Notch inhibition counteracts Paneth cell death in absence of caspase-8.

Opposing activities of Notch and Wnt signaling regulate mucosal barrier homeostasis and differentiation of intestinal epithelial cells. Specifically, Wnt activity is essential for differentiation of secretory cells including Wnt3-producing Paneth cells, whereas Notch signaling strongly promotes generation of absorptive cells. Loss of caspase-8 in intestinal epithelium (casp8∆int) is associated with fulminant epithelial necroptosis, severe Paneth cell death, secondary intestinal inflammation, and an increase in Notch activity. Here, we found that pharmacological Notch inhibition with dibenzazepine (DBZ) is able to essentially rescue the loss of Paneth cells, deescalate the inflammatory phenotype, and reduce intestinal permeability in casp8∆int mice. The secretory cell metaplasia in DBZ-treated casp8∆int animals is proliferative, indicating for Notch activities partially insensitive to gamma-secretase inhibition in a casp8∆int background. Our data suggest that casp8 acts in the intestinal Notch network.

Paneth cells secrete lysozyme via secretory autophagy during bacterial infection of the intestine.

Intestinal Paneth cells limit bacterial invasion by secreting antimicrobial proteins, including lysozyme. However, invasive pathogens can disrupt the Golgi apparatus, interfering with secretion and compromising intestinal antimicrobial defense. Here we show that during bacterial infection, lysozyme is rerouted via secretory autophagy, an autophagy-based alternative secretion pathway. Secretory autophagy was triggered in Paneth cells by bacteria-induced endoplasmic reticulum (ER) stress, required extrinsic signals from innate lymphoid cells, and limited bacterial dissemination. Secretory autophagy was disrupted in Paneth cells of mice harboring a mutation in autophagy gene Atg16L1 that confers increased risk for Crohn's disease in humans. Our findings identify a role for secretory autophagy in intestinal defense and suggest why Crohn's disease is associated with genetic mutations that affect both the ER stress response and autophagy.

Secreted Phospholipases A2 Are Intestinal Stem Cell Niche Factors with Distinct Roles in Homeostasis, Inflammation, and Cancer.

The intestinal stem cell niche provides cues that actively maintain gut homeostasis. Dysregulation of these cues may compromise intestinal regeneration upon tissue insult and/or promote tumor growth. Here, we identify secreted phospholipases A2 (sPLA2s) as stem cell niche factors with context-dependent functions in the digestive tract. We show that group IIA sPLA2, a known genetic modifier of mouse intestinal tumorigenesis, is expressed by Paneth cells in the small intestine, while group X sPLA2 is expressed by Paneth/goblet-like cells in the colon. During homeostasis, group IIA/X sPLA2s inhibit Wnt signaling through intracellular activation of Yap1. However, upon inflammation they are secreted into the intestinal lumen, where they promote prostaglandin synthesis and Wnt signaling. Genetic ablation of both sPLA2s improves recovery from inflammation but increases colon cancer susceptibility due to release of their homeostatic Wnt-inhibitory role. This "trade-off" effect suggests sPLA2s have important functions as genetic modifiers of inflammation and colon cancer.

Identification of the Paneth cells in chicken small intestine.

The Paneth cells are highly specialized cells in the epithelium of the small intestine of many vertebrate species. These cells reside at the base of crypts of the Lieberkühn and contain abundant secretory granules. Previous studies suggesting the existence of Paneth cells in the chicken (Gallus gallus) remained controversial. Here we seek to identify the Paneth cells in the chicken small intestine through morphological examination and specific gene expression. Histological staining and transmission electron microscope confirmed the presence of granulated secretory cells at the base of the crypts in the chicken small intestine. Western blotting experiment also manifested the expression of lysozyme protein, which is specifically secreted by the Paneth cells in the small intestine. Moreover, lysozyme c and lysozyme g mRNAs were expressed in the small intestine of chickens at different ages. Lysozyme c mRNA, in particular, was located at the base of the small intestinal crypts as displayed by in situ hybridization. Collectively, we provide evidences that the Paneth cells indeed exist in the small intestine of the chicken.

A20 controls intestinal homeostasis through cell-specific activities.

