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Colonic goblet cell-associated antigen passages mediate physiologic and beneficial translocation of live gut bacteria in preweaning mice

Nature Microbiology volume 10, pages 927–938 (2025)Cite this article

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Abstract

Gut-resident microorganisms have time-limited effects in distant tissues during early life. However, the reasons behind this phenomenon are largely unknown. Here, using bacterial culture techniques, we show that a subset of live gut-resident bacteria translocate and disseminate to extraintestinal tissues (mesenteric lymph nodes and spleen) in preweaning (day of life 17), but not adult (day of life 35), mice. Translocation and dissemination in preweaning mice appeared physiologic as it did not induce an inflammatory response and required host goblet cells, the formation of goblet cell-associated antigen passages, sphingosine-1-phosphate receptor-dependent leukocyte trafficking and phagocytic cells. One translocating strain, Lactobacillus animalisWU, showed antimicrobial activity against the late-onset sepsis pathogen Escherichia coli ST69 in vitro, and its translocation was associated with protection from systemic sepsis in vivo. While limited in context, these findings challenge the idea that translocation of gut microbiota is pathological and show physiologic and beneficial translocation during early life.

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Fig. 1: Live gut-resident bacteria spontaneously translocate to MLNs and spleens in preweaning mice.
Fig. 2: Early-life L. animalisWU translocation requires GCs, GAPs and host cells that express S1PR and are phagocytic.
Fig. 3: L. animalisWU translocation does not trigger a substantial systemic inflammatory response in preweaning mice.
Fig. 4: L. animalisWU translocation in early life helps protect against systemic E. coli ST69 infection.

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Data availability

The stool sequencing data and L. animalisWU whole-genome sequencing data are archived in Sequence Read Archive (BioProject: PRJNA1067122 and PRJNA1066880). The RNA-seq data are archived in Gene Expression Omnibus (GSE278303).

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Acknowledgements

This study was supported by National Institutes of Health grants R37AI112626, P30DK052574, R01DK097317, U01AI163073 and R01AI173220 awarded to R.D.N. B.A.R. was supported by K01DK125606 and P&F grant through DDRCC P30DK052574. V.J. was supported by the Crohn’s and Colitis Foundation grant number 902790. E.M.S. was supported by grants T32DK077653 and T32HD043010. cDC1 mice were a gift from K. Murphy, Washington University in Saint Louis School of Medicine.

Author information

Authors and Affiliations

  1. School of Biochemistry and Immunology, Trinity College Dublin, Dublin, Ireland

    Sreeram Udayan

  2. Division of Gastroenterology, Department of Internal Medicine, Washington University in Saint Louis School of Medicine, St. Louis, MO, USA

    Sreeram Udayan, Alexandria N. Floyd, Vini John, Bibiana E. Barrios, Keely G. McDonald, Ellen Merrick Schill, Andrew L. Martin, Rafael Gutierrez, Khushi B. Talati, Dalia L. Harris, Sushma Sundas, Kayla M. Burgess, Jocelyn T. Pauta, Elisabeth L. Joyce & Rodney D. Newberry

  3. Division of Gastroenterology Hepatology & Nutrition, Department of Pediatrics, Washington University in Saint Louis School of Medicine, St. Louis, MO, USA

    Brigida A. Rusconi, Jacqueline D. Wang & Phillip I. Tarr

  4. Division of Newborn Medicine, Department of Pediatrics, Washington University in Saint Louis School of Medicine, St. Louis, MO, USA

    Ellen Merrick Schill & Rafael Gutierrez

  5. Department of Medicine, University of California, Los Angeles, Los Angeles, CA, USA

    Devesha H. Kulkarni

  6. Division of Comparative Medicine, Washington University in Saint Louis School of Medicine, St. Louis, MO, USA

    Leslie D. Wilson

  7. Department of Immunology, Mayo Clinic, Rochester, MN, USA

    Kathryn A. Knoop

  8. Department of Pediatrics, Mayo Clinic, Rochester, MN, USA

    Kathryn A. Knoop

  9. Department of Molecular Microbiology, Washington University in Saint Louis School of Medicine, St. Louis, MO, USA

    Phillip I. Tarr

  10. Division of Rheumatology, Department of Internal Medicine, Washington University in Saint Louis School of Medicine, St. Louis, MO, USA

