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Dedicated macrophages organize and maintain the enteric nervous system

Nature volume 618, pages 818–826 (2023)Cite this article

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Abstract

Correct development and maturation of the enteric nervous system (ENS) is critical for survival1. At birth, the ENS is immature and requires considerable refinement to exert its functions in adulthood2. Here we demonstrate that resident macrophages of the muscularis externa (MMϕ) refine the ENS early in life by pruning synapses and phagocytosing enteric neurons. Depletion of MMϕ before weaning disrupts this process and results in abnormal intestinal transit. After weaning, MMϕ continue to interact closely with the ENS and acquire a neurosupportive phenotype. The latter is instructed by transforming growth factor-β produced by the ENS; depletion of the ENS and disruption of transforming growth factor-β signalling result in a decrease in neuron-associated MMϕ associated with loss of enteric neurons and altered intestinal transit. These findings introduce a new reciprocal cell–cell communication responsible for maintenance of the ENS and indicate that the ENS, similarly to the brain, is shaped and maintained by a dedicated population of resident macrophages that adapts its phenotype and transcriptome to the timely needs of the ENS niche.

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Fig. 1: MMϕ engulf synapses and neurons during early postnatal development.
Fig. 2: MMϕ undergo functional and transcriptional shifts from early postnatal development to adulthood.
Fig. 3: MMϕ subsets occupy distinct micro-anatomical niches in muscularis externa.
Fig. 4: ENS-derived TGFβ instructs the NA-MMϕ phenotype.

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Data availability

The scRNA-seq dataset generated and analysed during the current study is available in the ArrayExpress repository with accession no. E-MTAB-11453. We further analysed published datasets from Morarach et al.16, which are available at the GeoGene Expression Omnibus database under identifier GSE149524. In addition we performed analysis using the dataset published by Elmentaite et al.34, which is available at ArrayExpress with accession no. E-MTAB-9543, and by Drokhlyansky et al.33, which is deposited in the Single Cell Portal (SCP1038). Source data are provided with this paper. Should further clarification be required, requests can be directed to the corresponding author.

Change history

  • 24 July 2023

    In the version of this article initially published, there was an error in the metadata associated with Pieter Vanden Berghe’s name, which is now amended in the online version of the article.

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Acknowledgements

We thank P. A. Penttila and R. Chinnaraj from the KU Leuven FACS core for their excellent support and cell sorting. We also thank the Cell & Tissue Imaging Core (P. Vanden Berghe and T. Martens, KU Leuven) for confocal imaging (supported by AKUL 15/37 and FWO I001918N). We also thank A. Escamilla-Ayala of the VIB Leuven BioImaging Core for her support and assistance in this work. We thank K. Kierdorf, M. Prinz, A. Schlitzer, F. Ginhoux, Z. Liu and M. Azhar for providing mice. M.A. is supported by NIH grant nos. R01HL126705, R01 HL157017-01A1 and R01 HL145064-01. M.F.V. is supported by FWO PhD fellowship no. 11C2219N. G.B. is funded by ERC Advanced grant no. 833816-NEUMACS.

Author information

Author notes

  1. Simon Verheijden

    Present address: Janssen Research and Development, Janssen Pharmaceutica NV, Beerse, Belgium

  2. Sebastiaan De Schepper

    Present address: UK Dementia Research Institute, UCL Queen Square Institute of Neurology, Faculty of Brain Sciences, University College London, London, UK

  3. Deceased: Jens Van Herck

Authors and Affiliations

  1. Laboratory for Intestinal Neuro-Immune Interaction, Translational Research Center for Gastrointestinal Disorders, Department of Chronic Diseases, Metabolism and Ageing, KU Leuven, Leuven, Belgium

    Maria Francesca Viola, Marta Chavero-Pieres, Elodie Modave, Marcello Delfini, Nathalie Stakenborg, Maria Cuende Estévez, Naomi Fabre, Iris Appeltans, Simon Verheijden, Sebastiaan De Schepper & Guy Boeckxstaens

