XBP1 links ER stress to intestinal inflammation and confers genetic risk for human inflammatory bowel disease

Abstract

Inflammatory bowel disease (IBD) has been attributed to aberrant mucosal immunity to the intestinal microbiota. The transcription factor XBP1, a key component of the endoplasmic reticulum (ER) stress response, is required for development and maintenance of secretory cells and linked to JNK activation. We hypothesized that a stressful environmental milieu in a rapidly proliferating tissue might instigate a proinflammatory response. We report that Xbp1 deletion in intestinal epithelial cells (IECs) results in spontaneous enteritis and increased susceptibility to induced colitis secondary to both Paneth cell dysfunction and an epithelium that is overly reactive to inducers of IBD such as bacterial products (flagellin) and TNFalpha. An association of XBP1 variants with both forms of human IBD (Crohn's disease and ulcerative colitis) was identified and replicated (rs35873774; p value 1.6 x 10(-5)) with novel, private hypomorphic variants identified as susceptibility factors. Hence, intestinal inflammation can originate solely from XBP1 abnormalities in IECs, thus linking cell-specific ER stress to the induction of organ-specific inflammation.

Figure 1. Spontaneous enteritis and Paneth cell loss in XBP1^−/− mice

A. Small intestinal mucosal scrapings (n = 8 per group) from Xbp1-deleted (“XBP1^−/−”) and Xbp1-sufficient (“XBP1^+/+”) mouse intestinal epithelium were analyzed for cryptdin-1 (Defcr1), cryptdin-4 (Defcr4), cryptdin-5 (Defcr5), lysozyme (Lysz), mucin-2 (Muc2), cathelicidin (Camp1), and XBP1 (primers binding in the floxed region) mRNA expression. Data are expressed as fold decrease in XBP1^−/− compared to XBP1^+/+ specimens, normalized to β-actin (Student’s t test). B. Fold increase in grp78 mRNA expression in XBP1^−/− compared to XBP1^+/+ epithelium, normalized to β-actin (n = 3 per group, Student’s t test). C. Spontaneous enteritis in XBP1^−/− mice (upper panels and lower left panel), and normal histology of XBP1^+/+ mice (lower right panel). Upper left, cryptitis with villous shortening, crypt regeneration and architectural distortion; upper right, neutrophilic crypt abscesses; lower left, duodenitis with surface ulceration and granulation tissue. D. Paneth cells with typical eosinophilic granules on H&E stained sections at the base of crypts in XBP1^+/+, but not XBP1^−/− epithelium. Electron microscopy (EM) with only rudimentary electron-dense granules and a contracted ER in XBP1^−/− basal crypt epithelial cells, normal configuration in XBP^+/+ mice. Immunohistochemistry (IHC) for the granule proteins lysozyme and pro-cryptdin in XBP1^+/+ and XBP1^−/− epithelia. E. Enumeration of Paneth cells and goblet cells in small intestines (n = 5 per group, Student’s t test). F. Goblet cell staining by PAS in XBP1^+/+ and XBP1^−/− epithelia. EM exhibited smaller cytoplasmic mucin droplets and a contracted ER in XBP1^−/− goblet cells. No structural abnormalities were found in neighboring absorptive epithelia in XBP1^−/− mice. G. The marker for enteroendocrine cells, chromogranin, was detected by IHC in small intestines of XBP1^+/+ and XBP1^−/− mice. H. XBP1^+/+ and XBP1^−/− mice were orally administered FITC-dextran, and FITC-dextran serum levels assayed 4h later.

