IFN-γ-Induced intestinal epithelial cell-type-specific programmed cell death: PANoptosis and its modulation in Crohn's disease

Abstract

Background: Crohn's disease (CD) is a chronic inflammatory bowel disease (IBD) and is considered a Th1-mediated disease, supported by the over-expression of interferon-gamma (IFN-γ) in the intestinal lamina propria. IFN-γ has a pleiotropic effect on the intestinal epithelial cells (IECs), suggesting that IFN-γ-induced responses may differ between epithelial cell types.

Methods: We established human small intestinal organoids (enteroids) derived from non-IBD controls and CD patients. Using human enteroids, the major response of IECs induced by IFN-γ was evaluated, focusing on the IFN-γ-induced programmed cell death (PCD) pathway. Identified IFN-γ-induced responses were validated in surgically resected intestinal samples and publicly available single-cell RNA-sequencing datasets.

Results: IFN-γ stimulated programmed cell death (PCD) of IECs in both control and CD enteroids in a dose-dependent manner. Pyroptosis, apoptosis. and necroptosis were activated in enteroids, suggesting that PANoptosis was the main process of IFN-γ-induced PCD in IECs. The response to IFN-γ depends on the cell type of the IECs. IFN-γ induced depletion of enterocytes with upregulation of PANoptosis-associated genes, while leading to expansion of goblet cells without significant change in PANoptosis-associated gene expression. Individual PCD inhibitors were insufficient to block IFN-γ-induced cytotoxicity, whereas the selective JAK1 inhibitor (upadacitinib) effectively blocked IFN-γ-induced cytotoxicity and PANoptosis. Furthermore, PANoptosis was significantly activated in surgically resected tissues and in publicly available single-cell RNA-sequencing datasets of intestinal tissues from patients with CD.

Conclusion: IFN-γ induces PANoptosis in enterocytes, which can be treated with a selective JAK1 inhibitor in patients with CD.

Keywords: Crohn’s disease; PANoptosis; enteroid; interferon-gamma; intestinal epithelial cell; intestinal organoid; programmed cell death; selective JAK1 inhibitor.

Figure 1

Effect of IFN-γ on Ctrl and CD enteroids. (A) Bright field microscopic image of different concentrations of IFN-γ-treated Ctrl and CD enteroids. Scale bar, 100 μm. (B) Organoid-forming efficiency as measure by organoid reconstitution assay. Assays were performed in triplicate with seven Ctrl enteroids and five CD enteroids. (C) Cell viability as measured by MTT assay. Assays were performed in triplicate with seven Ctrl enteroids and five CD enteroids. GI50, 50% growth inhibition concentration. (D) Fluorescence microscopy image of the EdU assay. Scale bar, 25 μm. (E) Comparison of EdU-positive cells per enteroids. Assays were performed on three Ctrl enteroids and five CD enteroids. Differences were evaluated by one-way ANOVA with multiple comparisons; ***p < 0.001, ns, not statistically significant.

Figure 2

Programmed cell death (PCD) pathway in IFN-γ-treated Ctrl and CD enteroids. (A) Principal component analysis (PCA) plot of bulk RNA-seq data set (IFN-γ-free and IFN-γ-treated Ctrl enteroids, n = 6 pair; IFN-γ-free and IFN-γ-treated CD enteroids, n = 10 pair). (B) Heatmap with hierarchical clustering of apoptosis, necroptosis, pyroptosis, and PANoptosis -associated genes in human enteroids treated with and without IFN-γ 100 pg/mL. Differences were evaluated by paired t-test with Bonferroni correction; *p-value < 0.01, adjusted p-value < 0.01, and q-value < 0.05. (C) Gene expression level [fragments per kilobase of transcript per million mapped reads (FPKM)] for the key molecules of PCD pathways in IFN-γ-free and IFN-γ-treated Ctrl and CD enteroids. Paired t-test were used for comparison of the paired IFN-γ-free and IFN-γ-treated enteroids. Unpaired t-test were used for comparison of the IFN-γ-treated Ctrl and CD enteroids. (D) Western blotting for the key molecules of PCD pathways in IFN-γ-free and IFN-γ-treated Ctrl enteroids (n = 4) and CD enteroids (n = 3). (E) TUNEL staining in IFN-γ-free and IFN-γ-treated Ctrl enteroids (n = 3) and CD enteroids (n = 3). Scale bar, 25 μm. Differences of TUNEL-positive cells per orgnaoids in the IFN-γ-free and IFN-γ-treated Ctrl and CD enteroids were evaluated by one-way ANOVA with multiple comparisons; *p < 0.05, ***p < 0.001. ns, not statistically significant.