  2. Laboratory for Enteric NeuroScience, Translational Research Center for Gastrointestinal Disorders, Department of Chronic Diseases, Metabolism and Ageing, KU Leuven, Leuven, Belgium

    Tobie Martens & Pieter Vanden Berghe

  3. Laboratory of Reproductive Genomics, Department of Human Genetics, KU Leuven, Leuven, Belgium

    Katy Vandereyken, Jens Van Herck & Thierry Voet

  4. KU Leuven Institute for Single Cell Omics, KU Leuven, Leuven, Belgium

    Katy Vandereyken, Alejandro Sifrim, Thierry Voet & Guy Boeckxstaens

  5. Quantitative Systems Biology, LIMES-Institute, University of Bonn, Bonn, Germany

    Hannah Theobald & Andreas Schlitzer

  6. Institute of Neuropathology, Faculty of Medicine, University of Freiburg, Freiburg, Germany

    Philippe Petry, Katrin Kierdorf & Marco Prinz

  7. Faculty of Biology, University of Freiburg, Freiburg, Germany

    Philippe Petry

  8. Laboratory of Multi-Omic Integrative Bioinformatics, Department of Genetics, KU Leuven, Leuven, Belgium

    Alejandro Sifrim

  9. Leuven AI Institute, KU Leuven, Leuven, Belgium

    Alejandro Sifrim

  10. Shanghai Institute of Immunology, Shanghai Jiao Tong University School of Medicine, Shanghai, China

    Zhaoyuan Liu & Florent Ginhoux

  11. Singapore Immunology Network, Agency for Science, Technology & Research, Singapore, Singapore

    Florent Ginhoux

  12. Translational Immunology Institute, SingHealth Duke-NUS Academic Medical Centre, Singapore, Singapore

    Florent Ginhoux

  13. Department of Cell Biology and Anatomy, University of South Carolina School of Medicine, Columbia, SC, USA

    Mohamad Azhar

  14. Laboratory for Mucosal Immunology, Translational Research Center for Gastrointestinal Disorders, Department of Chronic Diseases, Metabolism and Ageing, KU Leuven, Leuven, Belgium

    Gianluca Matteoli

  15. Centre for Integrative Biological Signalling Studies, University of Freiburg, Freiburg, Germany

    Katrin Kierdorf

  16. Center for Basics in NeuroModulation (NeuroModulBasics), Faculty of Medicine, University of Freiburg, Freiburg, Germany

    Katrin Kierdorf & Marco Prinz

  17. Faculty of Medicine, University of Freiburg, Freiburg, Germany

    Marco Prinz

  18. Signalling Research Centres BIOSS and CIBSS, University of Freiburg, Freiburg, Germany

    Marco Prinz

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  1. Maria Francesca Viola
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Contributions

M.F.V. designed and conducted experiments and wrote the manuscript. M.P. performed histological analyses. E.M. and A. Sifrim performed analysis of scRNA-seq data. I.A. and N.F. provided excellent technical support throughout all experiments. M.D., N.S., J.V.H., H.T. and M.C.E. performed experiments. T.M. performed ex vivo time-lapse imaging. K.V. performed HCR experiments. S.V., S.D.S., P.V.B., T.V. and A. Sifrim provided intellectual input. K.K., M.P., A. Schlitzer, Z.L. and F.G. provided mice. P.P. performed genotyping and tamoxifen injections. M.A. generated Tgfb2fl/fl and Tgfb3fl/fl mice and genetically combined them to create Tgfb2-3fl/fl mice. G.M. performed histological evaluation of haematoxylin and eosin stainings. G.B. led the project and revised the manuscript.

Corresponding author

Correspondence to Guy Boeckxstaens.

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Extended data figures and tables

Extended Data Fig. 1 Postnatal maturation of the ENS.

