Abstract
Increased reactive oxygen species (ROS) levels are a hallmark of inflammatory bowel disease (IBD) and constitute a major mechanism of epithelial cell death. Approaches to broadly inhibit ROS have had limited efficacy in treating IBD. Here we show that lipid peroxidation contributes to the pathophysiology of IBD by promoting ferroptosis, an iron-dependent form of programmed cell death. Mechanistically, we provide evidence of heterocellular crosstalk between intestinal fibroblasts and epithelial cells. In IBD tissues and mouse models of chronic colitis, acyl-CoA synthetase long-chain family 4 (ACSL4) is overexpressed in fibroblasts. ACSL4 in fibroblasts reprograms lipid metabolism and mediates intestinal epithelial cell sensitivity to ferroptosis. In mouse models, overexpressing ACSL4 in fibroblasts results in increased intestinal epithelial ferroptosis and worsened colitis, while pharmacological inhibition or deletion of fibroblast ACSL4 ameliorates colitis. Our work provides a targeted approach to therapeutic antioxidant treatments for IBD.
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Data availability
The publicly available bulk RNA-seq data of IBD and non-IBD tissues (GSE57945 (ref. 18), GSE109142 (ref. 20), GSE117993 (ref. 47) and GSE199906 (ref. 21)) and the publicly available scRNA-seq data of IBD and non-IBD tissues (GSE114374 (ref. 25), GSE214695 (ref. 26), E-MTAB-8901 (ref. 27) and SCP1884 (ref. 28)) were reanalyzed. We provided the lipidomics dataset as Supplementary Information and most of the raw data as Source data. Any additional data and materials not publicly available can be provided upon reasonable request by contacting the corresponding author. Source data are provided with this paper.
Code availability
We used the Seurat (v.3.2.2) R package for the published dataset collection and reanalysis. The custom computer code and algorithms used in this study are available on Zenodo (https://doi.org/10.5281/zenodo.15191281)52.
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Acknowledgements
Schematic diagrams (Figs. 1d, 5a and 7a,m and Extended Data Fig. 1) were created with BioRender.com. This work was funded by NIH grants R01CA148828, R01CA245546 and R01DK095201 (Y.M.S.) and UMCCC Core Grant P30CA046592 (Y.M.S.). W.H. was supported by the NIH grant F30DK131851. Y.Z. was supported by the Rackham International Students Fellowship/Chia-Lun Lo Fellowship. J.S. was supported in part by NIH grant R37CA262209. J.A.C. was supported by NIH grants R01ES028802, P30 ES017885 and NCI R21 CA273646. J.R.S. was supported by NIDDK R01 DK137806 and NIDDK RC2 DK140862. S.P.H. was supported by the Crohn’s Colitis Foundation Senior Research Award. A.D.P. was supported by NIH grant S10OD021750, NIH/National Institute of Environmental Sciences grant R35 ES035027 and the US Department of Agriculture National Institute of Food and Federal Appropriations under project PEN047702 and accession number 7006412. A.S.G. was supported by USDA grants USDA 58-8050-9-004 and USDA NIFA 2022-67018-37186 and by NIH grant P30DK046200. A.N. was supported by grant DK59888.
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W.H., Y.Z., N.K.D. and Y.M.S. conceived of and designed the study. W.H., Y.Z., N.K.D., S.S., J.S., S.P.H., M.K.D., J.R.S., A.D.P. and J.A.C. developed the methodologies. W.H., Y.Z., N.K.D., S.S., C.J., M.O.E.-D., H.N.B., N.A., R.S., C.C., K.B., Y.Y., J.D., I.K. and A.S.G. acquired the data. W.H., Y.Z., N.K.D., M.O.E.-D., J.D., A.S., P.D.R.H., S.P.H., J.A.C., T.G.O., A.D.P., A.S.G., J.K.G. and A.N. analyzed and interpreted the data. W.H., Y.Z. and Y.M.S. supervised the study and wrote the paper; all authors edited and provided inputs to the paper.
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Extended data
Extended Data Fig. 1 Schematics for different colitis mouse models.
