Am J Physiol Gastrointest Liver Physiol 306: G594–G605, 2014. First published February 13, 2014; doi:10.1152/ajpgi.00393.2013.

The acetylome regulators Hdac1 and Hdac2 differently modulate intestinal epithelial cell dependent homeostatic responses in experimental colitis Naomie Turgeon, Julie Moore Gagné, Mylène Blais, Fernand-Pierre Gendron, François Boudreau, and Claude Asselin Département d’anatomie et biologie cellulaire, Faculté de médecine et des sciences de la santé, Pavillon de recherche appliquée sur le cancer, Université de Sherbrooke, Sherbrooke, Québec, Canada Submitted 19 November 2013; accepted in final form 6 February 2014

Turgeon N, Gagné JM, Blais M, Gendron F, Boudreau F, Asselin C. The acetylome regulators Hdac1 and Hdac2 differently modulate intestinal epithelial cell dependent homeostatic responses in experimental colitis. Am J Physiol Gastrointest Liver Physiol 306: G594–G605, 2014. First published February 13, 2014; doi:10.1152/ajpgi.00393.2013.—Histone deacetylases (Hdac) remove acetyl groups from proteins, influencing global and specific gene expression. Hdacs control inflammation, as shown by Hdac inhibitor-dependent protection from dextran sulfate sodium (DSS)-induced murine colitis. Although tissue-specific Hdac knockouts show redundant and specific functions, little is known of their intestinal epithelial cell (IEC) role. We have shown previously that dual Hdac1/Hdac2 IEC-specific loss disrupts cell proliferation and determination, with decreased secretory cell numbers and altered barrier function. We thus investigated how compound Hdac1/Hdac2 or Hdac2 IEC-specific deficiency alters the inflammatory response. Floxed Hdac1 and Hdac2 and villin-Cre mice were interbred. Compound Hdac1/Hdac2 IEC-deficient mice showed chronic basal inflammation, with increased basal disease activity index (DAI) and deregulated Reg gene colonic expression. DSS-treated dual Hdac1/ Hdac2 IEC-deficient mice displayed increased DAI, histological score, intestinal permeability, and inflammatory gene expression. In contrast to double knockouts, Hdac2 IEC-specific loss did not affect IEC determination and growth, nor result in chronic inflammation. However, Hdac2 disruption protected against DSS colitis, as shown by decreased DAI, intestinal permeability and caspase-3 cleavage. Hdac2 IEC-specific deficient mice displayed increased expression of IEC gene subsets, such as colonic antimicrobial Reg3b and Reg3g mRNAs, and decreased expression of immune cell function-related genes. Our data show that Hdac1 and Hdac2 are essential IEC homeostasis regulators. IEC-specific Hdac1 and Hdac2 may act as epigenetic sensors and transmitters of environmental cues and regulate IEC-mediated mucosal homeostatic and inflammatory responses. Different levels of IEC Hdac activity may lead to positive or negative outcomes on intestinal homeostasis during inflammation. Hdac1; Hdac2; inflammation; intestinal epithelial cell; colitis; DSS INFLAMMATORY BOWEL DISEASES (IBDs), namely Crohn’s disease and ulcerative colitis, occur in genetically predisposed patients in response to altered immunity against environmental luminal factors, including a dysbiotic microbiota (32, 39). The intestinal epithelium is central to intestinal homeostasis by its position between luminal microbiota and the mucosal immune system, and by its capacity to sense and to respond to signals emanating from both (16, 26). In addition, intestinal epithelial cells (IEC) form a physical barrier conferred by tight junctions (61) as well as a chemical barrier sustained by production of a

Address for reprint requests and other correspondence: C. Asselin, Département d’anatomie et biologie cellulaire, Faculté de médecine et des sciences de la santé, Université de Sherbrooke, Sherbrooke, Québec, Canada J1E 4K8 (e-mail: [email protected]). G594

mucus layer by goblet cells (34) and of antimicrobial proteins by Paneth cells, goblet cells, and enterocytes (19). Although many signaling pathways, including the Wnt and the Notch pathways (43, 64), regulate IEC determination and growth, little is known of the role of epigenetic modifications in the control of intestinal homeostasis. Lysine acetylation is one epigenetic posttranslational modification occurring on histones and nonhistone proteins as well, through opposite actions of histone acetyltransferases and histone deacetylases (Hdac) (68), and forming the acetylome. In addition to inducing relaxed histone interactions with DNA, lysine acetylation recruits bromodomain-containing proteins, leading to alterations in gene expression (47). Of the four different Hdac classes, class I includes Hdac1 and Hdac2, two similar enzymes that form homo- or heterodimers in multiprotein corepressor complexes such as Sin3, CoREST, and NuRD (12, 24). Whereas Hdac1 deficiency in mice is developmentally lethal as a result of decreased proliferation (35), Hdac2 deleted mice die from heart alterations (41). Conditional deletion of either Hdac1 or Hdac2 in various adult tissues does not lead to obvious phenotypes as opposed to dual deletion. For example, double Hdac1 and Hdac2 deletion in mouse embryo fibroblasts arrests adipocyte differentiation (21). Specific deletion of both genes in the epidermis results in epidermal differentiation and proliferation defects, in contrast to single knockouts (37), and deletion specifically in B lymphocytes blunts differentiation (67). Thus Hdac1 and Hdac2 exhibit both specific and equivalent functions, by regulating cell proliferation and cell differentiation, among others (22, 31). In recent years, global and class-selective HDAC inhibitors have been considered for treatment of various diseases, including cancer. Indeed, HDAC inhibitors (HDACi) induce apoptosis and cell cycle arrest and inhibit DNA repair in many cancer cell types, including colon cancer cells. Antineoplastic HDACi are currently used in the clinic against acute T cell lymphomas (3). In addition, mouse colitis models suggest that HDACi are anti-inflammatory, in part through their action as regulator of T-regulatory (T-reg) cell activation and development (6, 17). However, little is known of the role of HDACi targets, namely Hdac1 and Hdac2, and acetylation in IEC homeostasis and differentiation. We have previously reported that dual Hdac1 and Hdac2 IEC-specific deletion leads to increased proliferation and determination defects, with loss of goblet and Paneth secretory cells, altered antimicrobial gene expression, and increased Notch activation. This disturbed homeostasis is accompanied by increased intestinal permeability and deregulated inflammatory gene expression. Overall, normal barrier functions are replaced by novel protective barriers to control inflammation in mutant mice (60). However, the exact role of

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HDAC1 AND HDAC2 MODULATE INTESTINAL INFLAMMATORY RESPONSES

Hdac1 and Hdac2 during the intestinal inflammatory response is not known. We thus determined the response of compound Hdac1/Hdac2 and single Hdac2 conditional IEC-specific depleted mice to intestinal inflammation in the murine model of DSS-induced colitis. Here, we show that, whereas compound Hdac1/Hdac2 mutant mice displayed increased sensitivity to DSS-induced colitis, Hdac2 mutant mice exhibited reduced inflammatory responses. These results demonstrate that Hdac1 and Hdac2 are essential IEC homeostasis regulators and that differential Hdac activity levels may lead to positive or negative outcomes in the regulation of IEC homeostasis during inflammation. MATERIALS AND METHODS

