Markus Tschurtschenthaler

Paneth cells as a site of origin for intestinal inflammation

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 Atg16l1HM 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 (Xbp1ΔIEC) or autophagy function (Atg16l1ΔIEC or Atg7ΔIEC) 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. Xbp1ΔIEC 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.

ER stress transcription factor Xbp1 suppresses intestinal tumorigenesis and directs intestinal stem cells

X-box–binding protein 1 suppresses tumor formation in the gut by regulating Ire1α and Stat3-mediated regenerative responses in the epithelium as a consequence of ER stress.

Toll Like Receptor 4-mediated Endoplasmic Reticulum Stress in Intestinal Crypts Induces Necrotizing Enterocolitis

Background: Cellular cues that regulate intestinal stem cell (ISC) apoptosis are unknown. Results: Toll-like-receptor 4 (TLR4) activation on ISCs induces endoplasmic reticulum (ER) stress, leading to ISC apoptosis and necrotizing enterocolitis (NEC). Conclusion: TLR4-induced ER stress in ISCs leads to apoptosis and NEC. Significance: This is the first study revealing that ER stress in ISCs via immune receptors induces NEC. The cellular cues that regulate the apoptosis of intestinal stem cells (ISCs) remain incompletely understood, yet may play a role in diseases characterized by ISC loss including necrotizing enterocolitis (NEC). Toll-like receptor-4 (TLR4) was recently found to be expressed on ISCs, where its activation leads to ISC apoptosis through mechanisms that remain incompletely explained. We now hypothesize that TLR4 induces endoplasmic reticulum (ER) stress within ISCs, leading to their apoptosis in NEC pathogenesis, and that high ER stress within the premature intestine predisposes to NEC development. Using transgenic mice and cultured enteroids, we now demonstrate that TLR4 induces ER stress within Lgr5 (leucine-rich repeat-containing G-protein-coupled receptor 5)-positive ISCs, resulting in crypt apoptosis. TLR4 signaling within crypts was required, because crypt ER stress and apoptosis occurred in TLR4ΔIEC-OVER mice expressing TLR4 only within intestinal crypts and epithelium, but not TLR4ΔIEC mice lacking intestinal TLR4. TLR4-mediated ER stress and apoptosis of ISCs required PERK (protein kinase-related PKR-like ER kinase), CHOP (C/EBP homologous protein), and MyD88 (myeloid differentiation primary response gene 88), but not ATF6 (activating transcription factor 6) or XBP1 (X-box-binding protein 1). Human and mouse NEC showed high crypt ER stress and apoptosis, whereas genetic inhibition of PERK or CHOP attenuated ER stress, crypt apoptosis, and NEC severity. Strikingly, using intragastric delivery into fetal mouse intestine, prevention of ER stress reduced TLR4-mediated ISC apoptosis and mucosal disruption. These findings identify a novel link between TLR4-induced ER stress and ISC apoptosis in NEC pathogenesis and suggest that increased ER stress within the premature bowel predisposes to NEC development.

Type I interferon signalling in the intestinal epithelium affects Paneth cells, microbial ecology and epithelial regeneration

Objective Intestinal epithelial cells (IECs) at the internal/external interface orchestrate the mucosal immune response. Paneth cells secrete antimicrobial peptides and inflammatory mediators, protect from pathogens and shape the commensal microbiota. Prompted by the genetic association of the locus harbouring the type I interferon (IFN) receptor (IFNAR1) with Crohns disease, and a transcriptional signature for type I IFN signalling in Paneth cells, we studied the function of IFNAR1 in IECs. Design Type I IFN signalling was studied in mice with conditional deletion of Ifnar1 in IECs. Phenotype was characterised at baseline, and gut microbiota composition was assessed by 16S rDNA ribotyping. The role of IFNAR1 was also investigated in experimental colitis induced by dextran sodium sulfate (DSS) and colitis-associated cancer induced by DSS in conjunction with azoxymethane (AOM). Results Ifnar1−/−(IEC) mice displayed expansion of Paneth cell numbers and epithelial hyperproliferation compared with Ifnar1-sufficient littermates. While Ifnar1−/−(IEC) mice did not exhibit spontaneous inflammation or increased severity in DSS colitis compared with Ifnar1+/+(IEC) mice, they exhibited an increased tumour burden in the AOM/DSS model. Both hyperproliferation and tumour promotion were dependent on the microbial flora, as the differences between genotypes were marked upon separately housing mice, but disappeared when Ifnar1−/−(IEC) and Ifnar1+/+(IEC) mice were co-housed. Accordingly, ribotyping revealed marked differences between Ifnar1−/−(IEC) and Ifnar1+/+(IEC) mice that where diminished upon co-housing. Conclusions IFNAR1 in IECs, and Paneth cells in particular, contributes to the regulation of the host–microbiota relationship, with consequences for intestinal regeneration and colitis-associated tumour formation.