The transcription factor NF-κB is indispensable for intestinal immune homeostasis, but contributes to chronic inflammation and inflammatory bowel disease (IBD). A20, an inhibitor of both NF-κB and apoptotic signalling, was identified as a susceptibility gene for multiple inflammatory diseases, including IBD. Despite absence of spontaneous intestinal inflammation in intestinal epithelial cell (IEC) specific A20 knockout mice, we found additional myeloid-specific A20 deletion to synergistically drive intestinal pathology through cell-specific mechanisms. A20 ensures intestinal barrier stability by preventing cytokine-induced IEC apoptosis, while A20 prevents excessive cytokine production in myeloid cells. Combining IEC and myeloid A20 deletion induces ileitis and severe colitis, characterized by IEC apoptosis, Paneth and goblet cell loss, epithelial hyperproliferation and intestinal microbiota dysbiosis. Continuous epithelial cell death and regeneration in an inflammatory environment sensitizes cells for neoplastic transformation and the development of colorectal tumours in aged mice.

Innate lymphoid cells regulate intestinal epithelial cell glycosylation.

Fucosylation of intestinal epithelial cells, catalyzed by fucosyltransferase 2 (Fut2), is a major glycosylation mechanism of host-microbiota symbiosis. Commensal bacteria induce epithelial fucosylation, and epithelial fucose is used as a dietary carbohydrate by many of these bacteria. However, the molecular and cellular mechanisms that regulate the induction of epithelial fucosylation are unknown. Here, we show that type 3 innate lymphoid cells (ILC3) induced intestinal epithelial Fut2 expression and fucosylation in mice. This induction required the cytokines interleukin-22 and lymphotoxin in a commensal bacteria-dependent and -independent manner, respectively. Disruption of intestinal fucosylation led to increased susceptibility to infection by Salmonella typhimurium. Our data reveal a role for ILC3 in shaping the gut microenvironment through the regulation of epithelial glycosylation.

ADAM10 regulates Notch function in intestinal stem cells of mice.

BACKGROUND & AIMS: A disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) is a cell surface sheddase that regulates physiologic processes, including Notch signaling. ADAM10 is expressed in all intestinal epithelial cell types, but the requirement for ADAM10 signaling in crypt homeostasis is not well defined. METHODS: We analyzed intestinal tissues from mice with constitutive (Vil-Cre;Adam10(f/f) mice) and conditional (Vil-CreER;Adam10(f/f) and Leucine-rich repeat-containing GPCR5 [Lgr5]-CreER;Adam10(f/f) mice) deletion of ADAM10. We performed cell lineage-tracing experiments in mice that expressed a gain-of-function allele of Notch in the intestine (Rosa26(NICD)), or mice with intestine-specific disruption of Notch (Rosa26(DN-MAML)), to examine the effects of ADAM10 deletion on cell fate specification and intestinal stem cell maintenance. RESULTS: Loss of ADAM10 from developing and adult intestine caused lethality associated with altered intestinal morphology, reduced progenitor cell proliferation, and increased secretory cell differentiation. ADAM10 deletion led to the replacement of intestinal cell progenitors with 2 distinct, post-mitotic, secretory cell lineages: intermediate-like (Paneth/goblet) and enteroendocrine cells. Based on analysis of Rosa26(NICD) and Rosa26(DN-MAML) mice, we determined that ADAM10 controls these cell fate decisions by regulating Notch signaling. Cell lineage-tracing experiments showed that ADAM10 is required for survival of Lgr5(+) crypt-based columnar cells. Our findings indicate that Notch-activated stem cells have a competitive advantage for occupation of the stem cell niche. CONCLUSIONS: ADAM10 acts in a cell autonomous manner within the intestinal crypt compartment to regulate Notch signaling. This process is required for progenitor cell lineage specification and crypt-based columnar cell maintenance.

Caspase-8 regulates TNF-α-induced epithelial necroptosis and terminal ileitis.

