supplementary material - Gut

SUPPLEMENTARY MATERIAL

Supplementary Materials and Methods Construction of the targeting vector and production of mice A lambda phage containing 20 kb corresponding to the 3' region of the murine Itga6 gene was isolated by screening a 129Sv genomic library with a probe spanning the 3’ end of an Itga6 cDNA (exons A and B). Subclones of this region were used to construct a targeting vector with three loxP sites and a neomycin-resistance cassette driven by the phosphoglycerate-kinase (PGK) promoter (Figure 1A; Supplementary Figure S1A). Embryonic stem (ES) cells derived from the 129Sv mouse strain were electroporated with the targeting vector. Out of 279 clones screened for homologous recombination, seven had correctly recombined. We electroporated two of them with a Cre recombinase expression plasmid (pCMV-Cre), recovered two independent clones having excised the neomycin cassette but retained the α6 sequence, and one clone transmitting to the germ-line after injection into mouse blastocysts of the C57/Bl6J strain (Supplementary Figure S1A). Mice carrying the Itga6 floxed allele were bred to homozygosity and displayed normal viability and fertility. They were crossed with Villin-Cre (or Villin-CreERT2) transgenic mice1 in two steps, first generating α6fl/+; Villin-Cre (or

α6fl/+; Villin-CreERT2) mice, which were then crossed with α6fl/fl animals. Control mice had a α6fl/+ or α6fl/fl genotype. Genotyping of mice was performed by PCR on tail genomic DNA. Primers used for genotyping and detection of the deleted allele are listed in the Supplementary Table S2. To induce the specific Itga6 deletion in the α6ΔIEC-TAM model, tamoxifen (Sigma-Aldrich) (dissolved in 0.9 % NaCl and homogenized by sonication) was administered by gavage (2 doses of 10 mg/day, 1 day/2, during 5 days) to 8 week-old α6fl/fl; Villin-CreERT2 mutant and α6fl/fl control mice. The animals were analyzed 6, 10 and 15 days after the first tamoxifen gavage.

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All animal experimentations were performed in accordance with French national regulations and guidelines of the national committee for animal experimentation (Comité National de Réflexion Ethique sur l’Expérimentation Animale). Agreements were summarized in a “Study approval” section at the end of the Supplementary Materials and Methods.

Depletion of the gut microbiota by antibiotic treatment A combination of 4 antibiotics (streptomycin, 2g/L, Sigma-Aldrich; gentamicin, 200 mg/L, Transgram Oral, CEVA; enrofloxacin, 100 mg/L, Baytril, Bayer; bacitracin, 1g/L, Bacivet, Alpharma) was administered in the drinking water to 6-week old WT and α6ΔIEC mice, for 3 weeks before sacrifice. The same treatment was applied to 7-week old control and α6ΔIEC-TAM mice, starting 1 week before the first tamoxifen gavage.

Epithelial cell detachment assay Epithelial cells were isolated from intestines as described in Raul et al.2 Fragments of jejunum were washed in PBS and incubated under shaking 10 min in dissociation buffer (0.5 mM EDTA and 0.5 mM DTT in PBS). Dissociated cells were collected by centrifugation and lysed for 15 min in lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100). Cellular debris were removed by centrifugation at 10,000 g and proteins were quantified by Bicinchoninic Acid protein (BCA) assay kit (Thermo Scientific).

FITC-dextran assay In vivo intestinal permeability was determined as described previously.3 FITC-dextran of average molecular weight 3,000-5,000 (FD4; Sigma-Aldrich) was used. Three independent

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experiments were performed in duplicate using 6-week old WT and α6ΔIEC mice, or 10-week old tamoxifen-treated WT and α6ΔIEC-TAM mice.

Endoscopy Mice were anesthetized by intraperitoneal injection of a mixture of ketamine (100 mg/kg) and xylazine (10 mg/kg). Colonoscopy was then performed using the high resolution “Coloview system” from Karl Storz.

