Abstract

Small intestinal mononuclear cells that express CX3CR1 (CX3CR1+ cells) regulate immune responses1,2,3,4,5. CX3CR1+ cells take up luminal antigens by protruding their dendrites into the lumen1,2,3,4,6. However, it remains unclear how dendrite protrusion by CX3CR1+ cells is induced in the intestine. Here we show in mice that the bacterial metabolites pyruvic acid and lactic acid induce dendrite protrusion via GPR31 in CX3CR1+ cells. Mice that lack GPR31, which was highly and selectively expressed in intestinal CX3CR1+ cells, showed defective dendrite protrusions of CX3CR1+ cells in the small intestine. A methanol-soluble fraction of the small intestinal contents of specific-pathogen-free mice, but not germ-free mice, induced dendrite extension of intestinal CX3CR1+ cells in vitro. We purified a GPR31-activating fraction, and identified lactic acid. Both lactic acid and pyruvic acid induced dendrite extension of CX3CR1+ cells of wild-type mice, but not of Gpr31b−/− mice. Oral administration of lactate and pyruvate enhanced dendrite protrusion of CX3CR1+ cells in the small intestine of wild-type mice, but not in that of Gpr31b−/− mice. Furthermore, wild-type mice treated with lactate or pyruvate showed an enhanced immune response and high resistance to intestinal Salmonella infection. These findings demonstrate that lactate and pyruvate, which are produced in the intestinal lumen in a bacteria-dependent manner, contribute to enhanced immune responses by inducing GPR31-mediated dendrite protrusion of intestinal CX3CR1+ cells.

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The datasets generated during the current study are available from the corresponding author on reasonable request.

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Acknowledgements

We thank F. Sugiyama (University of Tsukuba) for his kind gift of B6J-S1UTR embryonic stem cell line; T. Kamisako, M. Kumai, Y. Izumi, H. Ishizaki and Y. Ono (KAN Research Institute) for their kind supply of Cx3cl1−/− mice; T. Kondo and Y. Magota for technical assistance; and C. Hidaka for secretarial assistance. This study was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (15H02511 and 16K08838), and Japan Agency for Medical Research and Development (J170701434).

Author information

Author notes

  1. These authors contributed equally: Naoki Morita, Eiji Umemoto

Affiliations

  1. Department of Microbiology and Immunology, Graduate School of Medicine, Osaka University, Suita, Japan

    • Naoki Morita
    • , Eiji Umemoto
    • , Yuichi Maeda
    • , Hisako Kayama
    • , Ryu Okumura
    •  & Kiyoshi Takeda
  2. WPI Immunology Frontier Research Center, Osaka University, Suita, Japan

    • Naoki Morita
    • , Eiji Umemoto
    • , Junichi Kikuta
    • , Hisako Kayama
    • , Ryu Okumura
    • , Masaru Ishii
    •  & Kiyoshi Takeda
  3. Core Research for Evolutional Science and Technology, Japan Agency for Medical Research and Development, Tokyo, Japan

    • Naoki Morita
    • , Eiji Umemoto
    • , Hisako Kayama
    • , Ryu Okumura
    •  & Kiyoshi Takeda
  4. Ono Pharmaceutical Co. Ltd., Exploratory Research Laboratories, Mishima-Gun, Japan

    • Setsuko Fujita
    • , Akio Hayashi
    •  & Ryusuke Nabeshima
  5. Department of Immunology and Cell Biology, Graduate School of Medicine and Frontier Biosciences, Osaka University, Suita, Japan

    • Junichi Kikuta
    •  & Masaru Ishii
  6. Department of Applied Biological Science, Graduate School of Agriculture, Tokyo University of Agriculture and Technology, Fuchu, Japan

    • Ikuo Kimura
  7. Department of Microbiology, School of Pharmacy, Kitasato University, Tokyo, Japan

    • Takeshi Haneda
    •  & Nobuhiko Okada
  8. KAN Research Institute, Kobe, Japan

    • Toshio Imai
  9. Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan

    • Asuka Inoue
    •  & Junken Aoki
  10. Department of Infection Microbiology, Research Institute for Microbial Diseases, Osaka University, Suita, Japan