Mice. Floxed Hdac1 and Hdac2, or Hdac2 mice (Dr. E. N. Olson, University of Texas Southwestern Medical Center, Dallas, TX) (41) were crossed with villin-Cre transgenic mice for IEC-specific gene deletion (38). Mouse genotypes were determined by PCR with genomic DNA recovered with the Spin Doctor genomic DNA kit (Gerard Biotech, Oxford, OH). Mice were raised in a transgenic mouse facility. Experiments were approved by the “Comité facultaire de protection des animaux” of the Faculty of Medicine and Health Sciences of the Université de Sherbrooke (protocol 074-12B). Histological analysis and immunofluorescence. We stained 5-␮m sections from paraffin-embedded colon tissues fixed in 4% paraformaldehyde (2) with hematoxylin and eosin for histological analysis. For immunofluorescence experiments, sections hydrated with graded ethanol solutions containing 100, 95, 80, and 70% xylene were boiled for 6 min in 10 mM citric acid. After blocking with a 2% BSA, 0.1% Triton X-100 and 10% goat serum supplemented PBS solution for 30 min, rabbit antibodies against Hdac1 and Hdac2 (1:600, Abcam, Toronto, ON, Canada) were added. Primary antibodies were recognized with fluorescein-coupled secondary antibodies (Vector Laboratories, Burlington, ON, Canada) incubated for 2 h at room temperature. DSS colitis model. Colitis was induced in 3- to 5-mo-old wild-type, compound Hdac1/Hdac2 and Hdac2 conditionally mutated mice by adding 3.5% dextran sulfate sodium (DSS) (molecular mass 40 –50 kDa) to the drinking water for 6 or 7 days ad libitum. Clinical assessment of colitis intensity was monitored daily by using a disease activity index (DAI) based on body weight, colon length, stool consistency, and the presence of fecal blood (9). Paraffin-embedded colon sections from DSS-treated mice were stained with hematoxylin and eosin to assess the severity of colonic damage on the basis of published histological criteria, including extent of inflammation, depth of injury, and crypt damage. Colon and small intestine weight and length were also measured at the end of the treatment. In vivo permeability assay. We introduced 60 mg/100 g body wt of 4-kDa fluorescein isothiocyanate (FITC)-labeled dextran (Sigma-Aldrich, Oakville, ON, Canada) by gavage. After 3 h, mice were killed and blood was recovered. FITC serum concentrations were determined with a RF-5301 PC spectrofluorophotometer (490/525 nm) (Shimadzu Scientific Instruments, Columbia, MD). For wild-type or mutant mice, a ratio between the fluorescence observed without or with DSS treatment was calculated to determine the increase in permeability after DSS treatment. Western blot analysis. Wild-type, compound Hdac1/Hdac2, and Hdac2 IEC-specific mutated colons were recovered in Laemmli buffer (Tissuelyser, Qiagen), or in a buffer containing 50 mM Tris·HCl pH 7.5, 15 mM NaCl, 1% Triton X-100, 5 mM EDTA, 5% glycerol, 40 mM ␤-glycerophosphate supplemented with protease inhibitors, 50 mM NaF and 200 mM Na3VO4. Protein concentrations were measured by BCA (Pierce BCA Protein Assay Kit, Thermo Scientific, Rockford, IL) or Bradford (Bio-Rad Protein Assay, Bio-Rad Laboratories, Mississauga, ON, Canada)

G595

methods. Thirty micrograms of total protein extracts were separated on a 10 or 15% SDS-polyacrylamide gel and transferred on a PVDF membrane (Roche Molecular Biochemicals, Laval, QC, Canada). Membranes were incubated for 1 h at room temperature with primary rabbit anti-Hdac1 and anti-Hdac2 (Abcam), sheep anti-Reg3b (R&D Systems, Minneapolis, MN), rabbit anti-cleaved caspase 3 (New England BioLabs, Burlington, ON, Canada), and mouse anti-actin (Millipore, Billerica, MA). Band intensity quantification was performed with the Image J software (developed by the U.S. National Institutes of Health). Cytokine and chemokine measurements. Wild-type and compound Hdac1/Hdac2 IEC-specific mutated distal colons were homogenized in PBS with protease inhibitors and 1% Triton X-100, and cellular debris was removed by centrifugation. Relative levels of 40 cytokines/ chemokines were measured with a Proteome Profiler Array (Mouse Cytokine Array Panel A, ARY006, R&D Systems), following the manufacturer protocol. Fluorescence intensity from two independent experiments was revealed by autoradiography. Pixel density of each spot was measured with Image J software. Proteins evaluated include Cxcl13, C5a, G-CSF, GM-CSF, Ccl1, CD54, Ifn␥, Il-1␣, Il-1␤, Il-1ra, Il-2, Il-3, Il-4, Il-5, Il-6, Il-7, Il-10, Il-13, Il-12p70, Il-16, Il-17, Il-23, Il-27, Cxcl10, Cxcl11, Cxcl1, M-CSF, Ccl2, Ccl12, Cxcl9, Ccl3, Ccl4, Cxcl2, Ccl5, Cxcl12, Ccl7, TIMP-1, Tnf␣, and TREM-1. RNA expression analysis. Total RNAs from colons of wild-type, compound Hdac1/Hdac2 and Hdac2 IEC-specific knockout mice were purified with Totally RNA kit (Life Technologies, Burlington, ON, Canada) and RNeasy Mini kit (Qiagen). cDNAs were synthesized from 1 ␮g of RNA, with oligo(dT15) and Superscript II reverse transcriptase (Life Technologies). For quantitative PCR (qPCR) analysis, 2 or 10 ng of cDNAs were used for amplification with the Brilliant III Ultra-fast SYBR Green QPCR Master Mix (Agilent Technologies, Mississauga, ON, Canada) and specific gene primers (Table 1). cDNA amplification started with a 95°C cycle for 10 min, followed by 40 cycles of 10 s at 95°C, 10 s at 60°C, and 20 s at 72°C. Porphobilinogen deaminase (Pbgd) amplification was performed to determine relative RNA amounts. Microarray analysis. Total RNAs from colons of three wild-type control and three Hdac2 IEC-specific knockout mice were isolated with the RNeasy kit (Qiagen). cDNA preparation and microarray assay were performed at the Microarray platform of the McGill University and Génome Québec Innovation Centre. An Affimetrix GeneChip mouse genome 430 2.0, displaying over 34,000 murine gene sequences, was used for hybridization. Data analysis, normalization average difference, and expression measurements were subsequently completed with Flexarray software version 1.6.1. Background correction and normalization were assessed with a multiarray average (RMA) algorithm. Significant statistical differences were calculated with Welch’s t-test, with the cutoff for statistical significance set to a P value below 0.05. Classification of genes according to their Gene Ontology biological processes was performed with the Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.7 (http://david.abcc.ncifcrf.gov/) (14) and the ToppGene suite for functional gene enrichment analysis and candidate gene selection (http:// toppgene.cchmc.org/) (8). Both analysis tools gave similar results regarding biological processes, and the highest gene count and lowest P value were selected. Microarray data obtained from Hdac2 IEC-specific deleted mice were compared with already published microarray data from compound Hdac1/Hdac2 IECspecific conditional mice (60). The microarray results for Hdac2 IEC-specific deleted mice have been deposited in the Gene Expression Omnibus database (GSE54785). Statistical analysis. All data were expressed as means ⫾ SE. Groups were compared via the Student’s t-test (unpaired), one-way ANOVA with Tukey multiple-comparisons test, or two-way ANOVA with Sidak multiple-comparisons test, unless otherwise indicated (GraphPad Prism 6, GraphPad Software, San Diego).

