Intestinal Inflammation is Linked to Hypoacetylation of Histone 3 Lysine 27 and can be Reversed by Valproic Acid Treatment in Inflammatory Bowel Disease Patients.

Benefits from biologic therapy for inflammatory bowel disease (IBD) come at a cost of side effects and antidrug antibodies. This necessitates novel anti-inflammatory therapies.

Histone acetylation is an important epigenetic gene expression regulator. Acetylation of histone-3 lysine-27 (H3K27ac), H3K9ac, and trimethylation of H3K4 (H3K4me3) mark active enhancers. H3K27ac can differentiate active from poised enhancers and is linked to gene expression. Trimethylation of H3K27 leads to gene repression. IBD-associated single-nucleotide polymorphisms overlap with regulatory elements marked by H3K27ac. H3K27ac is reduced in dextran sulfate sodium (DSS)-induced colitis in mice and broad-acting histone deacetylases (HDAC) inhibitors (eg, SAHA and valproic acid [VPA]) restore H3K27ac levels and inhibit inflammatory cytokine production.,

We extend findings from models to patients with IBD to evaluate the therapeutic potential of HDAC inhibitors. We assessed H3K27ac in patients with IBD and tested whether VPA could inhibit inflammatory cytokine production in IBD biopsies cultured ex vivo. VPA was selected because of its safety record in treating neurologic disorders. Methods and patient characteristics are given in the Supplementary Material.

To validate our approach, we confirmed a reduction in the percentage of H3K27ac positive (H3K27ac+) cells in conjunction with the development of inflammation in a DSS model (Figure 1A–C). Moreover, after removal of DSS, H3K27ac+ cells significantly increased during resolution of inflammation (Figure 1C). No significant changes in HDAC mRNA were detected during the inflammatory phase, despite an increase in interleukin (IL) 6 mRNA levels (positive control for DSS-treatment) (Figure 1D). During the resolution of inflammation (Day 17), expression of 4 Hdacs differed from baseline (downregulated, Hdac1, 5, and 10; upregulated, Hdac2) (Figure 1D).

Figure 1

( Representative Masson trichrome and H3K27ac stains. (B) Assessment of trichromatee stains confirmed an increase in inflammation markers post–DSS treatment, which lowered on DSS removal. (C) Conversely, percentage of H3K27ac+ cells decreased following DSS treatment and increased following DSS removal. (D) DSS treatment associated with increased IL6 mRNA levels. DSS did not alter HDAC mRNA levels, although these decreased during recovery following DSS removal. Significant changes: ∗< .05, ∗∗< .01.

A reduction in H3K27ac+ cells was confirmed in actively inflamed IBD biopsies relative to inactive IBD control subjects (Figure 2A and B) and was maintained in a paired subanalysis of the same samples between patient matched active and inactive segments (-16.25%; P = .0243; n = 11). This indicated that confounding factors, including disease duration and medications, could not account for the observed differences. A similar pattern of change was observed in patients with Crohn’s disease (CD) and ulcerative colitis (UC), although the reduction in H3K27ac+ cells in active biopsies was significant only in patients with UC (Figure 2B). As in the DSS model, active disease in patients with IBD was marked by increased IL6 mRNA but was not associated with changes in HDAC mRNA levels (Figure 2C). A subanalysis of patients with UC and CD (Supplementary Figure 1) found increased HDAC 9 in UC consistent with its inflammatory role.

Figure 2

( (C) No changes in HDAC mRNA were observed in IBD biopsies with active disease. (D) Culturing biopsies with an HDAC inhibitor (5 mM VPA) increased the percentage H3K27ac+ cells in IBD biopsies relative to patient-matched biopsies treated with a vehicle control (RPMI medium), and (E–G) decreased inflammatory cytokine mRNA levels and protein production. Significant changes: ∗< .05, ∗∗< .01, ∗∗∗< .001, and ∗∗∗∗< .0001. ELISA, enzyme-linked immunosorbent assay; IFNG, interferon gamma; NS, not significan; TNF, tumor necrosis factor.

