Light-entrained and brain-tuned circadian circuits regulate ILC3s and gut homeostasis

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Abstract

Group 3 innate lymphoid cells (ILC3s) are major regulators of inflammation, infection, microbiota composition and metabolism1. ILC3s and neuronal cells have been shown to interact at discrete mucosal locations to steer mucosal defence2,3. Nevertheless, it is unclear whether neuroimmune circuits operate at an organismal level, integrating extrinsic environmental signals to orchestrate ILC3 responses. Here we show that light-entrained and brain-tuned circadian circuits regulate enteric ILC3s, intestinal homeostasis, gut defence and host lipid metabolism in mice. We found that enteric ILC3s display circadian expression of clock genes and ILC3-related transcription factors. ILC3-autonomous ablation of the circadian regulator Arntl led to disrupted gut ILC3 homeostasis, impaired epithelial reactivity, a deregulated microbiome, increased susceptibility to bowel infection and disrupted lipid metabolism. Loss of ILC3-intrinsic Arntl shaped the gut ‘postcode receptors’ of ILC3s. Strikingly, light–dark cycles, feeding rhythms and microbial cues differentially regulated ILC3 clocks, with light signals being the major entraining cues of ILC3s. Accordingly, surgically or genetically induced deregulation of brain rhythmicity led to disrupted circadian ILC3 oscillations, a deregulated microbiome and altered lipid metabolism. Our work reveals a circadian circuitry that translates environmental light cues into enteric ILC3s, shaping intestinal health, metabolism and organismal homeostasis.

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Fig. 1: Intestinal ILC3s are controlled in a circadian manner.
Fig. 2: ILC3-intrinsic Arntl regulates gut homeostasis and defence.
Fig. 3: ILC3-intrinsic circadian signals regulate an enteric receptor postcode.
Fig. 4: Light-entrained and brain-tuned cues shape intestinal ILC3s.

Data availability

Source data for quantifications shown in all graphs plotted in the Figures and Extended Data Figures are available in the online version of the paper. The datasets generated in this study are also available from the corresponding author upon reasonable request. RNA-seq datasets analysed are publicly available in the Gene Expression Omnibus repository with accession number GSE135235.

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Acknowledgements

We thank the Vivarium, Flow Cytometry, Histology, Molecular Biology and Hardware platforms at the Champalimaud Centre for the Unknown; the Congento infrastructure for genetic model organisms; R. Pirzgalka, R. Klein Wolterink, S. Correia, I. Godinho, F. Cardoso, B. Garcia Cassani and K. Fischer for technical help and discussions; C. French for helping in behaviour analysis; N. Shemesh, T. Serradas Duarte and D. Nunes for MRI imaging; A. Silva for technical help with hardware; F. Rijo-Ferreira and L. Petreanu for discussions; and P. Faísca for pathology scoring. C.G.-S., R.G.D. and M.R. were supported by Fundação para a Ciência e Tecnologia (FCT), Portugal. N.L.B.-M. is supported by FCT, Portugal, and the European Molecular Biology Organisation (EMBO). H.V.-F. is supported by the ERC (647274), the EU, The Paul G. Allen Frontiers Group, US, and the FCT, Portugal.

Author information

C.G.-S. and R.G.D. designed, performed and analysed the experiments shown in Figs. 14 and Extended Data Figs. 110. M.R. performed circadian analysis. M.R. and H.R. provided technical assistance for the experiments shown in Fig. 2d–j and Extended Data Fig. 5f–h. M.R. provided technical assistance for the experiments shown in Fig. 3b and Extended Data Fig. 6b, e. B.R. provided technical assistance for the experiments shown in Figs. 1a–d, 3a, h, i, 4j–k and Extended Data Figs. 1, 3f, j, 6a, k, l, 8d, 9e–h. H.R. managed the animal colony. J.A.d.S. and R.M.C. helped to design the experiments shown in Fig. 4e and Extended Data Fig. 8b. A.V. provided technical assistance with flow cytometry. N.L.B.-M. analysed the experiments shown in Fig. 3b and Extended Data Fig. 6b. T.C. analysed the experiments in Fig. 2e, f and Extended Data Figs. 5a–c, 8d. H.V.-F. supervised the work, planned the experiments and wrote the manuscript.

Correspondence to Henrique Veiga-Fernandes.

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Competing interests

The authors declare no competing interests.

