Genome wide binding of Ahr in ILC3 [ChIP-seq]

Iron metabolism is pivotal for cell fitness in the mammalian host. However, its role in group 3 innate lymphoid cells (ILC3s) is unknown. Here we show that transferrin receptor CD71-mediated iron metabolism cell-intrinsically controls ILC3 maintenance, cytokine production and host protection against Citrobacter rodentium infection, and metabolically affects mitochondrial respiration by switching of oxidative phosphorylation toward glycolysis. Iron deprivation or Tfrc deficiency reduces the expression and/or activity of the aryl hydrocarbon receptor (Ahr), a ligand-dependent transcription factor and a key ILC3 regulator. Furthermore, consistent with its role in generation of Ahr ligand, microbiome exerts a negative impact on CD71 expression in an Ahr-dependent manner. Genetic ablation or activation of Ahr in ILC3s leads to CD71 upregulation or downregulation, respectively, suggesting an active suppression of CD71 by Ahr. Iron overload partially restores defective ILC3 compartment in the small intestine of Ahr-deficient mice, representing compensatory action of CD71 upregulation during Ahr deficiency. Mechanistically, Ahr directly binds to the promoter region of the Tfrc locus to inhibit Tfrc transcription. These data collectively demonstrate an under-appreciated role of the Ahr-CD71-iron axis in regulation of ILC3 maintenance and function. ILC3s (Lin–CD90hiCD45lo) were FACS-sorted from the gut LPLs (combined LI and SI) of Rag1−/− mice. For ChIP of Ahr, cells were treated with FICZ (200 nM) for 4 hours before harvest. Cells were cross-linked with 1% formaldehyde for 15 min. Chromatin was sheared by sonication with Bioruptor Pico (30 s on and 30 s off for 25 cycles) and immunoprecipitated with anti-Ahr (Enzo Life Science, BML-SA210-0100) using iDeal ChIP-Seq Kit for transcription factors (Diagenode). Libraries were generated using the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB). Samples were sequenced as 50 bp single-end reads on the Illumina HiSeq 2500 instrument.