Journal: Nature Communications

Article Title: Epithelial NF-κB shapes protective Treg cell responses to restrain intestinal inflammation

doi: 10.1038/s41467-026-78796-z

Figure Lengend Snippet: a Medium from freshly isolated organoids analysed by proteome array. n = 4 mice per group, pooled. Analysis: one-way ANOVA with Tukey’s correction. In bold: known NF-κB target genes. b Treg cell migration transwell assay: isolated MLN/ spleen primary Treg cells from Wt mice incubated with factors shown. NC (negative control – medium alone) or PC (positive control – Fetal bovine serum). Analysis: one-way ANOVA; Bonferroni’s multiple comparison test. Mean and SD shown. n = 9. c IHC against CCL22 ( n = 5 mice). Black triangles: CCL22+ immune cells. Purple triangles: CCL22+ epithelial cells. Scale: 10 μm. d Primary Treg cells from Wt mice cultured with organoid conditioned medium from Wt vs CCL22 −/− mice ( n = 3 mice), treated with LPS (25 μg/ml LPS over 48 h) or left untreated. Treg cell migration quantified via Transwell migration assay. Analysis: one-way ANOVA. Mean and SD shown. e Treg cell co-culture with conditioned organoid medium (SN), 30 Vol.-% and/or + 30 ng/mL murine IL-33, CM + 30 ng/mL IL-33 or CM + 100 ng/mL Amphiregulin. The fold change increase of expanded Treg cells on day 3 was measured in relation to the initial cell count. n = 3 mice. Analysis: as in a. Mean and SD shown. f Quantitation of CCR4+ and FoxP3+ cells from IF per image. n = 9. Mean and SD shown. g Isolated Wt ( n = 5) and IκBα IEC KO ( n = 3) CD4 + CD25+ Treg cells cultured for 6 h in medium supplemented with 50 ng/mL PMA and 1 µg/mL ionomycin. Supernatants analysed by ELISA. Analysis: unpaired t-test, two-sided. Mean and SD shown. h scRNA of colonic versus splenic immune cells from Wt and IκBα IEC KO mice. i Genes with significant changes in expression between colonic or splenic Treg cells in Wt versus IκBα IEC KO mice. j Differential expression of Gdpd3 and Ftl1 in spleen and colon Treg cells. Analysis: two-sided Wilcoxon rank-sum test k Colonic IF against GDPD3, CD4 and CDH1. Scale: 50 μm. n = 12. Zoom: double positive cells. l Quantitation of GDPD3+CD4+ cells per image. Analysis as in ( a ). Mean and SD shown. m Primary Treg cells from Wt mice cultured with organoid SN (supernatants). n = 3 mice. GDPD3+ cells visualized by IF. Analysis: as in ( a ). Mean and SD shown. Source data are provided as a Source Data file.

Article Snippet: 4.5 × 10 4 cells/well were seeded in RPMI medium without additives on a transwell insert (8μm pore size, Greiner Bio-One Cat. 662638) in a 24- well plate.

Techniques: Isolation, Organoids, Analysis, Migration, Transwell Assay, Incubation, Negative Control, Positive Control, Comparison, Cell Culture, Transwell Migration Assay, Co-culture Assay, Cell Characterization, Quantitation Assay, Enzyme-linked Immunosorbent Assay, Expressing, Quantitative Proteomics

Journal: Nature Communications

Article Title: Epithelial NF-κB shapes protective Treg cell responses to restrain intestinal inflammation

doi: 10.1038/s41467-026-78796-z

Figure Lengend Snippet: a scRNA dataset ( GSE116222 ) is based on samples from inflamed and non-inflamed colon regions of UC patients, as well as those from healthy controls. Visualization of IEC cells across the three conditions. b TRRUST analysis of top upregulated TF networks across the three conditions (Healthy, UC and UC NI (non-inflamed)) in IEC presented as a volcano plot. Text in blue: factors belonging to the NF-κB axis. A Gene-set enrichment was performed using Enrichr. Enrichment significance was assessed using Fisher’s exact test, and enrichment magnitude was represented by the odds ratio. Volcano plots generated with the Enrichr Appyter display odds ratio versus −log 10 ( P value). c TRRUST analysis of downregulated TF networks in UC-inflamed colon IEC versus healthy IEC. Text in blue: factors belonging to the NF-κB axis. Analysis as in ( b ). d H&E of colon sections from healthy and UC patients. Scale: 50 μm. e Colitis score (Nancy index) from ( d ). The patient number is shown in the graph. Analysis: Two-tailed t -test. Mean and SD shown. f Colon-derived organoids were derived from colon biopsies of UC patients or non-IBD controls, with at least n = 4 patients per group. Growth of colon organoids was measured over several days. Mean and SD shown. Analysis: t- test, two-tailed, unpaired. g At least 10 colon organoids per patient from healthy ( n = 3 persons) and UC colon-derived organoids ( n = 4 patients) treated with LPS over 48 h and stained with antibodies as indicated. Scale: 50 μm. h Quantification from g. Top panel: Ki67 (not significant). Lower panel: cleaved caspase 3. Analysis: One-way ANOVA with Tukey’s correction. Mean and SD shown. i Colon organoids were treated with TNF (TNF was removed, and the medium was changed) or left untreated. Medium from colon-derived organoids was analysed by proteome array, and the presence of secreted factors quantitated from bulk ( n = 4) samples per group and n = 2 technical replicates. Significance determined by one-way ANOVA with Tukey’s correction. Only significant results are shown. j Analysis of proteome data by DAVID v2023q4 followed by REVIGO for visualization of GO terms on semantic space. Bubble size indicates prevalence. Color: orange—terms enriched in medium of UC colon-derived organoids treated with TNF, purple—terms enriched in medium of healthy colon-derived organoids treated with TNF. SD and mean shown. Analysis: Welch’s t -test, two-sided – no significance. k Transwell recruitment with Jurkat cells incubated in medium from colon-derived organoids from UC patients or healthy controls. Analysis: one-way ANOVA with Tukey’s correction. n = 4 for healthy samples and n = 6 for UC samples (pooled). Individual points indicate technical replicates. Mean and SD shown. Source data are provided as a Source Data file.

Article Snippet: 4.5 × 10 4 cells/well were seeded in RPMI medium without additives on a transwell insert (8μm pore size, Greiner Bio-One Cat. 662638) in a 24- well plate.

Techniques: Analysis, Generated, Two Tailed Test, Derivative Assay, Organoids, Staining, Incubation, Significance Assay