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Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Peer review information Nature thanks Richard Locksley, Christoph Scheiermann and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Fig. 1 Clock genes in progenitors and gut ILCs.

a, Circadian clock gene expression in bone marrow ILC2Ps and small intestinal ILC1s and ILC2s. ILC2P n = 4; ILC1 n = 3; ILC2 n = 6. b, PER1–VENUS MFI analysis in bone marrow ILC2Ps and small intestine lamina propria ILC1s and ILC2s. ILC2P n = 6; ILC1 and ILC2 n = 4. c, Circadian clock gene expression in bone marrow CLPs, ILCPs and ILC2Ps; n = 3. d, Circadian clock gene expression in small intestine lamina propria ILC1s and ILC2s. ILC1 n = 3; ILC2 n = 6. bd, White, light period; grey, dark period. Data are representative of three independent experiments. a, c, d, n represents biologically independent samples; b, n represents biologically independent animals. Data shown as mean ± s.e.m. a, Two-way ANOVA followed by Tukey’s multiple comparison test. P values relative to differences in Per1 expression in ILC1s and ILC2s when compared with ILC2Ps. b–d, Cosinor regression was used to define circadian rhythmicity. *P < 0.05; **P < 0.01; ***P < 0.001; NS, not significant. Source Data

Extended Data Fig. 2 Circadian signals regulate gut ILC3.

a, Percentage of lamina propria ILC3s; CD4NCR, LTi CD4+ and NCR+ subsets; n = 6. b, Cell numbers of lamina propria group 3 ILCs; CD4NCR, LTi CD4+ and NCR+ ILC3 subsets; n = 6. c, Percentage of donor cells in ILC2s, CD4+ T cells and Th17 subsets of mixed bone marrow chimaeras. Arntlfl n = 6, ArntlΔVav1 n = 7. Data are representative of three independent experiments. ac, n represents biologically independent animals. Data shown as mean ± s.e.m. ac, Two-tailed Mann–Whitney U test. **P < 0.01; NS, not significant. Source Data

Extended Data Fig. 3 Cell-intrinsic Arntl signals control intestinal ILC3s.

a, Representative histogram of RFP expression in small intestine lamina propria ILC3s. Representative of three independent analyses. b, Number of Peyer patches. Arntlfl and ArntlΔVav1 n = 9; Arntlfl and ArntlΔRorgt n = 7. c, Percentage of small intestine lamina propria ILC3s; n = 5. d, Percentage of CCR6NCR, CCR6+ (LTi-like) and NCR+ ILC3 subsets; n = 5. e, Percentage of intestinal lamina propria CD4+ T cells and T helper (Th)17 cells; n = 5. f, Percentage and cell numbers of ILC3s, regulatory T cells (Tregs) and RORγt-expressing Tregs in the colonic lamina propria; n = 6. g, Percentage of lamina propria ILC3s, CCR6NCR, CCR6+ (LTi-like) and NCR+ ILC3 subsets; n = 3. h, Percentage of donor cells and cell numbers of intestinal ILC3s in mixed bone marrow chimaeras; n = 4. i, Percentage of donor cells in CCR6NCR, CCR6+ (LTi-like), and NCR+ ILC3 subsets of mixed bone marrow chimaeras; n = 4. j, Faecal Bacteroidetes and Firmicutes. Arntlfl n = 5; ArntlΔRorgt n = 6. j, White, light period; grey, dark period. Data are representative of at least three independent experiments. aj, n represents biologically independent animals. Data shown as mean ± s.e.m. bf, hi, Two-tailed Mann–Whitney U test; j, cosinor regression was used to define circadian rhythmicity; g, two-tailed unpaired Student’s t-test. *P < 0.05; **P < 0.01; ***P < 0.001; NS, not significant. Source Data

Extended Data Fig. 4 Effect of Nr1d1 in intestinal ILC3s.

a, Percentage and cell numbers of small intestine lamina propria ILC3s. N1d1+/+ n = 6; N1d1−/− n = 5. b, Percentage of CCR6NCR, CCR6+ (LTi-like) and NCR+ ILC3 subsets. N1d1+/+ n = 6; N1d1−/− n = 5. c, Schematic representation of mixed bone marrow chimaeras. d, Percentage and cell numbers of donor cells in mixed bone marrow chimaeras; n = 5. e, Percentage of CCR6NCR, CCR6+ (LTi-like), and NCR+ ILC3 subsets in the small intestine lamina propria of mixed bone marrow chimaeras; n = 5. Data are representative of at least three independent experiments. n represents biologically independent animals. Data shown as mean ± s.e.m. a, b, d, e, Two-tailed Mann–Whitney U test. **P < 0.01; NS, not significant. Source Data

Extended Data Fig. 5 ILC3-autonomous ablation of Arntl impairs intestinal defence.

