The liver–brain–gut neural arc maintains the Treg cell niche in the gut

Abstract

Recent clinical and experimental evidence has evoked the concept of the gut–brain axis to explain mutual interactions between the central nervous system and gut microbiota that are closely associated with the bidirectional effects of inflammatory bowel disease and central nervous system disorders1,2,3,4. Despite recent advances in our understanding of neuroimmune interactions, it remains unclear how the gut and brain communicate to maintain gut immune homeostasis, including in the induction and maintenance of peripheral regulatory T cells (pTreg cells), and what environmental cues prompt the host to protect itself from development of inflammatory bowel diseases. Here we report a liver–brain–gut neural arc that ensures the proper differentiation and maintenance of pTreg cells in the gut. The hepatic vagal sensory afferent nerves are responsible for indirectly sensing the gut microenvironment and relaying the sensory inputs to the nucleus tractus solitarius of the brainstem, and ultimately to the vagal parasympathetic nerves and enteric neurons. Surgical and chemical perturbation of the vagal sensory afferents at the hepatic afferent level reduced the abundance of colonic pTreg cells; this was attributed to decreased aldehyde dehydrogenase (ALDH) expression and retinoic acid synthesis by intestinal antigen-presenting cells. Activation of muscarinic acetylcholine receptors directly induced ALDH gene expression in both human and mouse colonic antigen-presenting cells, whereas genetic ablation of these receptors abolished the stimulation of antigen-presenting cells in vitro. Disruption of left vagal sensory afferents from the liver to the brainstem in mouse models of colitis reduced the colonic pTreg cell pool, resulting in increased susceptibility to colitis. These results demonstrate that the novel vago-vagal liver–brain–gut reflex arc controls the number of pTreg cells and maintains gut homeostasis. Intervention in this autonomic feedback feedforward system could help in the development of therapeutic strategies to treat or prevent immunological disorders of the gut.

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Fig. 1: Potential interaction between APCs and neurons in the gut.
Fig. 2: The hepatic vagal sensory afferent pathway is essential for NTS activation during colitis.
Fig. 3: The liver–brain–gut axis regulates colonic Treg cell homeostasis through muscarinic signalling in APCs.
Fig. 4: Perturbation of hepatic vagal afferents exacerbates mouse colitis in a muscarinic signalling-dependent manner.

Data availability

All raw and processed sequencing data in this paper have been deposited at NCBI Gene Expression Omnibus under accession number GSE140952.

Code availability

All computer code to analyse RNA-seq data is available at https://github.com/mikamiy/liver-brain-gut-neural-arc.

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Acknowledgements

We thank M. Tsuruta, T. Ishida, K. Shigeta, R. Seishima and the medical staff of the surgical department for collecting samples: H. Sato, R. Aoki, Y. Kohda, K. Ono, Y. Yoshimatsu, K. Yoshida, S. Tanemoto, Y. Takada, E. Nomura, S. Umeda, M. Ichikawa, Y. Wakisaka, R. Ishihara and E. Irie; K. Hagiwara for technical assistance; Y. Sagawa, C. Ido and E. Niikura for technical support and mouse handling; and K. Tanaka for suggestions and discussions. This study was supported by Japan Society for the Promotion of Science (JSPS) KAKENHI Grant-in-Aid (C) 16K09374 for N.N., (B) 20H03666 for Y.M., (A) 15H02534 and 20H00536 for T.K., and (S) JP17H06175 for A.Y.; Advanced Research and Development Programs for Medical Innovation (AMED-CREST; 16gm1010003h0001 for T.K., JP20gm1110009 for A.Y., and 20gm1210001h0002 for Y.M.); Takeda Science Foundation; Kanae Foundation for The Promotion of Medical Science; the Smoking Research Foundation; and Keio University Medical Fund.

