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Innate and adaptive lymphocytes sequentially shape the gut microbiota and lipid metabolism


The mammalian gut is colonized by numerous microorganisms collectively termed the microbiota, which have a mutually beneficial relationship with their host1,2,3. Normally, the gut microbiota matures during ontogeny to a state of balanced commensalism marked by the absence of adverse inflammation4,5. Subsets of innate lymphoid cells (ILCs) and conventional T cells are considered to have redundant functions in containment and clearance of microbial pathogens6,7, but how these two major lymphoid-cell populations each contribute to shaping the mature commensal microbiome and help to maintain tissue homeostasis has not been determined. Here we identify, using advanced multiplex quantitative imaging methods, an extensive and persistent phosphorylated-STAT3 signature in group 3 ILCs and intestinal epithelial cells that is induced by interleukin (IL)-23 and IL-22 in mice that lack CD4+ T cells. By contrast, in immune-competent mice, phosphorylated-STAT3 activation is induced only transiently by microbial colonization at weaning. This early signature is extinguished as CD4+ T cell immunity develops in response to the expanding commensal burden. Physiologically, the persistent IL-22 production from group 3 ILCs that occurs in the absence of adaptive CD4+ T-cell activity results in impaired host lipid metabolism by decreasing lipid transporter expression in the small bowel. These findings provide new insights into how innate and adaptive lymphocytes operate sequentially and in distinct ways during normal development to establish steady-state commensalism and tissue metabolic homeostasis.

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Figure 1: pSTAT3+ ILC3s and IECs induced by microbiota in small intestine of Rag1−/− mice.
Figure 2: Transient activation of ILC3s and IECs shortly after weaning in wild-type mice.
Figure 3: Role of CD4+ T cells in controlling ILC3 and IEC activation.
Figure 4: Suppression of ILC3 activation by Treg and TH17 cells.
Figure 5: Disrupted lipid metabolism in ILC3 and IEC-activated mice.

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We thank Y. Choi and D.M. Kobuley for providing germ free Rag1−/− mice and performing SFB mono-colonization; M. Oukka and S. K. Durum for providing mice; M. Mack, B. Gao and Y. Umesaki for providing anti-CCR2 antibody, IL-22 adenovirus and SFB faecal pellets; C. Eigsti, V. Nair and J. Davis for cell sorting, scanning electron microscopy and microbiota analysis; J. Zhu for discussions; and members of the Laboratory of Systems Biology for their comments during the course of these studies and input during preparation of this manuscript. Y.H. was supported by an NIAID K99 award (1K99AI123350-01A1). This research was supported by the Intramural Research Program of NIAID, NIH.

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Authors and Affiliations



K.M. designed and conducted most of the experiments and data analysis and prepared the manuscript; A.P.B., S.T. and Y.H. helped with cell isolation and transfer; L.Z. measured mouse body composition; N.B. performed the analysis of microbiota translocation; A.J.M. performed the RNA-seq and data analysis; M.Y.G. provided helpful suggestions regarding imaging and histo-cytometry; Y.B. provided helpful suggestions, discussed data interpretation and contributed to the manuscript; and R.N.G. designed experiments, interpreted data and helped to write the manuscript.

Corresponding authors

Correspondence to Kairui Mao or Ronald N. Germain.

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The authors declare no competing financial interests.

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Extended data figures and tables

Extended Data Figure 1 Quantification of pSTAT3+ ILC3s by histo-cytometry.

Gating strategy for analysis of pSTAT3+ ILC3s from small intestine of wild-type or Rag1−/− mice.

Extended Data Figure 2 Cellular and molecular mechanism of STAT3 activation in Rag1−/− small intestine.

a, Immunofluorescence staining of ileum from Rag1−/− (n = 4), Il23a−/−Rag1−/− (n = 5), Il22−/−Rag1−/− (n = 4) and Il6−/−Rag1−/− mice (n = 4). b, Percentage of pSTAT3+ ILC3s in a. cd, Immunofluorescence staining of ileum from Rorc(γt)GFP/+Rag1−/− and Rorc(γt)GFP/GFPRag1−/− mice (n = 4; c) and Il22-tdTomato Rag1−/− mice (n = 3; d). Results are representative of three independent experiments. Bars show mean; exact P values are given and calculated by one-way ANOVA.

