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. 2022;13(3):789-808.
doi: 10.1016/j.jcmgh.2021.10.010. Epub 2021 Oct 23.

TNFα Induces LGR5+ Stem Cell Dysfunction In Patients With Crohn's Disease (V体育2025版)

Chansu Lee  1 , Minae An  2 , Je-Gun Joung  3 , Woong-Yang Park  2 , Dong Kyung Chang  4 , Young-Ho Kim  4 , Sung Noh Hong  5
Affiliations

TNFα Induces LGR5+ Stem Cell Dysfunction In Patients With Crohn's Disease (V体育安卓版)

Chansu Lee et al. Cell Mol Gastroenterol Hepatol. 2022.

Abstract

Background & aims: Tumor necrosis factor alpha (TNFα) is considered a major tissue damage-promoting effector in Crohn's disease (CD) pathogenesis. Patient-derived intestinal organoid (enteroid) recapitulates the disease-specific characteristics of the intestinal epithelium VSports手机版. This study aimed to evaluate the intestinal epithelial responses to TNFα in enteroids derived from healthy controls and compare them with those of CD patient-derived enteroids. .

Methods: Human enteroids derived from patients with CD and controls were treated with TNFα (30 ng/mL), and cell viability and gene expression patterns were evaluated. V体育安卓版.

Results: TNFα induced MLKL-mediated necroptotic cell death, which was more pronounced in CD patient-derived enteroids than in control enteroids. Immunohistochemistry and RNA sequencing revealed that treatment with TNFα caused expansion of the intestinal stem cell (ISC) populations V体育ios版. However, expanded ISC subpopulations differed in control and CD patient-derived enteroids, with LGR5+ active ISCs in control enteroids and reserve ISCs, such as BMI1+ cells, in CD patient-derived enteroids. In single-cell RNA sequencing, LGR5+ ISC-enriched cell cluster showed strong expression of TNFRSF1B (TNFR2) and cyclooxygenase-prostaglandin E2 (PGE2) activation. In TNFα-treated CD patient-derived enteroids, exogenous PGE2 (10 nmol/L) induced the expansion of the LGR5+ ISC population and improved organoid-forming efficiency, viability, and wound healing. .

Conclusions: TNFα increases necroptosis of differentiated cells and induces the expansion of LGR5+ ISCs. In CD patient-derived enteroids, TNFα causes LGR5+ stem cell dysfunction (expansion failure), and exogenous PGE2 treatment restored the functions of LGR5+ stem cells VSports最新版本. Therefore, PGE2 can be used to promote mucosal healing in patients with CD. .

Keywords: Crohn’s Disease; Intestinal Organoid; Intestinal Stem Cell; Prostaglandin E2; Tumor Necrosis Factor-Alpha V体育平台登录. .

