Figure 1.
Experimental design, weight curves and disease activity index.
(A) One cycle of DSS comprises 7 days of DSS followed by a recovery period of 14 days with normal drinking water. The study set-up consists of 6 different groups (n = 10 for each group with exception of the control group (n = 7) and 3-cycles group (n = 9). Acute colitis mice (n = 10) received 7 days of DSS without a recovery period prior to sacrifice and/or scanning. In a second group (n = 10) colitis was induced by DSS for 7 days followed by a recovery period of two weeks with normal drinking water, and this was defined as one cycle of DSS. In a third and fourth group, the DSS cycle was repeated twice (n = 10) or three times (n = 10). A fifth group (n = 10) received two cycles of DSS followed by an additional recovery period of three weeks with normal drinking water. Control mice (n = 7) received normal drinking water throughout. All mice were 6-weeks old at time of start of the 9-weeks study period and in this way age-matched at the time of sacrifice and scanning (15-weeks old). (B) Weight curves of control mice, acute, 2-, and 3-cycles mice. Data are expressed as medians with interquartile range (IQR). (C) The disease activity index (DAI) was determined at the end of the 9-weeks study period, based on body weight loss (one point for each 5% loss of weight), stool consistency (0, normal; 2, formed but very soft; 4, liquid) and presence of gross blood in the stools (0, none; 1, present). Data are expressed as medians with IQR. Mann-Whitney U testing (*p<0.05, **p≤0.01, ***p≤0.001).
Figure 2.
(A) Representative pictures of the colon in control mice, acute, 2-, and 3-cycles DSS colitis mice. The black bar represents 5 cm. (B) Macroscopic damage score of the colon in the 6 groups. (C) Length of colon shortened significant after 7 days of DSS (acute DSS colitis) compared to the control mice. (D) Weight of colon after removal of the feces. (E) Weight/length ratio of the colon. All data are expressed as medians with IQR. Mann-Whitney U testing (*p<0.05, **p≤0.01, ***p≤0.001).
Figure 3.
Histological read-outs of colitis.
(A) Representative pictures of colon sections from mice after 2 and 3 cycles of DSS exposure. The repeated cycles of DSS induce persistent and dense infiltration of deeper layers with mononuclear cell aggregates in the subserosa (*), crypt dilatation (>), re-epithelialization (→) and crypt fission (dotted →). (B) Microscopic inflammation score. Three sections per animal were evaluated. The details of this score are shown in supplementary figure 1. Data are expressed as medians with IQR. (C) The lower degree of inflammation after 3 cycles and 2 cycles with prolonged recovery is associated with a lower histological active disease score. The histological active disease score comprised the sum of neutrophil infiltration and epithelial defects, expressing the activity of disease and the potential for acute tissue damage during the last 24 hours prior to tissue sampling. (D) Thickness of the mucosa and (E) thickness of the muscularis propria. Two sections per animal were evaluated. The mean is annotated as +. Data are expressed as box and whiskers and 5–95 percentile are plotted. Mann-Whitney U testing (*p<0.05, **p≤0.01, ***p≤0.001).
Figure 4.
Fibrogenic changes in the colon.
(A) Representative pictures of an Martius-scarlet-blue (MSB) staining of control mice, acute colitis and 3-cycles mice. A clear deposition of collagen in the submucosa is present in chronic DSS colitis. (B) Histological scoring of fibrosis using a MSB-staining shows that more cycles of DSS are associated with more fibrosis (surface of blue in mucosa and submucosa). More recovery is associated with a lower degree of fibrosis. (C) Hydroxyprolin assay. Data are expressed as µg collagen per mm wet colon. Data are expressed as medians with IQR. Mann-Whitney U testing (*p<0.05, **p≤0.01, ***p≤0.001). (D-E) Analysis of the mesenchymal cells in the submucosa shows the presence of myofibroblasts (α-SMA+ and vimentin+) in the submucosa after 2 and 3 cycles, indicated with the arrows. These cells are absent in control colon and in acute colitis.
Figure 5.
ECM changes in acute and chronic DSS colitis.
(A) Representative pictures of the collagen I staining of control, acute, 3-cycles and 2-cycles with recovery colon and corresponding MSB and picro-sirius red staining. The rectangle in the upper pictures is shown with higher magnification in the lower pictures. (B) Representative pictures of the collagen III and tenascin staining in control, acute, 2-cycles and 3-cycles colon (merge image, blue = DAPI, green = collagen III and red = tenascin). The rectangle in the picture of the acute and 2-cycles colon is shown with higher magnification in the next picture (merge of collagen III and tenascin staining). No tenascin is observed in control colon. In the acute model, tenascin stains as a dot-like pattern in the mucosa and submucosa, indicating the tenascin-producing cells. Also a linear deposition of tenascin is seen around some crypts (>) and in blood vessels present in the submucosa (→). In the 2-cycles colon, the same expression of tenascin is observed. More collagen III is present in the submucosa (*). In the 3-cycles colon, less tenascin-positive cells are observed.
Figure 6.
Unsupervised hierarchical cluster analysis based on log2 expression values of top 50 most variable gene probe sets.
