The intestinal microbiota regulates body composition through NFIL 3 and the circadian clock

Abstract

The intestinal microbiota has been identified as an environmental factor that markedly affects energy storage and body-fat accumulation in mammals, yet the underlying mechanisms remain unclear. Here we show that the microbiota regulates body composition through the circadian transcription factor NFIL3. Nfil3 transcription oscillates diurnally in intestinal epithelial cells, and the amplitude of the circadian oscillation is controlled by the microbiota through group 3 innate lymphoid cells, STAT3 (signal transducer and activator of transcription 3), and the epithelial cell circadian clock. NFIL3 controls expression of a circadian lipid metabolic program and regulates lipid absorption and export in intestinal epithelial cells. These findings provide mechanistic insight into how the intestinal microbiota regulates body composition and establish NFIL3 as an essential molecular link among the microbiota, the circadian clock, and host metabolism.

Figure 1. Nfil3^ΔIEC mice are resistant to high fat diet (HFD)-induced obesity

(A) qRT-PCR analysis of Nfil3 transcript abundance in small intestinal epithelial cells recovered by laser capture microdissection from conventional (conv) and germ-free mice. (B) Age-matched Nfil3^fl/fl and Nfil3^ΔIEC mice were co-housed and placed on a high fat diet (HFD) for 10 weeks. Body weight was measured before and after diet switching. (C) Body fat percentages of mice in (B). (D) Epididymal fat pad weight, (E) serum triglyceride concentration, (F) hematoxylin & eosin (H&E) staining of liver (scale bar=100 μm), and (G) glucose tolerance and insulin tolerance tests. (H) Body fat percentage of mice treated with or without antibiotics after switching to HFD. All data represent two independent experiments with 4–8 mice per group. Male mice were used in all experiments. Means±SEM are plotted; statistics were performed with Student’s t-test or one-way ANOVA. *p<0.05; **p<0.01; ***p<0.001; ns, not significant.

Figure 2. The microbiota induces epithelial NFIL3 expression through the circadian clock factor REV-ERBα and a DC-ILC3 signaling relay

(A–D) qRT-PCR analysis of Nfil3 (A) and Rev-erbα (C) transcript abundance in small intestinal epithelial cells from germ-free (dotted line) and conventional mice (solid line) across a 24-hour day-night light cycle. Western blot analysis of NFIL3 (B) and REV-ERBα (D) was performed on small intestinal epithelial cells isolated from conventional or germ-free mice, Lamin B is the loading control. (E) Chromatin immunoprecipitation assay on intestinal epithelial cells using immunoglobulin G (IgG) or anti-REV-ERBα antibody. Precipitated fragments of the Nfil3 promoter were detected by qRT-PCR. (F) qRT-PCR analysis of epithelial Nfil3 expression in conventional wild-type, antibiotic (Abx)-treated wild-type or Abx-treated Rev-erbα^−/− mice. (G) qRT-PCR analysis of epithelial Rev-erbα and Nfil3 expression in germ-free and conventional wild-type mice and conventional Myd88^fl/fl, Myd88^−/−, Myd88^ΔIEC (epithelial cell-specific knockout) and Myd88^ΔDC (DC-specific knockout) mice. (H) qRT-PCR analysis of epithelial Rev-erbα and Nfil3 expression in germ-free and conventional wild-type mice, conventional Cd11c-DTR mice that were untreated or treated with Diphtheria toxin (DT), Id2^gfp/gfp and Rag1^−/− mice. (I,J) qRT-PCR analysis of epithelial Nfil3 (I) and Rev-erbα (J) expression in Rorc^+/+ (solid line) and Rorc^gfp/gfp (dotted line) mice. (K) qRT-PCR analysis of epithelial Rev-erbα and Nfil3 expression in Myd88^−/− mice treated with recombinant IL-23, IL-22 or vehicle. Data in E,F,G,H, and K were collected at ZT4. N=3–8 mice per group. Means±SEM are plotted; statistics were performed with Student’s t-test or one-way ANOVA. *p<0.05; **p<0.01; ***p<0.001; ns, not significant; conv, conventional; ZT, Zeitgeber time.

Figure 3. STAT3 represses Rev-erbα transcription by binding directly to its promoter

(A) Schematic of the Rev-erbα gene promoter. (B) ChIP analysis of intestinal epithelial cells from conventional (conv) or germ-free mice using immunoglobulin G (IgG) or anti-STAT3 antibody. Precipitated fragments of the Rev-erbα promoter or control exon were detected by qRT-PCR. (C) Luciferase reporter assay. A 504 bp fragment of Rev-erbα promoter was fused to a firefly luciferase reporter. HEK-293T cells were transfected with reporters and either empty vector, a wild-type STAT3-encoding vector (Stat3-wt), or a dominant active STAT3-encoding vector (Stat3-c). (D) Western-blot of total STAT3, phosphorylated STAT3 (p-STAT3) in small intestinal organoids treated with IL-22 and/or the STAT3 inhibitor Stattic. Mito 70 is the loading control. (E,F) qRT-PCR analysis of Rev-erbα (E) and Nfil3 (F) expression in small intestinal organoids treated with IL-22 and/or Stattic. (G,H) qRT-PCR analysis of epithelial Rev-erbα (G) and Nfil3 (H) expression in Stat3^fl/fl and Stat3^ΔIEC mice at ZT4. N=3–8 samples per group. Means±SEM are plotted; statistics were performed with Student’s t-test or one-way ANOVA. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; ns, not significant.

Figure 4. Epithelial NFIL3 controls expression of a circadian lipid metabolic program and regulates lipid absorption in intestinal epithelial cells

(A) RNAseq analysis of epithelial cell transcripts in Nfil3^fl/fl and Nfil3^ΔIEC mice across a circadian cycle. The heatmap visualizes expression levels of the 33 genes that have altered expression in Nfil3^ΔIEC mice as compared to Nfil3^fl/fl mice. Genes encoding proteins that function in lipid metabolism are highlighted in blue. Top panels show sustained circadian expression of the core clock genes Bmal1 (Arnt1), Per2, and Nr1d1 (Rev-erbα) in Nfil3^ΔIEC mice. (B) qRT-PCR analysis of epithelial Cd36 and Scd1 expression in germ-free wild-type (wt) and conventional Nfil3^fl/fl and Nfil3^ΔIEC mice at ZT4. (C) Western blot of epithelial CD36 in germ-free (gf) and conventional wild-type (conv) mice, and in conventional Nfil3^fl/fl and Nfil3^ΔIEC mice. All mice were fed a HFD. Mice were sacrificed at ZT4. α-tubulin is the loading control. (D,E) qRT-PCR analysis of epithelial Cd36 and Scd1 expression in conventional wild-type (wt) and ID2-deficient (Id2^gfp/gfp) mice (D) and Stat3^fl/fl and Stat3^ΔIEC mice (E) at ZT4. (F) Oil red O detection of lipids in the small intestines of Nfil3^fl/fl and Nfil3^ΔIEC mice fed on HFD. Nuclei were stained with Methyl Green. Scale bar=40 μm. (G) Total lipid concentrations in isolated small intestinal epithelial cells from Nfil3^fl/fl and Nfil3^ΔIEC mice fed on HFD. (H) Total neutral lipid concentrations in feces of Nfil3^fl/fl and Nfil3^ΔIEC mice fed on HFD. Data in B,D,E,G,H have N=5-12 mice per group. Means±SEM are plotted; statistics were performed with Student’s t-test or one-way ANOVA. *p<0.05; **p<0.01; ns, not significant; ZT, Zeitgeber time