Neuronal density (A), ganglia (> 5 neurons)/mm2 (B) and single extra-ganglionic neurons/mm2 (C) in the myenteric plexus, corrected for tissue growth at P10 (n = 5-9), P14 (n = 6), P21 (n = 5) and P56 (n = 4-6). (D) NOS+ neurons, expressed as a percentage of HuC/D+ cells (n = 4-5). (E) ChAT+ area, normalised to HuC/D+ neurons (n = 4-5). (F) Synapsin-I volume/µm3, normalised to tissue growth (n = 4). Total length of MMϕ filopodia (G), number of dendrites (H) and cell volume (I) (Imaris) (n = 3). (J) Percentage of MMϕ with at least one phagocytic cup (n = 3). (K) Confocal images displaying MMϕ morphology. Scale bar: 10 µm. (L) Confocal image of the myenteric plexus at P14 of Synapsin-I or isotype control. Scale bar: 20 µm. (M) Percentage of synaptic engulfment per cell (Synapsin-I+ volume/MMϕ volume normalised to synaptic density) (n = 4). (N) Digital reconstruction depicting synapses within CD68+ lysosomes of MMϕ. Arrowheads indicate engulfed synapses. (O) Orthogonal view depicting MMϕ engulfing synaptic machinery at P14. (P) Confocal images displaying MMϕ engulfing neuronal cell bodies. Scale bar: 20 µm. (Q) Digital reconstructions of MMϕ engulfing enteric neurons. (R) MMϕ engulfing tdTomato+ neural debris (orthogonal view). (S) Mean fluorescence intensity of MMϕ engulfing neural cells at P14 (n = 7) and P56 (n = 6). (T) Graph and contour plot of tdTomato+ debris uptake by MMϕ in vivo (GFP+) and ex vivo (GFP-) (n = 4). (U) MMϕ engulfing Wnt1-tdTomato+ debris (orthogonal view). (V) MMϕ engulfing Baf53b-tdTomato+ neuronal debris (orthogonal view). Arrowhead indicates engulfed tdTomato+ debris. Scale bar: 5 µm unless otherwise indicated. Unpaired two-tailed t-tests (G-J, S) or Ordinary One-Way ANOVAs with Tukey’s multiple comparisons (A-E, M). Data are shown as Mean ± SD. Images shown are representative of at least 3 biological replicates, apart from L, R, U-V (2 biological replicates). Single points indicate biological replicates, apart from F-M (technical replicates). n = x biologically independent samples.

Source data

Extended Data Fig. 2 MMϕ mature transcriptionally after weaning.

(A) Dot plot in αCSF1R-treated mice and controls to show efficient depletion of MMϕ. Cells shown are pre-gated on fsc/ssc, single cells, live and CD45+. (B) Quantification of immune cells in αCSF1R-treated mice (n = 4) and controls (n = 5). Macrophages are defined as CD45+CD11b+CD64+, B cells as CD45+CD3e-CD64−CD11b−CD19+ and T cells as CD45+CD3e+. All populations were pre-gated on fsc/ssc and live cells. Unpaired two-tailed t-tests. Data are shown as Mean ± SD. Each data point indicates a separate biological replicate. *** p < 0.001; ns = not significant. (C) Gating strategy used to isolate Cx3cr1+ MMϕ from the muscularis externa for scRNAseq. (D) UMAP of data prior to exclusion of contaminating cluster (pink). (E) UMAP with heatmap colour-coding to show expression for canonical macrophage markers and CCR2 within the scRNAseq dataset. (F) Violin plots showing the upregulation of extracellular matrix genes in the contaminating cluster (pink). (G) Representative confocal image of the muscularis externa at P10, showing Cx3cr1+ MMϕ embedded within the extracellular matrix (Dcn, Decorin). Image is representative of 3 biological replicates. Scale bar is 20 µm. (H) Volcano plot depicting globally differentially expressed genes at P10 and P56. Coloured dots represent significantly (p < 0.05) upregulated genes over Log2FC > 0.5. (I) Dot plot to depict expression of top 10 upregulated genes within each cluster. Colour of dots represents expression level, while size of dots represents percentage of cells expressing the gene identified. Only genes significantly (p ≤ 0.05) upregulated (logFC ≥ 0.2) are shown. (J) Violin plots showing the expression of Bmp2 in the identified MMϕ clusters. n = x biologically independent samples.

