Abstract

Alterations in enteric microbiota are associated with several highly prevalent immune-mediated and metabolic diseases1,2,3, and experiments involving faecal transplants have indicated that such alterations have a causal role in at least some such conditions4,5,6. The postnatal period is particularly critical for the development of microbiota composition, host–microbe interactions and immune homeostasis7,8,9. However, the underlying molecular mechanisms of this neonatal priming period have not been defined. Here we report the identification of a host-mediated regulatory circuit of bacterial colonization that acts solely during the early neonatal period but influences life-long microbiota composition. We demonstrate age-dependent expression of the flagellin receptor Toll-like receptor 5 (TLR5) in the gut epithelium of neonate mice. Using competitive colonization experiments, we demonstrate that epithelial TLR5-mediated REG3γ production is critical for the counter-selection of colonizing flagellated bacteria. Comparative microbiota transfer experiments in neonate and adult wild-type and Tlr5-deficient germ-free mice reveal that neonatal TLR5 expression strongly influences the composition of the microbiota throughout life. Thus, the beneficial microbiota in the adult host is shaped during early infancy. This might explain why environmental factors that disturb the establishment of the microbiota during early life can affect immune homeostasis and health in adulthood.

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Acknowledgements

We thank D. Gütle, T. Albers, M. Nietschke, A. Smoczek, V. Tremaroli and M. Krämer for technical support and M. Rühlemann for discussions. The work was supported by the priority programs SPP1656 (Ho-2236/9-1 and BL953/5-1) and SPP1580 (Ho 2236/11-1, He 1964/18-2), SFB944 (P4 to M.H.) and SFB1182 (C2 to F.S. and P.R.) and the single grant Ho 2236/14-1 from the German Research Foundation (DFG), as well as the Lower Saxony-Israel Fond and the Niedersachsen-Research Network on Neuroinfectiology (N-RENNT) of the Ministry of Science and Culture of Lower Saxony, Germany, to M.F. and M.W.H. M.F. and K.v.V. were supported by the German Federal Ministry of Education and Research (BMBF) within the consortium InfectControl 2020 (Project NeoBiom, grant ID 03ZZ0829C). M.F. was supported by the Freie Universität Berlin within the Excellence Initiative of the DFG and P.R. by the ExC306 Inflammation at Interfaces. B.C. is supported by a Career Development Award from the Crohn’s and Colitis Foundation and an Innovator Award from the Kenneth Rainin Foundation. F.B. is Torsten Söderberg Professor in Medicine and recipient of ERC consolidator Grant 2013 (European Research Council, Consolidator grant 615362-METABASE).

Reviewer information

Nature thanks J. Kagan and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Author information

Author notes

  1. These authors contributed equally: Felix Sommer, Benoit Chassaing

Affiliations

  1. Institute for Medical Microbiology and Hospital Epidemiology, Hannover Medical School, Hannover, Germany

    • Marcus Fulde
    •  & Mathias W. Hornef
  2. Institute of Microbiology and Epizootics, Department of Veterinary Medicine at the Freie Universität Berlin, Berlin, Germany

    • Marcus Fulde
    •  & Kira van Vorst
  3. Department of Molecular and Clinical Medicine/Wallenberg Laboratory, Institute of Medicine, University of Gothenburg and Sahlgrenska University Hospital, Gothenburg, Sweden

    • Felix Sommer
    •  & Fredrik Bäckhed
  4. Institute of Clinical Molecular Biology (IKMB), Kiel University, Kiel, Germany

    • Felix Sommer
    •  & Philip Rosenstiel
  5. Neuroscience Institute, Georgia State University, Atlanta, GA, USA

    • Benoit Chassaing
  6. Institute for Biomedical Sciences, Georgia State University, Atlanta, GA, USA

    • Benoit Chassaing
    •  & Andrew T. Gewirtz
  7. Institute for Medical Microbiology, RWTH University Hospital Aachen, Aachen, Germany