Histological Analysis Intestinal samples were fixed in 4% paraformaldehyde and processed for paraffin embedding. For conventional histology, 5 µm paraffin sections were stained with Harris haematoxylin and eosin (H/E). For histological score assessment of inflammation/colitis, representative H/E stained cross-sections of proximal and distal colon were graded as indicated in Supplementary Table S3. For mucus analysis, 5 µm paraffin sections were stained with Periodic Acid-Schiff (PAS) and haematoxylin or Alcian blue (AB) (pH 2.5) using standard histology techniques. Sections stained with AB alone were counterstained with nuclear fast red. For proliferation assessment, BrdU was injected intraperitoneally to mice at 50 mg/kg body weight, 2h or 12h prior sacrifice.

Immunofluorescence (IF), immunohistochemistry (IHC) and Fluorescent In Situ Hybridization (FISH) Intestinal samples were either directly embedded for cryosectioning or fixed in 4% paraformaldehyde overnight at 4°C prior to paraffin embedding. Sections were processed following standard immunostaining procedures with the primary and secondary antibodies 3

listed in Supplementary Table S4. Nuclei were counterstained with 4',6-diamidino-2phenylindole (DAPI). For IHC, antigen retrieval using citrate buffer was performed prior incubation with primary antibodies. For BrdU staining, sections were incubated in 2N HCl (1h) prior incubation with the primary antibody. For each experiment, control stainings were made under the same conditions by omitting the primary antibody. Images displayed in the figures are representative of two independent experiments including at least 4-5 samples per group. Observations were made as described before.4 For FISH analysis, colon and rectum pieces were fixed in Methanol-Carnoy’s fixative and processed as described.5 Sections (5 µm) were hybridized to the universal bacterial probe Cy3–conjugated EUB338 (5’-GCTGCCTCCCGTAGGAGT-3’) at 10 nM. After hybridization, a co-immunostaining using the anti-Muc2 antibody (H-300, Santa Cruz Biotechnology) was performed as described.5 Nuclei were counterstained with DAPI.

FACS analysis Mononuclear immune cells from the lamina propria (LPMC) were isolated as described 6 and were subsequently analyzed by fluorescence activated cell sorting (FACS). LPMC (5.105) were stained with antibodies listed in Supplementary Table S5. Flow cytometry analyses were performed using Facs DIVA™ software on a BD LSR II flow apparatus (BD Biosciences) with automatic compensation. The number of positive cells was evaluated based on the total number of cells present in the tissue lysate.

Image analysis Images were captured using SP2 and SP8 confocal laser scanning microscopes, a DMRXA2 fluorescence microscope, or a DM-RB fluorescence microscope all from Leica. Digital

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slide scanner NanoZoomer (2.0-HT C9600-13) and the NDP.view software (Hamamatsu) were used for immune cell counting and gland size measurement. The assessment of the number and the area of the PAS+ goblet cells was performed using the image processing program Fiji (ImageJ). RGB images of the PAS/AB stained colon sections were submitted to a color deconvolution to visualize only the PAS+ content of goblet cells (Supplementary Figure S8B). PAS+ areas were selected and measured on individual goblet cells located in the upper ¾ portion of the colonic crypts (as exemplified by yellow highlighted cells in Supplementary Figure S8B). A total of 100 cells/animal (n=5/group) was evaluated on 2 non-adjacent sections. PAS+ goblet cell counting was realized on the whole height of the crypt (Total) or on its lower ¼ portion (Base, bar in Supplementary Figure S8B), from 10 crypts randomly selected on 2 non adjacent sections/animal (n=4-5 mice/group). The number of cells was normalized to the crypt height. The quantification of the CD4+ T cells detected on immunofluorescent stained colonic sections was performed using ImageJ software. The whole intestinal mucosa (corresponding to the region of interest) was selected on colonic transverse sections immunostained with DAPI which marks nuclei. Nuclei were then delineated using an automatic segmentation based on the optimal thresholding, Otsu, and the total occupied area was assessed (µm2). CD4+ cells were also automatically delineated with the optimal thresholding Otsu. The relative amount of CD4+ cells was defined as the ratio of CD4+ signal intensity/total area.

