Abstract
Host immunity and commensal bacteria synergistically maintain intestinal homeostasis and mediate colonization resistance against pathogens. However, the molecular and cellular mechanisms remain unclear. Here, with a mouse infection model of Citrobacter rodentium, a natural mouse intestinal pathogen that mimics human enteropathogenic Escherichia coli and enterohaemorrhagic Escherichia coli, we find that group 3 innate lymphoid cells (ILC3s) can protect the host from infection by regulating gut microbiota. Mechanistically, ILC3s can control gut dysbiosis through IL-22-dependent regulation of intestinal galactosylation in mice. ILC3 deficiency led to an increase in intestinal galactosylation and the expansion of commensal Akkermansia muciniphila in colonic mucus. The increased A. muciniphila and A. muciniphila-derived metabolic product succinate further promoted the expression of pathogen virulence factors tir and ler, resulting in increased susceptibility to C. rodentium infection. Together, our data reveal a mechanism for ILC3s in protecting against pathogen infection through the regulation of intestinal glycosylation and gut microbiota metabolism.
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Data availability
The data that support the findings of this study are available in the Article. The 16S rRNA-seq data are available in SRA under BioProject accession number PRJNA859678. Source data are provided with this paper.
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Acknowledgements
We thank D. R. Littman and I. Ivanov (New York University, NY) for Rorc-cre mice, A. Lasorella and A. Iavarone (Columbia University, NY) for Id2-floxed mice, E. Hartland (The University of Melbourne, Melbourne) for GFP-expressing C. rodentium, B. Liu (Naikai University, Tianjin) for EHEC O157:H7, F. Shao (National Institute of Biological Sciences, Beijing) for EPEC E2348169 and J. Zhang (Tsinghua University, Beijing) for plasmid pKD4. We also thank the Core Facility of the Institute for Immunology, the Animal Facility and Facility Center of Metabolomics and Lipidomics, National Center for Protein Sciences, Tsinghua University, for the support provided. We appreciate the support from the Beijing Natural Science Foundation (Z210015 to X.G.), National Key R&D Program of China (2023YFC2306202 and 2017YFA0103602 to X.G.) and National Natural Science Foundation of China (82141201, 82122030, 32170872, 82150104 and 31821003 to X.G.; 32370967 to W.W.). The Guo laboratory was also supported by the SXMU-Tsinghua Collaborative Innovation Center for Frontier Medicine and the Institute for Immunology, Tsinghua University.
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X.G. and W.W. conceived and designed the study and prepared the paper. W.W. and N.L. performed all the experiments and assisted in data analysis. H.X., S.W., Y.L., J.O., J.H., J.Z. and Y.Q. participated in some experiments. L.D. performed the experiments of GF mice. X.H. and Y.-X.F. provided critical materials and helpful suggestions.
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Extended data
Extended Data Fig. 1 The increased A. muciniphila in ILC3-deficient mice enhances the host’s susceptibility to C. rodentium infection.
(a) Bacteria within the colonic mucosa of Id2fl/fl and RorcCreId2fl/fl mice were determined by qPCR with specific primers. (b) Heatmap of differentially abundant bacteria at family level identified by 16S rRNA gene amplicon sequencing of feces from Id2fl/fl, RorcCreId2fl/fl and CD4CreId2fl/fl mice (n = 4). (c) Absolute abundances of A. muciniphila in feces from Id2fl/fl, RorcCreId2fl/fl and CD4CreId2fl/fl mice. (d) SPF WT mice were orally treated with A. muciniphila (1*108 CFU) daily for 7 days and then were infected with C. rodentium (5*106 CFU). The colon was harvested at day 7 post infection and the H&E staining and the pathological scores were shown (n = 5). Scale bars, 100 μm and 40 μm. (e, f) WT mice were orally administrated with A. muciniphila (1*108 CFU) daily for 3 days post 7 days antibiotics administration and then were infected with C. rodentium (1*105 CFU) (n = 5). (e) Fecal C. rodentium titers and (f) the colon histology changes at day 7 post infection by H&E staining and the pathological scores were shown. Scale bars, 100 μm and 40 μm. (g) WT mice were orally administrated with A. muciniphila (1*108 CFU) or Odoribacter splanchnicus (1*108 CFU) daily for 7 days and then were infected with C. rodentium (5*106 CFU). Fecal C. rodentium titers at day 1 and blood C. rodentium titers at day 5 post infection were shown (n = 5). (h) Heatmap of differentially abundant bacteria identified by 16S rRNA gene amplicon sequencing of feces from OMM11 and OMM11ΔA.m. mice (n = 6). Each dot (a, c–g) represents one individual mouse. Data (e, g) are representative of two independent experiments. Data (a, c, d, f) are pooled from two independent experiments. Statistical significance was tested by unpaired two-sided Student’s t-test (a, c, d–f) and two-sided one-way ANOVA with Tukey’s test adjusted for multiple comparisons (g). Error bars represent the mean ± SEM.
Extended Data Fig. 2 The influences of A. muciniphila-metabolites on the growth of C. rodentium.
(a) Growth curves of C. rodentium (1*103 CFU/ml) co-cultured with A. muciniphila (5*103 CFU) in BHI medium (n = 4). (b) Metabolomics analysis of the short chain fatty acids in the A. muciniphila culture medium and BHI medium. The significant different compounds (p < 0.01) were shown (red triangle, up-regulated compound). (c) C. rodentium (1000/ml) was cultured with isovalerate, propionate, methylmalonic acid, acetate (n = 2) and succinate (n = 3) at different concentrations and the growth were examined at 4 h and 8 h. Data are representative of at least two independent experiments. Statistical significance was tested by ordinary two-way ANOVA with Turkey’s multiple comparisons test (a, c) or by unpaired two-sided Student’s t-test. Error bars represent the mean ± SEM.