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Graphical abstract
Figure 1
Figure 1
Organoid-forming efficiency and cell viability of CD patient-derived enteroids and control enteroids under TNFα-enriched conditions. (A) Representative microscopic images of spheroids and enteroids. Scale bar = 100 μm. (B) Immunohistochemical staining for epithelial cell markers in spheroids and enteroids. Scale bar = 75 μm. (C) Morphology, (D) organoid-forming efficiency, and (E) cell viability (MTT assay) of organoids derived from controls (n = 12) and patients with CD cultured under TNFα-free and -treated conditions (n = 11 each). Morphologic observations were performed using bright-field microscopy. The measured optical density (O.D.) values are expressed as a ratio based on the O.D. values of TNFα-untreated control enteroids. Scale bar = 100 μm. Assays were conducted in triplicate. Differences were assessed using one-way analysis of variance (ANOVA) with Bonferroni multiple comparison test; ∗∗∗P < .001, ∗∗∗∗ P ≤ .0001.
Figure 2
Figure 2
Gene expression profile of enteroids derived from controls and patients with CD under TNFα-free and -treated conditions. (A) Hierarchical clustering heatmap and (B) principal component analysis using paired samples of TNFα-untreated and -treated enteroids derived from controls (n = 12 pairs) and patients with CD (n = 11 pairs). (C) Volcano plot of DEGs in TNFα-treated and -untreated enteroids. The 10 up- and down-regulated genes annotated with gene symbols are displayed. Volcano plot shows fold change (log2 ratio) plotted against absolute confidence (–log10P value). (D) Gene set enrichment analysis of DEGs. The top 10 significant terms of gene ontology biological process in TNFα-treated enteroids are listed, based on the normalized enrichment score. Enrichment plot of the most enriched signaling pathway (I-κB kinase/NF-κB signaling; GO:0007249) genes is illustrated. (E) Hierarchical clustering heatmap of TNFα signaling pathway and its related genes. DEGs were identified using the paired t test with Bonferroni correction (adjusted P value < .001 and Q value < 0.001). (F) Differences in expression levels (FPKM) of TNFα pathway genes in paired sample of TNFα-untreated and -treated enteroids derived from controls and patients with CD. Black dots with solid lines indicate changes in control enteroids, and blue dots with blue broken lines indicate changes in CD patient-derived enteroids. (G) Western blotting for RIPK3 and MLKL proteins in enteroids derived from controls and patients with CD. Enteroids derived from controls (n = 3) and patients with CD (n = 3) were treated with different concentrations of TNFα, infliximab, and Z-VAD-FMK. (H) Double immunofluorescence staining for CC3 and TUNEL. Distribution of CC3 (red fluorescence) and TUNEL (green fluorescence) was determined using co-immunofluorescence analysis. Cell nuclei were co-stained with DAPI (blue fluorescence). The number of cells was counted in 10 enteroids (size >100 μm) of each group. Differences were evaluated using one-way ANOVA with Bonferroni multiple comparison test. Scale bar = 50 μm; ∗∗P < .01, ∗∗∗P < .001, ∗∗∗∗ P < .0001. (I) Proposed cellular responses to TNFα in human enteroids.
Figure 3
Figure 3
TNFα treatment alters the epithelial lineage-specific gene expression in CD patient-derived enteroids. (A) Hierarchical clustering heatmap of epithelial cell lineage-specific markers and (B) differences in expression levels (FPKM) of signature genes in paired samples of TNFα-untreated and -treated enteroids derived from controls (n = 12 pairs) and patients with CD (n = 11 pairs). Wilcoxon matched-pairs signed-rank test was used to analyze differences in gene expression in paired samples (TNFα-untreated and -treated). Mann-Whitney test was used to analyze differences in gene expression between TNFα-treated control enteroids and TNFα-treated CD patient-derived enteroid; ∗P < .05, ∗∗P < .01. Immunohistochemistry for (C) E-cadherin, (D) LGR5, and (E) BMI1. The number of LGR5+ and BMI1+ cells was counted in 15 enteroids (size >100 μm) from each group (n = 3 for each group). Black arrows indicate LGR5+ or BMI1+ cells. (F) Alcian blue staining. Stained area was quantified using ImageJ and expressed as percentage of the average area stained in the TNFα-untreated control enteroids. Area was measured in 30 enteroids (size >100 μm) from each group (n = 3 for each group). Differences were evaluated using one-way ANOVA with Bonferroni multiple comparison test; ∗P < .05, ∗∗P < .01, ∗∗∗P < .001. Blue scale bar = 100 μm, black scale bar = 50 μm, and red scale bar = 25 μm.
Figure 4
Figure 4
