Paneth cells as a site of origin for intestinal inflammation

Abstract

The recognition of autophagy related 16-like 1 (ATG16L1) as a genetic risk factor has exposed the critical role of autophagy in Crohn's disease. Homozygosity for the highly prevalent ATG16L1 risk allele, or murine hypomorphic (HM) activity, causes Paneth cell dysfunction. As Atg16l1(HM) mice do not develop spontaneous intestinal inflammation, the mechanism(s) by which ATG16L1 contributes to disease remains obscure. Deletion of the unfolded protein response (UPR) transcription factor X-box binding protein-1 (Xbp1) in intestinal epithelial cells, the human orthologue of which harbours rare inflammatory bowel disease risk variants, results in endoplasmic reticulum (ER) stress, Paneth cell impairment and spontaneous enteritis. Unresolved ER stress is a common feature of inflammatory bowel disease epithelium, and several genetic risk factors of Crohn's disease affect Paneth cells. Here we show that impairment in either UPR (Xbp1(ΔIEC)) or autophagy function (Atg16l1(ΔIEC) or Atg7(ΔIEC)) in intestinal epithelial cells results in each other's compensatory engagement, and severe spontaneous Crohn's-disease-like transmural ileitis if both mechanisms are compromised. Xbp1(ΔIEC) mice show autophagosome formation in hypomorphic Paneth cells, which is linked to ER stress via protein kinase RNA-like endoplasmic reticulum kinase (PERK), elongation initiation factor 2α (eIF2α) and activating transcription factor 4 (ATF4). Ileitis is dependent on commensal microbiota and derives from increased intestinal epithelial cell death, inositol requiring enzyme 1α (IRE1α)-regulated NF-κB activation and tumour-necrosis factor signalling, which are synergistically increased when autophagy is deficient. ATG16L1 restrains IRE1α activity, and augmentation of autophagy in intestinal epithelial cells ameliorates ER stress-induced intestinal inflammation and eases NF-κB overactivation and intestinal epithelial cell death. ER stress, autophagy induction and spontaneous ileitis emerge from Paneth-cell-specific deletion of Xbp1. Genetically and environmentally controlled UPR function within Paneth cells may therefore set the threshold for the development of intestinal inflammation upon hypomorphic ATG16L1 function and implicate ileal Crohn's disease as a specific disorder of Paneth cells.

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1. PERK/eIF2α signaling induces autophagy in Xbp1-deficient intestinal epithelial cells

a, b, Immunoblot for LC3 conversion in isolated primary IECs (a) (n=5/4) and for autophagy proteins in primary IEC scrapings (b) (n=3). c, Transmission electron microscopy (TEM) of crypts. Note autophagic vacuoles in various stages of evolution in Xbp1ΔIEC hypomorphic Paneth cells. d, e, Crypt showing GFP-LC3 punctae (d), quantified in (e) (n=10; unpaired Student’s t-test). Bar, 5 μm. f, g, Immunoblot of silenced MODE-K cells (f) and primary IEC scrapings (g) for the PERK/eIF2α branch (n=3). h, i, Promoter sequence qPCR for Map1lc3b (LC3b) (h) and Atg7 (i) after anti-ATF4 ChIP (unpaired Student’s t-test). j, GFP-LC3 punctae per crypt after treatment with tamoxifen for 3 days and vehicle or salubrinal (n=10; one-way ANOVA with post-hoc Bonferroni). (k) Enteritis histology score after salubrinal and tamoxifen co-treatment (n=12/14/13; median shown; Kruskal-Wallis with post-hoc Holm’s-corrected Mann-Whitney U). Results represent three (a, f, g) or two (c, e, h, i) independent experiments. *P < 0.05, ***P < 0.001.