Extended Data Fig. 3 The effect of A. muciniphila-derived metabolites on C. rodentium and human pathogens.
(a) C. rodentium (1*107 CFU) was cultured with methylmalonic acid, propionate or acetate for 6 h and then the virulence factors were analyzed by qPCR (n = 3). Mice were pretreated with methylmalonic acid (b), propionate (c) or acetate (d) containing drinking water for 1 day, and then were infected with C. rodentium (5*106 CFU). Fecal C. rodentium burden at day 1 post infection were examined. EPEC (1*107 CFU, n = 5) (e) or EHEC (1*107 CFU, n = 3) (f) was grown with or without 40 mM succinate for 6 h in vitro and then the relative expression of virulence factors tir and ler were detected by qPCR. (g, h) Mice were treated with succinate-containing drinking water one day before infection, and then were infected with EHEC (2*108 CFU) (n = 5). Fecal EHEC titers at day 1 (g) and blood pathogen burden at day 3 (h) post-infection were shown. Each column contains three (a, f) or five (e) biological replicates. Each dot (b–d, g, h) represents one individual mouse. Data are representative of two (e–h) or three (a–d) independent experiments. Statistical significance was tested by unpaired two-sided Student’s t-test (b–h) or by two-sided one-way ANOVA with Tukey’s test adjusted for multiple comparisons (a). Error bars represent the mean ± SEM.
Extended Data Fig. 4 Construction of DcuB deficient C. rodentium.
(a) Workflow for construction of the DcuB-deficient strain of C. rodentium using lambda-derived Red recombination system. (b) PCR was performed to pick up the successful construction of mutant C. rodentium strain. (c) The relative expression of DcuB was identified by qPCR. Each column contains three biological replicates (c). Data are representative of three (b) or two (c) independent experiments. Statistical significance was tested by unpaired two-sided Student’s t-test (c). Error bars represent the mean ± SEM. Panel a was created with BioRender.com.
Extended Data Fig. 5 The pathological manifestation of OMM11 mice in the colon.
The colon was harvested at day 7 post infection and the histological manifestation was shown by H&E staining. The pathological scores were evaluated blind (n = 6). The scale bars shown in 100 μm and 40 μm. Each dot represents one individual mouse. Data are pooled from two independent experiments. Statistical significance was tested by two-sided one-way ANOVA with Tukey’s test adjusted for multiple comparisons. Error bars represent the mean ± SEM.
Extended Data Fig. 6 Galactose supports A. muciniphila growth and succinate production.
(a, b) A. muciniphila (1*105 CFU) were incubated in LB medium with or without galactose (25 mM) for 48 h and the growth curve of A. muciniphila was examined by OD600 measurement (a) and qPCR (b). Each time point contains at least five replicates. (c, d) The transcriptional expression of galactosidases and galactokinase in A. muciniphila were detected by regular RT-PCR (c) and qPCR (d). Amuc_1100 was detected as positive control. Each column contains six replicates. (e) A. muciniphila were cultured in BHI medium with galactose (25 mM) or 0.05% mucin, and the production of succinate in the supernatants was quantified by LC-MS (n = 3). Each dot represents one replicate in one column. Data are representative of two independent experiments (a–e). Statistical significance was tested by ordinary two-way ANOVA with Dunnett’s multiple comparisons test (a, b) and two-sided one-way ANOVA with Tukey’s test adjusted for multiple comparisons (e). Error bars represent the mean ± SEM.
Extended Data Fig. 7 The co-culture of sorted ILC3s and organoids.
(a) The gating strategy for sorting ILC3s from the small intestine by flow cytometry. The sorted ILC3s were further stained with anti-RORγt and anti-IL-22 antibodies. (b) Representative immunofluorescence staining for galactosylation (PNA staining, green) and DAPI (nuclear staining, blue) in organoids. The scale bars shown in 50 μm. (c) IL-22 in the supernatant of organoids with ILC3s co-cultured was detected by ELISA (n = 7). Each dot represents one biological replicate in one column (c). Data are representative of two independent experiments (b, c). Statistical significance was tested by unpaired two-sided Student’s t-test. Error bars represent the mean ± SEM.
Extended Data Fig. 8 ILC3 regulates galactosylation by C1galt1.
(a) C1galt1 expression in the small intestine of the Id2fl/fl (n = 8) and RorcCreId2fl/fl (n = 9) mice was detected by qPCR. (b) Representative flow cytometry analysis for MC38 cell line treated with different concentrations of itraconazole (ITZ) that inhibits the function of C1GALT1 protein for 48 h (n = 3). (c) C1galt1 expression in intestinal organoids treated with or without IL-22 (5 ng/ml) at day 4 was detected by qPCR (n = 8). Each dot represents one individual mouse (a, c) or one replicate in one column (b). Data are pooled from two independent experiments (a, c). Data are representative of two independent experiments (b). Statistical significance was tested by unpaired two-sided Student’s t-test (a, c) and two-sided one-way ANOVA with Tukey’s test adjusted for multiple comparisons (b). Error bars represent the mean ± SEM.
Supplementary information
Supplementary Table 1
The primers for the quantitative real-time PCR.
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Wang, W., Li, N., Xu, H. et al. ILC3s regulate the gut microbiota via host intestinal galactosylation to limit pathogen infection in mice. Nat Microbiol 10, 654–666 (2025). https://doi.org/10.1038/s41564-025-01933-9
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DOI: https://doi.org/10.1038/s41564-025-01933-9
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