AJP-Gastrointest Liver Physiol • doi:10.1152/ajpgi.00393.2013 • www.ajpgi.org

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Table 1. Oligonucleotides used for quantitative PCR analysis Gene

Up

Down

Ang 4 Reg3␤ Reg3␥ TNF␣ TGF␤ Cxcl1 Ccl2 Ccl4 Ccl5 CD11a CD11b CD11c Cd68 MRC1 PBGD

5=-GGTTGTGATTCCTCCAACTCTG-3= 5=-ACTCCCTGAAGAATATACCCTCC-3= 5=-ATGCTTCCCCGTATAACCATCA-3= 5=-ACGGCATGGATCTCAAAGAC-3= 5=-GACTCTCCACCTGCAAGACCA-3= 5=-CACTGCACCCAAACCGAAGT-3= 5=-CTTCTGGGCCTGCTGTTCA-3= 5=-TCTGCCCTCTCTCTCCTCTT-3= 5=-GAAGATCTCTGCAGCTGCCCT-3= 5=-ATGCACCAAGTACAAAGTCAGC-3= 5=-CTGAACATCCCATGACCTTCC-3= 5=-ACACAGTGTGCTCCAGTATGA-3= 5=-TGGACAGCTTACCTTTGGATTCA-3= 5=-CCCAAGGGCTCTTCTAAAGCA-3= 5=-CCTCCTGGCTTTACTATTGGA-3=

5=-CTGAAGTTTTCTCCATAAGGGCT-3= 5=-CGCTATTGAGCACAGATACGAG-3= 5=-GGCCATATCTGCATCATACCAG-3= 5=-GTGGGTGAGGAGCACGTAGT-3= 5=-GGGACTGGCGAGCCTTAGTT-3= 5=-GGACAATTTTCTGAACCAAGG-3= 5=-CCAGCCTACTCATTGGGATCA-3= 5=-GATCTGTCTGCCTCTTTTGG-3= 5=-GCTCATCTCCAAATAGTTGA-3= 5=-TTGGTCGAACTCAGGATTAGC-3= 5=-GCCCAAGGACATATTCACAGC-3= 5=-GCCCAGGGATATGTTCACAGC-3= 5=-TGTATTCCACCGCCATGTAGTC-3= 5=-CGCCGGCACCTATCACA-3= 5=-TAGCTGAGCCACTCTCCTCAG-3=

RESULTS

to wild-type mice (Fig. 1A). To confirm colonic gene expression changes in compound Hdac1/Hdac2 deficient mice, we verified by qPCR the expression of selected genes whose expression was decreased or increased, as assessed by microarray analysis (60). We confirmed decreased expression of the Ang4 bactericidal peptide gene, expressed in Paneth and goblet cells (5, 28) (Fig. 1B), as well as increased expression of antimicrobial C-type lectin genes Reg3␤ and Reg3␥ (45), expressed in enterocytes and in Paneth and goblet cells (5) (Fig. 1C). Western blot analysis confirmed increased expression of Reg3␤ proteins in compound IEC-specific mutant colon, as opposed to wild-type colons (Fig. 1D). Thus Hdac1/Hdac2 deficiency in IECs leads to basal chronic inflammation.

Conditional intestinal epithelial Hdac1/Hdac2 loss leads to basal inflammatory defects. We have previously found that dual intestinal epithelial specific gene deletion of Hdac1 and Hdac2 disrupts IEC growth and differentiation, with decreases in jejunal Paneth and goblet cell numbers. In the colon, although goblet cell numbers are decreased, increased expression of proximal enterocyte markers is observed. These changes result in increased immune cell infiltration and intestinal permeability, as well as inflammatory gene expression deregulation (60). On the basis of these data, we observed an increased DAI in 4-mo- and 1-yr-old Hdac1/Hdac2 mutant mice, with decreased body weight and shortened colon length, as well as looser stools, as opposed

A

C Wt

** ***

DAI

2

1

0

Four months

One year

B Relative mRNA expression

Fig. 1. Conditional intestinal epithelial Hdac1/Hdac2 (Hdac1/Hdac2 ⌬IEC) loss leads to basal inflammatory defects. Disease activity index (DAI), based on weight, colon length, presence of fecal blood, and stool consistency, of 3- to 5-mo-old (n ⫽ 18 –22) and 1-yr-old (n ⫽ 9 –11) wild-type (Wt) and Hdac1/ Hdac2 ⌬IEC mice was measured (A). Total RNAs were isolated from Wt and compound Hdac1/Hdac2 IEC-specific deficient distal colons. Relative mRNA expression levels of intestinal epithelial cell (IEC)regulated genes, namely Ang4 (B) (n ⫽ 5), and Reg3b and Reg3g (C) (n ⫽ 5) were determined by quantitative PCR (qPCR), with Pbgd as a control. Results represent means ⫾ SE (Student’s t-test, *P ⱕ 0.05; **P ⱕ 0.01; ***P ⱕ 0.005). D: total protein extracts from 3- to 5-mo-old Wt or Hdac1/Hdac2 ⌬IEC distal colons were separated on a 10% SDSPAGE gel, transferred to a PVDF membrane, and analyzed by Western blot for expression of Reg3␤, with actin as a loading control (n ⫽ 4-4).

