Abstract
The regenerative potential of mammalian peripheral nervous system neurons after injury is critically limited by their slow axonal regenerative rate1. Regenerative ability is influenced by both injury-dependent and injury-independent mechanisms2. Among the latter, environmental factors such as exercise and environmental enrichment have been shown to affect signalling pathways that promote axonal regeneration3. Several of these pathways, including modifications in gene transcription and protein synthesis, mitochondrial metabolism and the release of neurotrophins, can be activated by intermittent fasting (IF)4,5. However, whether IF influences the axonal regenerative ability remains to be investigated. Here we show that IF promotes axonal regeneration after sciatic nerve crush in mice through an unexpected mechanism that relies on the gram-positive gut microbiome and an increase in the gut bacteria-derived metabolite indole-3-propionic acid (IPA) in the serum. IPA production by Clostridium sporogenes is required for efficient axonal regeneration, and delivery of IPA after sciatic injury significantly enhances axonal regeneration, accelerating the recovery of sensory function. Mechanistically, RNA sequencing analysis from sciatic dorsal root ganglia suggested a role for neutrophil chemotaxis in the IPA-dependent regenerative phenotype, which was confirmed by inhibition of neutrophil chemotaxis. Our results demonstrate the ability of a microbiome-derived metabolite, such as IPA, to facilitate regeneration and functional recovery of sensory axons through an immune-mediated mechanism.
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Data availability
All RNA sequencing data are available from the NCBI GEO database under accession number GSE161342. Source data are provided with this paper.
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Acknowledgements
We thank I. Palmisano for reading the manuscript and providing feedback, K. Shkura for providing guidance on computational analysis and S. Mani for providing PXRKO sperm. This work was supported by start-up funds from the Department of Brain Sciences, Imperial College London (S.D.G.); Wings for Life (S.D.G.); Rosetrees Trust (S.D.G.); Spinal Research (S.D.G.); and the Imperial PhD Presidential Scholarship (J.S.C.). The laboratory of M.E.D. has received funding by METACARDIS (HEALTH-F4-2012-305312) and the UK Medical Research Council (MRC grants “µNeuroInf” MR/M501797/1 and “National Mouse Genetics Network Microbiome Cluster” MR/W022532/1), and by grants from the French National Research Agency (ANR-10-LABX-46 [European Genomics Institute for Diabetes]), from the National Center for Precision Diabetic Medicine – PreciDIAB, which is jointly supported by the French National Agency for Research (ANR-18-IBHU-0001), by the European Union (FEDER), by the Hauts-de-France Regional Council (Agreement 20001891/NP0025517) and by the European Metropolis of Lille (MEL, Agreement 2019_ESR_11) and by Isite ULNE (R-002-20-TALENT-DUMAS), also jointly funded by ANR (ANR-16-IDEX-0004-ULNE), the Hauts-de-France Regional Council (20002845) and by the European Metropolis of Lille (MEL). This research was also supported by the National Institute for Health Research (NIHR) Imperial Biomedical Research Centre (MED, S.D.G.). Diagrams in the figures were created with BioRender.com. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health.
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E.S. designed the study, performed experiments and data analysis and wrote the manuscript. J.S.C., L.L.-G., G.K., L.Z., G.C., A.M., A.B. and A.S.-V. performed experiments and data analysis. F.M. and A.T.B. performed data analysis. P.L. and F.D.V. performed experiments. M.E.D. performed data analysis and edited the manuscript; J.S., S.M. and D.D. provided experimental advice and edited the manuscript. S.D.G. designed experiments, provided funding and wrote the manuscript.
