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C. difficile intoxicates neurons and pericytes to drive neurogenic inflammation

Nature volume 622, pages 611–618 (2023)Cite this article

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Abstract

Clostridioides difficile infection (CDI) is a major cause of healthcare-associated gastrointestinal infections1,2. The exaggerated colonic inflammation caused by C. difficile toxins such as toxin B (TcdB) damages tissues and promotes C. difficile colonization3,4,5,6, but how TcdB causes inflammation is unclear. Here we report that TcdB induces neurogenic inflammation by targeting gut-innervating afferent neurons and pericytes through receptors, including the Frizzled receptors (FZD1, FZD2 and FZD7) in neurons and chondroitin sulfate proteoglycan 4 (CSPG4) in pericytes. TcdB stimulates the secretion of the neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP) from neurons and pro-inflammatory cytokines from pericytes. Targeted delivery of the TcdB enzymatic domain, through fusion with a detoxified diphtheria toxin, into peptidergic sensory neurons that express exogeneous diphtheria toxin receptor (an approach we term toxogenetics) is sufficient to induce neurogenic inflammation and recapitulates major colonic histopathology associated with CDI. Conversely, mice lacking SP, CGRP or the SP receptor (neurokinin 1 receptor) show reduced pathology in both models of caecal TcdB injection and CDI. Blocking SP or CGRP signalling reduces tissue damage and C. difficile burden in mice infected with a standard C. difficile strain or with hypervirulent strains expressing the TcdB2 variant. Thus, targeting neurogenic inflammation provides a host-oriented therapeutic approach for treating CDI.

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Fig. 1: SP-mediated and CGRP-mediated neurogenic inflammation in a TcdB caecum injection model and in a CDI mouse model.
Fig. 2: TcdB targets neurons through FZD1/2/7 and pericytes through CSPG4.
Fig. 3: Delivery of TcdB-GTD into peptidergic sensory neurons using the toxogenetic approach is sufficient to induce neurogenic inflammation.
Fig. 4: Inhibiting SP or CGRP signalling alleviates inflammation and reduces C. difficile colonization for endemic and hypervirulent epidemic strains.

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Data availability

All data generated or analysed during this study are included in this published article (and its supplementary data and source data files). All biological materials are available upon request from the co-corresponding authors. The following sources were utilized for this work: Allen Brain Atlas (https://mousespinal.brain-map.org/) and SILVA (https://www.arb-silva.de/no_cache/download/archive/release_132/). Mouse and human intestinal cell data are available from the Broad Institute Single Cell portal with the accession number SCP1038. The following datasets from the NCBI Expression Omnibus database were used: for stromal data, GSE132465, GSE132257 and GSE144735; for colon-innervating DRG neurons (https://hockley.shinyapps.io/ColonicRNAseq/) GSE102962. Source data are provided with this paper.

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Acknowledgements

We thank members of the Dong and Rao Laboratories for technical assistance and suggestions. This study was partially supported by grants from the National Institutes of Health (NIH): R01NS080833, R01NS117626, R01AI132387 and R01AI139087 to M.D.; K08DK110532, R01DK135707 and R01DK130836 to M.R.; DP2GM136652 to S.R.-N.; T32DK007477 for supporting M.A.M.; and R01HL150106 to K.Y. G.A.K. was supported by a NDSEG Fellowship and A.S. by a Schmidt Science Fellowship. We acknowledge support from the NIH-funded Harvard Digestive Disease Center (P30DK034854) and the Boston Children’s Hospital Intellectual and Developmental Disabilities Research Center (P30HD18655). M.R. was supported by an Odyssey Award from the Richard and Susan Smith Family Foundation. M.D. holds the Investigator in the Pathogenesis of Infectious Disease Award from the Burroughs Wellcome Fund. S.R.-N. acknowledges support from Mathers Foundation, Career Award for Medical Scientists from the Burroughs Wellcome Fund, and a Pew Biomedical Scholarship. Figure 3c was created using BioRender (https://biorender.com).