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Contributions

T.T., Y.M. and T.K. designed experiments and interpreted data. T.T., T. Suzuki, Y. Harada, Y. Hagihara, S.S., H.I., N.T., K.K., K.M., P.-S.C., T. Sujino., W.S., M.M., M.I., M. Tanida and Y.I. performed experiments. Y.M., K.M. and W.S. analysed genomic data and generated figures. T.K., N.N., K.O., T.O., T.Y., H.O., M.H., Y.K., A.Y. and M. Tsuda provided supervision and support. Y.M., T.T., T.K. wrote the manuscript with input from the other authors.

Corresponding authors

Correspondence to Yohei Mikami or Takanori Kanai.

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Competing interests

T. Suzuki and K.M. are current employees of Miyarisan Pharmaceutical.

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Peer review information Nature thanks John Bienenstock and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 Muscarinic signalling in colonic APC activates induction of Treg.

a, Three-dimensional reconstruction of CX3CR1+ APC (green) and enteric neurons (purpule) in colon in mice. b, Three-dimensional reconstruction of MHCII+ APC (green), eneric Tuj+ neurons (purple), and Foxp3+ Tregs (yellow). Scale. bar = 50 μm. c, d, Anatomical diagram (c) and Operative field (d) for subdiaphragmatic truncal vagotomy. e, f, Colonic T cell phenotypes in mice after VGx surgery. Representative contour plots with frequency of Foxp3+ cells (Treg) among CD4+ T cells in colonic LP (e) and RORγt+ pTregs in colonic Foxp3+ Tregs (f). g, Expression of Chrm1, Adrb2, Htr7 and Chrna7 mRNA in colonic and splenic APCs. h, Strategy for sorting colonic CD11b-CD11c+ (CD11c SP), CD11b+CD11c+ (DP) and CD11b+CD11c- (CD11b SP) subsets by FACS. i, Enteric neuro-spheroid derived neuron-induced retinol metabolism-related genes expression in colonic APC was dependent on muscarinic signalling. left, Schema of experience. right, Aldh1a1 and Aldh1a2 mRNA levels in colonic APC (n = 6/group). j, Muscarinic signalling in colonic APCs promoted induction of Treg. left, Schema of experience. right, Frequency of Foxp3+ Treg among CD4+ T cells. (left panel) Representative contour plots. (right panel) Quantification (n = 6/group). k, Enteric neuro-spheroid derived neuron enhanced Foxp3+ Treg induction via activation of muscarinic signalling in colonic APC. left, Schema of experience. right, Frequency of Foxp3+ Treg among CD4+ T cells. (left panel) Representative contour plots. (right panel) Quantification (n = 6/group). Representative of two (a, b, i-k) or three (e, f) independent experiments. P values obtained via one-way ANOVA with Tukey’s post hoc test. Data are shown as mean ± SEM (i-k).

Extended Data Fig. 2 Colitis activates liver-brain axis.

ac, WT mice were subjected to Sham or VGx and then were given DSS for 7 days, starting at day 2 after surgery. Graphs show pooled data of three independent experiments (n = 15/group). a, Relative body weight change during colitis. ** indicates P < 0.01. b, DAI. c, Representative HE staining of colon sections (left panel, bar: 200 μm) and histological scores (right panel). df, WT mice were given DSS or water for 6 days (n = 6/group). d, Representative images of immunofluorescence staining for pERK1/2 (green) in NG (upper panel, bar: 100 μm). Quantification of pERK1/2-experssing neurons (lower panel). e, Representative images of c-Fos immunoreactivity in NTS (left panel, bar: 200 μm). Number of c-Fos immunoreactive neurons (right panel). f, Representative images of immunofluorescence double-staining for pERK1/2 (green) and PGP9.5 (red) in murine liver sections. Co-stained sites are shown in yellow (left panel). Scale bar indicates 10 μm. Quantification of pERK1/2-experssing area in PGP9.5 positive nerve fibre (right panel). g. Hepatic phosphor-mTOR and total mTOR protein levels. WT mice were treated with Abx-cocktail for 3 weeks and then were given DSS for 4 days. h, i, WT mice were intravenously injected with Si-negative control (Si-Cont) or Raptor (Si-Raptor) and at day 3 after were subjected to sham or HVx (n = 6/group). Phenotypes of colonic T cell were analysed at day 2 after surgery. h, Frequency of Foxp3+ Treg among CD4+ T cells. (left panel) Representative contour plots. (right panel) Quantification. i, Frequency of RORγt+ cells among colonic Foxp3+ Treg. Representative contour plots (left panel). Quantification (right panel). Representative of two independent experiments (di). P values obtained via unpaired two-tailed Student’s t tests (af) and one-way ANOVA with Tukey’s post hoc test (h, i). Data are shown as mean ± SEM (bf, h, i).

