Microbiota-assisted iron uptake promotes immune tolerance in the intestine

**Background:** Iron deficiency is common in inflammatory bowel disease (IBD) patients and may precede disease onset, suggesting a causal role in immune tolerance. Regulatory T cells (Tregs) are essential for intestinal immune homeostasis, but how iron influences Treg function was unknown. This study investigates the role of iron uptake via Transferrin receptor 1 (TfR1) in Treg differentiation and the contribution of the gut microbiota to this process.

**Methods:** The authors generated Treg-specific TfR1 knockout mice (Tfrc cKO) by crossing Tfrc-floxed mice with Foxp3-YFP-IRES-Cre mice. They analyzed survival, systemic inflammation, Treg numbers, and effector function using flow cytometry, histology, and adoptive transfer models. Heterozygous female mice (Tfrc fl/fl Foxp3 YFP-Cre/+) and mixed bone marrow chimeras were used to study cell-intrinsic effects. Acute TfR1 deletion was achieved using Foxp3-eGFP-creERT2 Tfrc fl/fl mice (Tfrc iKO) treated with tamoxifen. Single-cell RNA-seq and bulk RNA-seq were performed on Tregs. In vitro iTreg differentiation assays with iron sulfate, iron chelator (DFO), and pentanoate were conducted. The role of HIF-2α was tested using an inhibitor (PT2385) and shRNA knockdown. Germ-free and antibiotic-treated mice were used to assess microbiota dependence. DSS-induced acute and chronic colitis models were used to test pentanoate's therapeutic effects. Human stool metabolomics data from HPM2 and PRISM cohorts were re-analyzed.

**Key Results:** Tfrc cKO mice died within 4 weeks from lethal autoimmunity, with systemic inflammation and T cell activation. Intracellular iron in TfR1-deficient Tregs was reduced by ~50% (8.58 vs 4.10 ng/10^6 cells). Iron dextran treatment rescued survival up to 200 days. In heterozygous female mice, TfR1-deficient Tregs showed a specific loss of effector Tregs (eTregs) and c-Maf+ Tregs (from ~15% to <1%). scRNA-seq identified c-Maf as the most downregulated gene in TfR1-deficient Tregs. Iron treatment in vitro increased c-Maf+ iTregs in a dose-dependent manner, while DFO repressed it. Mechanistically, iron promoted HIF-2α translation via iron-responsive elements (IREs) in its 5'UTR, and HIF-2α directly regulated c-Maf expression. PT2385 completely abrogated iron-induced c-Maf. In Tfrc iKO mice, colonic Tregs were reduced by >50% (from 39.6% to 14.4%) within 3 weeks, leading to colitis. Intestinal Tregs had higher TfR1 and labile iron than splenic Tregs. Germ-free and antibiotic-treated mice had reduced labile iron in colonic Tregs, which was restored by iron dextran. Pentanoate, a microbiota-produced SCFA, promoted iron uptake, HIF-2α and c-Maf expression in colonic Tregs, and partially restored Treg ratios in antibiotic-treated mice. In DSS-induced colitis, pentanoate treatment significantly improved anemia (increased RBC, hemoglobin, hematocrit), serum iron levels, colon length, and histological inflammation. In human cohorts, stool pentanoate levels were reduced in Crohn's disease (CD) and ulcerative colitis (UC) patients compared to controls, and positively correlated with intracellular iron marker FTH1 expression.

**Clinical Implications:** This study reveals a critical link between iron metabolism, the gut microbiota, and intestinal Treg homeostasis. It identifies pentanoate as a potential therapeutic agent that could simultaneously address both iron deficiency and intestinal inflammation in IBD patients, offering an advantage over direct iron supplementation which can cause oxidative stress. The findings also highlight TfR1 and the iron-HIF-2α-c-Maf axis as potential targets for modulating immune tolerance.