    • Hitomi Mimuro
  11. Division of Bacteriology, International Research Center for Infectious Diseases, Institute of Medical Science, The University of Tokyo, Tokyo, Japan

    • Hitomi Mimuro
  12. Department of Respiratory Medicine and Clinical Immunology, Graduate School of Medicine, Osaka University, Suita, Japan

    • Yuichi Maeda
  13. Department of Applied Chemistry, Graduate School of Engineering, Osaka University, Suita, Japan

    • Toshiyuki Kida
  14. Integrated Frontier Research for Medical Science Division, Institute for Open and Transdisciplinary Research Initiatives, Osaka University, Suita, Japan

    • Toshiyuki Kida
    •  & Kiyoshi Takeda

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Contributions

N.M. planned and performed experiments. E.U. planned and performed experiments and wrote the paper. S.F., A.H., T.K. and R.N. performed LC–MS analyses. I.K., A.I. and J.A. established the ligand-binding assay. J.K. and M.I. performed two-photon microscopic analyses. T.H., H.M. and N.O. prepared S. Typhimurium mutants. T.I. generated Cx3cl−/− mice. Y.M., H.K. and R.O. performed animal experiments. K.T. planned and directed the research, and wrote the paper.

Competing interests

S.F., A.H. and R.N. are employees of Ono Pharmaceutical Co. Ltd. This does not alter the authors’ adherence to all Nature policies on sharing data and materials.

Corresponding author

Correspondence to Kiyoshi Takeda.

Extended data figures and tables

  1. Extended Data Fig. 1 Roles of commensal bacteria in trans-epithelial dendrite protrusion of CX3CR1+ cells.

    a, A cocktail of antibiotics (Abx; ampicillin, neomycin, metronidazole and vancomycin) were orally administered to Cx3xr1gfp/+ mice raised under SPF conditions. Tissue segments of the distal ileum were stained with CMRA (red) and CX3CR1+ cell dendrites (green) were observed by confocal microscopy. The 2D projection of z-stack images is shown. The numbers of trans-epithelial dendrites per villus were quantified. Each symbol represents an individual villus. Data represent the mean ± s.d. from three mice (Abx: proximal, n = 24; distal, n = 35; untreated: proximal, n = 28; distal, n = 30 villi). Two-tailed Student’s t-test was performed for statistical analysis. b, Low-density cells were prepared from the small intestine of Myd88+/+ and Myd88−/− mice, and labelled with 2 μM CFSE. Sorted CX3CR1+ cells were incubated with a methanol-soluble fraction prepared from the small-intestinal luminal contents of SPF mice for 4 h. The cells with extended dendrites were quantified (n = 5 independent samples per group). One-way ANOVA with Tukey’s comparison was performed for statistical analysis. Data represent the mean ± s.d. from two biologically independent experiments. Scale bar, 10 μm. NS, not significant. Source data

  2. Extended Data Fig. 2 Expression of GPCRs in subsets of intestinal myeloid cells.

    a, Sorting strategy for separating subsets of myeloid cells in the small intestine, used for the analysis of GPCR gene expression. CD45+ cells were collected from Cx3cr1gfp/+ mice. b, GPCRs that are highly expressed in CX3CR1+ cells of the small intestine were picked from the Immunological Genome Project (ImmGen) database, and the expression levels of these GPCRs in R1 CD11c+CD11bCX3CR1 cells, R2 CD11c+CD11b+CX3CR1 cells and R3 CD11c+CD11b+CX3CR1+ cells were examined by quantitative PCR (n = 3 mice). Gpr31 expression shows the total expression level of three Gpr31 isoforms (Gpr31a, Gpr31b and an unnamed isoform). cg, Gating strategies of cell populations used for quantitative PCR analysis of Gpr31 expression. Circulating leukocytes were prepared from Cx3cr1gfp/+ mice (c). Low-density cells in the mesenteric lymph nodes (d), Peyer’s patches (e), large intestine (f) and small intestine (g) were prepared from Cx3cr1gfp/+ mice, and stained with fluorochrome-conjugated mAbs. CD45+ cells were gated and further analysed by using indicated mAbs. hj. Quantitative PCR of Gpr31b expression in subsets of leukocytes in the peripheral blood and the small intestine (h), myeloid-cell subsets and epithelial cells in the intestine (i), and CX3CR1+ cells in various tissues (j). MCs, monocytes; S.I., small intestine (hj, n = 3 independent samples per group). Values were normalized to the expression of Gapdh. Data represent the mean ± s.d. Source data

