Animals. Five- to eight-week-old C57BL6 and BALB/C male mice were purchased from Janvier Laboratory (Le Genest-St-Isle, France). Animals were maintained under specific pathogen-free conditions in the animal facility at the Institut Pasteur de Lille. B6-IL10tm1Cgn (IL10−/−) mice were bred in the animal facility of the University Hospital of Zurich. Animals had access to standard tap water and chow diet ad libitum. All animal experiments were approved by the local animal care program and were in accordance with the European convention on research animal protection.

Aluminum treatment. Aluminum phosphate (AlH 6 O 12 P 3 ; Sigma-Aldrich, Saint-Quentin Fallavier, France) or aluminum citrate (AlC 6 O 7 H 5 ) was diluted in PBS (Lifetechnologies, Saint Aubin, France) and administered once a day with a gavage needle at a concentration of 1.5 mg Al element per kg body weight, an amount equivalent to the high end of the total aluminum range ingested daily by humans living in contemporary urban society.16 The duration of aluminum treatment was dependent on the setting of each experiment and was detailed in each figure. A dose-response experiment was performed and AluP was given to mice at a concentration of 0.15, 1.5, and 15 mg Al element per kg body weight per day. Zinc phosphate (Zn 3 (PO 4 ) 2 ) diluted in PBS was used as a control and was orally administered to mice at a concentration of 1.5 mg Zn element per kg body weight per day. Stock solutions of aluminum salts or ZnP, adjusted to mice weight, were prepared weekly. In all experiments, control mice received PBS by gavage.

For experiments performed with IL10−/− mice, AluCi was diluted in their drinking water at a concentration of 0.015 mg Al element ml−1 to reach a daily exposure of 1.5 mg Al element per kg body weight.

Induction of TNBS and DSS colitis. TNBS colitis was induced in anesthetized C57BL6 mice by intrarectal administration of TNBS (150 mg kg−1; Sigma-Aldrich) diluted in a 1:1 (v/v) mix of 0.9% NaCl and 100% ethanol, as described previously.47 Control animals received an NaCl/ethanol mix using the same technique. Mice were euthanized 4 days after TNBS/ethanol administration.

Acute colitis was induced with 2.5% (w/v) DSS (45 kDa; TdB Consultancy, Uppsala, Sweden) dissolved in water for 7–9 days. For recovery experiments, colitis was induced by feeding mice with 2% (w/v) DSS for 7 days, followed by normal water until the end of the experiments, 10 or 12 days after DSS discontinuation. At the end of each experiment, mice were assessed for clinical score and euthanized.

Determination of clinical scores. For TNBS-induced colitis, animals were euthanized and the colon of each mouse was dissected and cut longitudinally to reveal the colonic mucosa. The intensity of colonic lesions was first evaluated macroscopically according to the Wallace score.48 The Wallace score rates macroscopic lesions on a scale from 0 to 10 based on features reflecting inflammation, such as hyperemia, thickening of the bowel, and the extent of ulceration. A colon specimen located within the ulceration was used for histological evaluation. The other parts of the colon were frozen for subsequent analysis of mRNA expression and MPO activity quantification.

For DSS-induced colitis, body weight was determined regularly during DSS and the water administration phase until the end of each experiment. At the end of each experiment, animals were assessed for clinical score by recording body weight variation, stool consistency, and occult blood before being euthanized. A DAI was determined as previously described and is summarized in Supplementary Table 1 online.49 Rectal bleeding was assessed with the ColoScreen III Lab Pack (Elitech, Salon-de-Provence, France). The DAI score ranged from 0 (healthy) to 12 (greatest level of colitis). After euthanasia, the colon was carefully dissected and its weight and size were measured. Circular sections of the colon were prepared for histological analysis. The other parts of the colon were frozen for subsequent analysis of mRNA expression and MPO activity quantification.

In the model of chronic colitis, IL10−/− animals were anesthetized intraperitoneally with a mixture of 100 mg ketamine (Vétoquinol, Bern, Swizerland) and 8 mg of Xylazine (Bayer, Lyssach, Switzerland) per kg body weight and examined as described previously with the Tele Pack Pal 20043020 (Karl Storz Endoskope, Tuttloingen, Germany).50 Colonoscopy was scored using the murine endoscopic index of colitis severity scoring system as described previously.51 After euthanasia, circular sections of the colon were prepared for histological analysis. The other parts of the colon were frozen for subsequent analysis of mRNA expression and MPO activity quantification.