a, b, Histopathology of colon and caecum in Rag1−/−ArntlΔRorgt and Rag1−/−Arntlfl littermate controls infected with C. rodentium. Pathological changes in colon and caecum of Rag1−/−ArntlΔRorgt mice included ulceration, loss of crypts and goblet cells, and inflammatory cell infiltration of the lamina propria by a granulocyte-rich population with a prominent and oedematous submucosa. Original magnification ×4 (colon: scale bars, 500 μm); ×1.25 (caecum: scale bars, 250 μm). c, Inflammation score in the caecum; n = 5. d, Total number of ILC3s and numbers of IL-17- and IL-22-producing ILC3s; n = 3. e, Whole-body imaging of Rag1−/−ArntlΔRorgt and their Rag1−/−Arntlfl littermate controls at day 7 after infection with luciferase-expressing C. rodentium. f, MacConkey plates of liver cell suspensions from Rag1−/−ArntlΔRorgt mice and their Rag1−/−Arntlfl littermate controls at day 13 after C. rodentium infection. g, h, Translocation of total bacteria (left) and C. rodentium (right) to the liver and mesenteric lymph nodes (mLN); n = 4. i, Epithelial reactivity gene expression in the colon of Rag1−/−ArntlΔRorgt mice compared with Rag1−/−Arntlfl littermate controls infected with C. rodentium; n = 3. j, Weight loss in C. rodentium-infected mice. Rag1−/−Arntlfl n = 6; Rag1−/−ArntlΔRorgt n = 7. km, Histopathology analysis of inflammatory infiltrates in the liver and gonadal and subcutaneous fat. Scale bars: 250 μm (liver); 100 μm (gonadal and subcutaneous fat); n = 4. n, Total body weight; n = 5. Data are representative of at least three independent experiments. n represents biologically independent animals. Data shown as mean ± s.e.m. c, g, h, Two-tailed Mann–Whitney U test; d, n, two-tailed unpaired Student’s t-test; j, two-way ANOVA and Sidak’s test. *P < 0.05; **P < 0.01; ***P < 0.001; NS, not significant. Source Data

Extended Data Fig. 6 ILC3 proliferation, apoptosis and gut homing markers.

a, ILC3-related gene rhythmicity in small intestinal lamina propria ILC3s; n = 4. b, RNA-seq analysis of lamina propria ILC3s at ZT23; n = 3. c, Percentage of Ki67 expression in small intestine lamina propria ILC3s; n = 4. d, Percentage of donor cells in mixed bone marrow chimaeras; n = 4. e, Number of LinCD127+RORγt+ cells in the blood; n = 4. f, Percentage of ILC3s in mLNs. Arntlfl n = 6; ArntlΔRorgt n = 8. g, Diurnal expression of Ccr9 transcripts in gut ILC3s; n = 4. h, i, Percentage of CCR9 expression in small intestinal lamina propria CCR6NCR, CCR6+ (LTi-like) and NCR+ ILC3 subsets, ILC2s and CD4+ T cells; n = 3. j, Percentage of α4β7 expression in small intestine ILC1s, ILC2s and CD4+ T cells; n = 4. k, Percentage of CCR9 expression in gut ILC1s, ILC2s and CD4 + T cells; n = 4. l, m, Diurnal analysis of α4β7 and CXCR4 expression in small intestine ILC3s; n = 4. a, e, g, l, m, White, light period; grey, dark period. Data shown as mean ± s.e.m. a, cm, n represents biologically independent animals. b, n represents biologically independent samples. a, g, Two-way ANOVA; c, d, hk, two-tailed Mann–Whitney U test; e, l, m, cosinor regression was used to define circadian rhythmicity; f, two-tailed unpaired Student’s t-test. *P < 0.05; **P < 0.01; ***P < 0.001; NS, not significant. Source Data

Extended Data Fig. 7 Light entrains intestinal ILC3 circadian oscillations.

a, Inverted feeding regimens in constant darkness. PER1–VENUS expression in gut ILC3s; n = 3. b, Circadian clock gene expression in hepatocytes of Per1Venus mice in inverted feeding regimens; n = 3. Acrophase mean ± s.e.m: Arntl: control 0.4 ± 0.5, inverted 11.5 ± 0.2; Per2: control 15.2 ± 0.6, inverted 3.9 ± 0.5; Nr1d1: control 7.1 ± 0.6; inverted 18.5 ± 0.8. c, Opposing light–dark cycles. PER1–VENUS in gut CD4NCR, LTi CD4+ and NCR+ ILC3 subsets; n = 3. Acrophase mean and s.e.m: CD4NCR: control 14.5 ± 0.5, inverted 2.5 ± 0.5; LTi CD4+: control 14.5 ± 0.6, inverted 2.5 ± 0.4; NCR+: control 14.5 ± 0.6, inverted 2.5 ± 0.4. d, PER1–VENUS MFI analysis of small intestine lamina propria ILC3s from mice maintained in constant darkness for 28 days; n = 3. b, White, light period; grey, dark period. Data are representative of three independent experiments. n represents biologically independent animals. Data shown as mean ± s.e.m. Cosinor regression. Standard errors with 95% confidence limits for amplitude (Amp) and acrophase (Acro) were extracted from the model and compared using two-tailed Student’s t-test. **P < 0.01; ***P < 0.001; NS, not significant. Source Data