Extended Data Fig. 3 Anatomy of the hepatic vagus nerve in mice.

a, Anatomical diagram. b, Operative field for hepatic vagotomy. c, A schematic view showing the ignition of liver-brain-gut neural arc during colitis. d, The common hepatic branch of the vagus does not contain sympathetic nerve. Operative field for electrical recording of hepatic sympathetic nerve (left). Electrical activity in common hepatic branch of the vagus and hepatic sympathetic nerve, respectively (middle panel). Representative images of immunofluorescence staining for Tyrosine hydroxylase (TH) in hepatic branch and DRG (right panel, bar: 100 μm). e, Fluorescent immunohistochemistry of TRPV1+ neuron in hepatic vagal branch at day 2 after capsaicin application (bar: 200 μm). fh, WGA retrograde tracing. f, g, WT mice were subjected to Sham or HVx and then were injected with Alexa Fluor 488 conjugated WGA at day 2 after surgery (n = 3/group). Fluorescence image of Alexa Fluor 488+ neuron (green) and DAPI (blue) in NG (f) and Th4 DRG (g) at 1 week after injection of WGA in liver. Representative images (left panel, bar: 50 μm). Number of WGA+ neurons (right panel). h, Fluorescence images of Alexa Fluor 488+ neuron (green) and DAPI (blue) in DRG (Th4-7 and Th13) at 1 week after injection of WGA in liver (bar: 100μm). Representative of two independent experiments (dh). P values obtained via unpaired two-tailed Student’s t tests. Data are shown as mean ± SEM (f, g).

Extended Data Fig. 4 Effects of vagotomy on maintenance and stability of colonic pTreg.

WT mice were subjected to Sham, VGx or HVx (n = 9/group). Phenotypes and gene expression of colonic immune cell were analysed at day 2 after surgery. a, Frequency of Foxp3+ cells among CD4+ cells in colon. Representative contour plots. b, Frequency of RORγt+ cells among Foxp3+ Treg in colonic LP. Representative contour plots (left panel). Quantification (right panel). c, Expression of Aldh1a1 and Aldh1a2 mRNA in colonic APC. d, Frequency of ALDH+ cells among MHC-II+ colonic APCs. Histograms of ALDH+ cells with colonic APCs (left panel). Quantification (right panel). e, f, WT mice were subjected to Sham or HVx. Colonic T cell phenotypes were analysed at the indicated time point following HVx (n = 9/group). Frequency of Foxp3+ cells among CD4+ T cells in colonic LP (e) and RORγt+ cells among Foxp3+ Treg in colonic LP (f). Representative contour plots (left panel) and quantification (right panel) are shown (e, f). gi, Frequency of Foxp3+ cells among CD4+ cells (gi) and RORγt+ cells among colonic Foxp3+ Treg (g, h) in colon at day 2 after surgery. g, B6 mice were subjected to Sham or HVx (n = 10/group). h, BALB/c mice were subjected to Sham or HVx (n = 4/group). i, Six-week-old WT rat were subjected to Sham or HVx (n = 4/group). j, k, Rag2−/− mice were subjected to sham-operation or HVx and then transferred with CD4+ CD45RBhi T cells (n = 8/group). Four weeks after transfer, mice were sacrificed and colonic Treg cells were analysed. j, Frequency of Foxp3+ cells among CD4+ cells in colon. Representative contour plots (left panel). Quantification (right panel). k, Frequency of RORγt+ cells among colonic Foxp3+ Treg. Representative contour plots (left panel). Quantification (right panel). l, WT mice were subjected to Sham or HVx. Colonic immune cell phenotypes were analysed 2 days later. Frequency of IFN-γ+, Gata3+ and IL-17A+ cells among CD4+ cells in colon (n = 9/group). Representative of two (e, f, h, i) independent experiments or pooled from three independent experiments (ad, g, jl). P values obtained via one-way ANOVA with Tukey’s post hoc test (bf) and unpaired two-tailed Student’s t tests (gl). Data are shown as mean ± s.e.m.