Wt

3000

Hdac1/Hdac2ΔIEC

Relative mRNA expression

3

*

Hdac1/Hdac2ΔIEC

2000

1000

* 0

Reg3β

Reg3γ

D 1.5

Wt

Wt

Hdac1/Hdac2ΔIEC

1.0

0.5

***

0.0

Ang 4 AJP-Gastrointest Liver Physiol • doi:10.1152/ajpgi.00393.2013 • www.ajpgi.org

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Reg3 β Actin

HDAC1 AND HDAC2 MODULATE INTESTINAL INFLAMMATORY RESPONSES

Mice with conditional intestinal epithelial Hdac1/Hdac2 loss show increased sensitivity to DSS-induced colitis. To characterize the possible associations between Hdac1/Hdac2 and the control of intestinal homeostasis during inflammation, mice were given DSS to induce colitis. Colitis symptoms were aggravated in mutant mice, as opposed to wild-type mice. Indeed, mutant mice exhibited 30% weight loss after 6 days (Fig. 2A), with increased mortality (Fig. 2B). Because of this very rapid weight loss, mutant mice were killed 6 days after beginning the DSS treatment. We then measured the inflammation score with a DAI based on body weight, presence of fecal blood, stool consistency and colon length. The data show that, indeed, compound mutant mice displayed increased colitis manifestations (Fig. 2C). Our previous data have shown increased basal permeability in mutant mice (60). To determine whether compound Hdac1/Hdac2 IEC-specific deletion increased permeability defects in response to DSS, mice were given 4-kDa FITC-labeled dextran by gavage after 6 days of DSS treatment. Compared with wild-type mice, results showed a striking increase in intestinal permeability in mutant mice, as assessed by fluorescence intensity recovered in blood (Fig. 2D). We then examined hematoxylin and eosin-stained colon sections for histological evaluation. In contrast to wild-type murine colon, dual Hdac1/Hdac2 colonic mutant epithelia contained larger disorganized cells, with disordered, enlarged, and unstained nuclei, expanded crypts, and increased immune cell numbers (Fig. 3A). DSS-treated colons from wild-type mice showed crypt abscesses, epithelial architectural perturbations, and increased immune cell recruitment, whereas DSStreated double Hdac1/Hdac2 deficient murine colons displayed complete surface epithelial loss, transmural inflammation, and increased immune cell infiltration (Fig. 3B). Histological colitis score, based on inflammation intensity and extent, as well as

Wt

**

Body Weight (%)

100

****

Hdac1/Hdac2ΔIEC

****

****

90

80

70

60

0

1

2

3

4

5

crypt damage, was significantly increased in mutant murine colons (Fig. 3C). Thus Hdac1/Hdac2 deficiency in IECs not only leads to chronic inflammation but also leads to increased sensitivity to DSS-induced colitis. DSS-treated Hdac1/Hdac2 IEC-specific deficient mice display increased inflammatory gene expression. We then verified cytokine and chemokine expression with a protein array, in colonic protein extracts. Compound Hdac1/Hdac2 IECdeficient mice displayed increased expression of C5, CD54, Il-16, Cxcl1, M-CSF, Ccl2, Cxcl9, Ccl5, and TIMP-1, as opposed to control mice (Fig. 4, A and B), confirming the increased inflammatory gene expression observed previously by microarray analysis (60). DSS treatment led to increased protein levels of G-CSF, GM-CSF, Ccl1, CD54, Il-1␤, Il-6, Cxcl10, Cxcl1, Ccl2, Cxcl9, Ccl4, Cxcl2, Ccl5, Cxcl12, TIMP-1 and TREM-1, compared with DSS-treated wild-type mice (Fig. 4, C and D). qPCR analysis showed increased expression of cytokines such as TNF␣ and TGF␤ (Fig. 5A), chemokines (Fig. 5B), and immune cell markers (Fig. 5, C and D) in DSS-treated double Hdac1/Hdac2 IEC-deficient mice, as opposed to DSS-treated wild-type mice. Thus, in addition to basal deregulation of the inflammatory response, compound Hdac1/Hdac2 mutant mice show an increased inflammatory response following DSSinduced colitis. Mice with conditional intestinal epithelial Hdac2 loss show decreased sensitivity to DSS-induced colitis. Phenotypic analysis of compound Hdac1/Hdac2 IEC-specific deficient mice has shown increased IEC proliferation and migration, increased Notch activation correlating with decreased numbers of Paneth and goblet secretory cells, defects in tissue structure correlating with permeability defects, and altered inflammatory gene expression patterns (60). Intriguingly, mutant mice survive for more than a year, suggesting the generation of unusual

C Wt

Disease activity index (DAI)

A

25

Hdac1/Hdac2ΔIEC

****

20 15

10 5

0

6

Days

B Hdac1/Hdac2ΔIEC

Survival (%)

100

Ratio of fluorescence intensity (DSS/water)

D Wt

105

60

90

*

Wt Hdac1/Hdac2ΔIEC

40

95

Fig. 2. Mice with Hdac1/Hdac2 ⌬IEC loss show increased sensitivity to DSS-induced colitis. Mice were given water without or with 3.5% dextran sulfate sodium (DSS) ad libitum. Wt and Hdac1/Hdac2 ⌬IEC % mouse body weight (n ⫽ 9 –15) (A) (2-way ANOVA, **P ⱕ 0.01; ****P ⱕ 0.001) and survival (B) (n ⫽ 20 –29) from 0 to 6 days of DSS treatment, as well as DAI (C) (n ⫽ 20 –26), were measured. Results represent means ⫾ SE. D: intestinal permeability of DSS-treated mice was evaluated by measuring fluorescence intensity in blood recovered 3 h after gavage of 4-kDa FITC-labeled dextran (n ⫽ 7– 8), with a RF-5301PC spectrofluorometer (Shimadzu Scientific Instruments, Columbia, MD). Results represent means ⫾ SE (Student’s t-test, *P ⱕ 0.05; ****P ⱕ 0.001).

20

85

80

0

1

2

3

4

5

6

G597

0

Days AJP-Gastrointest Liver Physiol • doi:10.1152/ajpgi.00393.2013 • www.ajpgi.org

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A

C Wt

Hdac1/Hdac2ΔIEC

Histological characterization

15

10

** Wt Hdac1/Hdac2ΔIEC

5

Fig. 3. Hdac1/Hdac2 ⌬IEC loss leads to increased histological inflammation in response to DSS treatment. Proximal colon sections (A) or distal colon sections from 4-mo-old Wt and Hdac1/Hdac2 ⌬IEC mice treated without or with DSS (B) were stained with hematoxylin and eosin. Magnification: ⫻20 (A) or ⫻10 (B). C: histological inflammation of colon sections of DSS-treated 3- to 5-mo-old Wt and Hdac1/Hdac2 ⌬IEC was determined by measuring inflammation intensity and extent, as well as crypt damage (n ⫽ 4 – 6). Results represent means ⫾ SE (Student’s t-test, **P ⱕ 0.01).