Cytokine Measurements Colon specimens (1 cm) were cultured at 37°C, 5% CO 2 for 24h in RPMI 1640 Medium, GlutaMAX™, HEPES (Invitrogen, Life Technologies) in presence of antibiotics. Medium was then recovered, and centrifuged at 5,000g at 4°C to eliminate cellular debris. Cytokines were measured on the resulting supernatant by Enzyme-Linked Immunosorbent Assay (ELISA)

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using DuoSet® ELISA Development Kits (R&D Systems) according to the manufacturer’s instructions, and for 11 designed cytokines, on a BioPlex analyser (BioRad) using the Mouse Cytokine/Chemokine Magnetic bead panel kit (MCYTOMAG-70K – Milliplex® Map, EMD Millipore). All values were normalized to the dry weight of the starting specimens. ELISA were performed to measure IL-1β, IL-6, IL-17, IL-18, IFNγ and TNFα and multiplex analysis to dose IL-1β, IL-4, IL-6, IL-10, IL-12, IL-13, IL-17, IL-22, IL-23, IFNγ and TNFα.

Preparation of colon protein extracts and Western Blotting Dissected colons were flushed with PBS and then processed to prepare protein extracts from whole colon segments (total colon lysates) or from an enriched epithelial fraction (enriched IECs) recovered from longitudinally opened colon by scraping the epithelial mucosa with a microscope glass slide. Tissue specimens were frozen and ground up in liquid nitrogen, and then lysed in RIPA buffer (150mM NaCl; 10mM Tris-HCl, pH7.4; 0.1% SDS; 0.01% Triton X100; 1% Sodium Deoxycholate; 5mM EDTA pH8; 1mM PMSF; 2µg/mL Leupeptin; 100µM Na 3 VO 4 ; 20mM NaF; 1X Protease Inhibitor Cocktail; Roche), 30 min at 4°C. Protein extracts were recovered after centrifugation at 13,000 rpm, for 10 min at 4°C. Protein concentrations were determined with BCA protein assay reagent (Thermo Fischer Scientific) and equal amounts (40µg) were loaded and separated by 8-18% SDS-polyacrylamide gel (SDSPAGE), then transferred onto nitrocellulose membranes. Ponceau red staining was performed to verify equal protein loading. The membranes were blocked in PBS, 0.05% Tween20, 5% non-fat dry milk, then incubated with the appropriate primary antibody (see Supplementary Table S4) overnight at 4°C, rinsed and incubated with the appropriate secondary HRP-coupled antibody (see Supplementary Table S4) for 1h at room temperature. Protein detection was performed by enhanced chemiluminescence (ECL) (Luminata™ Forte or Luminata™ Crescendo Western HRP Substrate, Millipore). Glyceraldehyde Phosphate

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Dehydrogenase (GAPDH) and β-tubulin were used as protein loading controls after stripping the membranes in Restore™ Western Blot Stripping Buffer (Thermo Scientific).

RT-qPCR For RT-qPCR, total RNA was extracted with the RNeasy kit (Qiagen) according to the manufacturer’s instructions. cDNAs were prepared from 1 µg of total RNA, using oligodT and the SuperScript™ II RT-PCR system (Invitrogen). PCR reactions were performed with SYBR® Green JumpStart™ Taq ReadyMix™ reaction mixture (SIGMA) or Universal Probe Library System (Roche Applied Science) according to the manufacturer’s instructions. Experimental values of gene expression levels were normalized by hypoxanthine phosphoribosyl-transferase (HPRT) mRNA levels. Primers sequences were listed in Supplementary Table S6.

Transcriptome analysis •

α6ΔIEC-TAM line

Total RNA was extracted with the RNeasy kit (Qiagen) according to the manufacturer’s instructions from the rectal mucosa of tamoxifen-treated control (n=3) and α6ΔIEC-TAM (n=4) mice, 15 days after the first TAM administration. Quantification and quality control of RNAs were assessed on Agilent 2100 Bioanalyzer. The transcriptome analysis was performed at the “IGBMC Microarray and Sequencing Platform” using the Affymetrix Mouse Gene 1.0 ST arrays. Raw data were processed with the Expression Console Software (Affymetrix). The Robust Multichip Analysis (RMA) algorithm was used to normalize the array signal data across chips and generate transcript level values. We used the Partek Genomics Suite software to obtain exon levels values from the CEL files. Student t-test was performed to obtain

transcript

(genes)

which

are

significantly 7

differentially

expressed

between

experimental groups. We used the Benjamini & Hochberg procedure for controlling the False Discovery Rate (FDR) due to the multiple testing. We used Ingenuity Pathway Analysis (IPA) for recovering links between selected genes. The α6ΔIEC-TAM microarray data from this manuscript have been submitted to the GEO database (http://www.ncbi.nlm.nih.gov/geo/) and assigned the identifier GSE70700. The following link has been created to allow review of record GSE70700: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=qjybsmewjzkpdof&acc=GSE70700

