Figure 1.

Gene expression in HpES- and cytokine-treated small intestinal organoids. Organoid cultures grown from duodenal crypt cells, taken from four individual C57BL/6 mice, were stimulated in the presence or absence of the type 1 cytokine IFN-γ, the type 2 cytokines IL-4 and IL-13 (IL-4/13), and/or the ES products of adult H. polygyrus parasites (HpES). After 24 h, RNA was extracted from each replicate, and all 24 samples were subjected to parallel RNA-seq analyses. Patterns of gene expression were then analyzed. (A) Principal component analysis plot showing transcriptomes of stimulated organoids projected onto two dimensions; each condition is represented by four replicates derived from four individual mice. Principal component analysis data were produced from log-transformed normalized counts in the DESeq2 package (Love et al., 2014). (B) Heat map of the top 1,000 DEGs, organized into seven gene clusters, and a group of unclustered genes. Z-scores of normalized count values are indicated by coloring from blue (lowest) to red (highest), based on data from four replicates per group, each individually presented. (C–E) MA plots showing log₂ fold change (M) plotted against log of mean normalized expression counts (A) for the comparisons of no stimulation versus HpES alone (C), IL-4/13 alone versus IL-4/13 + HpES (D), and IFN-γ alone versus IFN-γ + HpES (E). Data from four replicates per group were pooled; genes with adjusted P value < 0.05 are colored. The top 10 DEGs by log₂ fold change that have known function are annotated, with those increased in the presence of HpES shown in blue and those decreased in red. Additional genes of interest are shown in black. DEGs were calculated in DESeq2. (F) Expression levels of 12 genes from cluster 6 showing the most significant (by adjusted P value) differential expression in the presence of HpES. Graphs show normalized counts/log₂ fold change compared with no stimulation control from four independent biological replicates, for organoid cultures exposed to IL-4/13 alone (pink circles) or IL-4/13 + HpES (magenta triangles). Stim, stimulation.