Extended Data Fig. 8 SCN ablation shapes intestinal ILC3s.

a, Circadian clock gene expression in small intestinal lamina propria ILC3s. n = 2 or 3. b, Magnetic resonance imaging of sham and SCN-ablated Per1Venus mice. Sagittal slices. White arrows show SCN ablation. c, Rhythms of animal locomotor activity. Total distance travelled in metres. d, Nissl staining of coronal brain sections. Scale bars: 1 mm (top); 250 μm (bottom). n represents biologically independent animals. Data shown as mean ± s.e.m. Cosinor regression was used to define circadian rhythmicity; cosine fitted curves are shown; standard errors with 95% confidence limits for amplitude and acrophase were extracted from the model and compared using two-tailed Student’s t-test. *P < 0.05; NS, not significant. Source Data

Extended Data Fig. 9 Brain-tuned signals shape gut ILC3s.

a, b, Confocal images of coronal brain sections showing neurotrace and RFP expression in the SCN. Scale bar, 100 µm. Representative of three independent analyses. c, Representative histogram of RFP expression in small intestine lamina propria ILC3s. Representative of three independent analyses. d, Per1 expression in small intestinal lamina propria ILC3s. n = 3. e, Percentage of small intestine lamina propria ILC3s; n = 3. f, Number of small intestine lamina propria ILC3s; n = 3. g, h, Epithelial reactivity gene expression in the intestinal epithelium; n = 3. i, Rhythms of faecal Bacteroidetes and Firmicutes. Arntlfl n = 4, ArntlΔCamk2a n = 3. n represents biologically independent animals. Data shown as mean ± s.e.m. d, gi, Cosinor regression was used to define circadian rhythmicity; cosine fitted curves are shown; e, two-way ANOVA and Sidak’s test; f, two-tailed unpaired Student’s t-test. *P < 0.05; **P < 0.01; ***P < 0.001; NS, not significant. Source Data

Extended Data Fig. 10 Effect of Nr3c1 and Adrb2 deficiency in gut ILC3s.

a, PER1–VENUS MFI analysis of lamina propria ILC3s after dexamethasone administration; n = 3. b, Percentage and cell numbers of small intestine ILC3s; n = 3. c, Percentage of lamina propria CCR6NCR, CCR6+ (LTi-like), and NCR+ ILC3 subsets; n = 3. d, TH-expressing neurons (red) and RET-positive ILC3s (green) in cryptopatches. Scale bars, 40 μm. Representative of three independent analyses. e, Normalized expression of Adrb1, Adrb2 and Adrb3 in CCR6NCR, CCR6+ and NCR+ ILC3 subsets. f, Percentage and cell numbers of gut ILC3s in Adrb2ΔIl7ra mice and their littermate controls; n = 6. g, Percentage of lamina propria CCR6NCR, CCR6+ (LTi-like) and NCR+ ILC3 subsets in Adrb2ΔIl7ra mice and their littermate controls; n = 6. h, Percentage and cell numbers of gut ILC3s in Adrb2ΔRorgt mice and their littermate controls. Adrb2fl n = 3; Adrb2ΔRorgt n = 4. i, Percentage of lamina propria CCR6NCR, CCR6+ (LTi-like) and NCR+ ILC3 subsets in Adrb2ΔRorgt mice and their littermate controls. Adrb2fl n = 3; Adrb2ΔRorgt n = 4. j, Light cues and brain-tuned circuits shape gut ILC3 homeostasis. Arrhythmic ILC3s impact intestinal homeostasis, epithelial reactivity, microbiota, enteric defence and the host lipid metabolism. Thus, ILC3s integrate local and systemic entraining cues in a distinct hierarchical manner, establishing an organismal circuitry that is an essential link between diurnal light signals, brain cues, intestinal ILC3s and host homeostasis. ad, fi, n represents biologically independent animals. a, White, light period; grey, dark period. Data shown as mean ± s.e.m. a, Two-way ANOVA and Sidak’s test; b, c, fi, two-tailed Mann–Whitney U test. *P < 0.05; ***P < 0.001; NS, not significant. Source Data

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