Differential patterns of histone acetylation in inflammatory bowel diseases

Abstract

Post-translational modifications of histones, particularly acetylation, are associated with the regulation of inflammatory gene expression. We used two animal models of inflammation of the bowel and biopsy samples from patients with Crohn's disease (CD) to study the expression of acetylated histones (H) 3 and 4 in inflamed mucosa. Acetylation of histone H4 was significantly elevated in the inflamed mucosa in the trinitrobenzene sulfonic acid model of colitis particularly on lysine residues (K) 8 and 12 in contrast to non-inflamed tissue. In addition, acetylated H4 was localised to inflamed tissue and to Peyer's patches (PP) in dextran sulfate sodium (DSS)-treated rat models. Within the PP, H3 acetylation was detected in the mantle zone whereas H4 acetylation was seen in both the periphery and the germinal centre. Finally, acetylation of H4 was significantly upregulated in inflamed biopsies and PP from patients with CD. Enhanced acetylation of H4K5 and K16 was seen in the PP. These results demonstrate that histone acetylation is associated with inflammation and may provide a novel therapeutic target for mucosal inflammation.

Figure 1

Acetylation on histone 4 in the trinitrobenzene sulfonic acid (TNBS) rat model of inflammation. A: Sham (saline treated) operated and TNBS treated rat large intestine. Rats were Sham or TNBS treated for 7 days before sacrifice. Well-advanced inflammation is apparent in the colon of the TNBS rat model. B: Pan acetylation on histone 4 (H4). The Sham model was saline-treated and therefore less inflamed (control). Results were obtained by Western blotting. The ratio of the density of histone H4 bands over β-actin control bands was calculated. In order to evaluate changes in density from different Western blotting experiments control densitometry was denoted as 100% and differences were accounted as increase percentage of the control. Representative examples of bands obtained are also illustrated. Columns represent the densitometric evaluation of three independent experiments (mean ± SEM). (*p < 0.05 vs Sham proximal or Sham distal respectively).

Figure 2

Acetylation on histone 4 (H4) specific lysine residues 5 (K5) (A), 8 (K8) (B), 12 (K12) (C) and 16 (K16) (D) in a Sham (control) and trinitrobenzene sulfonic acid (TNBS) rat model of colitis. Results were obtained by Western blotting. In order to evaluate changes in density from different western blotting experiments control densitometry was denoted as 100% and differences were accounted as increase percentage of the control. Representative examples of bands obtained are also illustrated. Columns represent the densitometric evaluation of three independent experiments (mean ± SEM). (*p < 0.05 vs Sham proximal or Sham distal respectively).

Figure 3

Acetylation on histones 3 (H3) and 4 (H4) in Lewis and Sprague-Dawley dextran sulfate sodium (5% DSS) treated rats. Tissue samples were obtained from the sigmoid colon of the animals. A: Representative bands of H4 and H3 acetylation as obtained by Western blotting. β-actin levels were measured to ensure equal protein loading. The results are representative of three independent experiments. B, C: Graphical analysis of data Lanes represent: (1) non-DSS treated Lewis rats (control), (2) DSS-treated Lewis rats, (3) non-DSS treated Sprague-Dawley rats (control) (4) DSS-treated Sprague-Dawley rats. Columns represent the mean ± SEM of three independent experiments (*p < 0.05). D: Histone 3 (H3) and histone 4 (H4) localisation in Peyer's patches of dextran sulfate sodium (DSS) treated Lewis rats. H3 is acetylated mainly in the mantle zone and H4 is acetylated throughout the surface of Peyer's patches to both mantle zone and germinal centre cells. In Peyer's patches of untreated animals no acetylation on either histone 3 or 4 was apparent. Micrographs are representative of two individual experiments for each strain. Isotype controls show no staining.

Figure 4

Acetylation on histone 4 (H4) specific lysine residues 5 (K5), 8 (K8), 12 (K12) and 16 (K16) in Lewis and Sprague-Dawley dextran sulfate sodium (5% DSS). A: Representative bands of H4K5, K8, K12 and K16 acetylation. Lanes for Lewis rats represent: non-DSS treated (control) and DSS-treated. Likewise representative bands are illustrated for the Sprague-Dawley rats. Graphical representation of Western blotting data. H4 acetylation of K5 (B), K8 (C), K12 (D) and K16 (E). Columns represent the mean ± SEM (bar) of three independent experiments.

Figure 5

Acetylation on histone 4 (H4) and H4 lysine residues in Crohn's disease. Columns represent the mean ± SEM of three independent experiments. Four biopsies were pooled to obtain sufficient protein for one experiment (50 μg of protein) (*p < 0.05 vs control). Pan acetylation on H4 in Crohn's disease (A). Acetylation on histone 4 (H4) specific lysine residues 5 (K5) (B), K8 (C), K12 (D), and 16 (E), in non-inflamed, inflamed tissue and Peyer's patches of Crohn's disease patients. Results were obtained by Western blotting. Columns represent the mean ± SEM of three independent experiments. (*p < 0.05 vs control, #p < 0.005 vs non-inflamed CD). Representative images of the bands obtained are illustrated.