0

B

Wt

Hdac1/Hdac2ΔIEC

Water

DSS

inflammatory regulatory pathways to replace protective IEC barrier functions, notably from Paneth and goblet cells. For example, Reg bactericidal lectin expression is increased in mutant murine distal colon (60). We thus verified whether loss of one Hdac could reveal pro- or anti-inflammatory actions. Hdac2 floxed mice (41) were crossed with villin-cre transgenic mice (38) to ensure IEC-specific Hdac2 deletion. Hdac2 depletion was confirmed by Western blot analysis of IEC protein extracts (Fig. 6A) and by immunofluorescence (data not shown). As opposed to compound Hdac1/Hdac2 mutant mice, Hdac2-deficient IEC-specific jejunal and colonic epithelial architecture was normal, as assessed by hematoxylin and eosin staining (data not shown). Hdac2 deletion did not affect IEC proliferation, as determined by in vivo BrdU labeling, nor Paneth and goblet cell numbers (data not shown). However, Hdac2 IEC-specific deficient mice showed decreased susceptibility to DSS-induced colitis. Indeed, DSS-treated wild-type mice lost more weight, starting at day 5, as opposed to Hdac2 IEC-deficient mice (Fig. 6B). Accordingly, a higher DAI in DSS-treated wild-type mice was observed in contrast to mutant mice (Fig. 6C). This was confirmed by decreased histological colitis scores in DSS-treated mutant colons, as opposed to wild-type colons (Fig. 6D). DSS treatment led to increased intestinal permeability in DSS-treated wild-type mice compared with nontreated mice, as well as in DSS-treated Hdac2 IEC-deficient mice, as assessed after gavage of 4-kDa FITCdextran. However, the level of intestinal permeability was

much lower in DSS-treated mutant mice (Fig. 6E). Finally, Western blot analysis showed that DSS-treated Hdac2 IECspecific deficient mice displayed decreased caspase-3 cleavage, as opposed to DSS-treated wild-type mice, suggesting a reduction in DSS colitis-dependent activation of cell death pathways (Fig. 6F). Thus single Hdac2 deficiency protects from DSSmediated colitis symptoms. Conditional intestinal epithelial Hdac2 loss leads to differential regulation of a subset of inflammatory genes. We then assessed gene expression patterns by microarray analysis with total RNAs isolated from wild-type and Hdac2 IEC-specific deficient murine colons. Although expression of over 58 genes was significantly increased more than twofold [(log2) ⬎ 1], expression of 95 genes was decreased more than twofold [(log2) ⬍ ⫺1], as opposed to respectively 434 genes increased and 352 genes decreased in Hdac1/Hdac2 IEC-specific double knockout (60). Comparison of the expression levels of Hdac2 and Hdac1/Hdac2-regulated genes suggested that, whereas some genes show the same expression pattern, either positive or negative, such as Reg3b, with a log2 of 3.54 and 7.07, respectively, in Hdac2 and Hdac1/Hdac2 mutants, the expression of other genes shows a specific pattern (Supplemental Table S1; supplemental material for this article is available online at the Journal website). Functional Gene Ontology annotations with the ToppGene suite identified strongly enriched biological processes, including Regulation of immune system process and of immune response, as highly significantly

AJP-Gastrointest Liver Physiol • doi:10.1152/ajpgi.00393.2013 • www.ajpgi.org

HDAC1 AND HDAC2 MODULATE INTESTINAL INFLAMMATORY RESPONSES

A

C 1

Wt

9

Hdac1/Hdac2 Δ IEC

2 4

5

6

7

3 8

1-C5 2-CD54 3-Il-16 4-Cxcl1 5-M-CSF 6-Ccl2 7-Cxcl9 8-Ccl5 9-TIMP-1

1

Wt DSS

2 6 16

3

4

9

10

5 11

12

13

14

D

3.0 2.5 2.0 1.5 1.0 1.0

Wt Hdac1/Hdac2 Δ IEC

0.5

Mean Pixel Density

Mean Pixel Density

15

8

Hdac1/Hdac2 Δ IEC DSS

B

4

Mean Pixel Density

Wt DSS Hdac1/Hdac2 Δ IEC DSS

3 2 1 0

C5 CD54 Il-16 Cxcl1 M-CSF Ccl2 Cxcl9 Ccl5 TIMP-1

Mean Pixel Density

0.0

7

1-G-CSF 2-GM-CSF 3-Ccl1 4-CD54 5-Il-1β 6-Il-6 7-Cxcl10 8-Cxcl1 9-Ccl2 10-Cxcl9 11-Ccl4 12-Cxcl2 13-Ccl5 14-Cxcl12 15-TIMP-1 16-TREM-1

Cxcl1 Cxcl2 Cxcl9 Cxcl10 Cxcl12

5

Wt DSS Hdac1/Hdac2 Δ IEC DSS

4 3 2 1 0

Ccl1

Ccl2

25

Ccl4

Ccl5

Il-1β

Wt DSS Hdac1/Hdac2 Δ IEC DSS

20 15 10 5 0

Il-6

G599

Fig. 4. Hdac1/Hdac2 ⌬IEC loss leads to increased colonic protein expression of a subset of cytokines and chemokines in response to DSS treatment. Total protein extracts from colons of 3- to 5-mo-old Wt and Hdac1/ Hdac2 ⌬IEC mice treated without or with DSS were prepared and cytokine secretion was measured with a cytokine array. A: data from Wt and Hdac1/Hdac2 ⌬IEC colon protein extracts represent a 5-min exposure to X-ray film. Cytokine and chemokine signals displaying intensity variations between Wt and Hdac1/Hdac2 ⌬IEC colons are boxed and are indicated as C5 (1), CD54 (2), Il-16 (3), Cxcl1 (4), M-CSF (5), Ccl2 (6), Cxcl9 (7), Ccl5 (8), and TIMP-1 (9). B: histogram, from results obtained with untreated Wt and Hdac1/Hdac2 ⌬IEC colon protein extracts, shows a representative experiment of 2 independent experiments, measured by Image J software (mean pixel density). C: data from DSS-treated Wt and Hdac1/Hdac2 ⌬IEC colon protein extracts represent a 5-min exposure to X-ray film. Cytokine and chemokine signals displaying intensity variations between DSS-treated Wt and Hdac1/Hdac2 ⌬IEC colons are boxed and are indicated as G-CSF (1), GM-CSF (2), Ccl1 (3), CD54 (4), Il-1␤ (5), Il-6 (6), Cxcl10 (7), Cxcl1 (8), Ccl2 (9), Cxcl9 (10), Ccl4 (11), Cxcl2 (12), Ccl5 (13), Cxcl12 (14), TIMP-1 (15), TREM-1 (16). D: histograms, from results obtained with DSS-treated Wt and Hdac1/Hdac2 ⌬IEC colon protein extracts, represent the mean fluorescence intensity of a duplicate experiment, measured by Image J software (mean pixel density). Of note, DSS treatment of Wt mice led to C5, CD54, Il-1␣, Il-1␤, Il-6, Il16, Cxcl10, Cxcl1, M-CSF, Ccl2, Ccl12, Cxcl9, Ccl3, Cxcl2, Ccl5, TIMP-1, Tnf␣, and TREM-1 increased signals (compare A and C).