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Extended data figures and tables
Extended Data Fig. 1 Intermittent fasting promotes axonal regeneration and DRG neurite outgrowth. Assessment of macrophages, Schwann cells and neurotrophic factors in the nerve and DRG in IF vs AL following sciatic injury.
a. Schematic of the experimental design. Mice underwent either 10 days or 30 days of intermittent fasting regime, followed a sciatic nerve crush and 24 h of axonal regeneration. b. Micrographs showing representative longitudinal sections of sciatic nerves stained with SCG-10 and ßIII-Tubulin (ßIIITub) 24 h after SNC. The dashed lines indicate the crush site at 0 and the distances of 500 µm and 1,000 µm from the crush site. c. Quantification of the percentage of SCG-10 positive fibers and normalised to the number of fibers at the crush site. (N = 4 biologically independent animals per group with a bilateral sciatic nerve crush, two-way-ANOVA with Tukey’s multiple comparisons test, data are presented as mean values +/− SEM). d. Schematic of the experimental design. e. Representative images of DRG neurons immunostained with ßIII-Tubulin (ßIIITub). f. Quantification of DRG neurite outgrowth (N = 4 biologically independent animals per group, two-sided Student’s unpaired t-test, Scale bar: 100 µm, examined over two independent experiments, data are presented as mean values +/− SEM). g-h. Representative images of nerves dissected from 10 days AL or IF treated animals at 3 days post sciatic injury. Sections were immunostained for a) CD68 (macrophage marker, red) or b) SOX10 (Schwann cell marker, white). Scale bar 500 µm. Asterisk indicates the crush site. i. Quantification of CD68 (red) was conducted by intensity measurements 1,000 µm proximal and distal to the crush site (N = 6 nerves per group from 3 biologically independent animals per group, data are presented as mean values +/− SEM). j. Quantification of SOX10 (white) was conducted by intensity measurements 1,000 µm proximal and distal to the crush site (N = 5 nerves per group from 3 biologically independent animals per group, data are presented as mean values +/- SEM) k. ELISA measurements of NGF, BDNF, NT3 and NT4/5 from DRG tissue following 10 days IF or AL (N = 3 biologically independent animals per group, data are presented as mean values +/ − SEM). Samples were collected on the refed day.
Extended Data Fig. 2 O-PLS-DA model discriminating AL and IF groups with and without Vancomycin using GC-MS-based metabolic profiles. Faecal microbiota transplantation from IF mice promote axonal regeneration.
a. Score plot of the O-PLS-DA model of the IF group vs AL group. The number of components and the cumulative R2X, R2Y and Q2Y are indicated below the plot. b. Significance diagnostic of the OPLS-DA: the R2Y and Q2Y of the model are compared with the corresponding values obtained after random permutation (1,000) of the y response. c. Model overview: Inertia bar plot. d. Schematic of the experiment presented in b and c. Faecal transplantation of faeces from either IF or AL mice for 1 days, followed by sciatic nerve crush and 3 days of axonal regeneration time. e. Micrographs of representative longitudinal sections of sciatic nerves after faecal transplantation from AL and IF treated mice, 3 days post sciatic nerve crush, immunostained with SCG-10. Scale bar: 1,000 µm. The dashed line indicates the crush site. f. Quantification of the percentage of fibers past the crush site and normalised to the number of fibers at the crush site revealed increased regeneration in IF-FMT mice compared to AL-FMT (N = 4 biologically independent animals per group; ****p < 0.0001, two-way-ANOVA with Tukey’s multiple comparisons test, data are presented as mean values +/− SEM). g. Score plot of the PLS-DA model discriminating IF, AL, IF+V and AL+V. h. Permutation testing of the PLS-DA model: the R2Y and Q2Y of the original model are compared with the corresponding values obtained after random permutation of the group classes (n = 1,000 iterations). i. Model overview: Inertia bar plot. j. Table showing metabolites differentially enriched in the serum of IF vs AL treated mice. Shown are the log fold change (logFC), IFvsAL p-value, vancomycin vs non-vancomycin (Ab vs non-Ab) p-value and the interaction p-value between intermittent fasting and vancomycin treatment (interaction) (bold p < 0.001, partial least-squares discriminant analysis was used to identify the significantly differential metabolites between the groups). R-square (Rsq, the coefficient of determination) showed the highest predictability (79.4%) for indole-3-propionic acid.