Extended Data Fig. 5 Afferent vagal, but not spinal cord, activation from the liver is involved in colonic Treg homeostasis.

a-e, WT mice underwent either corn oil (Oil) or capsaicin (Cap) application to the hepatic vagal branch. Phenotypic analysis of T cell and APC in colon was performed at day 2 after application of capsaicin (c, d, n = 16/group; e, f, n = 8/group). a, b, Representative fluorescence images of TRPV1 (red) and DAPI (blue) in NG (a) and Th4-DRG (b). Scale bar indicates 100μm. c, Frequency of Foxp3+ cells among CD4+ cells in colon. Representative contour plots (left panel). Quantification (right panel). d, Frequency of RORγt+ cells among colonic Foxp3+ Tregs. Representative contour plots (left panel). Quantification (right panel). e, Frequency of ALDH+ cells among MHC-II+ colonic APCs. Histograms of ALDH+ cells with colonic APCs (left panel). Quantification (right panel). f, Expression of Aldh1a1 and Aldh1a2 mRNA in colonic APC. gp, Eight-week-old WT type were intrathecally injected with capsaicin (gj, n = 5/group) and resiniferatoxin (RTX) (kp, n = 4/group). TRPV1+ nerves in spinal cord (Th4-7 and Th13) and colonic immune cells were analysed at day 7 after administration. g, Fluorescent immunohistochemistry of TRPV1+ neuron in spinal cord (bar: 200μm). Frequency of Foxp3+ cells among CD4+ cells (h, n) and RORγt+ cells among colonic Foxp3+ Tregs (i, o) in the colon. Frequency of ALDH+ cells among MHC-II+ colonic APCs (j, p). k, l, Effects of intrathecal injection of RTX in DRG (k) and NG (l). Scale bar indicates 100 μm. m, Colonic CGRP levels. Representative of two independent experiments (ab, go) or pooled from two (e) or three (c, d, f) independent experiments. P values obtained via unpaired two-tailed Student’s t tests. Data are shown as mean ± s.e.m.

Extended Data Fig. 6 Hemi-subdiaphragmatic vagotomy revealed functional asymmetries of the vagus nerve.