GM-CSF G-CSF CD54 TIMP-1TREM-1

downregulated in Hdac2 IEC-specific deficient mice (P value: 2.402E⫺12, gene count: 21), and categories related to lipid metabolic processes, as significantly increased (Supplemental Table S2). The volcano plot of mutant Hdac2 gene expression shows the mostly decreased expression pattern of immune response related genes (Fig. 7). Analysis of the upregulated genes suggests enrichment for IEC-expressed genes, such as Reg genes. Indeed, colonic expression of antibacterial lectin genes Reg3b and Reg3g, whose basal expression is increased in compound Hdac1/Hdac2 mutant mice (60), is enhanced in Hdac2 IEC-deficient mice, as shown by qPCR analysis (Fig. 8). These results suggest that Hdac2 IEC-specific deletion leads to changes in the expression of a subset of genes that could play a protective role against colitis. DISCUSSION

We have previously observed that dual IEC-specific Hdac1/ Hdac2 deficiency leads to alterations of both physical and chemical barrier function, as well as secretory Paneth and goblet cell loss, and to dysregulated inflammatory gene expression (60). Here, we show that compound Hdac1/Hdac2 IECdeficient mice indeed display chronic inflammation with in-

creased DAI and altered colonic protein expression of a subset of inflammatory genes, including TIMP-1, an inhibitor of metalloproteinases (42); Cxcl9, a T cell chemoattractant interacting with the Cxcr3 receptor (55); and Il-16, a chemoattractant for CD4 expressing T lymphocytes and mononuclear cells (10). Of note, TIMP-1, Cxcl9, and Il-16 expression is increased in IBD patients (18, 40, 51). Although increased expression of inflammatory genes following altered barrier function and increased interaction of the mucosa with the gut microbiota creates a chronic inflammatory milieu, a novel homeostatic equilibrium in mutant Hdac1/Hdac2 IEC-deficient mice is achieved. Indeed, mutant mice live for more than a year without increased death. Thus novel homeostatic anti-inflammatory signals may be induced. One of the anti-inflammatory proteins induced is Reg3g, an antibacterial C-type lectin (45) that segregates the microbiota from the mucosal intestinal surface (62) and displays bactericidal activities selective for gram-positive bacteria (7). Reg3g is induced in IBD (44, 63). However, this novel homeostatic equilibrium is insufficient to control DSS-induced colitis. Indeed, as opposed to wild-type mice, Hdac1/Hdac2 IEC-specific mutant mice show an increased inflammatory response during colitis, as observed by

AJP-Gastrointest Liver Physiol • doi:10.1152/ajpgi.00393.2013 • www.ajpgi.org

HDAC1 AND HDAC2 MODULATE INTESTINAL INFLAMMATORY RESPONSES

A

D

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Fig. 5. Hdac1/Hdac2 ⌬IEC loss leads to differential regulation of colonic inflammatory gene mRNA expression in response to DSS treatment. Total RNAs were isolated from Wt and dual Hdac1/Hdac2 IEC-specific deficient colons. Relative expression levels of inflammatory genes, including cytokines such as TNF␣ and TGF␤ (A); chemokines such as Cxcl1, Ccl2, Ccl4, and Ccl5 (B); and lymphocyte/macrophage markers such as CD11a, CD11b, CD11c, Cd68 (C) and MRC1 (D) were determined by qPCR, with Pbgd as a control. The results shown are normalized with untreated Wt mice. Results represent means ⫾ SE (1-way ANOVA, *P ⱕ 0.05; **P ⱕ 0.01; ***P ⱕ 0.005; ****P ⱕ 0.001).

****

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augmented histological damage and inflammatory gene expression, resulting in early death. Intriguingly, the anti-inflammatory Tgf␤ cytokine, as well as other proinflammatory cytokines, are much increased in DSS-treated IEC-specific Hdac1/ Hdac2 mutant mice. Our previous results have shown increased basal levels of Tgf␤ and T lymphocyte CD4 mRNAs in the compound Hdac1/Hdac2 mutant mice (60). Tgf␤ levels are increased during acute (11) as well as chronic (58) DSS-

CD11c

CD68

induced colitis. Mice without the junctional adhesion molecule A F11r gene display increased gut permeability and inflammatory gene expression, as well as increased sensitivity to DSS-induced colitis (36, 66). Of note, it has been established that F11r-dependent barrier impairment triggers protective adaptive immune responses implicating CD4⫹ T cells, Tgf␤, and IgA (33). Thus barrier disruption in IEC-specific Hdac1/ Hdac2-deficient mice could lead to defensive compensatory

AJP-Gastrointest Liver Physiol • doi:10.1152/ajpgi.00393.2013 • www.ajpgi.org

HDAC1 AND HDAC2 MODULATE INTESTINAL INFLAMMATORY RESPONSES

Hdac2 ΔIEC

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Fig. 6. Mice with conditional intestinal epithelial Hdac2 (Hdac2 ⌬IEC) loss show decreased sensitivity to DSS-induced colitis. A: 30 ␮g of total protein extracts from 3- to 5-mo-old Wt (n ⫽ 2) or Hdac2 ⌬IEC (n ⫽ 3) colons were separated by SDS-PAGE and transferred to PVDF membranes for Western blot analysis of Hdac1, Hdac2, and actin, as a loading control. B: mice were given water without or with 3.5% DSS ad libitum. Wt and Hdac2 ⌬IEC % mouse body weight was measured for 7 days (n ⫽ 13) (2-way ANOVA, *P ⱕ 0.05). C: DAI, based on weight, colon length, presence of fecal blood, and stool consistency, of 3- to 5-mo-old (n ⫽ 13) Wt and Hdac2 ⌬IEC mice was measured. Results represent means ⫾ SE (Student’s t-test, *P ⱕ 0.05). D: histological appearance of DSS-treated 3- to 5-mo-old Wt and Hdac2 ⌬IEC colon sections was characterized for inflammation extent, as well as crypt damage (n ⫽ 5). Results represent means ⫾ SE (Student’s t-test, *P ⱕ 0.05). E: intestinal permeability of Wt and DSS-treated mice was evaluated by measuring fluorescence intensity in blood recovered 3 h after gavage of 4-kDa FITC-labeled dextran (n ⫽ 6), with a RF-5301PC spectrofluorometer (Shimadzu Scientific Instruments). The ratio of fluorescence intensity obtained in mice treated with or without DSS is indicated. Results represent means ⫾ SE (Student’s t-test, *P ⱕ 0.05). F: total protein extracts from 3- to 5-mo-old DSS-treated Wt or Hdac2 ⌬IEC colons separated on a 15% SDSPAGE gel were transferred to a PVDF membrane and analyzed by Western blot for expression of cleaved caspase 3, with actin as a loading control. The histogram indicates the ratio of band intensities normalized to actin. Quantification of band intensity was performed with the Image J software. Results represent means ⫾ SE (Student’s t-test, *P ⬍ 0.05).