Supplementary Figure 1

( Data are expressed as log2 fold-change in active biopsies relative to the mean level of expression in inactive biopsies. Differences between groups were determined using Student t test, assuming equal variance, and the P values for each comparison are given in the table. (B) The expression levels for IL6 and HDAC9 are also graphically represented (∗< .05, ∗∗< .01).

Inhibiting HDAC activity in mucosal IBD biopsies cultured ex vivo with 5 mM VPA (Figure 2D) increased H3K27ac levels and reduced expression of IL6, IL10, IL1B, and IL23 mRNA significantly relative to patient-matched biopsies treated with control media (Figure 2E). Reduced production of IL6, IL10, and IL23 protein into culture media was confirmed (Figure 2F), and a reduction in tumor necrosis factor and interferon gamma was found (Figure 2F). Analysis of IL6 in an expanded cohort again indicated that VPA effects were greater in biopsies from areas with active disease, and in patients with UC (Figure 2G); namely, groups that had the most pronounced reductions in H3K27ac+ cells (Figure 2B).

In biopsy cultures, we cannot discern the primary cellular source of IL6 or other inflammatory cytokines. VPA can suppress inflammation by inducing apoptosis of laminal propria mononuclear cells, and we observed a decrease in BLC3 mRNA and an increase in CASP9 expression in VPA-treated IBD biopsies (Supplementary Figure 2). Although not significant, there was an increase in the percentage of CASP+ cells in VPA-treated biopsies and the proportion of CASP+/CD3+ T cells (Supplementary Figure 2).

Supplementary Figure 2

( Paired Student t tests showed a significant decrease in BCL3 expression, coupled with an increase in CASP9 mRNA levels (D–G). There was also a small increase in the percentage of CASP9+ cells measured using immunofluorescence. This increase was relatively larger in CD3+ T cells. However, these differences did not reach statistical significance. Significant changes: ∗∗∗∗< .0001. N.S, not significant.

Other potentially relevant mechanisms of action for VPA include modulation of acetylation at inflammatory gene promoters and regulation of microRNAs. For example, in a model of colitis-accelerated colon carcinogenesis, DSS treatment increased HDAC activity and decreased H3K27ac levels in the intestine. However, promoters of inflammatory genes, including the IL6 promoter, were hyperacetylated. Conversely, anti-inflammatory treatment with aspirin increased global acetylation levels but reduced H3K27ac levels at the promoter regions of proinflammatory genes. Others have reported that in T cells, HDAC inhibitors promote FOXP3 expression by acetylating its promoter and inducing microRNA signatures associated with regulatory T cells. Thus, reducing IL6/STAT3/IL17 signaling in naive CD4+ T cells and blocking the polarization of Th17 cells. HDAC inhibitors also acetylate nonhistone proteins including STAT1, blocking their phosphorylation and inhibiting proinflammatory signalling. Increasing histone acetylation may alter other histone modifications (eg, H3K27me3), which is altered by DSS treatment.

VPA can have HDAC-independent effects, including regulating DNA methylation leading to gene repression and metabolic changes. Because chemically diverse HDAC inhibitors have similar effects, an HDAC-dependent mechanism seems likely. Indeed, SAHA (vorinostat), which is more potent than VPA in IBD models but currently only used for cutaneous T-cell lymphoma, is being trailed in patients with CD.

Our data identify H3K27ac levels in the mucosa as a potential biomarker of response to HDAC therapy and establish ex vivo biopsy cultures as an alternative screen for HDAC inhibitors. Although further work is needed to elucidate VPA mechanism of action, we highlight the potential to repurpose VPA, a widely used and inexpensive drug, to treat inflammation in IBD.

