Figure 5.
IFN-λ treatment produces a proliferative phenotype in GI stem cells. PEG-rIL-29 (5 μg) or PBS was given intraperitoneally for 3 days before gut harvest and evaluation of GI epithelial function. (A-C) Representative images (A), numbers (B), and size of GI organoids (C) grown from colonic crypt isolates (n = 9 per group, combined from 3 experiments). (D-E) Numbers of SI organoids (n = 5, combined from 3 experiments) (D) and number of stem cells (CD45.2–/EpCAM+/GFP+) (E) isolated from digested gut of Lgr5-EGFP-IREScreERT2 mice (n = 5, combined from 3 experiments). (F) Number of organoids grown from FACS sorted single stem cells (n = 4, combined from 3 experiments). (G) Number of colonic organoids from WT and IL-22–/–- mice treated with PBS or PEG-rIL-29 (n = 4, combined from 2 experiments). (H) Number of colon and SI organoids from Ifnar–/– mice treated with PBS or PEG-rIL-29 (n = 6, combined from 3 experiments). (I) RNA-seq from sort purified colonic epithelial cells and stem cells derived from either PEG-rIL-29–treated or PBS-treated mice. Heat map showing top 300 genes significantly differentially expressed in colonic epithelial cells (Lgr5–) and stem cells (Lgr5+) after in vivo PEG-rIL-29 vs PBS treatment (2420 genes total). Expression of the same genes from ISCs derived from PBS- or IL-29–treated mice included for comparison (n = 5 per group). (J) Normalized messenger RNA transcript counts for Ifnlr1, Il10rb, Ifnar1, and Ifnar2 in colonic epithelial cells (Lgr5–) and stem cells (Lgr5+). (K) Functional enrichment analysis of differentially expressed genes: canonical pathway enrichment analysis (log2 fold-change >0.58, and adjusted P value <.05) in sorted Lgr5+ and Lgr5– epithelial cells after in vivo PEG-rIL-29 treatment relative to genotype-matched PBS-treated samples using Ingenuity pathway analysis. Enrichment of canonical pathways associated with immune responses (left) and regulation of cellular proliferation (right). Bubbles represent significant pathway enrichment, as determined by Fisher’s exact test. Bubble diameter represents the log10P value as determined by Fisher’s exact test. Crosses signify a lack of significant pathway enrichment, color indicates predicted pathway activation (red) or predicted inhibition (blue), and white bubbles represent significant functional enrichment of pathways with no available prediction patterns. (L) Venn diagram of overlap of differentially expressed genes in Lgr5+ and Lgr5– cells as for panel K. Data are presented as mean ± SEM. P values were calculated by using the 2-tailed Mann-Whitney t test. For RNA-seq, differentially regulated genes were determined after filtering for genes with >5 cpm and fold change differences >1.2 and corrected P values (false discovery rate) <.05. *P <.05, **P < .01, ***P < .001.

IFN-λ treatment produces a proliferative phenotype in GI stem cells. PEG-rIL-29 (5 μg) or PBS was given intraperitoneally for 3 days before gut harvest and evaluation of GI epithelial function. (A-C) Representative images (A), numbers (B), and size of GI organoids (C) grown from colonic crypt isolates (n = 9 per group, combined from 3 experiments). (D-E) Numbers of SI organoids (n = 5, combined from 3 experiments) (D) and number of stem cells (CD45.2–/EpCAM+/GFP+) (E) isolated from digested gut of Lgr5-EGFP-IREScreERT2 mice (n = 5, combined from 3 experiments). (F) Number of organoids grown from FACS sorted single stem cells (n = 4, combined from 3 experiments). (G) Number of colonic organoids from WT and IL-22–/–- mice treated with PBS or PEG-rIL-29 (n = 4, combined from 2 experiments). (H) Number of colon and SI organoids from Ifnar–/– mice treated with PBS or PEG-rIL-29 (n = 6, combined from 3 experiments). (I) RNA-seq from sort purified colonic epithelial cells and stem cells derived from either PEG-rIL-29–treated or PBS-treated mice. Heat map showing top 300 genes significantly differentially expressed in colonic epithelial cells (Lgr5–) and stem cells (Lgr5+) after in vivo PEG-rIL-29 vs PBS treatment (2420 genes total). Expression of the same genes from ISCs derived from PBS- or IL-29–treated mice included for comparison (n = 5 per group). (J) Normalized messenger RNA transcript counts for Ifnlr1, Il10rb, Ifnar1, and Ifnar2 in colonic epithelial cells (Lgr5–) and stem cells (Lgr5+). (K) Functional enrichment analysis of differentially expressed genes: canonical pathway enrichment analysis (log2 fold-change >0.58, and adjusted P value <.05) in sorted Lgr5+ and Lgr5– epithelial cells after in vivo PEG-rIL-29 treatment relative to genotype-matched PBS-treated samples using Ingenuity pathway analysis. Enrichment of canonical pathways associated with immune responses (left) and regulation of cellular proliferation (right). Bubbles represent significant pathway enrichment, as determined by Fisher’s exact test. Bubble diameter represents the log10P value as determined by Fisher’s exact test. Crosses signify a lack of significant pathway enrichment, color indicates predicted pathway activation (red) or predicted inhibition (blue), and white bubbles represent significant functional enrichment of pathways with no available prediction patterns. (L) Venn diagram of overlap of differentially expressed genes in Lgr5+ and Lgr5– cells as for panel K. Data are presented as mean ± SEM. P values were calculated by using the 2-tailed Mann-Whitney t test. For RNA-seq, differentially regulated genes were determined after filtering for genes with >5 cpm and fold change differences >1.2 and corrected P values (false discovery rate) <.05. *P <.05, **P < .01, ***P < .001.

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