    Chyi-Song Hsieh

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  1. Sreeram Udayan
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Contributions

S.U. and R.D.N. conceptualized the project. S.U. designed the experiments. S.U., A.N.F., V.J., B.E.B., S.S., K.G.M., E.M.S., K.B.T., D.L.H., S.S., K.M.B., D.H.K., K.A.K., J.D.W., A.L.M., R.G. and E.L.J. conducted the studies. S.U. and B.A.R. conducted the bioinformatic analysis. L.D.W. performed the necropsy. S.U., B.A.R., J.T.P. and E.M.S. analysed the data. K.A.K., P.I.T., C.-S.H. and R.D.N. contributed reagents, materials and analysis tools. S.U. and R.D.N. wrote the paper with contributions from the other authors.

Corresponding author

Correspondence to Rodney D. Newberry.

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Competing interests

R.D.N., K.G.M. and K.A.K. are inventors in patent US11,241,480 Methods for Modulation of Dietary and Microbial Exposure With Compounds Comprising An EGFR Ligand. All other authors declare no competing interests.

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Nature Microbiology thanks Gerard Eberl and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Bionomial distribution of translocation, characterization of colon bacterial taxa at family level in preweaning and adult mice, luminal vs mucosal residence of L. animalisWU, and changes in gut barrier with EGF.

(a) Pattern of number of CFUs recovered from the MLN and spleen of DOL17 mice (relates to supplementary table 2. b) Relative frequency of bacterial taxa at family level compared by 16S v4 sequencing of specific pathogen free laboratory mice at DOL17 (n = 3) and DOL35 (n = 4). Lactobacillaceae family (denoted by red box) was one of the most common translocating taxa, is present at both ages. c) Culture of luminal vs mucosal scrapings reveals that L. animalisWU can be present in both compartments. d) Schematic of EGF treatment to inhibit GAPs and FTY720 treatment to inhibit trafficking. e) Assessment of gut barrier function in L. animalisWU colonized mice in the presence and absence of EGF using the 4kD FITC dextran leak assay. Graphs represent the mean +/− SEM. P value calculated using two-tailed Student’s t-test.

Extended Data Fig. 2 S1PR modulation alters immune cell trafficking to spleen in preweaning mice.

Percentage of immune cells expressing S1PR (%S1PRGFP+CD45+) assessed in colon and spleen of S1PR-GFP reporter mice (B6.129P2-S1pr1tm1Hrose/J) treated at DOL17 with pan-S1PR inhibitor (FTY720) (n = 3) or not treated (control) (n = 3). Statistical analyses were performed by two-tailed Student’s t test in GraphPad Prism. Data represented as mean with individual values. P values are as denoted. Figure created with BioRender.com.

Extended Data Fig. 3 CSF1R blockade, loss of cDC1 cells, and CX3CR1 deletion does not impair L. animalisWU translocation in preweaning mice.

(a) Flow cytometry plots and (b-e) graphs demonstrate that CSFR1 blockade in preweaning mice reduces the CD45 + MHCII+ CD11c- colonic LP cellular population which can also express CX3CR1 and F4/80. (f) CFU/organ of L. animalisWU recovered from intestinal and extraintestinal tissues of L. animalisWU fed preweaning mice that were nontreated controls (n = 4) or treated with anti-CSF1R (n = 3). (g) CFU/organ of L. animalisWU recovered from intestinal and extraintestinal tissues of L. animalisWU fed wildtype, cDC1 deficient (Irf8 delta 32), or CX3CR1 deficient preweaning mice. Graphs represent mean +/− SEM. Statistical analyses were performed by two- tailed Student’s t test for B – F, one-way ANOVA for intestinal tissues in G, and one-sided cumulative binomial distribution probability test for extraintestinal tissues in F and G.

Extended Data Fig. 4 Induction of Th1, Th2 and Th17 cytokines by MLN and splenic CD4+ T cells from preweaning mice given L. animalisWU with and without EGF.