For acute DSS-induced colitis, mice were placed on 3% DSS in the drinking water for 7 days. For chronic DSS-induced colitis, the mice were placed in 1% DSS water for 21 days, and the DSS concentration was increased by 0.5% DSS every 7 days until the mice lost 30% of their starting body weight (1% DSS for 21 days, 1.5% DSS for 7 days, 2% DSS for 7 days, etc.). For the T-cell colitis model, CD4+CD45RBhigh (experimental) and CD4+CD45RBlow (control) T cells from male and female C57BL/6 mice were isolated from the spleen using a MojoSort mouse CD4 T-cell isolation kit and sorted via flow cytometry using a 1:100 dilution of an anti-mouse CD4 PE-Cy7 antibody and a 1:100 dilution of an anti-mouse CD45RB APC antibody. IP was injected into male and female immunodeficient Rag1 KO mice. Notably, male or female donor mice can be generated from male Rag1 KO mice, while only female donor mice can be generated from female Rag1 KO mice. Mice were monitored weekly for weight loss and signs of diarrhea. Mice were sacrificed at the end of 8 weeks. Created in BioRender. Shah, Y. (2025) https://BioRender.com/l21i803.
Extended Data Fig. 2 Intestinal Gpx4 or Slc7a11 deletion exacerbates chronic, but not acute, colitis.
a, Representative H&E images and 4HNE images, b, inflammatory histology score, c, percentage positive (%) of 4HNE staining, and d, colon lengths from Rag1 KO; Gpx4f/f mice (n = 8) and Rag1 KO; VilERT2; Gpx4f/f mice (n = 5) under T cell transfer induced chronic colitis model. e, Relative gene expression of Slc7a11 in small intestine tissue and colon tissue from Slc7a11f/f mice (n = 3) and VilERT2; Slc7a11f/f mice (n = 3). f, representative H&E images of the small intestine and colon tissue of VilERT2; Slc7a11f/f mice and Slc7a11f/f mice. g, Representative H&E images, h, inflammatory histology scores, and i, colon lengths of VilERT2; Slc7a11f/f mice (n = 7) and Slc7a11f/f mice (n = 6) with acute DSS-induced colitis. j, Representative H&E images, k, inflammatory histology scores, and l, colon lengths of VilERT2; Slc7a11f/f mice (n = 11) and Slc7a11f/f mice (n = 7) with chronic DSS-induced colitis. Data are shown as mean ± SEM, statistical analysis was performed using two-tailed unpaired T-test (for two column comparison) or 2-way ANOVA (for groups comparison). All experiments were repeated at least twice independently, with similar results.
Extended Data Fig. 3 Intestinal FPN deletion has no effects on chronic colitis.
a, Relative gene expression and b, Protein expression of Ferroportin (Fpn) in colon tissue from Fpnf/f mice (n = 5) and VilERT2; Fpnf/f mice (n = 5). c, Representative H&E images, d, inflammatory histology scores, and e, colon lengths of VilERT2; Fpnf/f mice (n = 12) and Fpnf/f mice (n = 8) with chronic DSS-induced colitis. Data are shown as mean ± SEM, statistical analysis was performed using two-tailed unpaired T-test. All experiments were repeated at least twice independently, with similar results.
Extended Data Fig. 4 ACSL4 is expressed in epithelial cells.
a, IF staining for ACSL4 (red), EpCAM (cyan), and Hoechst (blue) in IBD and normal tissue. b, IF staining for ACSL4 (red), EpCAM (cyan), and Hoechst (blue) in basal, acute DSS, and chronic DSS conditions. The experiments were repeated three times independently, with similar results. These samples are the same ones shown in Fig. 4 with more details and with a different antibody displayed.
Extended Data Fig. 5 ACSL4 expression is increased in fibroblasts in IBD tissue.
a, UMAP and violin plots for ACSL4 expression in GSE114374, b, GSE214695, c, SCP1884, and d, E-MTAB-8901.