    • Aline Dupont
    •  & Mathias W. Hornef
  8. Division of Microbiology, University of Osnabrück, Osnabrück, Germany

    • Michael Hensel
  9. Institute for Laboratory Animal Science, Hannover Medical School, Hannover, Germany

    • Marijana Basic
    •  & André Bleich
  10. Institute of Veterinary Pathology, Department of Veterinary Medicine at the Freie Universität Berlin, Berlin, Germany

    • Robert Klopfleisch

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Contributions

M.F. and M.W.H. performed and analysed colonization and infection experiments. M.F., M.B., B.C., A.B. and A.T.G. planned and performed microbiota transfer experiments. M.F., M.B., B.C. and A.D. performed conventional and germ-free mouse breeding and sample collection. F.S., F.B., P.R., and M.F. analysed the enteric microbiota samples. K.v.V., M.F. and A.D. analysed faecal flagellin content. M.H. generated bacterial mutants. R.K. performed histopathological analysis. M.W.H., A.T.G. and F.B. supervised the study.

Competing interests

The authors declare no competing interests.

Corresponding author

Correspondence to Mathias W. Hornef.

Extended data figures and tables

  1. Extended Data Fig. 1 Role of TLR5 and flagellin expression in the pathogenesis of neonatal Salmonella infection.

    a, b, Intestinal colonization and organ dissemination evaluated by serial dilution and plating two (a) or three (b) days after infection of 1-day-old wild-type (WT, filled circles, n = 5) or Tlr5-deficient mice (Tlr5, open circles, n = 4 or n = 5, respectively) with approximately 102 CFU S. Typhimurium. Results from one litter at each time point. c, Intestinal colonization and organ dissemination evaluated by serial dilution and plating in small intestine (n = 6 and 11), colon (n = 7 and 11), liver (n = 10 and 12), spleen (n = 10 and 12) and lung (n = 10 and 12) 4 days after infection of 1-day-old WT (filled symbols) mice with approximately 102 WT S. Typhimurium (circles, two litters) or ΔfljB/fliC mutant S. Typhimurium (triangles, three litters). d, Intestinal colonization and organ dissemination evaluated by serial dilution and plating in small intestine (n = 6 and 4), colon (n = 9 and 4), liver (n = 9 and 4), spleen (n = 9 and 4) and lung (n = 9 and 4) 4 days after infection of 1-day-old Tlr5-deficient (open symbols) mice with approximately 102 WT S. Typhimurium (circles, 1 litter) or ΔfljB/fliC mutant S. Typhimurium (triangles, 1 litter). Data in ad represent the median (two-sided Mann–Whitney test). e, Survival of 1-day-old WT mice infected orally with wild-type (wt, dashed line, n = 20, three litters) or isogenic flagellin-deficient (ΔfljB/fliC, solid line, n = 22, three litters) S. Typhimurium. f, Cxcl2 mRNA expression analysed by quantitative RT–PCR in intestinal epithelial cells (IECs) isolated 4 days after infection of 1-day-old wild-type mice (WT, filled symbols, n = 13 and 11 from each three litters) or Tlr5-deficient mice (Tlr5, open symbols, n = 6 and 4 from each two litters) with wild-type S. Typhimurium (wt, circles) or an isogenic flagellin mutant (ΔfljB/fliC, triangles). Values were normalized to the expression of the housekeeping gene Hprt. Data represent the median (*P < 0.05; **P < 0.01, two-sided Mann–Whitney test). Source data

  2. Extended Data Fig. 2 Kinetics of counter-selection against flagellated Salmonella in Tlr5-proficient mice.

    Intestinal colonization (CI) at 3, 6, 12, and 24 h after oral infection of 1-day-old mice with a 1:1 ratio of wild-type and ΔfljB/fliC S. Typhimurium. Data represent the median; n = 7, 7, 7 (derived from 2 litters) and 26 (6 litters), respectively; **P < 0.01, Kruskal–Wallis test followed by Dunn’s post-test). Note that results from competition experiments with WT and flagellin-deficient (ΔfljB/fliC) S. Typhimurium in WT mice after 24 h presented in Fig. 1d, f, h and Extended Data Fig. 2 are identical and were pooled from six independent experiments conducted in two different laboratory animal facilities. Source data