ac, WT mice were subjected to Sham, ventral (left) subdiaphragmatic vagotomy (LVx) or dorsal (right) subdiaphragmatic vagotomy (RVx) (n = 4/group). Phenotypic analysis of T cell and APC in colon was performed at day 2 after surgery. a, Frequency of Foxp3+ cells among CD4+ cells in colon. Representative contour plots. b, Frequency of RORγt+ cells among Foxp3+ Treg in colonic LP. Representative contour plots (left panel). Quantification (right panel). c, Frequency of ALDH+ cells among MHC-II+ colonic APCs. Histograms of ALDH+ cells with colonic APCs (left panel). Quantification (right panel). dj, Ablation of sympathetic signalling via CG/SMG does not affect maintenance of Treg in colon. d, Operative field for CG/SMG ganglionectomy. e, Electrical activity in splanchnic nerve. The numbers in parentheses correspond to the nerves indicated in d. fi, WT mice were subjected to Sham (n = 4) or ganglionectomy of CG/SMG (n = 5). Phenotypes of immune cell in colon and spleen were analysed 2 days later. j, WT mice were injected with MLA (α7-antagonist, 150 μg/day, i.p.) for 2 days and then splenic T cells were analysed at 12 h after last injection (n = 5/group). f, i, j, Frequency of Foxp3+ cells among CD4+ cells in colon (f) and spleen (i, j). g, Frequency of RORγt+ cells among colonic Foxp3+ Tregs. h, Frequency of ALDH+ cells among MHC-II+ colonic APCs. km, WT mice were subjected to Sham or HVx and then injected daily with vehicle, salbutamol (β-agonist, 30 μg/day, i.p.) or propanolol (β-antagonist, 300 μg/day, i.p.) for 2 days (n = 6/group). Frequency of Foxp3+ cells among CD4+ cells (k), RORγt+ cells among colonic Foxp3+ Tregs (l), and ALDH+ cells among MHC-II+ colonic APCs (m) in the colon at 12 h after the last injection. n, WT mice were subjected to Sham or HVx. T cell phenotypes in colon, small intestine and spleen were analysed at day 2 after surgery. Frequency of Foxp3+ cells among CD4+ T cells in the colon, the small intestine, and the spleen (n = 4/group). Representative of two independent experiments (an). P values obtained via one-way ANOVA with Tukey’s post hoc test (b, c, k–n) and unpaired two-tailed Student’s t tests (f–j). Data are shown as mean ± s.e.m.

Extended Data Fig. 7 Effects of VGx and HVx on intrinsic enteric neuron.

ac, g, WT mice were subjected to Sham (n = 4) or VGx (n = 5). df, h, WT mice were subjected to Sham (n = 6) or HVx (n = 6). Activity of intrinsic enteric neuron were measured at day 2 after surgery. a, d, Representative images of immunofluorescence staining for HuC/D (white) and c-Fos (red) in colon. Scale bar indicates 100 μm. b, e, Quantification of c-Fos+ neurons. c, f, Expression of Hand2 mRNA in colon. g, h, Colonic acetylcholine, norepinephrine and CGRP levels. * and ** indicate P < 0.05 and P < 0.01, respectively. P values obtained via unpaired two-tailed Student’s t tests. Data are shown as mean ± s.e.m.

Extended Data Fig. 8 Effects of mAChR and α7nAChR on maintenance of colonic Treg.

a-g, WT mice were subjected to Sham or HVx and then injected daily with water or bethanechol (BETH; i.p. 300 μg/day) (ad) or GST-21 (α7-agonist, i.p. 300 μg/day) (eg) for 2 days (a, b, n = 5/group; c, d, n = 10/group; e-g, n = 6/group). hj, WT mice were subjected to Sham or HVx and then daily injected with water, BETH only or BETH plus MLA (α7-antagonist) for 2 days (n = 6/group). k, l, WT and mAchR TKO mice were subjected to Sham or HVx and then daily injected with water or BETH for 2 days (n = 4/group) related to Fig. 3e. Phenotypes of colonic immune cell were analysed at 12 h after last injection. a, g, j, l, Frequency of ALDH+ cells among MHC-II+ colonic APCs. b, Expression of Aldh1a1 and Aldh1a2 mRNA in colonic APC. c, e, h, Frequency of Foxp3+ cells among CD4+ cells in colon. d, f, i, k, Frequency of RORγt+ cells among colonic Foxp3+ Tregs. P values obtained via one-way ANOVA with Tukey’s post hoc. Data are shown as mean ± s.e.m.