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  1. These authors contributed equally: John Manion, Melissa A. Musser

Authors and Affiliations

  1. Department of Urology, Boston Children’s Hospital, Boston, MA, USA

    John Manion, Min Liu, Siyu Wang, Pyung-Gang Lee, Leo Zhao, Jie Zhang & Min Dong

  2. Department of Surgery, Harvard Medical School, Boston, MA, USA

    John Manion, Min Liu, Siyu Wang, Pyung-Gang Lee, Leo Zhao, Jie Zhang & Min Dong

  3. Department of Microbiology, Harvard Medical School, Boston, MA, USA

    John Manion, Gavin A. Kuziel, Min Liu, Siyu Wang, Pyung-Gang Lee, Leo Zhao, Jie Zhang, Seth Rakoff-Nahoum & Min Dong

  4. Division of Gastroenterology, Department of Pediatrics, Boston Children’s Hospital and Harvard Medical School, Boston, MA, USA

    Melissa A. Musser, Amy Shepherd, Seth Rakoff-Nahoum & Meenakshi Rao

  5. Division of Infectious Diseases, Department of Pediatrics, Boston Children’s Hospital and Harvard Medical School, Boston, MA, USA

    Gavin A. Kuziel & Seth Rakoff-Nahoum

  6. Department of Colorectal Surgery, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China

    Siyu Wang

  7. Center for Infectious and Inflammatory Diseases, Institute of Biosciences and Technology, Texas A&M Health Science Center, Houston, TX, USA

    Ravi K. R. Marreddy & Julian G. Hurdle

  8. Department of Pathology, Boston Children’s Hospital, Boston, MA, USA

    Jeffrey D. Goldsmith

  9. Division of Pulmonary Medicine, Department of Pediatrics, Boston Children’s Hospital and Harvard Medical School, Boston, MA, USA

    Ke Yuan

  10. Institute of Toxicology, Hannover Medical School, Hannover, Germany

    Ralf Gerhard

  11. Department of Physiology and Biophysics, School of Medicine, University of California Irvine, Irvine, CA, USA

    Rongsheng Jin

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  1. John Manion
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Contributions

J.M. and M.D. conceived the project. J.M., M.A.M., G.A.K., A.S., M.L., S.W., M.R. and M.D. participated in experimental design, execution and data analyses. G.A.K., M.L., A.S. and S.W. contributed equally. Cultured DRG studies were performed by J.M. and L.Z. Cultured pericyte studies were performed by J.M. and M.L. J.Z. provided essential training and established surgical models. M.A.M. and M.R. performed most of the immunohistochemistry and confocal imaging studies with assistance from J.M. and A.S. J.M., G.A.K. and S.R.-N. performed microbiome analyses and in vitro analyses of C. difficile. J.M. conceived the toxogenetic approaches. P.-G.L. designed and purified the toxogenetic proteins. K.Y. helped with pericyte culture and analysis. J.D.G. provided human tissue samples. R.G. and R.J. designed and provided essential reagents. R.K.R.M. and J.G.H. prepared C. difficile spores and strains. M.R. and M.D. supervised and coordinated the work.

Corresponding authors

Correspondence to Meenakshi Rao or Min Dong.

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Extended data figures and tables

Extended Data Fig. 1 Histopathology score analysis and time course of TcdB-induced inflammation in a mouse cecum injection model.

a, A list of criteria for assessing histopathology scores of colonic tissues. b-e, H&E micrographs illustrating oedema (b, black line indicates oedema extent); immune cell infiltration (c); epithelial disruption (d); haemorrhage or congestion (e). f, Representative histopathology of human CDI colon tissues compared to a normal control, illustrating neutrophilia, oedematous inflammation, and pseudomembranes. g-j, The subcategories for the histopathology scoring in Fig. 1b: oedema (g), immune cell infiltration (h), epithelial disruption (i), and haemorrhage or congestion (j). n = 5, 6, 9, 5 mice. k-l, SP (k) and CGRP (l) levels in colonic explants from the mouse cecal TcdB injection model at the indicated time (min) following TcdB injection. Vehicle was at 120 min. n = 6, 7, 7, 7, 7 mice. m-n, The levels of SP (m) and CGRP (n) in colonic explants from CDI mouse models infected with either C. difficile 630 or a toxin-null strain (630 A- B-) at 23 h (n = 5), 37 h (n = 5), and 48 h (n = 3), normalized to mice treated with antibiotics but gavaged with saline (time point 0, n = 5). g-j, assessed by one-way ANOVA with post hoc Dunnett’s test. k-l, assessed by Kruskall-Wallis test with post-hoc Dunn’s test. Centre line, mean; error bars reflect the standard error of the mean (SEM); n.s., not significant, p values are exact. Scale bar is 50 µm in b, c and 20 µm in d and e. n is described from left to right.

