Genetic and Small-Molecule Modulation of Stat3 in a Mouse Model of Crohn's Disease

Abstract

Crohn's disease (CD), is an inflammatory bowel disease that can affect any part of the gastro-intestinal tract (GI) and is associated with an increased risk of gastro-intestinal cancer. In the current study, we determined the role of genetic and small-molecule modulation of STAT3 in a mouse model of CD. STAT3 has 2 isoforms (α, β) which are expressed in most cells in a 4:1 ratio (α: β). STAT3α has pro-inflammatory and anti-apoptotic functions, while STAT3β has contrasting roles. We used an animal model of CD consisting of intrarectal administration of 2,4,6-trinitrobenzene sulfonic acid and examined the severity of CD in transgenic-mice that express only STAT3α (∆^β/∆^β), as well as in wild-type (WT) mice administered TTI-101 (formerly C188-9), a small molecule STAT3 inhibitor. We determined that clinical manifestations of CD, such as mortality, rectal-bleeding, colonic bleeding, diarrhea, and colon shortening, were exacerbated in ∆^β/∆^β transgenic versus cage-control WT mice, while they were markedly decreased by TTI-101 treatment of WT mice. TTI-101 treatment also increased apoptosis of pathogenic CD4⁺ T cells and reduced colon levels of IL-17-positive cells. Our results indicate that STAT3 contributes to CD and that targeting of STAT3 with TTI-101 may be a useful approach to treating CD.

Keywords: Crohn’s disease; STAT3; inflammatory bowel disease.

Figure 1

Effects of TNBS-induced CD on mortality, diarrhea, colonic bleeding, and colon shortening in +/+ versus ∆^β/∆^β mice. Effect of STAT3 deletion on TNBS-induced CD was studied in +/+ and ∆^β/∆^β mice. (A) Survival was assessed daily and was 74% in the TNBS ∆^β/∆^β mice group vs. 100% in the TNBS +/+ mice group 48 hrs. post-administration of TNBS (p < 0.05, Kaplan–Meier analysis, n = 9–19). (B) Diarrheal score, (C,D) colonic bleeding, (E,F), and colon length were assessed 48 hrs. post-administration of TNBS. Results are expressed as the mean ± SEM of two separate experiments [*, vehicle (+/+ mice) vs. TNBS (∆^β∆^β/mice)], [^†, TNBS (+/+ mice) vs. TNBS (∆^β∆^β/mice)], and [^‡, vehicle (∆^β/mice) vs. TNBS (∆^β∆^β/mice)]; all p < 0.05, ANOVA followed by Tukey’s post-test; n = 6–12.

Figure 2

Effect of TNBS-induced CD on apoptosis of CD4⁺ cells in colons of +/+ and ∆^β/∆^β mice. Representative photomicrographs of CD4-stained and TUNEL-stained sections of colons (magnification 400×) derived from; (A,B) TNBS +/+ mice and (C,D) TNBS ∆^β/∆^β mice. Red arrows depict cells positively stained for CD4 or TUNEL. Scale bar on each micrograph represents 50 microns. CD4⁺ cells (500–1000 per section) within colon sections IHC stained for CD4 were assessed on the adjacent slide for TUNEL positivity or negativity. (E) The percentage TUNEL-positive cells expressed as mean ± SD ((*, TNBS (+/+ mice) vs. vehicle (∆^β/∆^β mice), ^†, TNBS (+/+ mice) vs. TNBS (∆^β/∆^β mice), both p < 0.05, ANOVA followed by Tukey’s post-test; n = 3–4).

Figure 3

Effect of TTI-101 treatment on colon levels of pY-STAT3 protein, diarrhea, colonic bleeding, colon length, and colon inflammation in TNBS-induced CD. (A) pY-STAT3 levels were measured by Luminex assay in the colons of mice that received 50% ethanol or TNBS in 50% ethanol and treated with vehicle (DMSO) or TTI-101 in DMSO. pY-STAT3 levels were normalized to β-tubulin protein levels and the means ± SD of the ratios plotted. (B) Diarrheal score, (C,D) colonic bleeding, (E,F), colonic inflammation, and (G) colon length were assessed 48 hrs. post-administration of TNBS. Results are expressed as the mean ± SEM of two separate experiments ((A,B,D,G): *, Ethanol vs. TNBS, ^†, Ethanol+TTI-101 vs. TNBS, and ^‡, TNBS vs. TNBS+TTI-101, all p < 0.05; ANOVA followed by Tukey’s post-test; n = 3–12), (F): *, TNBS vs. TNBS+TTI-101. p < 0.05; ANOVA followed by Tukey’s post-test; n = 6–12).

