Gene expression changes in the colon epithelium are similar to those of intact colon during late inflammation in interleukin-10 gene deficient mice

Abstract

In addition to their role in absorption and secretion, epithelial cells play an important role in the protection of the colon mucosa from the resident microbiota and are important for the maintenance of homeostasis. Microarray analysis of intact colon samples is widely used to gain an overview of the cellular pathways and processes that are active in the colon during inflammation. Laser microdissection of colon epithelial cells allows a more targeted analysis of molecular pathways in the mucosa, preceding and during inflammation, with potentially increased sensitivity to changes in specific cell populations. The aim of this study was to investigate the molecular changes that occur in early and late inflammation stages in colon epithelium of a mouse model of inflammatory bowel diseases. Microarray analysis of intact colon samples and microdissected colon epithelial cell samples from interleukin-10 gene deficient and control mice at 6 and 12 weeks of age was undertaken. Results of gene set enrichment analysis showed that more immune-related pathways were identified between interleukin-10 gene deficient and control mice at 6 weeks of age in epithelial cells than intact colon. This suggests that targeting epithelial cells could increase sensitivity for detecting immune changes that occur early in the inflammatory process. However, in the later stages of inflammation, microarray analyses of intact colon and epithelium both provide a similar overview of gene expression changes in the colon mucosa at the pathway level.

Figure 1. Non-inflamed mouse colon.

Colon section for mouse 18, an Il10^−/− mouse sampled at 6 weeks of age, before microdissection of epithelial cells (A) and after microdissection of epithelial cells (B). The lamina propria was taken from this section along with the epithelial cells but is mostly connective tissue in the non-inflamed mice.

Figure 2. Inflamed mouse colon.

Colon section from mouse 17, an Il10^−/− mouse sampled at 12 weeks of age, before microdissection of epithelial cells. The aggregate of immune cells is visible in the mucosa, which is typical for CD-like colitis, such as that seen in this mouse model. The circle outlines epithelial cells in a colonic crypt, which were cut out for analysis of gene expression.

Figure 3. Overlap of differentially expressed genes between treatments.

Venn diagrams show the numbers of differentially expressed genes in each comparison and how many are shared between each comparison, for intact colon (A) and colon epithelium (B). Treatments are identified in the diagram as: Il10^−/− mice =  I; C57BL/6J mice =  C; weeks of age =  w. Each colour arbitrarily represents a different treatment. Numbers are of differentially expressed genes within each treatment comparison and each microarray experiment (intact colon and epithelial cells). Numbers in (B) are not subsets of the equivalent treatment comparison in (A), although there is overlap in the gene lists for each comparison between intact colon and colon epithelium.

Figure 4. Overall and immune-related gene expression profiles in intact colon and epithelium.

Heatmaps generated by unsupervised hierarchical clustering of differentially expressed genes. (A) shows all genes differentially expressed within any of the treatment comparisons, and the expression values are derived from individual mice. The bars above the heatmap indicate whether the data are from microdissected (white bar) or intact (black bar) tissue, while those below the heatmap show the age and genotype of the mice as indicated by the colour key. (B) shows a subset of differentially expressed immune related genes from the Il10^−/− mice at 12 weeks vs. C57BL/6J mice at 12 weeks comparison, showing the gene expression profile of each treatment comparison in both intact colon and colon epithelium. Expression values for each gene are averages from 6 arrays per treatment.

Figure 5. Network diagram showing gene expression in the colon epithelium.

Network diagram of genes from the top 5 differentially expressed canonical pathways in Il10^−/− mice at 12 weeks of age vs. 6 weeks of age in the colon epithelium. *denotes genes that are detected two or more times on an array. Genes or gene products are represented as nodes, and the biological relationship between two nodes is represented as a line. All relationships are supported by at least one reference from literature. Red and green coloured nodes indicate degree of fold change, with red indicating an increase in expression in Il10^−/− mice at 12 weeks of age relative to Il10^−/− mice at 6 weeks of age and green indicating a decrease in expression. Colour intensity is correlated with the degree of change in expression with greater intensity representing a higher expression level. Nodes are displayed with various shapes that represent the functional class of genes as shown in the legend. Fold changes are shown beneath each node.

Figure 6. Network diagram of gene expression in the intact colon.

Network diagram shown in Figure 5 overlaid with expression values for the same treatment comparison in intact colon.