Extended Data Fig. 3 16S rDNA amplicon sequencing reveals increased number of Clostridiales following intermittent fasting.
a. Shannon index reveals significant differences in Alpha diversity between vancomycin vs non-vancomycin treated groups (16S sequencing was carried out from cecum of AL, AL+vancomyin, IF, IF+vancomycin treated animals (N = 8 biologically independent animals per group, unpaired, two-sided Mann Whitney U test). The centre line shows the median value (50th percentile), while the box edges correspond to the 25th (Q1) to 75th (Q3) percentiles of dataset. The whiskers mark 1.5 x IQR (interquartile range) below Q1 and above Q3. b. Beta diversity reveals differences in microbiota composition between IF and AL groups c-d. Relative abundance (%) of bacterial phyla (c) and bacterial order (d) between AL, AL+ vancomycin, IF and IF+ vancomycin. e. Plot showing that the top 25 bacterial Amplicon sequence variants (ASVs) that are statistically different (p < 0.05, FDR corrected, plotted as log2FoldChange, data are presented as mean values +/ − SEM) between IF and AL belong to either Bacteroidetes or Firmicutes phyla, Bacteroida or Clostridia classes and Bacteroidales or Clostridiales orders.
Extended Data Fig. 4 Piphillin analysis reveals increased abundance of specific metabolic pathways.
Error bar blot showing the abundance of selected KEGG pathways compared between the four treatment groups (n=8 biologically independent animals per group, Tukey-Kramer post-hoc test, mean +/ − SD). The plot provides a p-value and effect size measure for each pair of groups. Of the pathways of interest we selected, “Xenobiotic biodegradation and metabolism”, “Drug resistance: antimicrobial” and “Metabolism of cofactors and vitamins” showed significantly higher abundance in IF vs AL, which was abolished in IF+V.
Extended Data Fig. 5 Indole-3-proprionate-dependent axonal regeneration.
a. Schematic of the experiment presented in b and c. Mice were gavaged with 10 mg/kg IPA daily for 10 days followed by 3 days of regeneration time post sciatic nerve crush. b. Micrographs of representative longitudinal sections of sciatic nerves immunostained with SCG-10. Scale bar: 1000 µm. The dashed line indicates the crush site. c. Quantification of the percentage of fibers past the crush site at the indicated distances and normalised to the number of fibers at the crush site. revealed no difference in regeneration potential when treated with 10 mg/kg/day. (N = 4 biologically independent animals per group with a bilateral sciatic nerve crush, two-way-ANOVA with Sidak multiple comparisons test, data are presented as mean values +/ − SEM). d. Quantification of IPA concentration in serum at 20 min, 1 h, 6 h and 12 h post gavage of 20 mg/kg IPA. e. Quantification of IPA concentration in naïve DRG tissue at 20 min, 1 h, 6 h and 12 h post gavage of 20 mg/kg IPA. f. Schematic of the experiment presented in g and h. Mice received daily intraperitoneal injections of 20 mg/kg IPA for 10 days, followed by 3 days of regeneration time post sciatic nerve crush. g. Micrographs of representative longitudinal sections of sciatic nerves immunostained with SCG-10. Scale bar: 1000 µm. The dashed line indicates the crush site. h. Quantification of the percentage of fibers past the crush site at the indicated distances and normalised to the number of fibers at the crush site in nerves of mice injected intraperitoneally with 20 mg/kg/day IPA (N = 4 biologically independent animals per group, ****P < 0.0001, two-way-ANOVA with Sidak test, data are presented as mean values +/ − SEM). i. DRG neurons were treated with 10 μM, 100 μM and 1000 μM IPA. Representative images of DRG neurons immunostained with ßIII-Tub. Scale bar: 50 µm. j. Quantification of neurite outgrowth by measuring the length of neurites (N = 4 independent experiments per group; one-way ANOVA, Dunnett’s multiple comparisons test, examined over two experiments, data are presented as mean values +/ − SEM). k+m. Micrographs of representative sections of DRG slices 72h post dextran injection to the nerve co-immunostained for neuronal markers NF200 and IB4. Scale bar: 200 µm. l+n. Quantification of the percentage of double positive NF200+ and IB4+ neurons in IPA and PBS post-injury treated sciatic DRG (N = 5 biologically independent animals per group, Student’s unpaired t-test, data are presented as mean values +/ − SEM).