Figure 2. Impairment of ER stress-induced compensatory autophagy results in severe transmural inflammation

a, TEM of crypts of Lieberkühn (n=2). Bar, 2 μm. b, Representative H&E stainings. Note transmural inflammation extending through muscularis propria (white arrow) into serosa (black arrow) in Atg7/Xbp1ΔIEC and Atg16l1/Xbp1ΔIEC mice scored in (c) and (e). Bar, 50 and 10 μm, respectively. c, Enteritis histology score (n=26/12/18/27; 10-18 weeks; median shown; Kruskal-Wallis with post-hoc Holm’s-corrected Mann-Whitney U). d, Crypt TEM in indicated genotypes (n=2). Bar, 2 μm. e, Enteritis histology score (n=11; 18 weeks; median shown; Kruskal-Wallis with post-hoc Holm’s-corrected Mann-Whitney U). f,Xbp1 mRNA splicing (Xbp1u, unspliced; Xbp1s, spliced) of crypts, densitometry in (g) (n=7; unpaired Student’s t-test). h, GRP78 (green) immunofluorescence, white arrows indicate GRP78+ crypts (DAPI, blue; n=5). Bar, 10 μm. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3. Autophagy restrains IRE1α-mediated NFκB activation in Xbp1-deficient epithelium

a,shCtrl or shXbp1 MODE-K cells were co-silenced for Atg16l1 (siAtg16l1) or with scrambled siRNA (siCtrl), and analyzed by flow cytometry for annexin V and propidium iodide (PI; one-way ANOVA with post-hoc Bonferroni). b, Immunoblot of primary IEC scrapings (n=3). c, Immunoblot of cytoplasmic extracts from shCtrl or shXbp1 MODE-K cells after TNF stimulation. d, TUNEL+ IECs per 100 crypts after BAY11-7082 or vehicle treatment (n=3/4/4; one-way ANOVA with post-hoc Holm’s-corrected unpaired Student’s t-test). e, Enteritis histology score of mice treated with BAY11-7082 or vehicle (n=10/10/9; median shown; Kruskal-Wallis with post-hoc Holm’s-corrected Mann-Whitney U). f, Immunoblot of IEC scrapings for (p-)IRE1α after IRE1α immunoprecipitation (IP). β-actin, loading control of whole lysates. g, Immunoblot of primary IEC scrapings (n=4). h, i, Enteritis histology score of indicated genotypes (h,n=15/16/14/15; i,n=5/10/12; median shown; Kruskal-Wallis with post-hoc Holm’s-corrected Mann-Whitney U). j, Enteritis histology score of specific pathogen free (SPF) and germ free (GF) housed mice (n=10/9/7/7; median shown; Kruskal-Wallis with post-hoc Holm’s-corrected Mann-Whitney U). (k) Representative images of p-IκBα immunoreactivity under conditions as in (j) (n=4). Bar, 20 μm. Results represent four (f), three (c) or two (a, b) independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4. ER stress-induced enteritis originates from Paneth cells and is alleviated through autophagy induction

a, Immunoblot of primary IEC scrapings from mice treated with or without rapamycin for 14 consecutive days (n=3). b, Enteritis histology score for experiment as in (a) (n=4; median shown; Mann-Whitney U). c, Representative images of EYFP-Rosa26/D6-Cre+/− reporter mice and EYFP-Rosa26 (controls). Co-localization of Defa6 Cre-driven EYFP expression (yellow) with lysozyme expressing Paneth cells (red; n=3). DAPI, blue; bar, 50 μm. d, Immunoblots of crypt IECs from Xbp1ΔPC and Wt controls (n=2). e, f, Representative confocal images of lysozyme (green) expressing Paneth cells (e) with quantification of crypts with indicated number of lysozyme+ granulated dots in (f) (n=5; unpaired Student’s t-test). DAPI, blue; bar, 10 μm. g, Immunohistochemistry for p-eIF2α (n=3). Bar, 20 μm. h, Representative H&E images of Xbp1ΔPC and Wt mice scored in (i). Bar, 50 μm. (i) Enteritis scoring in Xbp1fl/fl (Wtfl), Defa6 Cre+ (WtCre) and Defa6 Cre+;Xbp1fl/fl (Xbp1ΔPC) mice (n=21/26/29; median shown; Kruskal-Wallis with post-hoc Holm’s-corrected Mann-Whitney U). Results represent two (b, d) independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.