Dysfunction of the intestinal epithelium is believed to result in the excessive translocation of commensal bacteria into the bowel wall that drives chronic mucosal inflammation in Crohn's disease, an incurable inflammatory bowel disease in humans characterized by inflammation of the terminal ileum. In healthy individuals, the intestinal epithelium maintains a physical barrier, established by the tight contact of cells. Moreover, specialized epithelial cells such as Paneth cells and goblet cells provide innate immune defence functions by secreting mucus and antimicrobial peptides, which hamper access and survival of bacteria adjacent to the epithelium. Epithelial cell death is a hallmark of intestinal inflammation and has been discussed as a possible pathogenic mechanism driving Crohn's disease in humans. However, the regulation of epithelial cell death and its role in intestinal homeostasis remain poorly understood. Here we demonstrate a critical role for caspase-8 in regulating necroptosis of intestinal epithelial cells (IECs) and terminal ileitis. Mice with a conditional deletion of caspase-8 in the intestinal epithelium (Casp8(ΔIEC)) spontaneously developed inflammatory lesions in the terminal ileum and were highly susceptible to colitis. Casp8(ΔIEC) mice lacked Paneth cells and showed reduced numbers of goblet cells, indicating dysregulated antimicrobial immune cell functions of the intestinal epithelium. Casp8(ΔIEC) mice showed increased cell death in the Paneth cell area of small intestinal crypts. Epithelial cell death was induced by tumour necrosis factor (TNF)-α, was associated with increased expression of receptor-interacting protein 3 (Rip3; also known as Ripk3) and could be inhibited on blockade of necroptosis. Lastly, we identified high levels of RIP3 in human Paneth cells and increased necroptosis in the terminal ileum of patients with Crohn's disease, suggesting a potential role of necroptosis in the pathogenesis of this disease. Together, our data demonstrate a critical function of caspase-8 in regulating intestinal homeostasis and in protecting IECs from TNF-α-induced necroptotic cell death.

Phospholipase A2 group IIA expression correlates with prolonged survival in gastric cancer.

AIMS: The secreted phospholipase A2 type IIA (PLA2G2A) gene has been identified as a modifier of intestinal adenoma multiplicity in Apc(Min/+) mice. The aim of the present study was to analyse the clinical significance of PLA2G2A expression in human gastric cancer. METHODS AND RESULTS: Using immunohistochemistry, cytoplasmic immunoreactivity of PLA2G2A was observed in 27% (40 of 149) of gastric cancer tissues compared with negative staining in normal mucosa. The PLA2G2A expression rate in well-differentiated carcinoma was elevated significantly compared with that in poorly differentiated carcinoma (46% versus 19%, P = 0.001). Statistical analysis also revealed that PLA2G2A expression correlated negatively with depth of mural invasion, lymph node metastasis and tumour-node-metastasis (TNM) stage (P < 0.05). Patients with positive PLA2G2A expression showed higher 5-year overall survival than those with negative expression (P = 0.0004). In intestinal metaplasia, PLA2G2A was found to be abundant in Paneth cells. The coexistence of PLA2G2A and lysozyme was observed in Paneth cell-rich gastric cancer (P < 0.0001). CONCLUSIONS: PLA2G2A may predict survival and might be a potential biomarker for early detection and individualized therapy.

Further studies support the participation of stem cells in the cell turnover of duodenal adenomas.

BACKGROUND: In the normal duodenal mucosa, differentiated cells (enterocytes, goblet cells and endocrine cells) migrate from stem cells to the tip of the villi, but the lysozyme-producing Paneth cells migrate to the bottom of the crypts. The position of the Paneth cells within duodenal adenomas was investigated. PATIENTS AND METHODS: Sections from 83 duodenal adenomas were stained with hematoxylin-eosin (H&E) and with anti-lysozyme. Mature Paneth cells were those showing coarse brightly red cytoplasmic granules in H&E stain whereas their precursors were the lysozyme-positive cells that were undetected by H&E. RESULTS: The number of mature Paneth cells/high power field (x 40) varied in adenomas from 4 to 12 (mean 65) in H&E stain, while 32 to 62 cells/field (mean 46.5) were positive in anti-lysozyme immunostain (p < 0.05). The lysozyme-expressing cells were randomly distributed within the adenoma including the superficial cell layers. DISCUSSION AND CONCLUSION: Since mature Paneth cells and their precursors are positioned underneath stem cells, the presence of mature Paneth cells and their lysozyme-positive precursors in the surface epithelium of duodenal adenomas would imply that stem cells might have already exfoliated. An alternative explanation would mean that mutated stem cells, anchored in the bottom of the crypts of the adenoma would redirect, in an unparalleled fashion, the ontogenetic logistics of migration for Paneth cells. This stochastic molecular behaviour would require a reversal from the pre-determined migratory flow for Paneth cells to a paradoxical migration mode for these cells (from stem cells vertically along the villus, before exfoliation). Consequently, it is not inconceivable that stem cells might participate, together with other mature cells, in the cellular turnover of duodenal adenomas. If that is the case, the duodenal adenoma emerges as a suitable model to monitor the actual fate of mutated stem cells.