Figure 2. XBP1 deletion results in apoptotic Paneth cell loss, inflammation, a distorted villus:crypt ratio, and IEC hyperproliferation

A. Apoptotic nuclei were identified in XBP1^+/+ (XBP1^flox/floxVCre) and XBP1^−/− (XBP1^flox/flox) sections with anti-active (cleaved) caspase-3. Arrows point to apoptotic cells. B. XBP1^{floxneo/floxneo}VCre-ER^T2 mice were administered 5 daily doses of 1 mg tamoxifen to induce deletion of the XBP1^floxneo gene in the intestinal epithelium. XBP1, cryptdin-5 (Defcr5), and Chop mRNA (all expressed normalized to β-actin; left y axis) expression in epithelium during and after tamoxifen treatment. Percentage of crypts with Paneth cells on H&E staining is shown (right y axis). Representative experiment of 3 performed. C. TUNEL and H&E staining on small intestinal sections of tamoxifen-treated XBP1^{floxneo/floxneo}VCre-ER^T2 mice collected at the indicated days. D. TUNEL⁺ and caspase-3⁺ cells were enumerated by light microscopy (3 mice per time-point with ileal and jejunal sections each; P values indicate comparisons to time-point 0; Student’s t test). E. TNFα mRNA was quantified by qPCR in small intestinal epithelial scrapings from ileum harvested at the indicated time-points after start of tamoxifen administration from VCre-ER^T2 XBP1^{floxneo/floxneo} (n = 4 per time-point) or XBP1^{floxneo/floxneo} (n = 1 per time-point) mice. P values indicate comparisons to time-point 0; Student’s t test. F. Enteritis in the small intestine in VCre-ER^T2 XBP1^{floxneo/floxneo} mice on day 5 after TAM administration. Upper left panel, 100×; upper right panel, same section, 400×, arrow points to a crypt abscess; lower panel 100×, crypts with Paneth cells (arrows). G. Jejunal sections of XBP1^flox/flox (XBP1^+/+; n = 7) and XBP1^flox/floxVCre (XBP1^−/−; n = 8) mice were assessed for their villus:crypt ratio on H&E stainings (ratios of ≥ 4:1 are considered normal for jejunum). H. XBP1^flox/flox (XBP1^+/+) and XBP1^flox/floxVCre (XBP1^−/−) mice were administered bromodeoxyuridine (BrdU) i.p., and small intestinal sections harvested after 1 h and 24 h (n = 3 per genotype per time-point). The 1 h time-point labels the pool of proliferating IEC in the crypts (mostly transit amplifying IEC), whereas the 24 h time-point assesses the migration along the crypt-villus axis indicating the turn-over of the IEC compartment.

Figure 3. XBP1 deficiency in epithelium results in impaired antimicrobial function

A. Lower panel. Small intestinal tissue from XBP1^+/+ and XBP1^−/− mice was homogenized, resolved on SDS-PAGE and detected by anti-lysozyme IgG and GAPDH to ensure equal loading. Upper panel. Small intestinal crypts isolated from XBP1^+/+ and XBP1^−/− animals were stimulated with 10µM carbamyl choline (CCh). Supernatants were precipitated, resolved on SDS-PAGE and detected by anti-lysozyme IgG. Blots are representative of 2 independent experiments. B. Small intestinal crypts were stimulated with LPS for 30min, and supernatants assayed for bactericidal activity. Data are expressed as % killing compared to unstimulated crypts, and are representative of 2 independent experiments. C. Intestinal epithelial cell-specific XBP1^−/− mice (n = 9) and XBP1^+/+ littermates (n = 9; 5–10 weeks of age) were perorally infected with 3.6×10⁸ L. monocytogenes. Faeces was aseptically collected 10h after infection and colony forming units (c.f.u.) of L. monocytogenes determined. Data are presented as c.f.u. per mg dry weight of faeces. D. Oral infection with L. monocytogenes was performed as in (C), liver and spleen aseptically harvested 72h after infection, and c.f.u. of L. monocytogenes determined (XBP1^+/+ n = 20; XBP1^−/− n = 17). Data are expressed as c.f.u. per organ. Two-tailed Mann-Whitney test was performed for (C) and (D).