Individual samples are shown in columns and gene probe sets in rows. The log2 expression values for individual genes are indicated by color, as shown in the scale (color key), with yellow indicating a high level of expression and blue a low level of expression. The abbreviations of the individual genes and gene probe IDs are explained in Table S1.
Figure 7.
Identification of the comparisons between the differentially expressed probe sets of the different groups.
(A) Identification of the top 50 significantly upregulated genes of 1-, 2-, and 3-cycles DSS colitis (FDR<0.05, FC>2) (most upregulated compared to controls). The analysis of these genes is shown in Figure 8. (B) Identification of the top 50 significantly upregulated genes of acute colitis and made comparison with the genes identified in chronic colitis. The analysis of these genes is shown in Figure 9. (C) Identification of the top 50 significantly most upregulated genes (fold change compared to controls) after additional recovery. The analysis of these genes is shown in Figure 10A. (*after removal of duplicates, of gene probe sets with unmapped IDs and of genes coding for gammaglobulin chains).
Figure 8.
Colonic gene expression in chronic DSS colitis.
(A) Venn diagram of the differentially expressed probe sets of 72 genes identified in the top 50 significantly upregulated genes of 1-, 2-, and 3-cycles DSS colitis (FDR<0.05, FC>2). Among these, 27 genes were commonly upregulated in 1-, 2-, and 3-cycles DSS colitis. The biological function of these 27 genes is summarized in Table 1. The abbreviations of the individual genes and gene probe IDs are explained in the Table S2. (B) Individual fold change expression of these 72 significantly upregulated genes vs. controls in chronic DSS colitis.
Table 1.
Biological function of selected genes in chronic and acute colitis and after additional recovery.
Figure 9.
Colonic gene expression in acute DSS colitis.
(A) Venn diagram of the differentially expressed probe sets of the top 50 significantly upregulated genes in acute DSS colitis (fold change compared to controls) (Table S3) (FDR<0.05, FC>2). These genes were compared with the 72 genes identified in chronic colitis. In total, 30 genes were uniquely upregulated in acute colitis. The biological functions of these 30 genes uniquely upregulated in acute colitis are summarized in Table 1. Out of these 50 genes, 21 genes were common in acute and chronic colitis (underlined in Figure 7). Only 11 out of these 21 genes were common with the 27 common upregulated genes in chronic colitis. For simplicity of the figure, only these 27 genes are shown in the venn diagram for the chronic condition. (B) Individual fold change expression of the top 50 most upregulated genes in acute DSS colitis vs. controls. The abbreviations of the individual genes and gene probe IDs are explained in Table S3.
Figure 10.
Colonic gene expression in 2 cycles DSS colitis followed by an additional recovery period of 3 weeks.
(A) Venn diagram of the differentially expressed probe sets of the top 50 most upregulated genes (fold change compared to controls) after additional recovery (Table S4). These genes were compared with the 72 genes identified in chronic colitis. In total, 34 genes were uniquely upregulated in the recovery group. Only 16 genes were common with chronic colitis. Eleven out of these 16 genes were common with the 27 common upregulated genes in chronic colitis. (B) To isolate the impact of recovery on gene expression unrelated to the degree of chronicity, we identified all significantly upregulated genes after additional recovery compared to 2 cycles DSS colitis followed by immediate sacrifice. We started from all significantly upregulated genes after 2 cycles of DSS followed by additional recovery. Of these 376 genes, 255 were also upregulated after 2 cycles of DSS while 90 genes were uniquely upregulated after additional recovery (Table S5), accounting for 65 unique genes. Among these 65 genes, 8 genes coded for keratins (KRT 4, KRT5, KRT6, KRT13, KRT14, KRT16, KRT17, KRT84). The biological functions of the 65 genes uniquely upregulated after recovery are summarized in Table 1. (C) Individual fold change expression of the 90 unique genes significantly upregulated (fold change compared to controls) in 2 cycles DSS colitis with additional recovery vs. 2 cycles DSS colitis. The abbreviations of the individual genes and gene probe IDs are explained in the Table S4 and Table S5.
Figure 11.
(A) Example of a T2w image and corresponding T2 map of a control, acute and 2-cycles colon shows high T2 in acute colon and intermediate T2 after 2 cycles of DSS. (B) The T2 histogram of control colon (black) shifts to higher values after 7 days of DSS (red, acute model). With more cycles of DSS, the T2 map shifts back to lower values. In the 2-, and 3-cycles model, the histogram of the colon goes to an intermediate state in between the normal colon and the acute colitis. The statistical analysis of these profiles is shown in table 2. (C) Although the water content of acute colitis tissue vs. 2- and 3-cycles DSS colitis is the same, T2 values are clearly different. (D) As a proof of concept, eight mice were sequentially scanned through a 2-cycles model. Mice were scanned at day 0, day 7, day 21, day 28 and day 42. (E) In this set-up, the mean T2 values of the distal colon after 2 cycles (day 42) show a significant decrease compared to the 1-cycle (21 days) time point (*, p = 0.016). Data are expressed as the mean value per mouse of T2 of three images, non-parametric testing. (F) The histograms show more clearly the changes in T2 profiles of the different scan time points following the same trend as in the endpoint-scanning.
Table 2.
Statistical analysis of the MRI T2 profiles.