Analysis of the clonal architecture of the human small intestinal epithelium establishes a common stem cell for all lineages and reveals a mechanism for the fixation and spread of mutations.

Little is known about the clonal structure or stem cell architecture of the human small intestinal crypt/villus unit, or how mutations spread and become fixed. Using mitochondrial DNA (mtDNA) mutations as a marker of clonal expansion of stem cell progeny, we aimed to provide answers to these questions. Enzyme histochemistry (for cytochrome c oxidase and succinate dehydrogenase) was performed on frozen sections of normal human duodenum. Laser-capture microdissected cells were taken from crypts/villi. The entire mitochondrial genome was amplified using a nested PCR protocol; sequencing identified mutations and immunohistochemistry demonstrated specific cell lineages. Cytochrome c oxidase-deficient small bowel crypts were observed within all sections: negative crypts contained the same clonal mutation and all differentiated epithelial lineages were present, indicating a common stem cell origin. Mixed crypts were also detected, confirming the existence of multiple stem cells. We observed crypts where Paneth cells were positive but the rest of the crypt was deficient. We have demonstrated patches of deficient crypts that shared a common mutation, suggesting that they have divided by fission. We have shown that all cells within a small intestinal crypt are derived from one common stem cell. Partially-mutated crypts revealed some novel features of Paneth cell biology, suggesting that either they are long-lived or a committed Paneth cell-specific long-lived progenitor was present. We have demonstrated that mutations are fixed in the small bowel by fission and this has important implications for adenoma development.

Intestinal adenomas of Min-mice lack enterochromaffin cells, and have increased lysozyme production in non-Paneth cells.

BACKGROUND: Adenomatous polyposis coli (APC) are important in maintaining normal epithelial mucosa. Intestinal tissues with mutations in Apc have disturbed cell proliferation, differentiation and migration. Paneth and enterochromaffin cells were studied in the intestine and intestinal adenomas from Min-mice with heterozygote and homozygote mutations in Apc, respectively. MATERIALS AND METHODS: The presence of Paneth and enterochromaffin cells in normal intestine and adenomas from Min-mice was studied in sections stained with lysozyme/PAS and connexin32. RESULTS: Min-mice intestinal adenomas had an increased number of lysozyme-producing Paneth/goblet and non-Paneth cells and a reduced number of enterochromaffin cells. The large intestine had a significantly higher number of enterochromaffin cells than the small intestine and more were seen in the large intestine of Min- compared with wt-mice. CONCLUSION: Altered cell differentiation in adenomas might be caused by different response to Wnt-signalling, while an increased number of enterochromaffin cells in the large intestine is rather an effect of a heterozygous Apc(Min) mutation.

Source of group II phospholipase A2 in gastric juice.

Gastric juice contains both pancreatic group I phospholipase A2 (PLA2-I) and synovial-type group II phospholipase A2 (PLA2-II), which may play a crucial role in Helicobacter pylori infection and gastric mucosal injury. PLA2-I present in gastric juice is derived from pancreatic acinar cells. The cellular source of PLA2-II found in gastric juice is unknown. A specific cell type of the intestinal mucosa, the Paneth cell, is known to secrete PLA2-II. The purpose of the present study was to define the source of PLA2-II present in gastric juice. For this purpose, gastric juice was collected from 29 individuals during gastroscopy, and mucosal biopsies were taken from the antrum and body of the stomach and from the duodenum as well as from the jejunum of individuals with resected stomach, for immunohistochemical detection of PLA2-II. The concentration of bilirubin in the gastric juice samples was determined to identify duodenogastric regurgitation. The PLA2-II content was significantly higher in bilirubin-positive than in bilirubin-negative gastric juice samples. PLA2-II was localized by immunohistochemistry in Paneth cells in three patients with areas of intestinal metaplasia of the gastric mucosa and in Paneth cells of duodenal and jejunal mucosa in all patients, but not in any other epithelial cell type of the mucosa of the stomach or the small intestine. Inflammatory cells did not contain PLA2-II. The current results suggest that PLA2-II found in gastric juice is derived from the Paneth cells of the small intestinal mucosa.

Paneth cell trypsin is the processing enzyme for human defensin-5.