  3. Extended Data Fig. 3 Influence of TLR5 on microbiota composition and the metabolic phenotype in individually housed adult mice.

    a, Bacterial DNA was extracted from the faeces of individually housed 6–8-week-old homozygous Tlr5+/+ (WT, red, n = 8) and homozygous Tlr5−/− (Tlr5, blue, n = 8) mice. The microbiota composition was analysed by 16S rDNA sequencing and the overall difference is illustrated in a principal coordinate analysis (PCoA). b, Body weight was evaluated in 7-week-old male WT (red) and Tlr5-deficient (blue) mice. Data represent the median. Statistical analysis was performed using two-sided Mann–Whitney U-test (n = 6 and 12, respectively, **P < 0.01). c, Daily food intake was evaluated in 25-week-old female WT (red) and Tlr5-deficient (blue) mice. Data represent the median. Statistical analysis was performed using two-sided Mann–Whitney U-test (n = 3 and 4, respectively). d, e, Haematoxylin and eosin staining of liver (d) and pancreas tissue sections (e) from 18-week-old WT and Tlr5-deficient mice. Images were randomly selected from n = 2 (WT) and n = 3 (Tlr5-deficient) mice examined. d, In contrast to WT mice, liver tissue from Tlr5-deficient mice showed decreased eosinophilic staining intensity of hepatocytes located close to the central veins (black asterisk) that is caused by an accumulation of microvacuoles in their cytoplasm consistent with increased lipid storage (black arrows). Scale bars: i and iii, 100 µm; ii and iv, 50 µm. e, Pancreas tissue from WT mice contained more and larger islets of Langerhans (black asterisks) than tissue derived from Tlr5-deficient mice. In addition, exocrine epithelial cells from WT mice contained larger amounts of eosinophilic zymogen granules (black arrows) than the more basophilic cells of Tlr5-deficient mice. Scale bars: i and iii; 100 µm; ii and iv, 20 µm. Source data

  4. Extended Data Fig. 4 Neonatal but not adult epithelial Reg3g mRNA expression is influenced by TLR5 expression.

    IECs from non-infected wild-type (WT, red) and Tlr5-deficient (Tlr5, blue) mice were collected at 1 (n = 9 from three litters and n = 7 from three litters), 4 (n = 7 from two litters and n = 10 from two litters), 10 (n = 9 from one litter and n = 11 from two litters) and 21 days (n = 9 from one litter and n = 6 from one litter) after birth and Reg3g mRNA expression was analysed by qRT–PCR. Values were normalized to the expression of the housekeeping gene Hprt. Data represent the median (*P < 0.05; **P < 0.01; ***P < 0.001; two-sided Mann–Whitney U-test). Source data

  5. Extended Data Fig. 5 REG3γ expression influences the faecal bioactive flagellin load.

    Faecal pellets from age-matched WT (n = 10; 6 female, 4 male) and Reg3g-deficient (n = 13; 8 female, 5 male) 5–8-week-old mice housed in 3 and 4 separate isolator cages, respectively, were collected. The amount of bioactive, pro-inflammatory flagellin was determined using the HEK-Blue-mTLR5 cell line as described in the Methods section. Data represent the median from two independent measurements; *P < 0.05, two-sided Mann–Whitney U-test. Source data

Supplementary information

  1. Supplementary Tables

    This file contains Supplementary Table 1 (Salmonella enterica serovar Typhimurium strains used in this study) and Supplementary Table 2 (Oligonucleotides used in this study).

  2. Reporting Summary

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https://doi.org/10.1038/s41586-018-0395-5

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