The Oxysterol Synthesising Enzyme CH25H Contributes to the Development of Intestinal Fibrosis

Abstract

Intestinal fibrosis and stenosis are common complications of Crohn's disease [CD], frequently requiring surgery. Anti-inflammatory strategies can only partially prevent fibrosis; hence, anti-fibrotic therapies remain an unmet clinical need. Oxysterols are oxidised cholesterol derivatives with important roles in various biological processes. The enzyme cholesterol 25-hydroxylase [CH25H] converts cholesterol to 25-hydroxycholesterol [25-HC], which modulates immune responses and oxidative stress. In human intestinal samples from CD patients, we found a strong correlation of CH25H mRNA expression with the expression of fibrosis markers. We demonstrate reduced intestinal fibrosis in mice deficient for the CH25H enzyme, using the sodium dextran sulphate [DSS]-induced chronic colitis model. Additionally, using a heterotopic transplantation model of intestinal fibrosis, we demonstrate reduced collagen deposition and lower concentrations of hydroxyproline in CH25H knockouts. In the heterotopic transplant model, CH25H was expressed in fibroblasts. Taken together, our findings indicate an involvement of oxysterol synthesis in the pathogenesis of intestinal fibrosis.

Keywords: Fibrogenesis; cholesterol 25 hydroxylase [CH25H]; graft; intestinal fibrosis; mouse model; oxysterols; transplantation.

Figure 1.

Upregulation of CH25H mRNA expression in human fibrotic tissue of patients with CD. [A] Representative images of Sirius Red-stained human ileum samples from healthy controls [left panel] and CD patients in a non-fibrotic [middle panel] and in a fibrotic region [right panel]. Scale bar: 2.5 mm. White: CD fibrotic, [n = 6], grey: CD non-fibrotic [n = 7], black: healthy control [n = 6]. [B] Samples were analysed for CH25H mRNA expression and normalised to GAPDH. CH25H mRNA level was correlated with mRNA levels of [C] Col1, [D] Col3 [E] SMA, and [F] TGFB. Statistical analysis: B: Kruskal-Wallis test; **p <0.01. CD, Crohn’s disease. C-E: correlation analysis: Spearman R [non-parametric correlation].

Figure 2.

Reduced fibrosis in Ch25h^-/- mice in chronic DSS colitis. Ch25h^-/- and WT female mice were treated for four cycles with 2.5% DSS or water [controls]. [A] Representative transmission light images of Sirius Red-stained intestinal sections of wild-type [WT] and Ch25h^-/- dextran sodium sulphate [DSS]-treated mice and water littermate controls. Scale bar: 100 μm. [B] Image analysis for identification of collagen layer areas in the colon mucosa using Matlab custom-made scripts. Left panel: Original transmission light Sirius Red-stained image. Right panel: Collagen layer area. [C] Quantification of collagen layer area in DSS-treated animals using customised Matlab scripts. [D] Collagen layer thickness calculated from eight or more positions per graft in representative areas of Sirius Red-stained slides with transmission light at 200-fold magnification. The colon was analysed for mRNA expression of: [E] Tgf-beta, [F] Col3, [G] Col1, [H] Loxl2, [I] Timp1, and [J] Ch25h [normalised to Gapdh]. Expression levels are normalised to water-treated wild-type controls. Statistical analysis: Mann-Whitney U test; *p <0.05. n = 4–6 per group.

Figure 3.

Reduced levels of intestinal fibrosis in CH25H-deficient mice is not due to reduced inflammation in chronic DSS colitis. Analysis of colon inflammation in H/E-stained colon sections. [A] Score of the inflammatory infiltrate [left panel], score for epithelial damage [middle panel], and total histology score [sum of both partial scores, right panel]. [B] Murine endoscopic index of colitis severity [MEICS] score [left panel] and spleen weight [right panel]. [C] Representative H/E-stained sections of the distal colon of water control mice [left panel] and DSS-treated mice. DSS, dextran sodium sulphate; H/E: haematoxylin and eosin.

Figure 4.