Histology. Colons were fixed in 4% paraformaldehyde, and embedded in paraffin (Labonord, Templemars, France). Tissue sections were stained with May-Grünwald and Giemsa and evaluated blindly by two investigators. Histological lesions of mice with TNBS-induced colitis were quantified using the modification by Ameho of the histopathological grading system of Macpherson and Pfeiffer (ranging from 0 to 6).48 Briefly, histological findings identical to those of normal mice were scored as 0, mild mucosal and/or submucosal inflammatory infiltrate and edema, punctate mucosal erosions, and intact muscularis mucosae were scored as 1, the same histological findings involving 50% of the specimen were scored as 2, prominent inflammatory infiltrate and edema, deeper areas of ulceration extending through the muscularis mucosae into the submucosa, and rare inflammatory cells invading the muscularis propria but without muscle necrosis were scored as 3, the same histological findings involving 50% of the specimen were scored as 4, extensive ulceration with coagulative necrosis with deep extension of the necrosis into the muscularis propria were scored as 5, and the same histological findings involving 50% of the specimen were scored as 6.

For DSS-induced colitis and chronic colitis, histological lesions were assessed using a score quantifying the intensity of the inflammatory cell infiltrate (scores 0–3) and the tissue damage (scores 0–3) as previously described.49 Briefly, the presence of occasional inflammatory cells in the lamina propria was scored as 0, increased numbers of inflammatory cells in the lamina propria as 1, confluence of inflammatory cells extending into the submucosa as 2, and transmural extension of the infiltrate as 3. For tissue damage, scores were 0, no mucosal damage; 1, lymphoepithelial lesions; 2, surface mucosal erosion or focal ulceration; 3, extensive mucosal damage and extension into deeper structures of the bowel wall. The combined histological score ranged from 0 (no changes) to 6 (extensive infiltration and tissue damage).

MPO activity measurement. MPO activity was measured to monitor the degree of neutrophil infiltration in the colonic lesions during chemically induced and chronic colitis.52 Colon specimens were homogenized with an Ultra Turrax T8 (Ika-Werke, Staufen, Germany) in a phosphate buffer (pH 6.0) containing 0.5% hexadecyltrimethyl ammonium and subjected to two sonication and freeze-thaw cycles. The suspensions were centrifuged at 14,000 × g for 15 min at 4 °C and the supernatants were reacted with 1 mg ml−1 o-dianisidine hydrochloride and 0.0005% hydrogen peroxide. The optical density of each sample was read at 450 nm with a Versamax microplate reader (MDS Analytical Technologies, Saint-Grégoire, France). One unit of MPO activity was defined as the amount that degraded 1 μmol peroxidase per minute at 25 °C. The results were expressed as absorbance per total quantity of proteins determined by the Bradford method.

PCNA staining and TUNEL. Ex vivo cell proliferation was assessed by staining for PCNA. Colonic sections were boiled in 0.1 M sodium citrate buffer pH 6.0 for 6 min for antigen unmasking. After washing, sections were blocked for 30 min with 5% bovine serum albumin in PBS, stained overnight at 4 °C with anti-PCNA antibody (1/50) (Santa Cruz Biotechnology, Dallas, TX), and incubated with Alexa 488 conjugated secondary antibody (1/100) (Invitrogen, Saint-Aubin, France) for 1 h. Sections were counterstained with DAPI (Molecular Probes, Eugene, OR). To ensure specificity of immunostaining, control sections underwent simultaneous staining with isotype control antibody.

Detection of apoptosis was performed by TUNEL assay using the in situ cell death detection kit (Roche, Meylan, France). Sections were permeabilized with 1% Triton X-100, 0.1% sodium citrate, washed and stained for TUNEL according to the manufacturer’s instructions. Sections were counterstained with DAPI.

The quantification of positive TUNEL- or PCNA-stained cells was performed randomly using the ImageJ processing and analysis software (NIH, Bethesda, MD). Images were acquired with a DM5500B microscope equipped with a DFC 310 FX camera (Leica Microsystems, Nanterre, France) and mucosal layers were photographed at a magnification of × 10 to measure specific fluorescence intensity.