  3. Extended Data Fig. 3 Generation of Gpr31b−/− mice.

    a, The structure of Gpr31 gene clusters and a targeting strategy for Gpr31b gene depletion. The mouse Gpr31 gene forms a gene cluster consisting of four isoforms: Gpr31a, Gpr31b, Gpr31c and an unnamed gene adjacent to Gpr31a. These genes show 99% homology at a nucleotide level. Among the Gpr31 genes, Gpr31a and the unnamed gene generate truncated gene products, which lack the first 19 amino acids compared with the Gpr31b gene product; Gpr31c serves as a pseudogene. The targeting vector was constructed by replacement of a 1-kb fragment of the exon of Gpr31b with a neomycin-resistance gene cassette. Digestion of wild-type genomic DNA with PstI generates 5.2-kb DNA fragments in Gpr31a and Gpr31c, and a 6.6-kb fragment in Gpr31b whereas the digestion of the mutant genomic DNA with a neoR cassette produces a 7.6-kb fragment. b, Southern blot analysis of offspring of the heterozygote intercrosses. Genomic DNA extracted from mouse tails was digested with PstI, electrophoresed and hybridized with the 32P-labelled DNA probe indicated in a (bold bar). For gel source data, see Supplementary Fig. 1. The experiment was repeated two independent times with similar results. c, Quantitative PCR analysis of intestinal CX3CR1+ cells in Gpr31b−/− mice (n = 3 mice). PCR was performed using DNA polymerase and primer sets that discriminate between Gpr31b and other Gpr31 isoforms. d, The proportion and number of intestinal CX3CR1+ cells in Gpr31b+/+ and Gpr31b−/− mice (n = 3 mice each). e, Quantification of trans-epithelial dendrites of CX3CR1+ cells in the ileum (corresponding to Fig. 2a). Explants of the distal ileum were observed by two-photon microscopy. Data are from three mice per group (Gpr31b+/+: proximal, n = 23; distal n = 30; Gpr31b−/−: proximal, n = 20; distal, n = 21; Cx3cr1gfp/gfp: proximal, n = 26; distal, n = 26 villi). f, Quantification for dendrite extension of CX3CR1+ cells in response to the methanol-soluble fraction of intestinal luminal contents (corresponding to Fig. 2b). Data are from two biologically independent experiments (vehicle, n = 5; methanol-soluble fraction, n = 6 independent samples per group). Two-tailed Student’s t-test (d, e) and one-way ANOVA with Tukey’s comparison (f) were performed for statistical analysis. Data represent the mean ± s.d. Source data

  4. Extended Data Fig. 4 Role of GPR31 signalling in S. Typhimurium uptake by intestinal myeloid cells.

    a, Gpr31b+/+ and Gpr31b−/− mice were orally administered CFSE-labelled ΔinvA S. Typhimurium (1 × 108 CFU). Ten hours after the administration, the uptake of S. Typhimurium by CX3CR1+ cells and CD11b+CD103+ cells was analysed by flow cytometry (n = 3 mice). b, Role of GPR31 signalling in S. Typhimurium uptake by CX3CR1+ cells in vitro. Intestinal low-density cells (8 × 105 cells) prepared from Gpr31b+/+ or Gpr31−/− mice were incubated in a fibronectin-coated 12-well plate for 2 h. The cells were further cultured with CFSE-labelled ΔinvA S. Typhimurium (5 × 107 CFU) for 3 h. After being washed, the cells were collected and CX3CR1+ cells carrying S. Typhimurium were assessed by flow cytometry (n = 3 independent samples per group). c, Mice were orally administered ΔinvA S. Typhimurium, and bacterial loads in the indicated tissues were analysed (n = 12 mice). d, Effects of intravenous immunization of non-pathogenic S. Typhimurium on invasive S. Typhimurium infection in Gpr31b−/− mice. Gpr31b+/+ mice and Gpr31b−/− mice were intravenously administered with ΔinvA ΔaroA S. Typhimurium (5 × 104 CFU) for six weeks, and the survival rate after oral infection of invasive S. Typhimurium (1 × 109 CFU) was monitored (n = 10 mice). Two-tailed Student’s t-test (a, b), two-tailed Mann–Whitney test (c) and log-rank test were performed for statistical analysis. Data represent the mean ± s.d. NS, not significant. Source data