(a) Flow cytometry gating strategy for identifying Th1 cytokines (TNFα and IFNɣ) secreted by CD45+CD3+CD4+ T cells in MLNs and spleens. Frequency of TNFα+ and IFNɣ + CD4 + T cells in (b-c) MLNs and (d-e) spleens of nontreated (control), L. animalisWU fed and L. animalisWU + EGF fed mice. (f) Flow cytometry gating strategy for identifying Th2 cytokines (IL4 and IL13) secreted by CD45+CD3+CD4+ T cells in MLNs and spleens. Frequency of IL13+ and IL4 + CD4 + T cells in (g-h) MLNs and (i-j) spleens of nontreated (control), L. animalisWU fed and L. animalisWU + EGF fed mice. (k) Flow cytometry gating strategy for identifying Th17 cytokines (IL17 and IL22) secreted by CD45+CD3+CD4+ T cells in MLNs and spleens. Frequency of IL17+ and IL22 + CD4 + T cells in (l-m) MLNs and (n-o) spleens of nontreated (control), L. animalisWU fed and L. animalisWU + EGF fed mice. Statistical analyses were performed by one-way ANOVA with Dunnett’s post test. Graphs represent mean + /− SEM. P values are as denoted.

Extended Data Fig. 5 Peripheral blood neutrophils increase in preweaning mice infected with E. coli ST69.

Neutrophil (CD45+Ly6G/C+ cells) numbers in non-infected mice (n = 3) or mice infected with E. coli ST69 (n = 5) gavaged orally at DOL17. Statistical analyses were performed by two-tailed Student’s t test in GraphPad Prism. Graph represents mean +/− SEM. P values are as denoted.

Extended Data Fig. 6 L. animalisWU does not induce regulatory T cell subsets in preweaning mice.

(a) Graphs and b) representative flow plots of Foxp3+ and Foxp3+ regulatory T cell subsets (Foxp3+ percentages (%CD4+FOXP3+ and %CD4+FOXP3+RorɣT+) between mice not treated (Control) (n = 4) or fed with L. animalisWU (n = 4) from DOL10-20, in (A-B) MLN and Spleen. Statistical analyses were performed by one-way ANOVA in GraphPad Prism. Graphs represent mean +/− SEM. with a Dunnett’s post test.

Extended Data Fig. 7 Characterization of MLN and splenic bacterial taxa at family level in preweaning mice with and without L. animalisWU feeding.

Cellular populations from the a) MLN and b) spleen were isolated and treated with propidium monoazide and photoactivation, DNA was isolated and bacterial taxa were characterized by 16 s rRNA v4 sequencing. The number of different taxa identified were not dramatically altered by L. animalisWU feeding.

Extended Data Fig. 8 E. coli ST69 and L. animalisWU are sensitive to vancomycin, neomycin, ampicillin and metronidazole.

Colony forming units (CFU) of (A-D) E. coli ST69 and (E-H) L. animalisWU after plating bacteria treated with (A, E) Vancomycin (b, f) Neomycin (c, g) Ampicillin and (d, h) Metronidazole at the specified concentrations for 4 hours. Statistical analyses were performed by one way ANOVA with a Dunnett’s post test. Data represented as mean +/− SEM P values are as denoted.

Supplementary information

Supplementary Information

Supplementary Tables 1–4 and 8 and Supplementary Methods.

Reporting Summary

Supplementary Video 1

Video of the three-dimensional reconstruction of DOL17 colonic GC (UEA1+, green) containing bacteria (eubacteria FISH probe, red; nuclei, blue).

Supplementary Tables

Supplementary Tables 5–7.

Supplementary File 1

Sequence of regions in L. animalisWU.

Supplementary File 2

Sequence of regions in L. animalisWU.

Supplementary File 3

Sequence of regions in L. animalisWU.

Supplementary File 4

Sequence of regions in L. animalisWU.

Supplementary File 5

Sequence of regions in L. animalisWU.

Supplementary File 6

Sequence of regions in L. animalisWU.

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Udayan, S., Floyd, A.N., John, V. et al. Colonic goblet cell-associated antigen passages mediate physiologic and beneficial translocation of live gut bacteria in preweaning mice. Nat Microbiol 10, 927–938 (2025). https://doi.org/10.1038/s41564-025-01965-1

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