Extended Data Fig. 6 RNA sequencing from DRG following IPA treatment and injury and increased number of neutrophils in the DRG following IPA treatment.
a-b. Shown are heatmaps of differentially regulated genes (FPKMs, P < 0.05) following 10 days IPA vs PBS treatment as, preceding (a) and 3 days following SN crush (b). Green: downregulated, red: upregulated. c. Heatmap showing the logarithmic expression fold change (logFC (P < 0.05)) of all genes belonging to “Neutrophil-Endothelial interaction” or “BP Neutrophil chemotaxis” GO class for the comparisons IPA-SNCvsPBS, PBS-SNCvsPBS and IPAvsPB. d. Representative image of a DRG stained for Ly6G (red), ßIIITubulin (ßIIITub, green) and DAPI (blue). Scale bar: 100 µm. e. Quantitative analysis of neutrophils, shown as number of neutrophils per 1 mm2 (N = 4 biologically independent animals per group, One-way Anova with Holm-Sidak multiple comparison test, if not indicated otherwise p-value compares to PBS group, data are presented as mean values +/ − SEM). f. Representative images of Ly6G+ cells in DRG tissue. Scale bar: 50 µm. g. Images of the sciatic nerve crush site, immunostained for Ly6G (red) after IPA and PBS treatment. Asterisk indicates the crush site. Scale bar: 250 µm. Magnified image scale bar: 50 µm. h. Quantitative analysis showing the number of neutrophils 1000 µm proximal and distal to the crush site (N = 4 biologically independent animals per group, data are presented as mean values +/ − SEM). i. FACS analysis of the nerve crush site following 10 days of IPA treatment and 3 days SNC. j+k. Bar graphs showing Ly6G+ cell counts (g) and percentage of Ly6G+ cells of the total CD45+ cells (h) in nerve crush site 3 days following SNC (N = 3 biologically independent animals per group, data are presented as mean values +/ − SEM).
Extended Data Fig. 7 FACS gating strategies.
a. Gating strategy for the quantification of Ly6G+hi neutrophils (as % of CD45+ and total cell counts) from the nerve crush site following IPA or PBS treatment (corresponding to Extended Data Fig. 6i–k). b. Gating strategy for the verification of neutrophil depletion following αLy6G or αIgG2A treatment (corresponding to Extended Data Fig. 8h, i). Gr-1+ neutrophils were quantified from the spleen as percentage of CD45+.
Extended Data Fig. 8 Immune cell quantification following IPA and PBS treatment including 3 days after SNC.
a. CD8, CD4, B220, CD68 and NK1.1 (red) coimmunostained with βIIITubulin (green) and DAPI (blue) in DRG sections from IPA and PBS treated groups 3 days after SNC. Scale bar: 50 µm. Scale bar in magnification: 10 µm. b. Quantification of CD8 T-cells, CD4 T-cells, B-cells (B220), Macrophages (CD68) and NK cells (NK1.1) number in IPA and PBS treated DRG 3 days after SNC (N = 4 biologically independent animals per group, Student’s unpaired t-test, data are presented as mean values +/− SEM).