  5. Extended Data Fig. 5 Step-by-step purification of GPR31-reacting molecules.

    a, The ligand-binding activity of GPR31 was assessed by intracellular cAMP concentration in HEK293 transfectants, in which human GPR31 expression is inducible by doxycycline (Tet-on system). A methanol (MeOH)-soluble fraction prepared from luminal contents of the intestine (red in b), which induced dendrite extension of CX3CR1+ cells, increased the concentration of cAMP in GPR31-expressing cells (n = 3 independent samples per group). b, Schematic of strategy to purify GPR31-activating molecules (n = 3 independent samples per group). c, The methanol-soluble fraction was washed with a mixture of methyl tert-butyl ether (MTBE) and methanol (MTBE-1), and then washed with a MTBE:methanol:acetic acid solution and saturated saline (MTBE-2) (green in b). The MTBE-2 activated GPR31 (n = 3 independent samples per group). d, The MTBE-2 was separated into the neutral and basic fraction (orange in b), and acidic fraction, by anion-exchange chromatography. The neutral and basic fraction increased cAMP concentration (n = 3 independent samples per group). e, Gel permeation chromatography was then applied to the neutral and basic fraction. Fraction number 5 and fraction number 6 increased cAMP concentration (blue in b) (n = 4 independent samples per group). f, The mixture of fraction number 5 and fraction number 6 was then separated by HILIC. The fraction at 8–10-min retention time activated GPR31 (pink in b) (n = 3 independent samples per group). Data represent the mean ± s.d. Source data

  6. Extended Data Fig. 6 Analysis of gel permeation chromatography fractions prepared from intestinal luminal contents.

    a, HILIC-LC–MS analysis of gel permeation chromatography fractions, which activated (fraction number 5 and fraction number 6) and failed to activate (fraction number 2) cells that express human GPR31. LC–MS chromatograms (at m/z 89.022–89.026) of fraction number 5 and fraction number 6 showed a retention time that was almost the same as that of lactic acid. The experiment was repeated two independent times with similar results. b, Reactivity of mouse GPR31 to enantiomers of lactic acid (n = 6 mice). Intracellular cAMP levels in HEK293 Tet-on inducible cells were assessed in the presence or absence of doxycycline (Dox). Data represent two biologically independent experiments. Two-tailed Student’s t-test was performed for statistical analysis. Data represent the mean ± s.d. Source data

  7. Extended Data Fig. 7 Effects of antibiotics on transepithelial dendrites of CX3CR1+ cells.

    a, Antibiotics with different spectra of activity were orally administered to SPF mice for four weeks. The methanol-soluble fractions prepared from intestinal luminal contents of these mice were added to cells that express human GPR31, and cytosolic cAMP levels were evaluated (n = 5 independent samples per group). bd, Concentrations of d-lactate (b), l-lactate (c) and pyruvate (d) in the intestinal luminal contents were determined (n = 6 mice). e, Wild-type mice were orally administered with the indicated antibiotics for four weeks, and the methanol-soluble fraction was prepared from intestinal luminal contents. Intestinal CX3CR1+ cells were incubated with the methanol-soluble fraction for 4 h, and the cells with extended dendrites were quantified (green, GFP; blue, DAPI). Data were from two biologically independent experiments (control and Abx groups; n = 7; other groups, n = 8 independent samples per group). Scale bar, 10 μm. f, Cx3cr1gfp/+ mice were orally received the indicated antibiotics for four weeks. Tissue segments of the distal ileum were stained with CMRA (red) and CX3CR1+ cells (green) were observed by confocal microscopy. The 2D projection of z-stack images is shown. Arrows indicate trans-epithelial dendrites. Scale bar, 100 μm. Abx, a mixture of ampicillin, neomycin, metronidazole and vancomycin; Gnt, gentamicin; Vnc, vancomycin; Amp, ampicillin; Neo, neomycin; Mtz, metronidazole. Two-tailed Student’s t-test was performed for statistical analysis (ae). Data represent the mean ± s.d. Source data