Figure 3

IFN-γ-induced epithelial cell differentiation in Ctrl and CD enteroids. (A) Expression of intestinal epithelial lineage-specific markers. Single-cell RNA-sequencing analyzed 11,316 cells derived from IFN-γ-free and IFN-γ-treated Ctrl enteroids (n = 2 pair) and IFN-γ-free and IFN-γ-treated CD enteroids (n = 2 pair). To identify different intestinal epithelial cell types, lineage-specific markers were applied: OLFM4 for intestinal stem cells (ISCs), MKI67 for proliferating cells, VIL1 for enterocytes (ECs), MMP7 for Paneth cells (PCs), and MUC4 and TFF3 for goblet cells (GCs). (B) FeaturePlot annotation based on epithelial cell type. (C) Effect of IFN-γ on epithelial cell types in Ctrl and CD enteroids. (D) Number and percentage of epithelial cell types occupying Ctrl and CD enteroids treated with IFN-γ. (E) Trajectory analysis of IFN-γ treated Ctrl and CD enteroids using Slingshot. (F) Hematoxylin and eosin (H&E) staining of Ctrl and CD enteroids treated with and without IFN-γ to measure the area of enteroids. H&E staining, immunohistochemistry, and alcian blue assay were performed on the IFN-γ-free and IFN-γ-treated Ctrl enteroids (n = 3 pair) and IFN-γ-free and IFN-γ-treated CD enteroids (n = 3 pair). (G) IHC for OLFM4 to compare OLFM4-stained intestinal stem cells in Ctrl and CD enteroids treated with and without IFN-γ. (H) IHC for PCNA to compare PCNA-stained proliferating cells in Ctrl and CD enteroids treated with and without IFN-γ. (I) Alcian blue staining to compare the percentage of Alcian blue-stained area to the total area of enteroids in Ctrl and CD enteroids treated with and without IFN-γ. (J) IHC for lysozyme to compare lysozyme-stained Paneth cells in Ctrl and CD enteroids treated with and without IFN-γ. Scale bar 100 μm. Differences were evaluated by one-way ANOVA with multiple comparisons; *p < 0.05, **p < 0.01, ***p < 0.001****, p < 0.0001, ns, not statistically significant.

Figure 4

IFN-γ-induced epithelial celltype-specific responses of PCD-associated genes (A) FeaturePlot for PCD-associated gene expression. The red dashed circle indicates the goblet cell (GC) population. (B) DotPlot for PCD-associated gene expression in Ctrl and CD treated with and without IFN-γ. (C) Protein-protein interaction network of DEGs in PCD pathways.

Figure 5

Validation of PANoptosis and Epithelial Cellular Responses in Human CD Samples. (A) Immunohistochemical staining for cleaved CASP3 (c-CASP3), phospho-MLKL (p-MLKL), and cleaved GSDMD (c-GSDMD) in surgically resected intestinal tissue from patients with CD (n=5). (B) FeaturePlot annotation based on epithelial cell type of publicly available datasets from normal and CD patients (https://www.gutcellatlas.org/). (C) FeaturePlot splited by disease status and epithelial cell types. (D) DotPlot of PCD-related gene expression in publicly available datasets from normal and CD patients. (E) ViolinPlot of key PCD molecules splited by disease status in publicly available datasets from normal and CD patients. (F) FeaturePlot of key PCD molecules splited by epithelial cell types in publicly available datasets from normal and CD patients. Scale bar: black 200 μm, orange 100 μm. Publicly available datasets was downloaded from Gut Cell Atlas (https://www.gutcellatlas.org/).

Figure 6

Effects of PCD inhibitors [pan-caspase inhibitor (Z-VAD-FMK), RIPK1 inhibitor (necrostatin-1), MLKL inhibitor (necrosulfonamide), caspase-1 inhibitor (AC-YVAD-CMK), GSDMD inhibitor (LDC7559), and JAK1 inhibitor (upadacitinib)] on IFN-γ-induced cytotoxicity. (A) Bright-field microscopic image of human enteroids treated with IFN-γ 0, 100 pg/mL, and 400 pg/mL in combination with PCD inhibitors. Black scale bar: 100 μm. (B) Organoid-forming efficiency. (C) Cell viability as measured by MTT assay. Assays were performed in triplicate with three Ctrl enteroids. Differences were evaluated by one-way ANOVA with multiple comparisons; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ns, not statistically significant..

Figure 7

Effects of upadacitinib on IFN-γ-treated Ctrl and CD enteroids. (A) Western blotting for the key molecules of PCD signaling pathways to identify the effect of upadacitinib in Ctrl enteroid (n=3) and CD enteroid (n=3) treated with and without IFN-γ stimulation at concentrations of 100 pg/mL and 400 pg/mL. (B) Bright-field microscopic image of human enteroids treated with IFN-γ 0, 100, and 1000 pg/mL accompanied by 0, 2, and 20 μM upadacitinib. Scale bar: 100 μm. (C) Organoid-forming efficiency. (D) Cell viability as measured by MTT assay. Assays were performed in triplicate with three Ctrl enteroids. Differences were evaluated by one-way ANOVA with multiple comparisons; **p < 0.01, ***p < 0.001, and ****p < 0.0001. ns, not statistically significant.