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Extended Data Fig. 2 Histological scoring for cecum injections of TcdB in the indicated mouse models as described in Fig. 1c,d.

a-e, Histopathology total scores and subscores of TcdB injected Tac1 KO mice, n = 7, 8 mice. f-j, Histopathology total scores and subscores of TcdB injected Nk1r KO mice, n = 7, 7 mice. k-o, Histopathology total scores and subscores of TcdB injected Calca KO mice, n = 8, 10 mice. p-t, Histopathology total scores and subscores of TcdB injected Calcb KO mice, n = 3, 5 mice. u, Representative histopathology of Tac1 KO mice injected with TcdB compared to Tac1 KO mice injected with TcdB and SP in the cecal TcdB injection model (120 min incubation). v, Histopathology scores of experiments described in u, n = 3, 4 mice. Assessed by Student’s T-test, two tailed, relative to WT control mice. Centre line: mean; error bars: SEM; n.s., not significant, p values are exact. n is described from left to right.

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Extended Data Fig. 3 Histological scoring for CDI in the indicated KO mouse models as described in Fig. 1e,f.

a-e, Histopathology total scores and subscores of CDI in Tac1 KO mice, n = 11, 20 mice. f-j, Histopathology total scores and subscores of CDI in Nk1r KO mice, n = 7, 10 mice. k-o, Histopathology total scores and subscores of CDI in Calcb KO mice, n = 6, 9, 7 mice. a-j, assessed by Student’s T-test, two tailed, relative to WT control mice. k-o, assessed by one-way ANOVA with post hoc Dunnett’s test. Centre line: mean; error bars: SEM; n.s., not significant, p values are exact. n is described from left to right.

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Extended Data Fig. 4 FZD1/2/7 are expressed in DRG and enteric neurons.

a, Single cell RNA-seq analysis of FZD1, 2, and 7 expression in different cell types within colonic tissues in mice58. Size of dot indicates proportion expressing whereas the colour indicates expression level (red high, blue low). Putative excitatory motor neuron (PEMN), putative inhibitory motor neuron (PIMN), putative sensory neuron (PSN), putative sudomotor and vasodilator neurons (PSVN), putative interneuron (PIN). b, FZD1, 2, and 7 expression in DRG neurons that innervate colonic tissues are displayed as violin plots72. c, In situ hybridization of spinal cord sections shows enriched expression of FZD1/2/7 in dorsal root ganglia areas. Data are from Allen Brain Atlas73. d, Phase-contrast images show that DRG neurons exposed to TcdB (1 pM, 60 min) exhibit no morphological changes. Scale bar is 50 µm. n = 3 independent replicates. e, RNAscope in situ hybridization showing localization of Pdgfrb expression (magenta) within Cspg4+ cells (cyan, Cspg4-dsRed mice) in colonic tissues. Panels in bottom row show regions in yellow boxed area at higher magnification. Scale bar: 50 µm and lower panel is 20 µm.