Source data

Extended Data Fig. 3 MMϕ subset phenotype and function prior to and after weaning.

(A) Representative gating strategy used to identified MMϕ subsets via flow cytometry. (B) Heatmap depicting expression of genes identified by Chakarov et al., within each cluster, cells pooled. (C) Representative contour plots of identified MMϕ subsets at P10 and P56. (D) Relative frequency of Lyve1+ MMϕ quantified via immunohistochemistry, in the different micro-anatomical niches of the muscularis externa (n = 4). (E) Phagocytic index of each MMϕ subset phagocytosing pHRodo-labelled apoptotic thymocytes, and representative histograms from the same sample incubated at 4 °C and at 37 °C with phRodo-labelled apoptotic cells (n = 5). (F) Representative confocal images showing CD68+ lysosomes in NA-MMϕ and Lyve1+ MMϕ. Images shown are representative of two independent experiments. Scale bar is 20 µm. (G) Relative frequency of NA-MMϕ quantified via immunohistochemistry, in the different micro-anatomical niches of the muscularis externa (n = 4). (H) Dot plot depicting expression of top 5 transcription factors identified via SCENIC within each cluster. Colour of dots represents expression level, while size of dots represents percentage of cells expressing the transcription factor identified. Only genes significantly (p ≤ 0.05) upregulated (logFC ≥ 0.2) are shown. (I) Quantification of the percentage of Ms4a3+ Cx3cr1+ MMϕ in the muscularis externa throughout early postnatal life and adulthood (n = 5-6). (J) Quantification of the percentage of Ms4a3+ Cx3cr1+ MMϕ in the identified MMФ subsets in the muscularis externa throughout early postnatal life and adulthood (n = 5-6). (K) Quantification via flow cytometry of population frequency at 8 weeks (P56) and 20 weeks (n = 5). Data are shown as Mean ± SD, analysed by multiple unpaired t-tests (D-G) or two-way ANOVA followed by Tukey’s (J) or Šídák’s (K) multiple comparison. Each data point indicates a separate biological replicate. n = x biologically independent samples.

Source data

Extended Data Fig. 4 NA-MMϕ phenotype is instructed by ENS-derived TGFβ.

(A) Gene expression of TGFβ within the muscularis externa at P10 (n = 4) and P56 (n = 7). (B) Percentage of enteric neurons (Wnt1+Cd49b-) and enteric glia (Wnt1+ Cd49b+) expressing TGFβ (n = 4). (C) Representative contour plot showing TGFβ expression in immune cells (CD45+) and Wnt1-CD45- cells. Only live cells are shown. The graph depicts the percentage of cells of each population expressing TGFβ (n = 4). (D) Expression of Tgfb1, 2 and 3 in enteric neurons, assessed in the published dataset of Morarch et al., 2021. (E) Gene expression of Tgfb1, Tgfb2 and Tgfb3 in immune cells (CD45+) at P10, P14, P28 and P56, FACS-sorted from the muscularis externa of Wnt1cre/wt Rosa26tdT/WT mice (n = 3-4). (F) Hybridization Chain Reaction (HCR) combined with immunohistochemistry showing transcripts of Tgfb2 and Tgfb3, or no probe, in the ENS. Images are representative of 3 biological replicates. Scale bar is 5 µm. (G) Heatmap indicating expression of key marker genes identified in MMϕ subsets in BMDMs stimulated for 24 h with TGFβ1, 2 or 3. Coloured line indicates the MMϕ subset (n = 6-10). Data is representative of two pooled experiments. (H) Frequency of NA-MMϕ throughout early postnatal development to adulthood (n = 5-6). Dotted line indicates time of weaning. (I) Frequency of identified MMϕ subsets in 8 week-old germ-free mice compared to conventionally colonised mice (n = 5). Data are shown as Mean ± SD, analysed using unpaired two-tailed T-tests or ANOVA followed by Šídák (A, I) or Holm-Šídák (E, H) multiple comparisons. Each data point indicates a separate biological replicate. n = x biologically independent samples.

