experimental colitis in young adult mice and is reduced in IBD [12]. However, no inflammatory defect was reported to occur in animals that were not chemically induced to develop colitis leaving questionable whether Hnf4a was functionally important to maintain appropriate inflammatory homeostasis during adult life. Herein, we aimed to investigate whether epithelial Hnf4a was solely involved in the control of intestinal inflammatory homeo- stasis and to explore the nature of the mechanisms implicated. Our results show that Hnf4a deletion spontaneously triggered chronic inflammatory response in the colon characterized by disrupted crypt architecture, proliferative changes, apoptosis, increased inflammatory mediator secretion and increased immune cell infiltration. We propose that the loss of mucosal homeostasis is initiated by an early reduction of mucosal ion transport, which was not associated with a significant change of barrier permeability. Claudin-15, a paracellular ion transporter, was confirmed to be a novel and direct gene target for Hnf4a. These findings identify Hnf4a as a transcriptional regulator of ion transport and support a novel functional role for this transcriptional regulator in colonic inflammatory homeostasis. Materials and Methods Animals and Ethics Statement 12.4 KbVilCre/Hnf4aloxP-loxP mutant mice were obtained exactly as described previously [11]. Mice were kept under pathogen free conditions and were tested negative for helicobacter, pasteurella and murine norovirus. Mice were treated in accor- dance with a protocol approved by the Institutional Animal Research Review Committee of the Universite´ de Sherbrooke. RNA Isolation and Quantitative PCR Tissue samples were harvested, total RNA was isolated and quantitative RT-PCR was performed as described previously [13]. Target expressions were normalized using either hypoxanthine- guanine phosphoribosyl transferase (HPRT) or TATA binding protein (TBP) expression depending on target mRNA abundance. All primer sequences and cycling conditions are available upon request. Microarray Screening and Data Analysis Probes for microarray analysis were generated from RNA isolated from the proximal colon of 3 control and 3 Hnf4a mutant mice at 12 months of age. Affymetrix GeneChipH Mouse Genome 430 2.0 arrays were screened with six generated probes via the microarray platform of McGill University and Ge´nome Que´bec Innovation Center as previously described [14] (data are accessible through GEO series accession number GSE11759). To test for statistically significant changes in signal intensity (p values of#0.05; SAM test), compiled data (RMA analysis) were screened using FlexArray 1.1 (Genome Quebec). Data were also analyzed through the use of Ingenuity Pathways Analysis (IPA) to generate networks of genes associated with biological functions and/or diseases (Ingenuity Systems, www.ingenuity.com). Immunoblots Total protein extracts were obtained and separated by protein electrophoresis as described previously [13]. The following antibodies were used and incubated at 4uC O/N: anti-b-actin (#sc1615, 10 ng/ml) and anti-HNF4a (#sc6556, 100 ng/ml) from Santa Cruz; anti-cleaved caspase-3 (#9664, 1:500), anti-IkBa (#9242, 1:1000), anti-phospho- IkBa (Ser32) (#2859, 1:1000) from Cell Signalling and anti-claudin-4 (#36-4800, 500 ng/ml), anti-claudin-8 (#40-0700Z, 32 ng/ml), anti-claudin- Hnf4a and Spontaneous IBD 15 (#38-9200, 250 ng/ml) and anti-E-cadherin (#33-400, 30 ng/ ml) from Zymed. Electronic Microscopy Tissues intended for electronic microscopy analysis were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer and processed as described previously [13]. Immunohistochemistry, Immunofluorescence and Cytological Stains For histological analysis, tissues were fixed in a swiss roll position using 10% neutral buffered formalin and paraffin embedded. Antigen retrieval was performed using 10 mM citrate buffer, pH 6.0 and the DAKO EnVision+ System-HRP according to the manufacturer’s instructions (DakoCytomation). Antibodies were used under the following conditions: anti-MPO (#RB-373, 1:100) and anti-Ki67 (#RB-1510-R7) (Neomarkers). For HNF4a indirect immunofluorescence, anti-HNF4a (1 mg/ml) was used. For claudin immunodetection, 5 mm thick OCT cryosections were fixed in 100% methanol for 10 minutes at – 20uC followed by incubation with anti-claudin-4 (1 mg/ml), anti-claudin-8 (0.2 mg/ ml) and anti-claudin-15 (1 mg/ml). Primary antibodies were visualized with the appropriate FITC-coupled secondary antibody (Vector Laboratories) and samples were mounted with Vectashield Hard Set mounting medium with DAPI (Vector Laboratories). Alcian blue staining was done as described elsewhere [15]. All images were captured on a Leica DMLB2 microscope using a Leica DFC300 FX camera. Cytokine Array and ELISA Cytokine production by colonic tissues of normal and Hnf4a mice was determined using the RayBioH Mouse Cytokine Antibody Array G series 1000 (RayBiotech, Norcross, GA). Colons of 1 year old mice were harvested and protein lysates prepared as previously described [16]. The cytokine antibody array was performed with 40 mg of protein and scanned in the Cy3 channel using a ScanArray Express dual-color confocal laser scanner (Perkin Elmer). Results were normalized and analyzed as recommended by the manufacturer [17]. CXCL1 expression was quantified using the R&D Systems duoSet ELISA development kit for mouse CXC chemokine KC (R&D Systems). ELISA assays were performed using 30 mg of whole colon protein extract per well following the manufacturer’s recommendation. DSS Samples and UC Samples 2.5% DSS mild treatment was given to CD1 mice for a period of 10 days and the colon was harvested. TissueScan Real-Time colitis disease panels were purchased from Origene Technologies. Each panel was composed of total isolated RNA from 6 control samples and 21 UC samples collected all from different individuals. Lyophilised cDNAs were resuspended and qRT- PCR was performed as described above and normalized against b- actin. PCR conditions and primers used are available upon request. EMSA and Chromatin Immunoprecipitation EMSA were performed exactly as described previously [18]. For chromatin immunoprecipitation, whole colon was removed, fixed in 1% formaldehyde and the epithelial cells harvested with a cell recovery solution (BD bioscience). Cells were then sonicated and ChIP performed with Ez-Chip as described by the manufacturer (Millipore). PLoS ONE | www.plosone.org 2 October 2009 | Volume 4 | Issue 10 | e7609