Extended Data Fig. 5 Cspg4+ pericytes wrap around blood vessels and form close contact with CGRP-containing afferent neurons.

a, Colon tissues from Cspg4-DsRed mice were harvested and subjected to whole mount immunofluorescence analysis, using an antibody against CD31 (a marker for blood vessel endothelial cells; magenta). DsRed (cyan) signals were detected directly. DsRed-positive cells surround CD31-positive capillaries in colonic crypts. Maximum intensity projection of the whole stack is presented in Fig. 2e. Scale bar is 50 µm. b, Experiments were carried out as in panel a, except that lymphatic endothelial cells were labeled with an antibody against Lyve-1 (magenta) and DsRed signals were detected using an antibody against DsRed. DsRed-positive cells are clearly separate from lymphatic endothelial cells. Scale bar is 50 µm. c-f, Experiments were carried out as described in panel a. Enteric glial cells were visualized with GFP fluorescence in examining proteolipid protein 1 (PLP1)-GFP/-Cspg4-dsRed dual reporter mice (panel c). Fibroblasts were labeled using an antibody against platelet-derived growth factor receptor alpha (PGDFRA, magenta, panel d). Myofibroblasts were labeled using an antibody against smooth muscle actin (SMA, magenta, panel e). Mast cells and interstitial cells of Cajal were labeled using an antibody against cKit (magenta, panel f). Scale bar: 10 µm. g, TcdB induced cell-rounding of cultured human brain vascular pericytes. Scale bar: 25 µm. h, The indicated TcdB mutants were injected into mouse ears via intradermal injection37. Pericytes surrounding ear arterioles were labeled and visualized through DsRed. TcdB and TcdB-FzM induced morphological changes of Cspg4+ pericytes surrounding ear arterioles, whereas TcdB-Cspg4M showed no effect. Scale bar, 50 µm. n = 3 mice/group. i, Experiments were carried out as described in panel h, except that WT mice were utilized and pericytes were detected by immunostaining using an antibody against smooth muscle actin (labeling pericytes). TcdB-FzM disrupted pericytes around ear arterioles, whereas TcdB-Cspg4M has no effect. Scale bar: 50 µm. n = 3 mice/group. j, Experiments were carried out as in panel a, except that neuronal processes were labeled using an antibody against Tubb3 (magenta, a marker for neuronal processes), showing that neuronal processes are extending alongside DsRed-positive pericytes. Scale bar: 10 µm. k, Experiments were carried out as in panel a, except that an antibody against CGRP was added to detect CGRP-positive nerve terminals. 3-dimensional reconstruction of images showed that DsRed+ pericytes (cyan) surround the vasculature (CD31 endothelial marker; magenta) and contact CGRP-expressing nerve terminals (CGRP; yellow). Scale bar: 10 µm. l, Immunoblot analysis of cell lysates showed expression of CGRP receptors (CALCRL: calcitonin receptor like receptor; and RAMP1: receptor activity modifying protein 1) in primary cultured human brain vascular pericytes. Two human cell lines, HeLa and U87, were analyzed in parallel as controls, which do not express detectable levels of CGRP receptors. Total protein staining with Coomassie blue was used as a control for protein loading. For gel source data, see Supplementary Fig. 1. N = 2 replicates.

Extended Data Fig. 6 AAV-DTR mediates selective expression of DTR in ENS and DRG neurons in Tac1-Cre mice.

a, AAV-DTR was administered to Tac1-Cre mice via tail vein injection. Colon tissues were harvested 3-weeks later and subjected to whole mount immunofluorescence analysis. DRG were harvested and their cryosections were analyzed. HuC/D (magenta) marks neuron soma in colon tissues. An antibody against β3-tubulin (TUBB3, magenta) was utilized to label DRG neurons. DTR-GFP was labeled using an antibody against GFP (cyan). Scale for DRG: 100 µm; for myenteric plexus: 50 µm. Images were collected using a confocal microscope and maximal intensity projection is presented. GFP is directly fused to the C-terminus of DTR, thus cyan color represents DTR expression. n = 4 mice. b, Experiments were carried out as described in upper panel. 5-HT (magenta) labels enterochromaffin cells in colonic tissues, showing no co-expression of DTR-GFP (cyan). Scale bars represent 100 µm (50 µm for right panel, which shows boxed region at higher magnification). c-d, Experiments were carried out as described in Fig. 3e. Colonic tissues (panel c) and DRG (panel d) were isolated and subjected to analysis as described in panel a. WT mice not exposed to AAV-DTR were analyzed in parallel as a negative control (right panels). HuC/D labels myenteric plexus neurons within the proximal colon and TUBB3 labels DRG neurons of a wildtype mouse injected with AAV-DTR. No GFP positive cell bodies or fibers were detected in WT mice. Scale bar is 50 µm. e-h, Sub-scoring of histopathology, related to Fig. 3f, n = 7, 6, 4 mice. One-way ANOVAs were applied with post hoc Tukey’s test as indicated. Centre line: mean; error bars: SEM; n.s., not significant, p values are exact. n is described from left to right.