Supplementary Table 1

Clinical Characteristics of the FFPE Cohort

Patient ID	Age	Gender	CD/UC	Disease extent/Montreal	CRP	Endoscopic evidence active disease	Medications
1	26	F	CD	A2L3B2p	<5	Yes	Steroids
2	37	F	CD	A?L3B3p	9	Yes	None
3	26	F	CD	A1L1B3	52	Yes	Thiopurine
4	38	F	CD	A?L3B3p	25	Yes	Adalimumab and thiopurine
5	19	M	CD	A1L1B2	7	Yes	Adalimumab
9	18	F	CD	A1L3B3	<5	Yes	Steroids
10	23	M	CD	A2L3B2p	35	Yes	Adalimumab and thiopurine
11	36	M	CD	A1L3B2p		Yes	Adalimumab and thiopurine
12	64	F	CD	A3L3B3	53	Yes	Adalimumab and steroids
6	35	F	UC	E3		Yes	None
7	50	F	UC	E3	37	Yes	None
8	34	M	UC	E3	<5	Yes	Infliximab
13	47	M	UC	E1	34	Yes	None
14	14	F	UC	E3	31	Yes	Steroids
15	28	M	UC	E3	9	Yes	Biologics and steroids
16	16	M	UC	E3	10	Yes	Immunomodulator and steroids
17	28	F	UC	E3	32	Yes	Tacrolimus

CD, Crohn’s disease; CRP, C-reactive protein; FFPE, formalin fixed and paraffin embedded; UC, ulcerative colitis.

Supplementary Table 2

Clinical Characteristics of Patients Used for Biopsy Cultures

Sample	Patient	Age	Gender	CD/UC	Disease extent UC/Montreal	CRP	Endoscopic evidence active disease	Medications
cf01	1	44	M	CD	A1L1L4B2	13	Yes	Thiopurine and steroids
cf02	2	20	F	CD	A1L2L4B1	6	Yes	Adalimumab and thiopurine
cf03	3	41	F	CD	A2L2B1p		No	None
cf04	4	25	M	CD	A2L2B1p	<5	Yes	Thiopurine and steroids
cf05	5	55	M	CD	A2L3B1P	<5	Yes	Thiopurine
cf06	6	59	F	UC	E2	<5	Yes	Steroids
cf07	7	59	F	UC	E1	<5	No	None
cf08	8	58	M	UC	E3	<5	Yes	None
cf09	9	68	M	UC	E3	<5	No	None
cf10	10	41	M	UC	E3	<5	Yes	None
cf11	11	35	M	UC	E2	19	Yes	None
cf12	12	37	M	CD	A1L3B2	<5	Yes	None
cf13	13	17	F	CD	A1L3B2	<5	Yes	Thiopurine
cf14	14	40	F	UC	E3	<5	Yes	None
cf15	15	39	F	UC	E3	<5	No	None
cf16	16	21	M	UC	E3	<5	No	Thiopurine and steroids
cf17	17	21	M	UC	E3	<5	Yes	Methotrexate and steroids
cf21	18	50	F	UC	E2	<5	Yes	None
cf23	19	41	M	UC	E3	<5	Yes	Thiopurine
cf24	20	51	F	UC	E1	<5	Yes	None
cf25	21	20	M	CD		16	Yes	Infliximab
cf26	22	21	M	UC	E3	<5	No	None
cf28	23	27	F	CD	A2L3B2p	<5	No	Steroids
cf32	24	59	M	CD	A3L2B1	<5	No	None
cf33	25	40	F	UC	E3	<5	No	None
cf34	26	29	M	CD	A2L1	<5	Yes	Methotrexate
cf36	27	46	F	CD	A1L3B2	7	Yes	None
cf37	28	46	F	CD	A1L3B2	7	Yes	None
cf38	29	43	F	CD	A2L3B2p	<5	Yes	Adalimumab and methotrexate

CD, Crohn’s disease; CRP, C-reactive protein; UC, ulcerative colitis.