Extended Data Fig. 9 The effects of gut-microbiota on colonic Treg maintenance of the liver-brain-gut axis.

ac, WT mice were subjected to Sham or HVx (n = 4/group). Faecal samples were collected on pre-treatment and days 2 post-surgery from the identical mice. a, α-diversity in the faecal microbiota. b, Principal coordinate analysis (PCoA) based on the weighted UniFrac analysis of bacterial community structures (black, pre-treatment; red, sham; blue, HVx). The two components of the weighted PCoA plot explained 45% and 22% of the variance. Dissimilarities between two groups were evaluated by permutational multivariate analysis of variance (PERMANOVA). c, Phylum-level taxonomic distribution. d, e, Faecal samples derived from sham-operated or HVx mice were inoculated into five-week-old male GF mice, followed by immunological phenotyping at day 21 post-inoculation (n = 5/group). f, g, WT mice were subjected to Sham or HVx and co-housed in our SPF facility for 2 days. Graphs show pooled data of three independent experiments (n = 14/group). hk, At day 14 post-parabiosis, mice were subjected to sham or HVx (n = 10/group). Phenotypes of colonic immune cell were analysed 2 days later (h). l, m, WT mice were treated with Abx-cocktail (metronidazole, vancomycin, ampicillin and neomycin) for 3 weeks and subjected to Sham or HVx. Colonic T cell phenotypes were analysed 2 days later. Graphs show pooled data of three independent experiments (n = 10/group). d, f, i, l, Frequency of Foxp3+ cells among CD4+ T cells in colon. e, g, j, m, Expression of RORγt+ in colonic Foxp3+ Treg. k, Frequency of ALDH+ cells among MHC-II+ colonic APCs. P values obtained via one-way ANOVA with Tukey’s post hoc test (a, d, e, l, m) or unpaired two-tailed Student’s t tests (f, g, ik). Data are shown as mean ± s.e.m.

Extended Data Fig. 10 Effects of HVx on colitis.

ac, WT mice were sensitized with TNBS. After 5 days, mice were subjected to Sham or HVx, and treated with TNBS by intrarectal administration at the same time. Graphs show pooled data from three independent experiments (n = 20/group). df, Eight-week-old male Rag2−/− mice were subjected to sham-operation or HVx and then were given DSS for 7 days, starting at day 2 after surgery (n = 12/group). g, h, Sham-operated and HVx mice were cohoused and orally challenged with 2.0% DSS (w/v) for 7 days. Graphs show pooled data of two independent experiments (n = 8/group). i, j, Abx treated mice were subjected to sham and HVx and after 2 days, orally challenged with 2.0% DSS (w/v) for 7 days. Graphs show pooled data of two independent experiments (n = 10/group). k, l, Sham operated and HVx Myd88-deficient mice were orally challenged with 2.0% DSS (w/v) for 7 days (n = 13/group). mo, Sham-operated and hepatic vagotomized mice were orally challenged with 2.0% DSS (w/v) and daily treated with BETH for 7 days. Graphs show pooled data of two independent experiments (n = 10/group). a, d, g, i, k, m, Relative body weight change during acute colitis. b, e, h, j, l, n, DAI. c, f, o, Representative HE staining of colon sections (left panel, bar: 200 μm) and histological scores (right panel). Each experiment was repeated at least twice with similar results P values obtained via unpaired two-tailed Student’s t tests. Error bars represent the mean ± s.e.m. p, A schematic view of the liver-brain-gut neural arc. A mouse in the supine position is illustrated. The liver senses the gut microenvironment and relays the sensory inputs to the left NTS of the brainstem, and ultimately to the left vagal parasympathetic nerves and enteric neurons. Gut APCs, activated by the liver-brain-gut neural arc, show enhanced ALDH expression and RA synthesis through mAChRs and maintain a reservoir of peripheral regulatory T cells.

Supplementary information

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Supplementary Table 1

| List of antibodies used in this study.

Supplementary Table 2

| List of qPCR primers used in this study.

Supplementary Table 3

| List of reagents used in this study.

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Teratani, T., Mikami, Y., Nakamoto, N. et al. The liver–brain–gut neural arc maintains the Treg cell niche in the gut. Nature (2020). https://doi.org/10.1038/s41586-020-2425-3

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