Cell line stimulation assay. Caco-2 and HT-29 epithelial cells were cultured in 12-well plates (density of 5 × 105 cells per well) with Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (Eurobio, Courtaboeuf Cedex, France) and 1% penicillin-streptomycin (Invitrogen), at 37 °C in 5% CO 2 /95% humidified air. Cells were treated with AluP at different concentrations (10–100 μg Al element ml−1) with or without LPS (1 μg ml−1; Sigma-Aldrich) for 3–6 h. After the incubation period, cells were washed twice with sterile PBS, then lysed with RA1 buffer containing 1% β-mercaptoethanol (Macherey-Nagel, Düren, Germany).

For the determination of cytotoxicity and proliferation, cells were incubated with aluminum for 5 days. Supernatants were collected for lactate deshydrogenase measurement (Cytotoxicity detection kit; Roche) and cell proliferation was assessed using a colorimetric MTT cell proliferation assay (Interchim, Montluçon Cedex, France). Optical density was read at 500 and 570 nm, respectively, with a Versamax microplate reader (MDS Analytical Technologies).

RNA extraction and real-time qPCR. Total RNA was extracted from colonic samples with the NucleoSpin RNAII commercial kit (Macherey-Nagel), as described by the manufacturer. cDNA was prepared with the High Capacity cDNA Archive kit and RT–qPCR was performed with SyBrGreen (Applied Biosystems, Saint-Aubin, France). Beta-actin was used as a reference gene and primer sequences are listed in Supplementary Table 2.

Microbiologic analysis. Colon samples and MLN were introduced into pre-weighed vials containing 1.5 ml of cysteinated Ringer’s solution. After physical disruption of the colon specimens, 10-fold dilutions were performed in the same diluent (decimal dilutions from 10–2 to 10–5). Each dilution was spread onto plates of non-selective blood agar (modified Columbia agar) incubated at 37 °C for 1 week under anaerobic conditions, McConkey plates (BioMerieux, Marcy l’Etoile, France) incubated at 37 °C for 48 h under aerobic conditions, and Man, Rogosa, Sharpe plates incubated at 37 °C for 48 h under CO 2 -enriched conditions. Total counts were performed, and different types of colonies were subcultured and identified following established morphological and biochemical criteria. Quantitative results are expressed in log colony forming unit(CFU) g−1. The threshold of detection is 104 CFU g−1.

After disruption of MLN in the Ringer’s solution, 1 ml was grown in brain-heart enrichment broth and 0.1 ml was spread onto plates of non-selective blood agar and incubated at 37 °C for 1 week under anaerobic conditions. If the brain-heart enrichment broth became turbid, then 0.1 ml was spread onto plate of non-selective blood agar and incubated at 37 °C for 1 week under anaerobic conditions. Subcultured bacteria were identified as above. The threshold of detection was 102 CFU g−1. All samples were analyzed in a blind manner.

In vitro granulomas formation. Fresh human blood from healthy volunteers was obtained from the Etablissement Français du Sang and was diluted 1/1 (v/v) with RPMI (Invitrogen), layered gently onto a ficoll-paque solution (Amersham, Courtaboeuf Cedex, France) and then centrifuged for 40 min at 1,800 r.p.m. PBMCs were collected and washed three times in RPMI medium by 10 min centrifugation at 1,800 r.p.m. Cells were counted with a Malassez cell and diluted to a concentration of 1 × 106 cells ml−1 in RPMI media supplemented with 7.5% heat-inactivated human AB serum (Sigma-Aldrich). PBMCs were then incubated in 24-well plates with increasing concentrations of AluP or alone for 4–7 days, at 37 °C in a 5% CO 2 atmosphere. In each well, the number of granulomas was counted. Granulomas enumeration was performed using an inverted microscope and a × 4 objective (Olympus CK40, Olympus, Rungis, France). Pictures were taken using an inverted microscope with a × 4, × 10, or × 40 objective (Nikon TE300 Eclipse, Champigny-sur-Marne, France). For experiments with bacterial stimulation, PBMCs were incubated with 1 × 103 non-pathogenic E. coli K-12 strain DH5α, AIEC strain LF82 or 1 × 104 BCG, alone or with AluP (5 ng Al element ml−1) for 4 days at 37 °C in a 5% CO 2 atmosphere. Granulomas enumeration was performed as previously described. Results are represented as the total number of granulomas per well and the percentage of small size (index 1) and big size granulomas (indexes 2–4).

Statistical analysis. Data are presented as the mean±s.e.m. Comparison between different treatment groups for quantitative variables was performed using the Wilcoxon–Mann–Whitney test. Two-tailed significance tests were used. Kaplan–Meier analysis with log-rank statistics was performed in survival during DSS-induced colitis. A P-value of <0.05 was considered as statistically significant.