The generation of XBP1flox/flox
VCre and VCreERT2
transgenic mice is detailed in Supplementary Data
. All mouse protocols were approved by the Harvard Standing Committee on Animals.
The source of antibodies, proteins and inhibitors are as follows: rabbit phospho-JNK, total-JNK, active (cleaved) caspase-3 (Cell Signaling Technology), anti-lysozyme (DakoCytomation), anti-procryptdin (Ayabe et al., 2002
) generously provided by A. Ouellette (UC Irvine), flagellin (Invivogen), TNFα (Peprotech). The JNK-1,-2,-3 inhibitor SP600125 (Sigma), p38 inhibitor SB203580, MEK inhibitors PD98059 and U0126 (Calbiochem) were dissolved in DMSO as recommended. Carbamyl choline and lipopolysaccharide (LPS; from Escherichia coli
0111:B4) (Sigma), were used at final concentrations of 10µM and 1µg/ml, respectively.
Immunohistochemistry, TUNEL and Electron microscopy
Tissues were handled by standard methods as detailed in Supplementary Methods
. Apoptotic cells were detected on paraffin embedded small intestine using TUNEL-POD kit (Roche Applied Sciences). Small intestinal tissue from sex-matched XBP1+/+
littermates was fixed as previously described (see Supplemental Methods
) and observed with a JEOL 1200EX TEM at 60 kV operating voltage.
Oral L. monocytogenes infection
Sex and age matched groups of XBP1+/+
littermates were infected under BL2 conditions using gastric gavage at 3.6×108 L. monocytogenes
strain 10403s per mouse. CFU assays (faeces c.f.u./mg dry weight; liver and spleen c.f.u./organ) were performed as in (Kobayashi et al., 2005
) and Supplementary methods
Dextran sodium sulphate colitis
Sex and age-matched littermates (8 to 12 wks) received 4.5% DSS (ICN Biomedicals Inc.) in drinking water for 5 days then regular water thereafter, or neomycin sulfate and metronidazole (1.5 g/L) (Sigma). Antibiotic treated mice received 7% DSS. Weight was recorded daily and rectal bleeding assessed as (0, absent; 1, traces of blood at anus or the base of the tail; 2, clearly visible rectal blood). Histological and mRNA expression studies on RNeasy kit isolated colon RNA (Qiagen) used mice sacrificed on day 8 after DSS treatment. Histological scoring of colons was as in (Garrett et al., 2007
Crypt isolation, stimulation, and bactericidal activity assays
Small intestinal crypts were isolated, stimulated with 10µM CCh or 1µg/ml LPS and lysozyme levels and bactericidal activity against 1 × 103
c.f.u. Salmonella typhimurium
cs015 following published protocols (Ayabe et al., 2000
) and Supplementary methods
Bromodeoxyuridine (BrdU) incorporation
XBP1+/+ and XBP1−/− littermates were injected with 1mg BrdU (Becton Dickinson) in 500µl PBS, and small intestinal tissue harvested after 1h or 24h and paraffin embedded tissue sectioned and stained with anti-BrdU antibody (Becton Dickinson).
Epithelial RNA isolation and quantification
intestines were opened longitudinally, rinsed with cold PBS, everted on a plain surface, RNAlater added and epithelium immediately scraped off using RNAse-free glass slides. Total RNA isolated using RNAeasy columns (Qiagen) was reverse transcribed and quantified by SYBR green PCR (Biorad). For microarray analysis, RNAs isolated from 3 specimens per genotype were pooled, and microarray carried out at the Biopolymers Core Facility (Harvard Medical School) with mouse genome 430 2.0 array (Affymetrix, Santa Clara, CA). Data analysis was performed with Agilent GeneSpring GX and Affymetrix GCOS software under default parameter setting. Quantitative PCR was performed as in (Lee et al., 2003b
). See Supplementary Table 5
for PCR primers.
XBP1 splicing assay
XBP1 splicing was measured by specific primers flanking the splicing site yielding PCR product sizes of 164 and 138bp for human XBP1u and XBP1s, 171bp and 145bp, for mouse XBP1. Products were resolved on 2% agarose gels, and band intensity determined densitometrically (Optiquant Software, Perkin Elmer).
