The TNFΔARE Mouse as a Model of Intestinal Fibrosis

Abstract

Crohn disease (CD) is a highly morbid chronic inflammatory disease. Although many patients with CD also develop fibrostenosing complications, there are no medical therapies for intestinal fibrosis. This is due, in part, to a lack of high-fidelity biomimetic models to enhance understanding and drug development, which highlights the need for developing in vivo models of inflammatory bowel disease-related intestinal fibrosis. This study investigates whether the TNF^ΔARE mouse, a model of ileal inflammation, also develops intestinal fibrosis. Several clinically relevant outcomes were studied, including features of structural fibrosis, histologic fibrosis, and gene expression. These include the use of a new luminal casting technique, traditional histologic outcomes, use of second harmonic imaging, and quantitative PCR. These features were studied in aged TNF^ΔARE mice as well as in cohorts of numerous ages. At >24 weeks of age, TNF^ΔARE mice developed structural, histologic, and transcriptional changes of ileal fibrosis. Protein and RNA expression profiles showed changes as early as 6 weeks, coinciding with histologic changes as early as 14 to 15 weeks. Overt structural fibrosis was delayed until at least 16 weeks and was most developed after 24 weeks. This study found that the TNF^ΔARE mouse is a viable and highly tractable model of ileal fibrosis. This model and the techniques used herein can be leveraged for both mechanistic studies and therapeutic development for the treatment of intestinal fibrosis.

Graphical abstract

Figure 1

Aged (>24 weeks) TNF^{ΔARE +/–} mice develop clinically analogous structural fibrosis, including stricture and luminal narrowing, while age-matched littermate control wild-type mice do not. A: Terminal ileum that has been filled with agarose casts. B: Casts from (A) that have been removed from the ileum. C: Quantification of gross ileal strictures in aged TNF^{ΔARE +/–} mice and wild-type controls. D: Manual inspection of gross strictures in aged TNF^{ΔARE +/–} mice and wild-type controls. E: Ratio of the width of the widest section to the narrowest section of the terminal ileum of wild-type controls and aged TNF^{ΔARE +/–} mice. F: Luminal cast area/length was measured. Dotted line outlines the ileum in (A) and the ileal casts in (B) to assist in visualization of the size and shape. Data were analyzed by using binomial test (C), Welch’s t-test (D and E), or unpaired t-test (F) based on normality and variance of the sample and are expressed as means ± SD. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗∗P < 0.0001.

Figure 2

Aged (>24 weeks) TNF^{ΔARE +/–} mice develop histologic fibrosis, whereas age-matched littermate control wild-type mice do not. A: Representative histology of the terminal ileum of wild-type and aged TNF^{ΔARE +/–} mice using hematoxylin and eosin staining. B: Wall width measurements of wild-type and aged TNF^{ΔARE +/–} mice. C: Representative histology of the terminal ileum of wild-type and aged TNF^{ΔARE +/–} mice using picrosirius red staining. Top panel was under white light, lower panel was under polarized light. D: Fibrosis score of wild-type and aged TNF^{ΔARE +/–} mice based on histologic scoring. E: Enzyme-linked immunosorbent assay quantification of collagen type I. Data were analyzed by using Welch’s t-test (B), U-test (D), or unpaired t-test (E) based on normality and variance of the sample and are expressed as means ± SD. n = 13 wild type and n =16 TNF^{ΔARE +/–} (A). ∗∗∗∗P < 0.0001. Scale bars: 100 μm (A and C).

Figure 3

The increased collagen content deposition of aged TNF^{ΔARE +/–} mice in the terminal ileum wall is presented using second harmonic generation on a subset of our >24-week–old age-matched littermate controls and aged TNF^{ΔARE +/–} mice. Representative images are shown. These data are inclusive of 10 age-matched littermate control wild-type mice and 10 aged TNF^{ΔARE +/–} mice. A: Representative images of the terminal ileum of wild-type and aged TNF^{ΔARE +/–} mice. B: Collagen content in the ileal wall quantified using second harmonic generation and reported as pixels of collagen (count) per length. Data were analyzed by using Welch’s t-test (B) based on normality and variance of the sample and are expressed as means ± SD. ∗∗P < 0.01. Scale bar: 100 μm (A).

Figure 4

Aged TNF^{ΔARE +/–} mice express increases in several pro-inflammatory and profibrotic genes in the terminal ileum, and these changes occur as early as 6 to 7 weeks. A: Gene expression in mice >24 weeks old. B: Gene expression in 6- to 7-week–old mice. Data were analyzed by using U-test, Welch’s t-test, or unpaired t-test based on normality and variance of the sample and are expressed as means ± SD. ∗∗P < 0.01; ∗∗∗P < 0.001; ∗∗∗∗P < 0.0001.

Figure 5

Age-related histologic changes of intestinal fibrosis. A: Representative histology of the terminal ileum of wild-type and aged TNF^{ΔARE +/–} mice using hematoxylin and eosin staining. B: Representative histology of the terminal ileum of wild-type and TNF^{ΔARE +/–} mice of varying ages using picrosirius red staining. Top panel was under white light, lower panel was under polarized light. C: Wall width measurements of wild-type and TNF^{ΔARE +/–} mice aged 7 to 10 weeks, aged 14 to 15 weeks, and aged 18 to 24 weeks. D: Fibrosis score based on histologic scoring of wild-type and TNF^{ΔARE +/–} mice aged 7 to 10 weeks, aged 14 to 15 weeks, and aged 18 to 24 weeks. Data were analyzed by using U-test (C) or U-test test, Welch’s t-test, unpaired t-test based on normality, and variance of the sample (D) and are expressed as means ± SD. ∗P < 0.05; ∗∗P < 0.01. Scale bars: 100 μm (A and B).

Figure 6

TNF^ΔARE+/– mice do not develop robust clinically analogous structural fibrosis until after 24 weeks of age. These cohorts are aged 7 to 10 weeks (A–E) or aged 16 to 24 weeks (F–J). (A and F) Terminal ileum that has been filled with agarose casts at ages 7 to 10 weeks (A) and 16 to 24 weeks (F). Once removed, the casts of the same mice are shown at ages 7 to 10 weeks (B) and 16 to 24 weeks (G). C–E: Quantification of gross ileal strictures, ileal narrowing, and smaller luminal area volumes in wild-type and 7- to 10-week–old TNF^{ΔARE +/–} mice. H–J: Quantification of gross ileal strictures, ileal narrowing, and luminal area volumes in wild-type and 16- to 24-week–old TNF^{ΔARE +/–} mice. Dotted line outlines the ileum in (A and F) and the ileal casts in (B and G) assist in visualization of the size and shape. Data were analyzed by using an unpaired t-test (C, E, H, and J) or the U-test (I) and are expressed as means ± SD. ∗P < 0.05.

Figure 7

TNF^ΔARE+/– mice develop structural and histologic changes of ileal fibrosis over time. These data have been presented in other figures and are re-organized here for direct comparison. All wild-type data across age groups have been pooled. These show the progression of number of ileal strictures (A), ileal narrowing (B), luminal cast area (C), wall width (D), and fibrosis score (E) in wild-type and TNF^{ARE +/–} mice at 7 to 10, 16 to 20, and >24 weeks. Data were analyzed by using a Kruskal-Wallis test (A and E) or ordinary one-way analysis of variance (with Welch’s correction when variance was significant) (B–D) and are expressed as means ± SD. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; ∗∗∗∗P < 0.0001.