Figure 3

Effect of TTI-101 treatment on colon levels of pY-STAT3 protein, diarrhea, colonic bleeding, colon length, and colon inflammation in TNBS-induced CD. (A) pY-STAT3 levels were measured by Luminex assay in the colons of mice that received 50% ethanol or TNBS in 50% ethanol and treated with vehicle (DMSO) or TTI-101 in DMSO. pY-STAT3 levels were normalized to β-tubulin protein levels and the means ± SD of the ratios plotted. (B) Diarrheal score, (C,D) colonic bleeding, (E,F), colonic inflammation, and (G) colon length were assessed 48 hrs. post-administration of TNBS. Results are expressed as the mean ± SEM of two separate experiments ((A,B,D,G): *, Ethanol vs. TNBS, ^†, Ethanol+TTI-101 vs. TNBS, and ^‡, TNBS vs. TNBS+TTI-101, all p < 0.05; ANOVA followed by Tukey’s post-test; n = 3–12), (F): *, TNBS vs. TNBS+TTI-101. p < 0.05; ANOVA followed by Tukey’s post-test; n = 6–12).

Figure 4

Effect of TTI-101 treatment on TNBS-induced apoptosis in CD4 cells. (A,B) Photomicrographs of CD4 and TUNEL stained sections of colon tissue derived from TNBS mice without TTI-101 treatment and from (C,D) TNBS mice with TTI-101 treatment, respectively (magnification 400×). Red arrows depict cells positively stained for CD4 or TUNEL. Scale bar on each micrograph represents 50 microns. (E) Percentage of apoptosis in colonic CD4+cells in response to TNBS, with and without TTI-101 treatment. Results are expressed as mean ± SD (*, Ethanol+TTI-101 vs. TNBS and ^†, TNBS vs. TNBS+TTI-101; all p < 0.05, ANOVA followed by Tukey’s post-test; n = 3–4).

Figure 5

Effect of TTI-101 treatment on colonic Th17 cell infiltration. (A) Photomicrographs of Th17-stained sections of colons derived from control mice without TTI-101 treatment or (B) control mice with TTI-101 treatment or (C) TNBS mice without TTI-101 treatment, and (D) TNBS mice with TTI-101 treatment (magnification 400×). Red arrows depict cells positively stained for Th17. Scale bar on each micrograph represents 50 microns. (E) TTI-101 treatment significantly decreased levels of TNBS-induced colonic Th17 cell infiltration. Results are expressed as the mean ± SEM (average Th17 number in *, Ethanol vs. TNBS, ^†, Ethanol+TTI-101 vs. TNBS and ^‡, TNBS vs. TNBS+TTI-101, all, p < 0.05, ANOVA followed by Tukey’s post-test) (n = 3–4).

Figure 5

Effect of TTI-101 treatment on colonic Th17 cell infiltration. (A) Photomicrographs of Th17-stained sections of colons derived from control mice without TTI-101 treatment or (B) control mice with TTI-101 treatment or (C) TNBS mice without TTI-101 treatment, and (D) TNBS mice with TTI-101 treatment (magnification 400×). Red arrows depict cells positively stained for Th17. Scale bar on each micrograph represents 50 microns. (E) TTI-101 treatment significantly decreased levels of TNBS-induced colonic Th17 cell infiltration. Results are expressed as the mean ± SEM (average Th17 number in *, Ethanol vs. TNBS, ^†, Ethanol+TTI-101 vs. TNBS and ^‡, TNBS vs. TNBS+TTI-101, all, p < 0.05, ANOVA followed by Tukey’s post-test) (n = 3–4).