’s post hoc test (b), one-way ANOVA with Dunnett’s post hoc test (c), or two-way ANOVA with Sidak’s post hoc test (d). *p<0.05, **p<0.01, ****p<0.0001. Data represent the mean ± s.e.m. For detailed statistical information, see Supplementary Table 2.

Source data

Extended Data Fig. 3 ET-induced analgesia does not show significant sex-dependent effects.

Breakdown of results in male and female mice from (a) Fig. 2b, (b) Fig. 2c, (c) Fig. 2f, (d) Fig. 2g, (e) Fig. 2h and (f) Fig. 2i. The sample sizes denoted in each panel represent the number of mice. The ‘*’ symbol compares Vehicle versus ET in all panels. Statistical significance was assessed by two-way RM ANOVA with Dunnett’s post hoc test (a, b) or two-tailed unpaired t-test (c-f). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Data represent the mean ± s.e.m. For detailed statistical information, see Supplementary Table 2.

Extended Data Fig. 4 ANTXR2 ablation from Nav1.8+ neurons attenuates cAMP signaling in DRG culture but causes minimal effects on baseline sensory function.

(a) qPCR analysis of Antxr2 expression in DRGs harvested from Nav1.8cre/+/Antxr2fl/fl (Cre+) or Nav1.8+/+/Antxr2fl/fl (Cre-) mice (n=5 mice/group). (b) cAMP levels normalized to protein concentration in DRG neurons that were harvested from Nav1.8cre/+/Antxr2fl/fl (Cre+) or Nav1.8+/+/Antxr2fl/fl (Cre-) mice and treated with 10 nM PA and the indicated concentrations of EF for 2 h (n=3 wells). (c) Baseline pain behaviors of Nav1.8cre/+/Antxr2fl/fl mice (Cre+) and Nav1.8+/+/Antxr2fl/fl littermates (Cre-). (von Frey, n=14 mice for Cre- and n=19 mice for Cre+; Randall-Selitto, n=19 mice/group; Hargreaves, n=10 mice for Cre- and n=14 mice for Cre+; Hot plate, n=10 mice for Cre- and n=11 mice for Cre+; Cold plate, n=11 mice for Cre- and n=18 mice for Cre+.) (d) (Left) Acute pain-like behaviors measured in 5 min intervals in Nav1.8cre/+/Antxr2fl/fl (Cre+; n=10 mice) or Nav1.8+/+/Antxr2fl/fl (Cre-; n=11 mice) mice following intraplantar injection of 5% formalin. (Right) Cumulative responses during Phase I (0 – 5 min) or Phase II (15 – 35 min) of formalin-induced pain. (e) Mechanical sensitivity in Nav1.8cre/+/Antxr2fl/fl (Cre+; n=6 mice) or Nav1.8+/+/Antxr2fl/fl (Cre-; n=5 mice) mice following intraplantar injection of 20 μL of 2% carrageenan. Statistical significance was assessed by two-tailed unpaired t-test (a, c, 4d-right), two-way ANOVA with Sidak’s post hoc test (b), or two-way RM ANOVA with Sidak’s post hoc test (d-left, e). n.s, not significant, *p<0.05, **p<0.01, ****p<0.0001. Data represent the mean ± s.e.m. For detailed statistical information, see Supplementary Table 2.

Extended Data Fig. 5 Intraplantar administration of Edema Toxin induces mechanical allodynia and edema.

(a) Mechanical sensitivity after intraplantar administration of vehicle (PBS; n=27 mice), PA (2 μg; n=17 mice), LT (2 μg PA + 2 μg LF; n=10 mice) or ET (2 μg PA + 2 μg EF; n=9 mice). (b) Paw thickness after intraplantar administration of vehicle (PBS) or ET (2 μg PA + 2 μg EF) (n=6 mice/group). (c) cAMP levels in the footpad, DRG or spinal cord after intraplantar administration of vehicle (PBS) or ET (2 μg PA + 2 μg EF) (n=4 mice/group). (d) Mechanical sensitivity after intraplantar administration of ET (2 μg PA + 2 μg EF) to Nav1.8cre/+/Antxr2fl/fl (Cre+; n=7 mice) or Nav1.8+/+/Antxr2fl/fl (Cre-; n=5 mice) mice. Statistical significance was assessed by two-way RM ANOVA with Dunnett’s post hoc test (a), mixed-effects model with Sidak’s post hoc test (b), one-way ANOVA with Dunnett’s post hoc test (c), or two-way RM ANOVA with Sidak’s post hoc test (d). *p<0.05, **p<0.01, ****p<0.0001. Data represent the mean ± s.e.m. For detailed statistical information, see Supplementary Table 2.

Extended Data Fig. 6 Edema Toxin induces PKA signaling in DRG neurons but not non-neuronal cells.