Figure 4. XBP1 deficiency results in increased inflammatory tone of the epithelium

A. Small intestinal and colonic epithelial mRNA scrapings from XBP1^+/+, XBP1^+/−, and XBP1^−/− mice were analyzed for XBP1 mRNA splicing status. B. Small intestinal formalin-fixed sections were stained with rabbit anti-phospho-JNK antibody, and revealed a patchy staining pattern in XBP1^−/−, but not XBP1^+/+ sections. Control rabbit mAb was negative (not shown). Representative of n = 5 per group. C. MODE-K.iXBP and MODE-K.Control were stimulated for the indicated time periods with flagellin (1 µg/ml) and TNFα (50 ng/ml) and analyzed for P-JNK and total JNK by Western. D. MODE-K.iXBP (filled circles) and MODE-K.Ctrl (open circles) cells were stimulated for 4h with flagellin, and supernatants assayed by ELISA for CXCL1. E. Experiment as in (D), with TNFα. F. MODE-K.iXBP (circles) and MODE-K.Ctrl (diamonds) cells were stimulated with either 10µg/ml flagellin (filled symbols) or media alone (open symbols) for 4h, with the JNK inhibitor SP600125 and supernatants assayed for CXCL1. G. As in (F), MODE-K cells were stimulated with 50 ng/ml TNF-α (filled symbols) or media alone (open symbols). H. MODE-K.iXBP (filled circles) and MODE-K.Ctrl (open circles) cells loaded with the glycolipid antigen α-galactosylceramide (αGC), fixed, and co-cultured with the CD1d-restricted NKT cell hybridoma DN32.D3 and antigen presentation measured as IL-2 release from DN32.D3.

Figure 5. XBP1 deficiency increases susceptibility to DSS colitis

A. 4.5% DSS was administered in drinking water for 5 days and then replaced by regular drinking water in XBP1^+/+ (n = 9), XBP1^+/− (n = 9) and XBP1^−/− (n = 12) littermates (age 6–12 weeks). Wasting is presented as % of initial weight. One-tailed Student’s t test was performed. B. Presence of rectal bleeding during DSS colitis was assessed daily and scored as in Methods. Mean ± s.e.m.; XBP1^+/+ (n = 9), XBP1^+/− (n = 9) XBP1^−/− (n = 12). Two-tailed Mann-Whitney test was peformed. C. Individual signs of inflammation of colonic tissue harvested on day 8 of DSS colitis were scored blindly. Two-tailed Mann-Whitney test was performed. D. Typical colonic histology on day 8 of DSS colitis. Arrows, borders of ulcers. E. mRNA expression (normalized to βactin) of inflammatory mediators was quantified by qPCR in colonic specimens on day 8 of DSS colitis. n = 4 per group. Mean ± s.e.m analyzed by two-tailed Mann-Whitney test. F. Human ileum in Crohn’s disease exhibits signs of ER stress. Inflamed (“CD-I”, n = 3) and non-inflamed (“CD-NI”, n = 3) ileal biopsies from CD patients and healthy control (“Ctrl”, n = 4) subjects were analyzed for grp78 mRNA expression (levels in Controls were arbitrarily set at 1, and CD-I and CD-NI levels expressed as ratio to Controls; left y axis). XBP1 mRNA splicing is expressed as ratio of XBP1s/XBPu (right y axis). G. Human colon mucosa in Crohn’s disease (“CD”) and ulcerative colitis (“UC”) exhibits signs of ER stress. Colonic biopsies from inflamed (“−I”) and non-inflamed (“−NI”) CD and UC patients (n = 3 each) and healthy control subjects (“Ctrl”, n = 4) were analyzed for grp78 mRNA expression and XBP1 splicing as described in (F).

Figure 6. Rare XBP1 variants are hypomorphic

A. MODE-K cells were transfected with UPRE-luciferase and unspliced hXBP1u expression plasmids encoding the rare, IBD-associated minor alleles XBP1snp8 (M139I) and XBP1snp17 (A162P) and treated with 1 µg/ml Tm. Values represent luciferase activities normalized to cotransfected Renilla reporter. B. Experiments as in (a), with indicated amounts of spliced hXBP1s cDNA variants C. Transduction efficiency measured by FACS of XBP1^−/− MEF cells reconstituted with human XBP1 wildtype or SNP variants bi-cistronic retroviral vectors (RV_GFP) (MFI, mean fluorescence intensity). D. XBP1s protein levels were determined by western blot of Tm-treated cells (*, non-specific band). E. ERdj4 and EDEM mRNA levels (normalized to β-actin mRNA expression) in untreated (NT) or Tm (1 µg/ml)-treated cells for 6 hrs.