  8. Extended Data Fig. 8 Effects of oral administration of lactate- and pyruvate-producing bacteria on trans-epithelial dendrite protrusion of CX3CR1+ cells.

    ac, Several species of bacteria were incubated in GAM medium supplemented with 1% glucose for 45 h, and concentrations of d-lactate (a), l-lactate (b) and pyruvate (c) were measured (n = 3 independent samples per group). The precise P values are described in Source Data. *P < 0.05, **P < 0.01, ***P < 0.001. df, SPF mice were administered with L. helveticus (2 × 109 CFU) by oral gavage for eight days. Concentrations of d-lactate (d), l-lactate (e) and pyruvate (f) in luminal contents of the small intestine were measured (n = 6 mice). g, Cx3cr1gfp/+Gpr31b+/+ and Cx3cr1gfp/+Gpr31b−/− mice were orally administered L. helveticus for eight days, and tissue segments of the distal ileum were observed by two-photon microscopy (green, GFP; red, CMRA). The experiment was repeated three independent times with similar results. Arrowheads indicate trans-epithelial dendrites. Scale bar, 50 μm. Two-tailed Student’s t-test was performed for statistical analysis (af). Data represent the mean ± s.d. Source data

  9. Extended Data Fig. 9 Protective effect of lactate and pyruvate on bacterial dissemination and tissue damage during S. Typhimurium infection.

    a, Quantification of the extended dendrites of CX3CR1+ cells treated with lactic acid or pyruvic acid (corresponding to Fig. 4a). Gpr31b+/+ and Gpr31b−/− CX3CR1+ cells were treated with lactic acid, pyruvic acid or propionic acid (100 μM each), and their morphology was observed under a microscope and quantified (n = 6 independent samples per group). Data represent two biologically independent experiments. b, Quantification of trans-epithelial dendrite protrusion of CX3CR1+ cells in mice that were administered lactate or pyruvate (corresponding to Fig. 4b). Trans-epithelial dendrites in explants of the distal ileum were observed by two-photon microscopy after administration of 100 mM lactate or pyruvate for three weeks. Data are from three mice per group (untreated: Gpr31b+/+ proximal, n = 29; distal, n = 33; Gpr31b−/− proximal, n = 28; distal, n = 27; lactate-treated: Gpr31b+/+ proximal, n = 32; distal, n = 32; Gpr31b−/− proximal, n = 24; distal, n = 25; pyruvate-treated: Gpr31b+/+ proximal, n = 29; distal, n = 29; Gpr31b−/− proximal, n = 27; distal n = 28 villi). c, Time course of trans-epithelial dendrite induction upon oral administration of lactate or pyruvate. Cx3cr1gfp/+ mice were orally administered 50 mM lactate or pyruvate. Explants of the ileum were collected at the indicated time, and the dendrite extension of CX3CR1+ cells was monitored by confocal microscopy (green, GFP; red, CMRA). The experiment was repeated two independent times with similar results. Arrowheads indicate trans-epithelial dendrites. de, GPR31-independent dendrite protrusion of intestinal CD103+ cells. Low-density cells prepared from Cx3cr1gfp/+ mice were stained with Alexa Fluor 594 anti-CD103 mAb and treated with methanol-soluble fraction, lactic acid (100 μM) or pyruvic acid (100 μM) for 4 h. The morphology of these cells was observed by microscopy (d) and quantified (e) (CX3CR1+ cells, n = 6; CD103+ cells, n = 4 independent samples per group). Data represent the mean ± s.d. from two biologically independent experiments. f, In non-infectious conditions, Gpr31b+/+ or Gpr31b−/− mice were intravenously injected with APC anti-CD103 mAb (20 μg), Alexa Fluor 594 anti-CD4 mAb (10 μg), Alexa Fluor 594 anti-CD8a mAb (10 μg) and Hoechst 33342 (100 μg). Thirty minutes later, explants of the distal ileum were observed by confocal microscopy. Note that CD103+CD4CD8a cells adjacent to epithelial cell layers (indicated by arrows) were comparably observed in Gpr31b+/+ mice and Gpr31b−/− mice. In infection experiments, mice were administered with S. Typhimurium (1 × 109 CFU) by oral gavage. Ninety minutes after the administration, APC anti-CD103 mAb (20 μg) and Hoechst 33342 (100 μg) were intravenously injected. These experiments were repeated two independent times with similar results. Arrowheads indicate trans-epithelial dendrites. gi, Wild-type mice were fed lactate or pyruvate for three weeks, treated with non-pathogenic S. Typhimurium for six weeks, and then orally administered 1 × 109 CFU invasive S. Typhimurium. At day 40 after the invasive S. Typhimurium infection, bacterial loads in the liver and spleen (g), histology in the liver (haematoxylin and eosin staining) (h) and serum AST levels (i) were analysed (n = 6 mice). In h, tissue damage with noticeable haemorrhage (arrows) and leukocyte infiltration after S. Typhimurium infection was suppressed by oral pretreatment of mice with lactate or pyruvate. One-way ANOVA with Tukey’s comparison (a), two-tailed Student’s t-test (b, e) and two-tailed Mann–Whitney test (g, i) were performed for statistical analysis. Data represent the mean ± s.d. Scale bar, 10 μm (c, d, f), 100 μm (h). NS, not significant. Source data