Reduced levels of intestinal fibrosis in CH25H-deficient mice in the heterotopic transplantation model. Wild-type and Ch25h^-/- animals were tested in a heterotopic transplantation model for intestinal fibrosis. [A] Left panels: Overview [low-resolution image] of Sirius Red-stained intestinal grafts of WT and Ch25h^-/- mice at Day 7 after transplantation. Scale bar: 1 mm. Middle panels: Representative transmission light images demonstrating increased collagen layer thickness in grafts at Day 7 compared with freshly isolated intestines at Day 0. Upper panels: WT littermate controls. Lower panels: Ch25h^-/-. Scale bar: 50 μm. Right panels: High-resolution inserts illustrating measurements of collagen layer thickness. [B] Collagen layer thickness calculated from eight or more positions per graft in representative areas of Sirius Red-stained slides with transmission light at 200-fold magnification. [C] Image analysis for identification of collagen layer areas using Matlab custom-made scripts. Left panel: Original polarised 200x light microscopy image. Middle panel: Collagen layer area. Right panel: Remaining non-collagen tissue. Scale bar: 50 μm. [D] Quantification of collagen layer area at Day 7 post transplantation, using the same strategy as in [C]. [E] Collagen quantification with hydroxyproline assay. Day 0, freshly isolated intestine. Day 7, intestine 7 days post transplantation [n_WT day 0 = 3, n_KO day 0 = 9, n_WT day 7 = 8, n_WT day 7 = 11]. Statistical analysis: Mann-Whitney U test; *p <0.05, **p <0.01. Bars indicate mean ± standard error of the mean [SEM]. WT, wild type; CH25H, cholesterol 25 hydroxylase; HYP, hydroxyproline.

Figure 5.

Expression of intestinal fibrosis markers in wild-type and Ch25h^-/- mice. Wild-type and Ch25h^-/- mice were tested in a heterotopic intestinal transplant model. Freshly isolated intestines [Day 0] and grafts 7 days after transplantation were analysed for mRNA expression of: [A] Ch25h, [B] Col1, [C] Mmp9, and [D] Timp1 [normalised to Gapdh]. [E, F] Analysis of protein expression of TGF-β by Western blot. Statistical analysis: A-D: Mann-Whitney U test; *p <0.05, **p <0.01 [n_WT day 0 = 3, n_KO day 0 = 9, n_WT day 7 = 8, n_WT day 7 = 11]. E-F: n = 4. Unpaired t test.

Figure 6.

Cells infiltrating the graft do not differ between wild-type and CH25H-deficient mice. Lamina propria infiltrating cells from grafts of wild-type and Ch25h^-/- mice were harvested 7 days after surgery and analysed by CyTOF. [A] Dimensionality-reduced projection of the entire phenotypical landscape was calculated using the tSNE algorithm with Barnes-Hut approximation [bhSNE]. The colour coding represents staining intensity of the specified marker. [B] t-SNE maps of each experimental group; 250 000 randomly selected points are plotted. Overlaid in colour are cluster designations computed by the Phenograph clustering algorithm. The represented clusters were manually constructed by merging the initial cluster output based on phenotypical similarity until the final number of 11 identifiable clusters was reached. [C] Bar plot showing the mean cluster frequencies and error bars representing standard error of the mean [SEM] [nWT = 5, nKO = 5].

Figure 7.

Expression of Ch25h in fibroblasts in intestinal grafts. [A] Representative images of the in situ hybridisation [RNAscope] analysis of wild-type small intestine. Negative control [a probe for the bacterial gene dihydrodipicolinate reductase, Dapb, left panel] and Ch25h mRNA [middle panel] are demonstrated with the RNAscope signal shown in red. Right panel: High resolution of inserts of the RNAscope signal. Scale bar: 25 μm. [B] Representative images of the Ch25h RNAscope analysis of the intestinal grafts of wild-type [WT] mice at Day 7 after transplantation, demonstrating accumulation of the CH25H signal in fibroblasts in the former mucosa layer. Scale bar left panel: 20 μm, middle panel: 50 μm. The right panel shows inserts of the middle panel. Fibroblasts are indicated by arrows, neutrophils by a double arrow. [C] Overlay of Ch25h RNAscope analysis and the α-SMA staining from two representative images with enlarged inserts. Scale bar: 100 μm, scale bar insert: 20 μm. [D] 3T3 cells [left panel] and primary mouse intestinal fibroblasts [right panel] were treated for 72 h with different concentrations of 25-HC and/or TGF-β as indicated. Samples were analysed for α-SMA protein levels by Western blot. Expression levels are normalised relative to the negative control and shown as mean ± standard error of the mean [SEM]. Statistical analysis: unpaired t test, the * is relative to the negative control; *p <0.05, **p <0.01, n = 2.