Cleaved caspase 3

changes, not only in adaptive but also in innate responses through, for example, immune suppressive M2 macrophage polarization (53). This would allow a novel homeostatic control in the setting of chronic inflammation. However, when challenged with DSS, IEC-specific Hdac1/Hdac2-deleted mice show more inflammation, in response to a leakier basal epithelial barrier, with augmented expression of pro- as well as anti-inflammatory genes, such as Tgf␤, and Mrc1 associated with M2 macrophages. Interestingly, in contrast to Hdac1/Hdac2 mutant mice, Hdac2 IEC-specific deficient mice show a decreased inflammatory response to DSS-induced colitis, as evidenced by decreased DAI and intestinal permeability, as opposed to DSS-treated wild-type mice. Interestingly, although Hdac2 IEC-specific deficient mice do not exhibit basal tissue architecture disorganization, dysplasia, hyperplasia, or differentia-

tion defects, Hdac2 deficiency leads to basal deregulated expression of a subset of inflammatory genes, either decreased or increased, such as Reg3g. Of note, Hdac2 mutated mice display increased expression of mostly IEC-specific genes while immune cell function-related genes are downregulated. This suggests that aberrant signaling from Hdac2-deficient IECs may be critical to establish the basal mucosal milieu associated to the mutant phenotype. These basal gene expression differences emerging from Hdac2 or Hdac1/Hdac2 deficiency could affect the intestinal response to colitis, for example by altering the microbiota. Indeed, recent data have shown the importance of epithelial antimicrobial proteins in shaping the gut microbial communities (19, 48). Thus novel inflammatory gene expression patterns observed in mutant mice could alter the microbiota and its subsequent response with the intestinal mucosa. In addition, Hdac2 or Hdac1/Hdac2 IEC-

AJP-Gastrointest Liver Physiol • doi:10.1152/ajpgi.00393.2013 • www.ajpgi.org

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Fig. 7. Volcano plot of gene expression in Hdac2 depleted murine colons, as measured by microarray analysis. Genes with P value ⬍ 0.05 and fold change (log2) ⬎ 1 or fold change (log2) ⬍ ⫺1 were classified to biological processes from GO database. Biological processes with best P value and higher gene counts were displayed in the Volcano plot. Genes in the Regulation of immune system process/defense response/inflammatory response categories are indicated by squares, and genes in the Digestion and lipid metabolic process category are labeled with triangles.

specific deficiency may alter differently immune cell recruitment and activation. Recent data support our hypothesis that Hdac1/Hdac2 and Hdac2 IEC-specific deletion may modify the microbiota as well as immune cell recruitment. Indeed, IEC-specific deletion of another Hdac class I member, namely Hdac3, results in barrier and Paneth cell disruption, increased IEC proliferation without apparent tissue architectural or goblet cell defects, changes in microbiota composition, and increased sensitivity to DSS-induced colitis (1). These data, and our own, suggest that class I Hdac, through similar and different mechanisms, contribute to mucosal homeostasis by regulating IEC function and the intestinal inflammatory response. In addition to verifying microbiota and immune cell mucosal changes, it will be important to establish the in vivo IECspecific gene expression pattern from enriched IEC populations and to determine the intrinsic Hdac1- and/or Hdac2-dependent IEC phenotype in organoid cultures (49). Nevertheless, our results suggest that intrinsic changes in IEC Hdac activity, thus of acetylation, through Hdac1 and/or

Relative mRNA expression

20 Wt

*

15

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* 5

0

Reg3 β

Reg3 γ

Fig. 8. Hdac2 ⌬IEC loss leads to differential regulation of a subset of inflammatory genes. Total RNAs were isolated from 3- to 5-mo-old Wt and Hdac2 IEC-specific deficient colons (n ⫽ 8 –13). Relative expression levels of selected genes, namely Reg3b and Reg3g, were verified by qPCR, with Pbgd as a loading control. Results represent means ⫾ SE. Statistical significance was determined by Mann-Whitney test (*P ⱕ 0.05).

Hdac2 genetic ablation, result in different intestinal homeostatic responses. Although genomewide association studies have not revealed specific HDAC1 or HDAC2 alterations, one important conclusion from our studies is that the dosage and the regulation of Hdac1 and Hdac2 activities may well be important for intestinal homeostasis. Indeed, Hdac1 and Hdac2, though having some redundant functions, are not equivalent, as shown recently in thymocytes. In this case, although single Hdac1 and Hdac2 deficiency did not result in overt phenotypes, dual Hdac1/Hdac2 deficiency in thymocytes resulted in lymphomagenesis defects. Of interest, expression of one Hdac2 allele, in the absence of both Hdac1 alleles, led to increased thymocyte proliferation, suggesting the importance of Hdac activity dosage (15, 25). A second conclusion from our work is that different levels of IEC acetylation could lead to intrinsic IEC alterations as well as intestinal homeostatic modifications. Recent data have shown how changes in cell metabolism, from intrinsic or extrinsic sources, could alter Hdac activity and the acetylome. Indeed, acetylation levels are regulated both by endogenous and exogenous signals emanating from environmental physiological changes. For example, intrinsic variations in nuclear acetyl-CoA levels modify the acetylome, linking cellular metabolism to histone acetylation (69). In addition, acetylation levels are regulated by endogenous Hdac inhibitors such as sphingosine phosphate (23), ␤-hydroxybutyrate (52), and carnitine (29), whose levels vary according to cell metabolism. IEC acetylation levels are also regulated by exogenous Hdac inhibitors, including bacterial metabolite products such as butyrate (13), as well as diet-derived products (27). Recently, microbial short-chain fatty acids, including butyrate, have been shown to regulate colonic T-reg cell homeostasis, by regulating in part Hdac activity (4, 56). Finally, the microbiota establishes acetylated protein patterns in murine liver and intestine, when reimplanted in germ-free mice (54). Thus acetylation, as an epigenetic modification, is at the junction of the environment, diet, and microbiota: the acetylome

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HDAC1 AND HDAC2 MODULATE INTESTINAL INFLAMMATORY RESPONSES

integrates exogenous and endogenous signals affecting both metabolism and gene expression (46). Pharmacological inhibitors of epigenetic enzymes, including HDACi, are being considered for treatment of various diseases, such as cancer and inflammatory diseases. Cancer cells are notoriously sensitive to HDACi, which induce growth arrest, differentiation, and apoptosis by activating or repressing gene expression in a cell- and gene-specific manner. HDACi antiinflammatory properties have been demonstrated in mouse models of colitis (6, 17). For example, HDACi vorinostat administration in mice reduces DSS- and TNBS-induced colitis, by suppressing proinflammatory cytokines and inducing mucosal lymphocyte apoptosis (20). HDAC inhibition in mice, with the global HDACi trichostatin A, increases T-reg cell activation, correlating with diminished DSS-induced colitis symptoms (59). Our results, suggesting that Hdac1 and Hdac2 regulate IEC functions, support the possibility that HDACi may influence the intestinal inflammatory response through IECs and that selective Hdac isoform inhibitors may be considered for anti-inflammatory therapies in IBD. As novel and more selective HDACi are being developed, there is an urgent need to identify the specific roles of Hdac isoforms in intestinal homeostasis and inflammatory responses. Genomewide association studies have led to the identification of more than 163 genetic loci associated with IBD and have confirmed the importance of the interaction between mucosal defenses and the environment, such as the microbiota (30). Since most of these variant loci do not change proteincoding sequences, it has been postulated that DNA alterations may affect gene expression, in part through epigenetic signals, a novel emerging concept to explain some IBD-associated predisposition. Likewise, the ever-increasing IBD incidence in human populations and IBD discordance in monozygotic twins have revealed an important role for environmental factors that could not be attributed to genetic alterations. Many recent reviews have acknowledged the fact that epigenetic regulation, including histone acetylation, histone methylation, and DNA methylation, could play important roles in IBD, by reading and translating environmental modifications, from diet-derived and bacterial products, as well as microbiota, in a gene-specific context (50, 57, 65). We have shown here that different levels of Hdac activity in IEC lead to differential homeostatic outcomes, and that Hdac1 and Hdac2 are implicated in intestinal cell homeostasis and in the response to inflammation. Acetylation and the regulation of Hdac activity in IEC may well be important epigenetic events regulating the intestinal inflammatory response. ACKNOWLEDGMENTS We thank Dr. E. N. Olson for providing the Hdac1 and Hdac2 conditional knockout mice, and Dr. F. Boudreau and N. Perreault for the villin-Cre transgenic mouse. We thank Dr. J. Carrier for histological evaluation of intestinal sections. GRANTS This work was supported by the Crohn’s and Colitis Foundation of Canada. C. Asselin, F.P. Gendron, and F. Boudreau are members of the Fonds de recherche du Québec-Santé-funded Centre de recherche Clinique ÉtienneLebel. DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the author(s).