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Extended Data Fig. 7 Extrinsic afferent neuron-dependent secretion of SP is necessary and sufficient for TcdB mediated pathology.

a-d; Histopathology subscoring of experiments following TcdB administration to RTX treated mice. Related to Fig. 3h, n = 10, 10 mice. e-j, Histopathology of RTX treated/vehicle mice treated with TcdB alone or TcdB and SP for 120 min, demonstrating that SP can restore the effects of TcdB in RTX-treated mice. Representative histopathology in e, and histopathology scores f-j, n = 4, 4, 6, and 6 mice. The scale bar is 50 µm. k, CGRP secretion from RTX treated mice is reduced in the cecal TcdB injection model, and is restored by co-administration of SP with TcdB. n = 2, 4, 6, 4, and 6 mice. l-m, Nav1.8Cre/+ Rosa-LSL-Tdtomato mice have no evident Tomato expression (magenta) in the ENS (myenteric neurons marked with HuC/D, cyan) with only sparse Tomato+ nerve fibers from extrinsic neurons labeled in the colon (l). In contrast, there are many Tomato+ neurons in DRG (m). Scale bar: 50 µm. n-q, Histopathology subscores of Nav1.8Cre/+, Rosa-LSL-DTR mice treated with GTD-iDT related to Fig 3i, j; n = 7, 9 mice. For a-d and n-q, significance was assessed by student’s two-tailed t-test. For f-k, significance was assessed by one-way ANOVA with post hoc Dunnett’s test. Centre line: mean; error bars: SEM. p values are exact. n is described from left to right.

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Extended Data Fig. 8 Inhibiting SP or CGRP signaling reduces severity of C. difficile colitis.

a, CDI models were carried out on WT versus Tac1 KO mice, using either C. difficile 630 Δerm or a toxin-null mutant strain (A-/B-). C. difficile CFUs from faeces were analyzed at 2 days post infection. The toxin-null strain showed reduced colonization on WT mice compared with the standard 630 Δerm, suggesting that the presence of toxins facilitates C. difficile colonization. In contrast, 630 Δerm showed similar levels of CFUs as the toxin-null strain in Tac1 KO mice, suggesting that the toxin’s contribution to C. difficile colonization relies on intact SP signaling. n = 7, 6, 4, 3 mice. b, CDI models were carried out on WT versus Calcb KO mice (using C. difficile 630 Δerm). C. difficile CFUs from faeces showed reduced colonization in Calcb KO mice compared with WT mice, n = 4, 5 mice. c, Representative H&E images for the experiments described in Fig. 4c. Scale bar is 50 µm. d-g, Sub-categories of histopathological scoring of Fig. 4c; n = 15, 12, 11, 3. h-l, Histopathology scoring of mice administered vehicle or aprepitant at the indicated times (0, 30 or 60 min following cecum injection and wound closure); n = 7, 6, 6, 7 mice. Significance was assessed for a – Two-Way ANOVA with post hoc Sidak’s test and by T-test for b. One Way ANOVA with post hoc Dunnett’s test was used for d-l. Centre line: mean; error bars: SEM. p values are exact. n is described from left to right.