Extended Data Figure 3 Mononuclear phagocyte subpopulation responsible for IL-23 production and pSTAT3 activation.

a, Flow cytometry of total live cells from the small intestine lamina propria, showing the gating strategy for sorting different myeloid-cell subsets: CD103+CD11b and CD11b+ conventional dendritic cells, CD64+CCR2 macrophages and CCR2+ monocytes and monocyte-derived dendritic cells. b, Expression of Il23a (n = 3) and Il12b (which encodes the p40 subunit of IL-12) in different cell populations sorted as in a (n = 2). c, Immunofluorescence staining of ileum from Rag1−/− mice and Rag1−/− mice treated with anti-CCR2 or anti-Gr1 antibody for two weeks (n = 4). Results are representative of three independent experiments. Mean ± s.d.; exact P values are given and calculated by two-way ANOVA.

Extended Data Figure 4 Microorganisms in the small intestine of co-housed wild-type, Rag1−/− and Il23a−/−Rag1−/− mice.

a, Quantification of indicated bacteria species in the ileum of co-housed wild-type (n = 6), Rag1−/− (n = 7) and Il23a−/−Rag1−/− (n = 8) mice by real-time PCR with primers specific to 16S rRNA genes. Results are pooled from two independent experiments. b, Scanning electron microscopy of terminal ileum of co-housed mice as in a (n = 3). c, Quantification of the length of SFB filaments in b. Results are representative of two independent experiments. Bars show mean (a) and mean ± s.d. (c); exact P values are given and calculated by one-way ANOVA.

Extended Data Figure 5 Lack of ILC3 activation in SFB negative Tcra−/− mice.

a, Immunofluorescence staining of ileum from wild-type (n = 6), Rag1−/− (n = 5), Tcra−/− (n = 5) and Ighm−/− mice (n = 6). b, Percentage of pSTAT3+ ILC3s in a. c, Quantification of SFB in distal small intestine of non-co-housed Rag1−/− and Tcra−/− or co-housed Rag1−/− and Tcra−/− mice (n = 4). Results are representative of three (a, b) or two (c) independent experiments. Bars show mean; ****P < 0.0001; otherwise exact P values are shown and calculated by one-way ANOVA.

Extended Data Figure 6 Dysregulation of lipid metabolism by IL-22.

a, Expression of indicated genes in the ileum from age-matched male Tcra−/− mice and Tcra−/− mice co-housed with Rag1−/− mice (n = 8). Results are pooled from two independent experiments. b, Serum triglyceride and free fatty acid levels from Tcra−/− mice and Tcra−/− mice co-housed with Rag1−/− mice (n = 5). cf, Expression of indicated genes in the ileum (c, e) and serum triglyceride and free fatty acid levels (d, f) from wild-type (c, d) or Tcra−/− mice (e, f) injected with adenovirus expressing IL-22 (n = 5) or GFP (n = 5). Results are representative of two independent experiments. Bars show mean (a, c, e) and mean ± s.d. (b, d, f); exact P values are given and calculated by two-tailed Student’s t-test.

Extended Data Figure 7 STAT3 activation in IECs by IL-22 adenovirus.

Immunofluorescence staining of ileum from C57BL/6 mice injected with adenoviruses expressing either IL-22 or GFP for two weeks. Images are representative of three sections from three mice of each group and results are representative of two independent experiments.

Extended Data Table 1 Primers and probes for quantitative PCR

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Mao, K., Baptista, A., Tamoutounour, S. et al. Innate and adaptive lymphocytes sequentially shape the gut microbiota and lipid metabolism. Nature 554, 255–259 (2018).

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