XBP1 silencing in MODE-K cells
The SV40 large T antigen-immortalized small intestinal epithelial cell line MODE-K (gift of D. Kaiserlian, Institute Pasteur) was transduced as described (Iwakoshi et al., 2003
) with an XBP1-specific RNAi vector and a control vector identical to (Lee et al., 2003a
) except that SFGΔU3hygro was used, and knockdown confirmed by qPCR. MODE-K.iXBP and MODE-K.Ctrl were seeded for CXCL1 experiments as described (Song et al., 1999
) at 1×105
cells/well in 96 well plates, adhered for 2–4 hours, supernatant removed and stimulated with flagellin and TNFα for 4h or preincubated for 30min with JNK, p38, and MEK inhibitors, supernatants removed and cells stimulated in fresh media with flagellin and TNFα CD1d-restricted antigen presentation by MODE-K cells (van de Wal et al., 2003
) is in Supplementary methods
. JNK phosphorylation was assessed in MODE-K cells seeded at 1×106
per well 6 well plates, allowed to form confluent mono-layers over 48–72h, stimulated with flagellin and TNFα for the indicated time periods, washed in ice-cold PBS and lysed in 500µl RIPA buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with protease (Complete®, Roche Applied Science) and Ser/Thr and Tyr phosphatase (Upstate) inhibitors.
Protein content of lysates was determined by BCA assay, and equal amounts of lysates containing Laemmli buffer were boiled at 95°C for 5min, resolved on 10% SDS-PAGE (for MODE-K cell lysates) or 12% SDS-PAGE (for TCA precipitates of purified crypts), transferred to Protran membranes (Whatman), blocked with 5% milk in TBS-T, incubated with primary antibody in 3–5% BSA in TBS-T at 4°C overnight, washed, and incubated with a 1:2,000 dilution of HRP-conjugated anti-rabbit secondary antibody in 3–5% milk in TBS-T for 45min at room temperature. Bands were visualized using SuperSignal chemoluminescent substrate (Pierce).
Human biopsy samples
Ileal and colonic biopsies were obtained from randomly selected patients with clinically, endoscopically and histologically confirmed diagnosis of CD and UC, as well as healthy control patients without any signs of intestinal inflammation. The diagnosis of CD and UC was confirmed by established criteria of clinical, radiological and endoscopic analysis, and from histology reports. Informed consent was obtained and procedures performed according to the approval by the local ethics committee of the Innsbruck Medical University. Biopsies were collected in RNAlater (Ambion), RNA isolated using RNAeasy columns (Qiagen), reverse transcribed, and used for quantitative PCR and XBP1 splicing assays.
German patients and controls in panels 1 and 2 almost completely overlap with the panels termed A and B in two recently published studies (Franke et al., 2007
; Hampe et al., 2007
). Panel 3 is unpublished. All patients were recruited at the Charité University Hospital (Berlin, Germany) and the Department of General Internal Medicine of the Christian-Albrechts-University (Kiel, Germany), with support from the German Crohn and Colitis Foundation and BMBF competence network “IBD”. Clinical, radiological and endoscopic (i.e. type and distribution of lesions) studies unequivocally confirmed the diagnosis of CD or UC, with confirmative or compatible histological findings. In the case of uncertainty, patients were excluded from the study. German healthy control individuals were obtained from the popgen biobank (Krawczak et al., 2006
). Informed written consent was obtained from all study participants. All collection protocols were approved by the Charité University Hospital and the Department of General Internal Medicine of the Christian-Albrechts-University ethics committees.
Genotyping and Sequencing
Genomic DNA was prepared and amplified as in Supplementary Methods
, genotyping performed using the SNPlex™ Genotyping System (Applied Biosystems, Foster City, CA) on an automated platform, genotypes generated by automatic calling using the Genemapper 4.0 software (Applied Biosystems) and all cluster plots reviewed manually. Prior to statistical analyses, quality checks (PHWE
>1%, callrate≥90%) were applied to the SNPs under study. Single-marker association and haplotype analyses, permutation tests, calculation of pairwise LD, and SNP selection were performed using Haploview 4.0 (Barrett et al., 2005
). Haplotype blocks were automatically defined as in (Gabriel et al., 2002
). Only haplotypes with population frequencies >1.0 % were included in the final association analysis.
Single-marker disease associations and possible marker-marker interactions were assessed for statistical significance by means of logistic regression analysis (forward selection), as implemented in the procedure LOGISTIC of the SAS software package (SAS Institute, Cary, NC). Prior to analyses, individuals with missing data were removed and genotypes coded numerically.
UPRE reporter assays
Expression plasmids hXBP1u and hXBP1s were engineered to incorporate the XBP1snp17
(M139I) and XBP1snp22
(P15L) minor variants using the GeneTailor site directed mutagenesis system (Invitrogen). See Supplementary Data
for primers used. Transient transfection of MODE-K cells followed by luciferase assays was performed as in (Lee et al., 2003b
). Reconstitution of XBP1−/−
MEF cells was with bicistronic retroviral vectors expressing GFP and human XBP1 constructed by inserting PCR amplified cDNAs for human wildtype and XBP1snp17
variants into RVGFP
vector between BglII
sites, as described previously (Iwakoshi et al., 2003
) and transduced as described (Lee et al., 2003b