  10. Extended Data Fig. 10 Regulation of GPR31 expression by the CX3CL1–CX3CR1 axis.

    a, Targeted disruption of the Cx3cl1 gene. The targeting vector was constructed by replacement of exon 2 with a genomic fragment containing loxP-flanked exon 2 and a neomycin-resistance gene cassette. The floxed allele was generated by homologous recombination and Cre-mediated recombination resulted in a deleted allele of the Cx3cl1 gene in embryonic stem cells. b, c, Intestinal CX3CR1+ cells isolated from Cx3cr1gfp/+ or Cx3cr1gfp/gfp mice were treated with lactic acid or pyruvic acid (100 μM each). Each symbol represents the percentage of GFP-positive cells with dendrites that are over 5 μm in length in a single well, from two biologically independent experiments (n = 6 independent samples per group). The morphology of CX3CR1+ cells was observed under a microscope (b) and their dendrite extension was quantified (c). d, Intestinal CX3CR1+ cells isolated from Cx3cr1gfp/+ mice, Cx3cr1gfp/gfp mice or Cx3cl1−/− mice were incubated with 500 nM CX3CL1 for 24 h, and mRNA expression of Gpr31b was analysed by quantitative PCR (Cx3cr1gfp/+ mice and Cx3cr1gfp/gfp mice, n = 3; Cx3cl1−/− mice, n = 4 independent samples per group). e, f, Intestinal CX3CR1+ cells isolated from Cx3cr1gfp/+Cx3cl1−/− mice were incubated in the presence or absence of CX3CL1 for 30 h, and further stimulated with lactic acid or pyruvic acid for 4 h. The dendrite extension of CX3CR1+ cells was observed by microscopy (e) and quantified (f) (n = 8 independent samples per group). Data represent the mean ± s.d. from two biologically independent experiments. Scale bar, 10 μm. One-way ANOVA with Tukey’s comparison (c, d) and two-tailed Student’s t-test (f) were performed for statistical analysis. Data represent the mean ± s.d. NS, not significant. Source data

