Fig 1.
Murine intestinal organoids elicit a preemptive response to virulent S. flexneri through NLRC4.
Murine ileal organoids were infected with virulent S. flexneri M90T or avirulent BS176 to assess NLRC4-dependent responses. (A) Western blot analysis of WT organoids infected with M90T or BS176. (B) Western blot of Nlrc4+/+ and Nlrc4-/- organoids treated with 1 nM anthrax PA and 1 nM NeedleTox for 3.5 hours. (C) Western blot of Nlrc4+/+ and Nlrc4-/- organoids infected with M90T or BS176, or treated with filtered bacterial supernatants. (D) CFUs recovered from Nlrc4+/+ and Nlrc4-/- organoids following infection with M90T. (E) Western blot of Nlrc4+/+ and Nlrc4-/- organoids primed with IFNγ (20 ng/mL, 18 h) before infection with M90T. Asterix denotes a non-specific band that appears with S. flexneri lysate. (F) Cellular ATP levels measured in Nlrc4-/- organoids infected with M90T or BS176. (G) Western blot analysis of Nlrc4-/- organoids infected with M90T or BS176 assessing apoptosis markers. Statistical significance in D was assessed by Student’s t-test, and F by One-way ANOVA. Data represent mean ± SD, with p < 0.05 considered statistically significant. All infections were performed at an MOI of 50 for 4 hours. Data are representative of at least three independent biological replicates.
Fig 2.
Murine intestinal organoids exhibit non-selective bacterial uptake.
(A) CFUs from WT HeLa cells infected with M90T or BS176. (B) CFUs from Nlrc4-/- organoids infected with M90T or BS176. (C) Confocal immunofluorescence imaging of Nlrc4-/- organoids infected with M90T or BS176. (D) CFUs from Nlrc4-/- organoids infected with DH5α E. coli. transformed with an afimbrial adhesin protein AfaE. (E-F) Confocal immunofluorescence imaging of Nlrc4-/- organoids infected with DH5α E.coli (E) or fluorescent beads (F). (G) WT ileal organoids were stimulated for 4 hours with LPS. Where indicated, organoids were pretreated with IFNγ (20ng/mL) for 18 hours prior to stimulation. Protein expression was assessed by western blot. Confocal Images in C, E and F show an x–y projection (center), with orthogonal z–x and z–y views on the sides, compiled from a z-stack. Arrows point to cells that contain bacteria (C, E) or beads (F). Scale bar is 20 μM. All infections were performed at an MOI of 50 for 4 hours. Statistical analysis in panels A and B was performed using two-way ANOVA, and D by Student’s t-test. Data represent mean ± SD, with p < 0.05 considered statistically significant. Data are representative of at least three independent biological replicates.
Fig 3.
RIPK2 does not alter the transcriptome of murine intestinal organoids after acute S. flexneri M90T infection.
(A) Relative expression (FPKM) of NLRs in WT murine ileal organoids under basal conditions, based on previously published bulk RNA sequencing data [GEO accession: GSE297523]. Nod1 and Nod2 are highlighted in green. (B) Uniform Manifold Approximation and Projection (UMAP) visualization of single-cell RNA sequencing data from WT murine ileal crypt-enriched epithelial cells, annotated by epithelial lineage identity [GEO accession GSE195742]. (C) Schematic representation of experimental design for bulk RNA sequencing of ileal organoids infected with S. flexneri M90T. (D) PCA of the top 500 differentially expressed genes, stratified by genotype and treatment condition. (E) Sample-to-sample correlation heatmap of the top 500 differentially expressed genes, showing clustering based on transcriptional relatedness. (F) qPCR validation in Nlrc4-/-Ripk2+/+ and Nlrc4-/-Ripk2-/- ileal organoids of selected genes from that were statistically significant by p-value but did not pass FDR correction. Statistical significance in F was assessed using two-way ANOVA. Data represent mean ± SD, with p < 0.05 considered statistically significant. Panel C was created in BioRender. PHILPOTT, D. (2026) https://BioRender.com/yz630ln. All infections were performed using S. flexneri M90T at an MOI of 50 for 4 hours. Data are representative of at least four independent biological replicates.
Fig 4.
Neither NOD1 nor NOD2 stimulation with PGN elicits a broad pro-inflammatory response in murine intestinal organoids.