Defective ATG16L1-mediated removal of IRE1α drives Crohn’s disease–like ileitis

ATG16L1T300A, a major risk polymorphism in Crohn’s disease (CD), causes impaired autophagy, but it has remained unclear how this predisposes to CD. In this study, we report that mice with Atg16l1 deletion in intestinal epithelial cells (IECs) spontaneously develop transmural ileitis phenocopying ileal CD in an age-dependent manner, driven by the endoplasmic reticulum (ER) stress sensor IRE1&agr;. IRE1&agr; accumulates in Paneth cells of Atg16l1&Dgr;IEC mice, and humans homozygous for ATG16L1T300A exhibit a corresponding increase of IRE1&agr; in intestinal epithelial crypts. In contrast to a protective role of the IRE1&bgr; isoform, hyperactivated IRE1&agr; also drives a similar ileitis developing earlier in life in Atg16l1;Xbp1&Dgr;IEC mice, in which ER stress is induced by deletion of the unfolded protein response transcription factor XBP1. The selective autophagy receptor optineurin interacts with IRE1&agr;, and optineurin deficiency amplifies IRE1&agr; levels during ER stress. Furthermore, although dysbiosis of the ileal microbiota is present in Atg16l1;Xbp1&Dgr;IEC mice as predicted from impaired Paneth cell antimicrobial function, such structural alteration of the microbiota does not trigger ileitis but, rather, aggravates dextran sodium sulfate–induced colitis. Hence, we conclude that defective autophagy in IECs may predispose to CD ileitis via impaired clearance of IRE1&agr; aggregates during ER stress at this site.

Intestinal epithelial cell endoplasmic reticulum stress promotes MULT1 up-regulation and NKG2D-mediated inflammation

Endoplasmic reticulum (ER) stress is commonly observed in intestinal epithelial cells (IECs) and can, if excessive, cause spontaneous intestinal inflammation as shown by mice with IEC-specific deletion of X-box–binding protein 1 (Xbp1), an unfolded protein response–related transcription factor. In this study, Xbp1 deletion in the epithelium (Xbp1&Dgr;IEC) is shown to cause increased expression of natural killer group 2 member D (NKG2D) ligand (NKG2DL) mouse UL16-binding protein (ULBP)–like transcript 1 and its human orthologue cytomegalovirus ULBP via ER stress–related transcription factor C/EBP homology protein. Increased NKG2DL expression on mouse IECs is associated with increased numbers of intraepithelial NKG2D-expressing group 1 innate lymphoid cells (ILCs; NK cells or ILC1). Blockade of NKG2D suppresses cytolysis against ER-stressed epithelial cells in vitro and spontaneous enteritis in vivo. Pharmacological depletion of NK1.1+ cells also significantly improved enteritis, whereas enteritis was not ameliorated in Recombinase activating gene 1−/−;Xbp1&Dgr;IEC mice. These experiments reveal innate immune sensing of ER stress in IECs as an important mechanism of intestinal inflammation.

Paternal chronic colitis causes epigenetic inheritance of susceptibility to colitis.

Inflammatory bowel disease (IBD) arises by unknown environmental triggers in genetically susceptible individuals. Epigenetic regulation of gene expression may integrate internal and external influences and may thereby modulate disease susceptibility. Epigenetic modification may also affect the germ-line and in certain contexts can be inherited to offspring. This study investigates epigenetic alterations consequent to experimental murine colitis induced by dextran sodium sulphate (DSS), and their paternal transmission to offspring. Genome-wide methylome- and transcriptome-profiling of intestinal epithelial cells (IECs) and sperm cells of males of the F0 generation, which received either DSS and consequently developed colitis (F0DSS), or non-supplemented tap water (F0Ctrl) and hence remained healthy, and of their F1 offspring was performed using reduced representation bisulfite sequencing (RRBS) and RNA-sequencing (RNA-Seq), respectively. Offspring of F0DSS males exhibited aberrant methylation and expression patterns of multiple genes, including Igf1r and Nr4a2, which are involved in energy metabolism. Importantly, DSS colitis in F0DSS mice was associated with decreased body weight at baseline of their F1 offspring, and these F1 mice exhibited increased susceptibility to DSS-induced colitis compared to offspring from F0Ctrl males. This study hence demonstrates epigenetic transmissibility of metabolic and inflammatory traits resulting from experimental colitis.