(a) Time-course of pRII intensity in DRG neurons stimulated with Ctrl (0.1% BSA), PA (10 nM), LF (10 nM) or the combination of both factors (n=3 experiments, >2500 neurons/condition). (b) Dose-response curve of pRII intensity in DRG neurons exposed to LF (0 - 50 nM, 2 h) in the presence of a constant concentration of PA (10 nM) (n=3 experiments, >2500 neurons/condition). (c) Single cell data of DRG neurons stimulated with control solvent (0.1% BSA) or EF and PA (10 nM each) for 2 h. The DRG neurons were stained for UCHL1, pRII, and the indicated subgroup markers (>5000 neurons per marker). (d) Changes in the percentage of pRII positive cells with ET treatment over Ctrl in each neuronal subgroup from (c). The % response with Ctrl treatment was subtracted from the % response with ET treatment. (e) Representative images of mouse DRG neurons showing the modified cell identification to analyze PKA-II activation in non-neuronal cells (green), but not neurons or associated cells (red). Scale bar, 50 μm. (f) Time-course of pRII intensity in the nuclei of UCHL1-negative non-neuronal cells stimulated with Ctrl (0.1% BSA), forskolin (10 µM), PA (10 nM), EF (10 nM), and LF (10 nM) or the combination of these factors (n=4 experiments). Statistical significance was assessed by two-way ANOVA with Bonferroni’s post hoc test (d). *p<0.05, **p<0.01, ***p<0.001. Data represent the mean ± s.e.m. For detailed statistical information, see Supplementary Table 2.

Extended Data Fig. 7 Edema Toxin induces PKA signaling in human iPSC-derived sensory neurons.

(a) Representative HCS microscopy images of human iPSC-derived nociceptors stimulated with vehicle control (Ctrl) or EF + PA (10 nM each) for 2 h. Cultures were labeled with fluorescent Nissl to identify the cells, and pRII and RIIβ to quantify PKA-II signaling activity. Green or red encircled cells indicate automatically selected or rejected objects, respectively (see methods section). Scale bar, 100 µm. (b) Single cell data of human iPSC-derived nociceptors stimulated with control solvent (0.1% BSA) or EF (2 nM) and PA (10 nM) for 2 h. (c) Dose-response curve of pRII intensity in human iPSC-derived nociceptors exposed to EF (0 - 50 nM, 2 h) in the absence or presence of a constant concentration of PA (10 nM) (n=8 wells from one culture of differentiated neurons analyzed in four replicate experiments. >8000 neurons/condition). Data represent the mean ± s.e.m.

Extended Data Fig. 8 Edema Toxin treatment enhances excitability of small-diameter DRG neurons.

Excitability of small diameter DRG neurons was examined after treatment with control media (Untreated) or ET (10 nM PA + 10 nM EF) for 2 – 10 h at 37°C. (a) Action potential firing elicited by a 1-s 20-pA current injection in a control small DRG neuron. (b) Action potential firing elicited by the same current injection in a representative ET-treated neuron. (c) Number of action potentials during 1-s current injections as a function of injected current (Untreated group: n=11 cells; ET group: n=12 cells). Data plotted as mean ± s.e.m. (d) Resting potential of untreated (n=11 cells) and ET-treated (n=12 cells) neurons. (e) Input resistance of untreated (n=11 cells) and ET-treated (n=12 cells) neurons. Statistical significance was assessed by unpaired two-tailed Mann-Whitney test (c-e). n.s, not significant, *p<0.05. For detailed statistical information, see Supplementary Table 2.

Extended Data Fig. 9 Intrathecal administration of ET induces transcriptional changes in the DRG.

(a) Mice received intrathecal injection of vehicle (PBS) or ET (2 µg PA + 2 µg EF) (n=6/group). Lumbar DRGs were harvested at 2 hours post-injection. Poly A-selected libraries were prepared from isolated RNA and sequenced on a NextSeq 500 sequencer. (b) Pathway analysis based on gene expression changes. (c) Pharmacological inhibition of DUSP1 does not affect ET-induced analgesia. The DUSP1/DUSP6 inhibitor BCI (1.6 µg) or its vehicle (1% DMSO) was injected intrathecally 15 min prior to ET (2 µg PA + 2 µg EF) or its vehicle (PBS). (n=7 mice for Veh, Veh and BCI, Veh groups; n=8 mice for Veh, ET and BCI, ET groups.) Two-way repeated measures ANOVA with Tukey’s post hoc test. **p<0.01, Veh, Veh vs. Veh, ET; ++p<0.01, BCI, Veh vs. BCI, ET. Data represent the mean ± s.e.m. For detailed statistical information, see Supplementary Table 2.

Extended Data Fig. 10 ET-induced pain blockade does not show sex-dependent effects in mouse models of pain.

Breakdown of results in male and female mice from (a) Fig. 6d and (b) Fig. 6e. The sample sizes denoted in each panel represent the number of mice. The ‘*’ symbol in (b) compares i.th Veh, i.pl Car vs. i.th ET, i.pl Car. Statistical significance was assessed by two-way RM ANOVA with Sidak’s (a) or Tukey’s (b) post hoc tests. *p<0.05, **p<0.01. Data represent the mean ± s.e.m. For detailed statistical information, see Supplementary Table 2.

Supplementary information

Supplementary Information

Supplementary Figs. 1–11 and Table 1.

Reporting Summary

Supplementary Table 2

Detailed statistical information for Figs. 1–7, Extended Data Figs. 1–10 and Supplementary Figs. 1–10.

Source data

Source Data Fig. 7

Unprocessed western blots for Fig. 7d.

Source Data Extended Data Fig. 2

Unprocessed western blots for Extended Data Fig. 2a,b.

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Yang, N.J., Isensee, J., Neel, D.V. et al. Anthrax toxins regulate pain signaling and can deliver molecular cargoes into ANTXR2+ DRG sensory neurons. Nat Neurosci 25, 168–179 (2022). https://doi.org/10.1038/s41593-021-00973-8

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