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AUTHOR CONTRIBUTIONS N.T., F.-P.G., F.B., and C.A. conception and design of research; N.T. and J.M.G. performed experiments; N.T., J.M.G., M.B., F.-P.G., F.B., and C.A. analyzed data; N.T. and M.B. prepared figures; N.T., M.B., F.-P.G., F.B., and C.A. edited and revised manuscript; M.B., F.-P.G., F.B., and C.A. interpreted results of experiments; C.A. drafted manuscript; C.A. approved final version of manuscript. REFERENCES 1. Alenghat T, Osborne LC, Saenz SA, Kobulay D, Ziegler CGK, Mullican SE, Choi I, Grunberg S, Sinha R, Wynosky-Dolfi M, Snyder A, Giacomin PR, Joyce KL, Hoang TB, Bewtra M, Brodsky IE, Sonnenberg GF, Bushman FD, Won KJ, Lazar MA, Artis D. Histone deacetylase 3 coordinates commensal-bacteria-dependent intestinal homeostasis. Nature 504: 153–157, 2013. 2. Babeu JP, Darsigny M, Lussier CR, Boudreau F. Hepatocyte nuclear factor 4␣ contributes to an intestinal epithelial phenotype in vitro and plays a partial role in mouse intestinal epithelium differentiation. Am J Physiol Gastrointest Liver Physiol 297: G124 –G134, 2009. 3. Balasubramanian S, Verner E, Buggy JJ. Isoform-specific histone deacetylase inhibitors: the next step? Cancer Lett 280: 211–221, 2009. 4. Bollrath J, Powrie F. Feed your TREGS more fiber. Science 341: 463–464, 2013. 5. Burger-van Paassen N, Loonen LMP, Witte-Bouma J, Korteland-van Male AM, de Bruijn AV, van der Sluis M, Lu P, Van Goudoever JB, Wells JM, Dekker J, Van Seuningen I, Renes IB. Mucin Muc2 deficiency and weaning influences the expression of the innate defense genes Reg3␤, Reg3␥ and angiogenin-4. PLoS One 7: e38798, 2012. 6. Cantley MD, Haynes DR. Epigenetic regulation of inflammation: progressing from broad acting histone deacetylase (HDAC) inhibitors to targeting specific HDACs. Inflammopharmacology 21: 301–307, 2013. 7. Cash HL, Whitham CV, Behrendt CL, Hooper LV. Symbiotic bacteria direct expression of an intestinal bactericidal lectin. Science 313: 1126 – 1130, 2006. 8. Chen J, Bardes EE, Aronow BJ, Jegga AG. ToppGene Suite for gene list enrichment analysis and candidate gene prioritization. Nucleic Acids Res 37: W305–W311, 2009. 9. Cooper HS, Murthy SN, Shah RS, Sedergarn DJ. Clinicopathologic study of dextran sulfate sodium experimental murine colitis. Lab Invest 69: 238 –249, 1993. 10. Cruikshank W, Little F. Interleukin-16: the ins and outs of regulating T-cell activation. Crit Rev Immunol 28: 467–483, 2008. 11. Da Silva APB, Ellen RP, Sorensen ES, Goldberg HA, Zohar R, Sodek J. Osteopontin attenuation of dextran sulfate sodium-induced colitis in mice. Lab Invest 89: 1169 –1181, 2009. 12. Davie JR. Inhibition of histone deacetylase by butyrate. J Nutr 133: 2485S–2493S, 2003. 13. Delcuve GP, Khan DH, Davie JR. Roles of histone deacetylases in epigenetic regulation: emerging paradigms from studies with inhibitors. Clin Epigenetics 4: 5, 2012. 14. Dennis G Jr, Sherman BT, Hosack DA, Yang J, Gao W, Lane HC, Lempicki RA. DAVID: database for annotation, visualization, and integrated discovery. Genome Biol 4: P3, 2003. 15. Dovey OM, Foster CT, Conte N, Edwards SA, Edwards JM, Singh R, Vassiliou G, Bradley A, Cowley SM. Histone deacetylase 1 and 2 are essential for normal T-cell development and genomic stability in mice. Blood 121: 1335–1344, 2013. 16. Dupaul-Chicoine J, Dagenais M, Saleh M. Crosstalk between the intestinal microbiota and the innate immune system in intestinal homeostasis and inflammatory bowel disease. Inflamm Bowel Dis 19: 2227–2237, 2013. 17. Edwards AJP, Pender SLF. Histone deacetylase inhibitors and their potential role in inflammatory bowel diseases. Biochem Soc Trans 39: 1092–1095, 2011. 18. Egesten A, Eliasson M, Olin AI, Erjefalt JS, Bjartell A, Sangfelt P, Carlson M. The proinflammatory CXC-chemokines GRO-alpha/CXCL1 and MIG/CXCL are concomitantly expressed in ulcerative colitis and decrease during treatment with topical corticosteroids. Int J Colorectal Dis 22: 1421–1427, 2007. 19. Gallo RL, Hooper LV. Epithelial antimicrobial defence of the skin and intestine. Nat Rev Immunol 12: 503–516, 2012. 20. Glauben R, Batra A, Fedke I, Zeitz M, Lehr HA, Leoni F, Mascagni P, Fantuzzi G, Dinarello CA, Siegmund B. Histone hyperacetylation is

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The acetylome regulators Hdac1 and Hdac2 differently modulate intestinal epithelial cell dependent homeostatic responses in experimental colitis.

Histone deacetylases (Hdac) remove acetyl groups from proteins, influencing global and specific gene expression. Hdacs control inflammation, as shown ...
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