Single-cell RNA sequencing for TNFα-untreated and -treated enteroids derived from controls and patients with CD. (A) Heatmap and clustering of DEGs. Rows represent the top 5 DEGs, and columns represent clusters. (B) UMAP plot of the aggregated samples and individual samples. The number indicates a cell cluster consisting of cells mainly originating from individual sample. Ctrl, control enteroids, CD, CD patient-derived enteroids. (C) Heatmap and hierarchical clustering of intestinal epithelial lineage-specific genes based on the single-cell clusters. EC, enterocyte; EEC, enteroendocrine cell; GC, goblet cell; PC, Paneth cell. (D) UMAP plot visualizing intestinal epithelial cell types of human enteroids based on marker gene expression. EC, enterocyte; EEC, enteroendocrine cell; GC, goblet cell; PC, Paneth cell; rISC, reserve intestinal stem cell. (E) Trajectory analysis of enterocyte differentiation from LGR5+ ISCs and reserve ISCs. (F) Heatmap for TNFRSF1A (TNFR1)/TNFRSF1B (TNFR2) expression ratio and the expression level of TNFα signaling pathway-related genes according to epithelial cell types. The number represents average expression level of single-cell RNA. aISC, active intestinal stem cell.
Figure 5
Figure 5
Effect of infliximab (IFX) and PGE2 on TNFα-treated CD patient-derived enteroids. (A) Effect of infliximab on viability in TNFα-treated control enteroids. Control enteroids (n = 3) were cultured in different concentrations of infliximab (0, 1, 10, and 20 μg/mL) and TNFα (0, 30, and 100 ng/mL). Assays were conducted in triplicate. Differences were evaluated using one-way ANOVA with Bonferroni multiple comparison test; ∗∗P < .01,∗∗∗P < .001. Scale bar = 200 μm. (B) Quantitative reverse transcription polymerase chain reaction for assessing LGR5 expression in CD patient-derived enteroids treated with TNFα, infliximab, and PGE2 (n = 3). Differences were evaluated using Wilcoxon signed-rank test; ∗P < .05, ∗∗∗P < .001. (C) Fluorescence-activated cell sorting analysis for LGR5+ cells in CD patient-derived enteroids treated with TNFα, infliximab, and PGE2 (n = 3). Differences were evaluated using Wilcoxon signed-rank test; ∗∗∗P < .001. (D) MTT assay for CD patient-derived enteroids treated with TNFα, infliximab, and PGE2 (n = 3). Differences were evaluated using Wilcoxon signed-rank test; ∗P < .05, ∗∗P < .01, ∗∗∗P < .001. (E) Organoid-forming efficiency of control and CD patient-derived enteroids treated with TNFα, infliximab, and PGE2 (n = 3 each). The number of reconstituted organoids is expressed as percentage based on the value for TNFα-untreated control enteroids with no infliximab and PGE2. Differences were evaluated using Wilcoxon signed-rank test; ∗∗∗P < .001. (F) Wound healing assay for CD patient-derived enteroids treated with TNFα, infliximab, and PGE2. Non-healed wound areas were evaluated in 3 different fields. Differences were evaluated using ordinary two-way ANOVA with Bonferroni multiple comparison test; ∗P < .05, ∗∗∗P < .001. O.D., optical density.
Figure 6
Figure 6
RNA-seq analysis for ER stress- and autophagy-related genes in control enteroids and CD patient-derived enteroids. Bulk RNA-seq. (A) Heatmap with hierarchical clustering for DEGs (paired t test with Bonferroni multiple comparison, adjusted P < .01, q < 0.05). (B) Differences in expression levels (FPKM) of ER stress- and autophagy-related genes in paired sample of TNFα-untreated and -treated enteroids derived from controls and CD patients. Single-cell RNA-seq. Expression of ER stress- and autophagy-related genes using heatmap (C) in the enteroids derived from controls and CD patients and (D) in the intestinal epithelial lineages.
Figure 7
Figure 7
Changes in the expression of epithelial intracellular junctional molecules in TNFα-untreated and -treated enteroids. FPKM of paired TNFα-untreated and -treated enteroids derived from controls and patients with CD were analyzed using the paired t test. Black dotted lines indicate changes in control organoids, and white dotted and dashed lines indicate changes in CD patient-derived enteroids.
Figure 8
Figure 8
Intestinal organoid generation and subculture derived from inflamed or non-inflamed mucosa of patients with CD.
Figure 9
Figure 9
TNFα signaling pathway and its related genes expression in the inflamed and non-inflamed mucosa obtained from the same patients with active CD. (A) Hierarchical clustering heatmap of TNFα signaling pathway and its related genes in 13 pairs of inflamed and non-inflamed mucosa obtained from the same patients with active CD. DEGs were identified by using the Wilcoxon matched-pairs signed-rank test (P < .05). (B) Differences in expression levels (FPKM) of TNFα pathway genes in paired sample of inflamed and non-inflamed mucosa of same patients with active CD. Differences were evaluated using paired t test; ∗P < .05, ∗∗P < .01.
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