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Extended Data Fig. 9 Inhibiting SP or CGRP reduces histopathological scores in cecum injection assays and in CDI models.

a, Representative H&E images for the experiment described in Fig. 4d. Scale bar is 50 µm. b-e, Sub-categories of histopathological scoring of Fig. 4d, n = 13,7, 8, 8 mice. f, C. difficile 630 Δerm fitness (measured by OD600) was not affected by treatment with aprepitant or olcegepant (20 µM) in vitro. Representative of 2 independent experiments (3 wells/group). g-h, CDI experiments (with C. difficile 630) were carried out on WT and Tac1 KO mice (co-housed from weaning), followed by treatment with vehicle or aprepitant. C. difficile colonization (panel g) and histopathological scores (panel h) were shown, n = 7, 7, 6, 7 mice. i-j, CDI experiments (with C. difficile 630) were carried out on WT and Calcb KO mice (littermates, co-housed from birth), followed by treatment with vehicle or olcegepant. C. difficile colonization (panel i) and histopathological scores (panel j) were shown. n = 4, 4, 5, 7 mice. k, TcdA induced oedema in footpad injection assays in mice, which is prevented by pre-treatment with aprepitant. Boiled TcdA solution did not induce any oedema. n = 7,4, 3 mice. Significance was assessed by one-way ANOVA with post hoc Dunnett’s test. Centre line: mean; error bars: SEM. n.s., not significant, p values are exact. n is described from left to right.

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Extended Data Fig. 10 Inhibiting SP or CGRP reduces histopathological scores and colonization of hypervirulent strains that express TcdB2.

a, Representative H&E images for the experiments described in Fig. 4i. Scale bar is 50 µm. b-e, Sub-categories of histopathological scoring of Fig. 4i, n = 3, 9, 7 mice. f, Representative H&E images for the experiment described in Fig. 4j. Scale bar is 50 µm. g-j, Sub-categories of histopathological scoring of Fig. 4j, n = 21, 10, 10, 12, 10, 5 mice. Significance was assessed by one-way ANOVA with post hoc Dunnett’s test. Centre line: mean; error bars: SEM; n.s., not significant; p values are exact. n is described from left to right.

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Supplementary information

Supplementary Information

Antibody information (Supplementary Table 1) and source western blot scans and Coomassie staining related to Extended Data Fig. 5l (Supplementary Fig. 1).

Reporting Summary

Supplementary Video 1

Pericytes envelope the colonic vascular network. Mouse colonic tissue from CSPG4–DsRed mice was collected, processed by whole mount and subjected to immunostaining with BABB optical clearing of tissue. Z-stack images were captured on a Zeiss LSM 700 confocal microscope with Zen Black software. Z-stack images were processed and rendered into 3D image movies using Aivia image analysis software. In the 3D image movie, an initial view is seen peering down at epithelium lining bottom of intestinal crypts (background nonspecific green fluorescence signal). Removal of epithelial background reveals CD31-immunolabelled capillary networks and a deeper arteriole (green). DsRed antibody immunolabelled CSPG4–DsRed reporter pericytes (magenta) are found in close juxtaposition to the vascular network throughout.

Supplementary Video 2

Pericytes wrap around mucosal capillaries. Mouse colonic tissue from CSPG4–DsRed mice was collected, processed by cryosection and subjected to immunostaining. Z-stack images were captured on a Zeiss LSM 880 confocal microscope with Zen Black software. Z-stack images were processed and rendered into 3D image movies using Aivia image analysis software. The movie demonstrates a 3D view of two CSPG4-reporter labelled pericytes (magenta). CD31-immunolabelled capillaries (green) are found nestled within the pericyte bodies and wrapped within pericyte cellular processes.

Supplementary Video 3

Colonic neurovascular unit comprised pericyte, capillary and CGRP+ neuron processes. Mouse colonic tissue from CSPG4–DsRed mice was collected, processed by cryosection and subjected to immunostaining. Z-stack images were captured on a Zeiss LSM 880 confocal microscope with Zen Black software. Z-stack images were processed and rendered into 3D image movies using Aivia image analysis software. The movie demonstrates a 3D image of a colonic neurovascular unit. A single CSPG4–DsRed reporter-labelled pericyte (magenta) is shown. A CD31-immunolabelled vessel (green) is surrounded by the pericyte body and its processes. CGRP-immunolabelled neuron processes (grey) appear to interact with the pericyte at multiple points.

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Manion, J., Musser, M.A., Kuziel, G.A. et al. C. difficile intoxicates neurons and pericytes to drive neurogenic inflammation. Nature 622, 611–618 (2023). https://doi.org/10.1038/s41586-023-06607-2

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