154 ER Stress Transcription Factor XBP1 Suppresses Intestinal Tumorigenesis

Background. The unfolded protein response (UPR) is a response mechanism to endoplasmic reticulum (ER) stress, which a cell is typically exposed upon protein misfolding. Intestinal epithelial cell (IEC) homeostasis critically depends upon an intact UPR and its perturbation due to deficiency of the key UPR transcription factor X-box binding protein-1 (XBP1) in IECs results in ER stress und spontaneous intestinal inflammation. Rare functional variants of XBP1 are associated with inflammatory bowel disease (IBD) and IBD patients typically exhibit unresolved ER stress in IECs. Long standing inflammation in IBD patients predisposes for colitis-associated cancer and cancer cells typically exhibit ER stress due to increased risk for protein misfolding as a consequence of the tumour microenvironment characterized by low oxygen, nutrient deprivation and pH changes. Aims. We hypothesised XBP1-deficiency and subsequent unresolved ER stress might affect intestinal regeneration, which may impact on tumourigenesis. Methods. NFκB and Stat3 activation was analysed by immunoblot and immunohistochemistry. Specific inhibitors of Stat3 (S3I-201) and NF κB (BAY11-7082), respectively, were administered to Xbp1flox/flox VillinCre mice. Epithelial proliferation was assessed by bromodeoxyuridine (BrdU) staining. Xbp1flox/flox VillinCre mice were analyzed at day 61 of azoxymethane (AOM) followed by three cycles of dextran sodium sulphate (DSS). Results. ER stress due to XBP1-deficiency results in a numerical increase of transit amplifying (TA) cells and consequently in an increased epithelial turn over. Xbp1-/-(IEC) mice activate Stat3, which localizes to TA cells, and its blockade via administration of the Stat3 inhibitor S3I-201 abrogates epithelial hyperregeneration. Xbp1-deletion in IECs leads to activation of NFκB and increased IL-6 secretion, a known activator of Stat3. Blockade of NFκB signaling via administration of the IKK2 inhibitor BAY11-7082 abrogated Stat3 activation in XBP1-deficient mice. NFκB and Stat3 promote colitis-associated cancer and XBP1deficient mice exhibit an increase in tumour burden in a model of colitis-associated cancer characterized by a 10-fold increase in tumour area as well as tumour number. Conclusions. Our data unravels XBP1 as an unexpected tumour suppressor in the intestinal epithelium, thereby opposing the current paradigm that cancer cells require an unimpaired UPR to survive in a stressful tumour microenvironment. Promotion of colitis-associated tumourigenesis is functionally linked to activation of NFκB and Stat3, which are potently engaged in XBP1deficient IECs. Our data show that unresolved ER stress in the intestinal epithelium increases the propensity to develop intestinal tumours.

ATG16L1 orchestrates interleukin-22-signaling in the intestinal epithelium via cGAS/STING

A coding variant of the inflammatory bowel disease (IBD) risk gene ATG16L1 has been associated with defective autophagy and deregulation of endoplasmic reticulum (ER) function. IL-22 is a barrier protective cytokine by inducing regeneration and antimicrobial responses in the intestinal mucosa. We show that ATG16L1 critically orchestrates IL-22 signaling in the intestinal epithelium. IL-22 stimulation physiologically leads to transient ER stress and subsequent activation of STING-dependent type I interferon (IFN-I) signaling, which is augmented in Atg16l1&Dgr;IEC intestinal organoids. IFN-I signals amplify epithelial TNF production downstream of IL-22 and contribute to necroptotic cell death. In vivo, IL-22 treatment in Atg16l1&Dgr;IEC and Atg16l1&Dgr;IEC/Xbp1&Dgr;IEC mice potentiates endogenous ileal inflammation and causes widespread necroptotic epithelial cell death. Therapeutic blockade of IFN-I signaling ameliorates IL-22–induced ileal inflammation in Atg16l1&Dgr;IEC mice. Our data demonstrate an unexpected role of ATG16L1 in coordinating the outcome of IL-22 signaling in the intestinal epithelium.