Supplementary information

  1. Supplementary Figure 1

    This file contains the Southern blot analysis from Gpr31b−/− mice

  2. Reporting Summary

  3. Video 1 Two-photon microscopic imaging of an intestinal explant prepared from Cx3cr1gfp/+ Gpr31+/+ mice. Explants of the ileum prepared from Cx3cr1gfp/+ Gpr31+/+ mice were stained with CMRA (red) and vertically monitored with a z-step interval of 0.5 μm by two-photon microscopy. The experiment was repeated three independent times with similar results. Scale bar, 10 μm. Video corresponds to Fig. 2d

  4. Video 2 Two-photon microscopic imaging of an intestinal explant prepared from Cx3cr1gfp/+ Gpr31-/-mice. Explants of the ileum prepared from Cx3cr1gfp/+ Gpr31-/- mice were stained with CMRA (red) and vertically monitored with a z-step interval of 0.5 μm by two-photon microscopy. The experiment was repeated three independent times with similar results. Scale bar, 10 μm. Video corresponds to Fig. 2d

  5. Video 3 Two-photon microscopic imaging of an intestinal explant prepared from Cx3cr1gfp/gfp Gpr31+/+ mice. Explants of the ileum prepared from Cx3cr1gfp/gfp Gpr31+/+ mice were stained with CMRA (red) and vertically monitored with a z-step interval of 0.5 μm by two-photon microscopy. The experiment was repeated three independent times with similar results. Scale bar, 10 μm. Video corresponds to Fig. 2d

  6. Video 4 Two-photon microscopic imaging of an intestinal explant prepared from untreated Cx3cr1gfp/+ Gpr31+/+ mice. Ileal explants were stained with CMRA (red) and vertically monitored with a z-step interval of 0.5 μm by two-photon microscopy. The experiment was repeated three independent times with similar results. Scale bar, 10 μm. Video corresponds to Fig. 4c

  7. Video 5 Two-photon microscopic imaging of an intestinal explant prepared from Cx3cr1gfp/+ Gpr31+/+ mice treated with lactate. Mice were orally administered with 100 mM sodium lactate for 3 weeks. Ileal explants were stained with CMRA (red) and vertically monitored with a z-step interval of 0.5 μm by two-photon microscopy. The experiment was repeated three independent times with similar results. Scale bar, 10 μm. Video corresponds to Fig. 4c

  8. Video 6 Two-photon microscopic imaging of an intestinal explant prepared from Cx3cr1gfp/+ Gpr31+/+ mice treated with lactate. Mice were orally administered with 100 mM sodium lactate for 3 weeks. Ileal explants were stained with CMRA (red) and vertically monitored with a z-step interval of 0.5 μm by two-photon microscopy. The experiment was repeated three independent times with similar results. Scale bar, 10 μm. Video corresponds to Fig. 4c

  9. Video 7 Two-photon microscopic imaging of an intestinal explant prepared from untreated Cx3cr1gfp/+ Gpr31-/- mice. Ileal explants were stained with CMRA (red) and vertically monitored with a z-step interval of 0.5 μm by two-photon microscopy. The experiment was repeated three independent times with similar results. Scale bar, 10 μm. Video corresponds to Fig. 4c

  10. Video 8 Two-photon microscopic imaging of an intestinal explant prepared from Cx3cr1gfp/+ Gpr31-/- mice treated with lactate. Mice were orally administered 100 mM sodium lactate for 3 weeks. Ileal explants were stained with CMRA (red) and vertically monitored with a z-step interval of 0.5 μm by two-photon microscopy. The experiment was repeated three independent times with similar results. Scale bar, 10 μm. Video corresponds to Fig. 4c

  11. Video 9 Two-photon microscopic imaging of an intestinal explant prepared from Cx3cr1gfp/+ Gpr31-/- mice treated with pyruvate. Mice were orally administered 100 mM sodium pyruvate for 3 weeks. Ileal explants were stained with CMRA (red) and vertically monitored with a z-step interval of 0.5 μm by two-photon microscopy. The experiment was repeated three independent times with similar results. Scale bar, 10 μm. Video corresponds to Fig. 4c

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https://doi.org/10.1038/s41586-019-0884-1

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