The antimicrobial peptide human alpha-defensin 5 (HD5) is expressed in Paneth cells, secretory epithelial cells in the small intestine. Unlike other characterized defensins, HD5 is stored in secretory vesicles as a propeptide. The storage quantities of HD5 are approximately 90 450 microg per cm2 of mucosal surface area, which is sufficient to generate microbicidal concentrations in the intestinal lumen. HD5 peptides isolated from the intestinal lumen are proteolytically processed forms--HD5(56-94) and HD5(63-94)--that are cleaved at the Arg55-Ala56 and Arg62-Thr63 sites, respectively. We show here that a specific pattern of trypsin isozymes is expressed in Paneth cells, that trypsin colocalizes with HD5 and that this protease can efficiently cleave HD5 propeptide to forms identical to those isolated in vivo. By acting as a prodefensin convertase in human Paneth cells, trypsin is involved in the regulation of innate immunity in the small intestine.

Identification of xanthine dehydrogenase/xanthine oxidase as a rat Paneth cell zinc-binding protein.

Paneth cells are zinc-containing cells localized in small intestinal crypts, but their function has not been fully elucidated. Previously, we showed that an intravenous injection of diphenylthiocarbazone (dithizone), a zinc chelator, induced selective killing of Paneth cells, and purified a zinc-binding protein in Paneth cells. In the present study, we further characterized one of these proteins, named zinc-binding protein of Paneth cells (ZBPP)-1. Partial amino acid sequences of ZBPP-1 showed identity with rat xanthine dehydrogenase (XD)/xanthine oxidase (XO). Anti-rat XD antibody (Ab) recognized ZBPP-1, and conversely anti ZBPP-1 Ab recognized 85 kDa fragment of rat XD in Western blotting. Messenger RNA and protein levels of XD were consistent with our previous data on the fluctuation of Paneth cell population after dithizone injection. Thus, ZBPP-1 is an 85 kDa fragment of XD/XO in Paneth cells. XD/XO in Paneth cells may play important roles in intestinal function.

Absence of group II phospholipase A2, a Paneth cell marker, from the epididymis.

Paneth cell-like metaplasia has been reported in the epithelium of the epididymis and prostatic adenocarcinomas. We studied the expression of group II phospholipase A2 (PLA2), a marker of Paneth cell differentiation, in six orchiectomy specimens with Paneth cell-like metaplasia. Both immunohistochemistry for group II PLA2 protein and in situ hybridization for the mRNA of group II PLA2 gave negative results in all six cases but positive reaction for lysozyme. The results show that the cells of the Paneth cell-like metaplasia are not true Paneth cells.

Cellular localization of group IIA phospholipase A2 in rats.

It has been known that group II phospholipase A2 (PLA2) mRNA and protein are present in the homogenates of the spleen, lung, liver, and kidney in normal rats, but the cellular origin of this enzyme has not been yet identified. At present, five subtypes of group II PLA2 have been identified in mammals. Antibodies or mRNA probes previously used for detecting group II PLA2 need to be evaluated to identify the subtypes of group II PLA2. In this study we tried to identify group IIA PLA2-producing cells in normal rat tissues by in situ hybridization (ISH) using an almost full-length RNA probe for rat group IIA enzyme. Group IIA PLA2 mRNA was detected in megakaryocytes in the spleen and Paneth cells in the intestine by ISH. These cells were also immunopositive for an antibody raised against group IIA PLA(2) isolated from rat platelets. Group IIA PLA2 mRNA-positive cells were not detected in lung, liver, kidney, and pancreas. Under normal conditions, group IIA PLA2-producing cells are splenic megakaryocytes and intestinal Paneth cells in rats.

The antibacterial properties of secreted phospholipases A(2).

There is a considerable body of evidence to support the antibacterial properties of the group IIa phospholipase A(2) as an important physiological function. This enzyme is able to act as an acute phase protein and may be part of the innate defence system of the body, acting in concert with other antibacterial proteins and peptides. The enzyme is most effective against Gram-positive bacteria whereas penetration of the lipopolysaccharide coat of Gram-negative bacteria requires bactericidal/permeability-increasing protein (BPI) as an additional permeabilizing factor. The global cationic nature of this protein (pI>10.5) appears to facilitate penetration of the anionic bacterial cell wall. In addition, the considerable preference of the enzyme for anionic phospholipid interfaces provides specificity toward anionic bacterial membranes as opposed to zwitterionic eucaryotic cell membranes.