Extended Data Fig. 9 Reduced regeneration following neutrophil depletion and in PXR KO mice.
a. Schematic of the experiment presented in b and c. Mice were cotreated for 10 days with either IPA (20 mg/kg/day) or PBS and anti-IgG or anti-Ly6G, followed by a sciatic nerve crush and 3 days of regeneration time. b. Quantification of the percentage of fibers past the crush site at the indicated distances and normalised to the number of fibers at the crush site. (N = 4 biologically independent animals per group with a bilateral sciatic nerve crush; ****p < 0.0001, two-way-ANOVA with Tukey’s multiple comparisons test, data are presented as mean values +/ − SEM). c. Micrographs of representative longitudinal sections of sciatic nerves immunostained with SCG-10. Scale bar: 1000 µm. The dashed line indicates the crush site. d. Representative images of Ly6G immunostaining in sciatic nerve sections (crush site) of mice treated with IPA or PBS and anti-Ly6G or anti-IgG monoclonal antibody 3 days after SNC. Scale bar: 250 µm. Scale bar in magnification: 50 µm. e. Quantification of the number of Ly6G+ cells in the sciatic nerve crush site of mice treated with IPA or PBS and anti-Ly6G or anti-IgG monoclonal antibody 3 days after SNC (N = 4 biologically independent animals per group with a bilateral sciatic nerve crush, One-way Anova with Holm-Sidak multiple comparisons test, data are presented as mean values +/ − SEM). f. Representative images of Ly6G immunostaining in sciatic DRG sections of mice treated with IPA or PBS and anti-Ly6G or anti-IgG monoclonal antibody 3 days after SNC. Scale bar: 50 µm. g. Quantification of the number of Ly6G+ cells in sciatic DRG sections of mice treated with IPA or PBS and anti-Ly6G or anti-IgG monoclonal antibody 3 days after SNC (N = 4 biologically independent animals per group, one way Anova with Tukey’s multiple comparisons test. Data are presented as mean values +/− SEM). h. Representative flow cytometry plots showing Ly6G+ neutrophil depletion following anti-Ly6G monoclonal antibody vs control IgG. i. Quantification of the percentage of neutrophils of the total CD45+ cells in spleen following control IgG or anti-Ly6G monoclonal antibody treatment (N = 4 biologically independent animals per group, two-sided Student’s unpaired t-test, data are presented as mean values +/− SEM). j. Representative images of Ly6G immunostaining in sciatic DRG sections of PXR WT (PXR+/+) and PXR KO (PXR−/−) mice treated with IPA or PBS for 3 days following SNC. Scale bar: 50 µm. k. Quantification of the number of Ly6G+ cells in sciatic DRG sections of PXR WT (PXR+/+) and PXR KO (PXR-/-) mice treated with IPA or PBS for 3 days following SNC (N = 4 biologically independent animals per group, One-way Anova with Tukey’s multiple comparisons test, data are presented as mean values +/ − SEM). l. Micrographs of representative longitudinal sections of sciatic nerves of PXR WT (PXR+/+) and PXR KO (PXR−/−) mice treated with IPA or PBS for 3 days following SNC immunostained with SCG-10. Scale bar: 1000 µm. The dashed line indicates the crush site. m. Schematic of the experiment presented in l and n. n. Quantification of the percentage of fibers past the crush site at the indicated distances and normalised to the number of fibers at the crush site. (N = 4 biologically independent animals per group with a bilateral sciatic nerve crush, ****p < 0.0001, two-way-ANOVA with Tukey’s multiple comparison test, data are presented as mean values +/− SEM).
Extended Data Fig. 10 Increased number of CXCR2+ neutrophils following IPA treatment and depletion with anti-CXCR2 antibody in nerve tissue.
a. Representative images of CXCR2+Ly6G+ cells in DRG tissue. Scale bar: 20 µm. b. Quantitative analysis of CXCR2+Ly6G+ cells in DRG tissue (N = 4 biologically independent animals per group, two-sided Student t-test, p-value compares CXCR2+Ly6G+ groups, data are presented as mean values +/ − SEM). c. Representative images of Ly6G immunostaining in sciatic nerve sections (crush site) of mice treated with IPA or PBS and anti-CXCR2 or anti-IgG monoclonal antibody 3 days after SNC. Scale bar: 250 µm. Scale bar in magnification: 50 µm. d. Quantification of the number of Ly6G+ cells in the sciatic nerve crush site of mice treated with IPA or PBS and anti-CXCR2 or anti-IgG monoclonal antibody 3 days after SNC (N = 4 biologically independent animals per group with a bilateral sciatic nerve crush; data are presented as mean values +/− SEM).