Extended Data Fig. 6 Overexpression ACSL4 in fibroblasts worsens chronic DSS induced colitis.
a, Relative gene expression of ferroptosis-related genes (Chac1, Ptgs2, Fsp1 and Fsp27). b, Representative H&E colon tissue, c, inflammatory histology scores, d, colon length, and lipid ROS levels in e, epithelium and f, fibroblasts, as well as cell death (%) in g, epithelium and h, fibroblasts from LSL-Acsl4 mice and PdgfrαERT2; LSL-Acsl4 mice treated with or without liproxstatin-1 (10 mg/Kg, i.p., daily) (LSL-Acsl4 + vehicle (n = 7), LSL-Acsl4 + liproxstatin-1 (n = 7), PdgfrαERT2; LSL-Acsl4 + vehicle (n = 7) and PdgfrαERT2; LSL-Acsl4 + liproxstatin-1 (n = 7)). Data are shown as mean ± SEM, statistical analysis was performed using 2-way ANOVA. All experiments were repeated at least twice independently, with similar results.
Extended Data Fig. 7 ACSL4 in fibroblast sensitizes epithelial ferroptosis through secreted PUFA.
a, Cell death (%) of NCM-460 cell treated with CM from ACSL4-Con and ACSL4-OE fibroblast (n = 3 for each group). b, Cell death (%) of NCM-460 cell treated with Control (0.5% DMSO), aqueous phase and lipid phase metabolite extraction from ACSL4-OE fibroblast CM, with and without liproxstatin-1 (1 μM) (n = 3 for each group). c, Cell death (%) of NCM-460 cell treated with RSL3 (0.5 μM), Control (BSA 5 μM), AA (20 μM), EPA (20 μM) and DHA (20 μM) with and without liproxstatin-1 (1 μM) (n = 3 for each group). Data are shown as mean ± SEM, statistical analysis was performed using two-tailed unpaired T-test (for two column comparison) or 2-way ANOVA (for groups comparison). All experiments were repeated at least twice independently, with similar results.
Extended Data Fig. 8 ACSL4 inhibitors protected DSS induced chronic colitis.
a, Representative H&E colon tissue, b, inflammatory histology scores, c, colon length, d, lipid ROS levels in epithelium and fibroblast, and e, cell death (%) in epithelium and fibroblast from WT mice treated with Control diet (n = 8) and Pioglitazone diet (n = 8). Data are shown as mean ± SEM, statistical analysis was performed using two-tailed unpaired T-test (for two column comparison) or 2-way ANOVA (for groups comparison). All experiments were repeated at least twice independently, with similar results.
Extended Data Fig. 9 No sex differences were observed in the key experiment.
a, histology scores, b, colon length and relative lipid ROS levels in c, epithelial cells and d, fibroblasts of LSL-Acsl4 and PdgfrαERT2, LSL-Acsl4 mice treated with acute DSS colitis ± liproxstatin-1 (10 mg/Kg, i.p. injection, daily) (n = 5 (male, LSL-Acsl4, vehicle), n = 6 (female, LSL-Acsl4, vehicle); n = 5 (male, PdgfrαERT2; LSL-Acsl4, vehicle), n = 7 (female, PdgfrαERT2; LSL-Acsl4, vehicle); n = 5 (male, LSL-Acsl4, liproxstatin-1), n = 4 (female, LSL-Acsl4, liproxstatin-1); n = 5 (male, PdgfrαERT2; LSL-Acsl4, liproxstatin-1), n = 6 (female, PdgfrαERT2; LSL-Acsl4, liproxstatin-1).). Data are shown as mean ± SEM, statistical analysis was performed using two-tailed unpaired T-test (for two column comparison) or 2-way ANOVA (for groups comparison). All experiments were repeated at least twice independently, with similar results.
Supplementary information
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Lipidomics dataset.
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Huang, W., Zhang, Y., Das, N.K. et al. Fibroblast lipid metabolism through ACSL4 regulates epithelial sensitivity to ferroptosis in IBD. Nat Metab 7, 1358–1374 (2025). https://doi.org/10.1038/s42255-025-01313-x
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DOI: https://doi.org/10.1038/s42255-025-01313-x
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