(A, B) Time-course qPCR of pro-inflammatory gene expression in WT murine ileal organoids treated with 1 µg/mL C12-iE-DAP (A) or L18-MDP (B). TNFα (10 ng/mL, 4 hours) was included as a positive control. (C) qPCR of WT ileal organoids co-treated with C12-iE-DAP (1 µg/mL) and a titration of IFNγ (0.1, 1, 10,100ng/mL) for 4 hours. TNFα (10 ng/mL, 4 hours) was included as a positive control. (D) qPCR of WT ileal organoids co-treated with L18-MDP (1 µg/mL) and a titration of IFNγ + TNFα (0.1, 1, 10,100ng/mL) for 4 hours. (E, F) qPCR analysis of HCT116 cells treated with 1 µg/mL L18-MDP (E) or 1 µg/mL C12-iE-DAP (F) for 4 hours. (G) qPCR of CSC28 human colorectal cancer-derived cells treated with 1 µg/mL L18-MDP or 1 µg/mL C12-iE-DAP for 4 hours. (H) qPCR of BMDMs from WT mice stimulated with L18-MDP (1 µg/mL) for 4 hours. (I) Relative expression by qPCR of Nod2 and Ripk2 in matched WT BMDMs and ileal organoids derived from the same mouse. Statistical analyses were performed using one-way ANOVA for panels A, B, G, and I; two-way ANOVA for panels C and D; and Student’s t-test for panels E, F, H. Data represent mean ± SD, with p < 0.05 considered statistically significant. Data are representative of at least three independent biological replicates.
Fig 5.
S. flexneri suppresses inflammatory gene expression in IECs irrespective of virulence.
(A) Volcano plots showing differentially expressed genes between uninfected (UI) and S. flexneri M90T-infected Nlrc4-/- ileal organoids. Key pro-inflammatory genes are highlighted in red. (B) Volcano plots showing differentially expressed genes between S. flexneri M90T-infected and M90T supernatant-treated Nlrc4-/- ileal organoids. Key pro-inflammatory genes are highlighted in red. (C) Pathway enrichment analysis of the top downregulated pathways in Nlrc4-/- organoids infected with M90T versus UI controls, highlighting a suppression of inflammatory signaling terms. (D) Heatmap of genes within the “Hallmark TNFα signaling via NF-κB” gene set, based on variance-stabilizing transformed counts from UI and M90T-infected Nlrc4-/- organoids. (E) qPCR validation of selected inflammatory genes in Nlrc4-/- organoids infected with either M90T or BS176. (F) CXCL1 levels measured by ELISA in supernatants from Nlrc4-/- organoids infected with M90T or BS176. All infections were performed at an MOI of 50 for 4 hours. Statistical analysis in panels E-F were performed using one-way ANOVA. Data represent mean ± SD, with p < 0.05 considered statistically significant. Data are representative of at least four independent biological replicates.
Fig 6.
Terminally differentiated EECs are enriched in response to S. flexneri infection.
(A) GSEA of IEC type signatures defined by Haber et al. [38], comparing Nlrc4-/- organoids infected with M90T versus UI controls. (B) Pathway enrichment analysis of the top upregulated gene sets in Nlrc4-/- organoids following M90T infection versus UI controls. Arrow points to the “Hallmark Pancreas Beta Cells” gene set. (C) Gene-concept network plot showing individual genes contributing to top enriched pathways from panel B, with the “Hallmark Pancreas Beta Cells” gene set circled. (D) Volcano plot of differentially expressed genes between UI and S. flexneri M90T-infected Nlrc4-/- ileal organoids, with key EEC markers highlighted in red. (E) GSEA showing differential enrichment of EECs at various stages of maturation defined by Gehart et al [39] between M90T-infected and UI Nlrc4-/- enteroids. (F) qPCR validation of selected EEC markers in Nlrc4-/- organoids infected with M90T or BS176. (G, H) Confocal immunofluorescence staining for ChgA (G) and 5-HT (H) in Nlrc4-/- organoids infected with M90T or BS176, with quantification of number of ChgA+ cells (G) and 5-HT+ cells (H) per organoid. Scale bar represents 20 um. (I) qPCR analysis of EEC markers in Nlrc4-/- organoids treated with live M90T, sonicated M90T, or heat-inactivated M90T. Bacterial input was stoichiometrically matched across all conditions. All infections were performed at an MOI of 50 for 4 hours. Statistical analysis in panels F, G, H and I was performed using one-way ANOVA. Data represent mean ± SD, with p < 0.05 considered statistically significant. Data are representative of at least three independent biological replicates.
Table 1.
qPCR primer sequences.