•

α6ΔIEC line

In order to define a specific molecular signature associated with tumor development in α6ΔIEC rectal prolapses, a transcriptome analysis was performed as described above, by comparing gene expression levels in the adenocarcinoma to that of the adjacent non-cancerous inflamed rectal mucosa, and that of the normal rectal mucosa obtained from matched WT mice. Briefly, the rectal prolapse from 53-80-week old α6ΔIEC mice (n=5) was excised, flushed with PBS and quickly frozen in methyl butane cooled in liquid nitrogen. The regions comprising adenocarcinoma were identified and precisely delineated by a pathologist (A. M.N.) on transverse 5 µm-thick cryosections extemporaneously stained with H/E. The identified tumors were then macrodissected from 50 to 100 µm-thick cryosections. Adjacent noncancerous inflamed rectal mucosa was similarly macrodissected and recovered. The normal rectal mucosa used as reference was recovered by scraping of the recto-anal mucosa of matched control WT mice (n=4). Total RNA (from 5 adenocarcinomas, 4 flanking inflamed rectal mucosa and 4 normal control rectal mucosa) was extracted using the RNeasy® Micro Kit (Qiagen), according to the manufacturer’s instructions. Quantification and quality control of RNA were assessed on Agilent 2100 Bioanalyzer and a transcriptome analysis was performed using the Affymetrix Mouse Gene 1.0 ST platform as described above.

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The

tumor

microarray

data

have

been

submitted

to

the

GEO

database

(http://www.ncbi.nlm.nih.gov/geo/) and assigned the identifier GSE37749. The following link has been created to allow review of record GSE37749: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=whmnmquodfwznqd&acc=GSE37749

FDG-microPET imaging Tumor progression was assessed in WT and α6ΔIEC animals aged over one year by fluorodeoxyglucose (FDG)-microPET scan. Mice were anesthetized by intraperitoneal injection of a mixture of ketamine (200 mg/kg) and xylazine (10 mg/kg). All microPET scans were performed with the IRIS PET small animal imaging system (inviscan SAS, Strasbourg, France). Mice were injected with 7 MBq in mean (range from 5 to 8 MBq) [18F]-FDG (IBA Radiopharma Solutions, Belgium) intravenously in the jugular vein. To perform whole body scans, mice were placed in a bed with the long axis of the mouse parallel to the long axis of the scanner, and scanned with one single position in the PET system at 1 h after injection of radiotracer. The acquisition time was 10 min. Image reconstructions were performed using model based 3D ordered subsets expectation maximization (3D-OSEM) algorithm with 6 subsets and 8 iterations. Images were reconstructed with matrix sizes of 201x201x120 and voxel sizes of 0.425x0.425x0.855 mm3. Decay, random, dead time and normalization were corrected in the reconstruction process.

Study approval All animal experimentations were performed in accordance with French national regulations and guidelines of the national committee for animal experimentation (Comité National de Réflexion Ethique sur l’Expérimentation Animale) with the following agreement numbers: 2012-012; 2012-165; ADA-2012-09-28; AL-05-12-02-13. 9

Statistical analysis

All data (unless specified) were analyzed using the Graphpad Prism 5.0 software and are displayed as scattered dot plots; error bars correspond to standard deviation. Significance among samples (mutants versus controls) was determined using the two tailed nonparametric test of Mann-Whitney offered in Prism (except for histological scores and quantification of SLC26A3 levels, see below). For the assessment of the gland height, of the percentage of BrdU+ cells relative to the total cells per gland, and of infiltrating CD11b+ cells, we scored positive cells within at least 8 non-adjacent colon sections and 10-25 crypts, averaged those counts and then used the averages originating from distinct animals for statistical comparisons. Histological scores were assessed using the non-parametric KruskalWallis test for categorical data offered in statXact 7.0. Statistical significance for the quantification of the SLC26A3 expression levels (measurement of the fluorescence intensity on colon sections) was assessed using the non-parametric Kolmogorov-Smirnov test offered in statXact 7.0. A p value