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Extended Data Fig. 5 Characterisation of the TGFβ-TGFBR axis in the GI tract.

(A) Expression of Uchl1 and Gfap in the muscularis externa 5 days after BAC treatment compared to SHAM (n = 5). (B) Live Cx3cr1+ MMϕ and (C) relative percentages of Lyve1+ MMϕ and differentiating MMϕ in the muscularis externa in SHAM (n = 4) and BAC-treated (n = 5) mice. (D) Transfection efficiency (GFP+ HUC/D+/HUC/D+ cells) in ganglia of TGFb2-3wt (n = 5) and TGFb2-3fl (n = 6) mice after infection with AAV9-Cre-GFP. (E) Number of live Cx3cr1+ MMϕ in Cx3cr1creERT2/WT TGFbr2fl/fl (Cre+) (n = 7) and Cx3cr1WT/WT TGFbr2fl/fl (Cre−) (n = 5) mice. (F) Frequency of MMϕ populations and (G) Cell counts of MMϕ populations in Cre+ (n = 7) and Cre− (n = 5) mice 4 weeks after tamoxifen administration. (H) Body weight of Cre+ (n = 7) and Cre− (n = 5) mice over 4 weeks following tamoxifen administration expressed as a percentage of baseline body weight. (I) Disease activity index (DAI) of Cre+ (n = 7) and Cre− (n = 5) mice over 4 weeks following tamoxifen administration. (J) Histopathological score of colonic tissue of Cre+ (n = 7) and Cre− (n = 5) mice 4 weeks after tamoxifen administration. (K) Brightfield images of colonic tissue stained with hematoxylin and eosin obtained from Cre+ and Cre− mice 4 weeks after tamoxifen administration. Images are representative of 5-7 biological replicates. Scale bar is 100 μm. (L) Expression of TGFBR1 and TGFBR2 in human myeloid cells, obtained from Elmentaite et al., 2021. (M) Expression of TGFB1, TGFB2 and TGFB3 in human enteric neurons, obtained from Drokhlyansky et al., 2020. Data are shown as Mean ± SD, analysed using unpaired two-tailed T-tests (A-E, J) or Two-way ANOVA followed by Šídák’s multiple comparisons (F-G) or RM Two-way ANOVA with Geisser-Greenhouse correction and Bonferroni’s multiple comparisons (H–I). Each data point indicates 1 biological replicate. ns = not significant. n = X biologically independent samples.

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Extended Data Fig. 6 Low magnification images of the ENS.

(A) Representative low magnification confocal images of the developing ENS, from P10 to P56. (B) Representative low magnification confocal images of the ENS in in Cx3cr1WT/WT TGFbr2fl/fl (Cre−) and in Cx3cr1creERT2/WT TGFbr2fl/fl (Cre+) littermate controls. Images shown are representative of at least 2 independent experiments. Scale bars are 100 μm.

Supplementary information

Supplementary Tables

This file contrains Supplementary Tables 1–3.

Reporting Summary

Supplementary Video 1

Stitched time-lapse of ex vivo live imaging of muscularis externa of 14-day-old Wnt1Cre/WT Rosa26tdT/WT Cx3cr1GFP/WT mice. Scale bar, 20 µm.

Supplementary Video 2

Stitched time-lapse of ex vivo live imaging of muscularis externa of 56-day-old Wnt1Cre/WT Rosa26tdT/WT Cx3cr1GFP/WT mice. Scale bar, 20 µm.

Supplementary Video 3

Stitched time-lapse of ex vivo live imaging of Cx3cr1+ MMФ engulfing tdTomato+ neural debris, recorded in 14-day-old Wnt1Cre/WT Rosa26tdT/WT Cx3cr1GFP/WT mice. White arrowhead indicates engulfment of tdTomato+ neural debris. Scale bar, 10 µm.

Source data

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Viola, M.F., Chavero-Pieres, M., Modave, E. et al. Dedicated macrophages organize and maintain the enteric nervous system. Nature 618, 818–826 (2023). https://doi.org/10.1038/s41586-023-06200-7

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