Extended Data Fig. 11 IFNy is required for IPA-dependent axonal regeneration after SNC and increases RAG phosphorylation and expression in DRG neurons.
a. Table showing IPA dependently upregulated genes assigned to GO BP cellular response to interferon gamma before and after injury. b-c. Mice were treated for 10 days with either IPA (20 mg/kg/day) or PBS (by gavage) and injected (IP) with anti-IFNγ/ anti-IgG/PBS (IFNγ neutralisation). Axonal regeneration was assessed at 3 days post sciatic nerve crush. b. Micrographs of representative longitudinal sections of sciatic nerves for all groups immunostained with SCG-10 following. Scale bar: 1000 µm. The dashed line indicates the crush site. c. Quantification of the percentage of fibers past the crush site at the indicated distances and normalised to the number of fibers at the crush site. (N = 4 biologically independent animals per group with a bilateral sciatic nerve crush, ****p < 0.0001, by two-way-ANOVA with Tukey’s multiple comparisons test, comparing IPA-αIFNγ and IPA-αIgG, data are presented as mean values +/− SEM). d-e. Ex vivo culture of DRG neurons 48 h after IFNγ i.p. injection. d. Representative images of DRG neuron for each treatment, immunostained with ßIII-Tubulin (ßIIITub, white). Scale bar: 100 μm. e. Quantification of DRG neurite outgrowth (n = 3 independent experiments per group, two-sided Student’s t-test, data are presented as mean values +/ − SEM). f-g. DRG neurons were treated with 5 ng/ml of IFNγ and co-treated with increasing concentrations of αIFNγR (0 ng/ml, 100 ng/ml, 200 ng/ml) in vitro and neurite outgrowth was assessed. f. Representative images of DRG neurons for each treatment were immunostained with ßIII-Tubulin (ßIIITub). Cultured for 12 h. Scale bar: 100 μm. g. Quantification of DRG neurite outgrowth (N = 3 independent experiments per group; one-way ANOVA with Dunnett’s multiple comparisons test, data are presented as mean values +/− SEM). h. Immunofluorescence of DRG sections from mice at three days post sciatic nerve crush for NCAM and IFNγR reveal expression of IFNγ receptor in DRG neurons, Scale bar: 50 µm. i. Immunofluorescence of the sciatic crush site at three days post sciatic nerve crush for bIIITubulin and IFNγR reveal the lack of expression of IFNγ receptor in DRG peripheral axons. Scale bar: 200 µm. Scale bar in magnified image: 50 µm. j. Immunohistochemistry of DRG sections from IFNγ or vehicle for regeneration associated genes (RAGs, red): MYC, pMYC, GAP43, ATF3, pERK, pAKT, cJUN, p-cJUN, co-stained for bIIITubulin (green). k. Quantification of RAGs as intensity or % of positive neurons versus total of IFNγ compared to vehicle treated (N = 4 biologically independent animals per group with a bilateral crush, Student’s t-test, data are presented as mean values +/− SEM). Scale bar: 50 µm.
Supplementary information
Supplementary Data 1
16S-Seq DESeq2 data
Supplementary Data 2
16S-Seq percentage abundances
Supplementary Data 3
IPA RNAseq DE genes
Supplementary Data 4
IPA-dependent DE genes
Supplementary Data 5
IPA vs PBS RNAseq GOBP
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Serger, E., Luengo-Gutierrez, L., Chadwick, J.S. et al. The gut metabolite indole-3 propionate promotes nerve regeneration and repair. Nature 607, 585–592 (2022). https://doi.org/10.1038/s41586-022-04884-x
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DOI: https://doi.org/10.1038/s41586-022-04884-x
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