技术资料

Rotavirus,a leading cause of severe gastroenteritis and diarrhoea in young children,accounts for around 215,000 deaths annually worldwide. Rotavirus specifically infects the intestinal epithelial cells in the host small intestine and has evolved strategies to antagonize interferon and NF-κB signalling,raising the question as to whether other host factors participate in antiviral responses in intestinal mucosa. The mechanism by which enteric viruses are sensed and restricted in vivo,especially by NOD-like receptor (NLR) inflammasomes,is largely unknown. Here we uncover and mechanistically characterize the NLR Nlrp9b that is specifically expressed in intestinal epithelial cells and restricts rotavirus infection. Our data show that,via RNA helicase Dhx9,Nlrp9b recognizes short double-stranded RNA stretches and forms inflammasome complexes with the adaptor proteins Asc and caspase-1 to promote the maturation of interleukin (Il)-18 and gasdermin D (Gsdmd)-induced pyroptosis. Conditional depletion of Nlrp9b or other inflammasome components in the intestine in vivo resulted in enhanced susceptibility of mice to rotavirus replication. Our study highlights an important innate immune signalling pathway that functions in intestinal epithelial cells and may present useful targets in the modulation of host defences against viral pathogens. View Publication

Intestinal tuft cells are one of 4 secretory cell linages in the small intestine and the source of IL-25,a critical initiator of the type 2 immune response to parasite infection. When Raptor,a critical scaffold protein for mammalian target of rapamycin complex 1 (mTORC1),was acutely deleted in intestinal epithelium via Tamoxifen injection in Tritrichomonas muris (Tm) infected mice,tuft cells,IL-25 in epithelium and IL-13 in the mesenchyme were significantly reduced,but Tm burden was not affected. When Tm infected mice were treated with rapamycin,DCLK1 and IL-25 expression in enterocytes and IL-13 expression in mesenchyme were diminished. After massive small bowel resection,tuft cells and Tm were diminished due to the diet used postoperatively. The elimination of Tm and subsequent re-infection of mice with Tm led to type 2 immune response only in WT,but Tm colonization in both WT and Raptor deficient mice. When intestinal organoids were stimulated with IL-4,tuft cells and IL-25 were induced in both WT and Raptor deficient organoids. In summary,our study reveals that enterocyte specific Raptor is required for initiating a type 2 immune response which appears to function through the regulation of mTORC1 activity. View Publication

Type I and type III interferons (IFNs) are crucial components of the first-line antiviral host response. While specific receptors for both IFN types exist,intracellular signaling shares the same Jak-STAT pathway. Due to its receptor expression,IFN-λ responsiveness is restricted mainly to epithelial cells. Here,we display IFN-stimulated gene induction at the single cell level to comparatively analyze the activities of both IFN types in intestinal epithelial cells and mini-gut organoids. Initially,we noticed that the response to both types of IFNs at low concentrations is based on a single cell decision-making determining the total cell intrinsic antiviral activity. We identified histone deacetylase (HDAC) activity as a crucial restriction factor controlling the cell frequency of IFN-stimulated gene (ISG) induction upon IFN-λ but not IFN-β stimulation. Consistently,HDAC blockade confers antiviral activity to an elsewise non-responding subpopulation. Second,in contrast to the type I IFN system,polarization of intestinal epithelial cells strongly enhances their ability to respond to IFN-λ signaling and raises the kinetics of gene induction. Finally,we show that ISG induction in mini-gut organoids by low amounts of IFN is characterized by a scattered heterogeneous responsiveness of the epithelial cells and HDAC activity fine-tunes exclusively IFN-λ activity. This study provides a comprehensive description of the differential response to type I and type III IFNs and demonstrates that cell polarization in gut epithelial cells specifically increases IFN-λ activity. View Publication

Sterol regulatory element-binding protein-2 (SREBP-2) activates transcription of all genes needed for cholesterol biosynthesis. To study SREBP-2 function in intestine,we generated a mouse model (Vil-BP2(-/-) ) in which Cre recombinase ablates SREBP-2 in intestinal epithelia. Intestines of Vil-BP2(-/-) mice had reduced expression of genes required for sterol synthesis,in vivo sterol synthesis rates,and epithelial cholesterol contents. On a cholesterol-free diet,they displayed chronic enteropathy with histological abnormalities of both villi and crypts,growth restriction,and reduced survival that was prevented by supplementation of cholesterol in the diet. Likewise,SREBP-2-deficient enteroids required exogenous cholesterol for growth. Blockade of luminal cholesterol uptake into enterocytes with ezetimibe precipitated acutely lethal intestinal damage in Vil-BP2(-/-) mice,highlighting the critical interplay in the small intestine of sterol absorption via NPC1L1 and sterol synthesis via SREBP-2 in sustaining the intestinal mucosa. These data show that small intestine requires SREBP-2 to drive cholesterol synthesis that sustains the intestinal epithelia when uptake of cholesterol from the gut lumen is not available,and provide a unique example of cholesterol auxotrophy expressed in an intact,adult mammal. View Publication

The current in vitro or in vivo intestinal fibrosis models have many limitations. Recent advancements in the isolation and culturing of organoids has led to development of various three-dimensional (3D) intestinal disease models with in vivo physiology. In this study,we generated an organoid-based epithelial to mesenchymal transition (OEMT) model,which could be used as a novel intestinal fibrosis model. Intestinal epithelial organoids (IEOs) were isolated and cultured from the small intestines of normal mice. IEOs were treated with transforming growth factor- β1 (TGF-β1) or Tumor necrosis factor-α (TNF-α) to evaluate their phenotypic change. Raw 264.7 cells (macrophage) stimulated with lipopolysaccharide were co-cultured with IEOs in growth media with or without TGF-β1. TGF-β1 alone slightly induced epithelial to mesenchymal transition (EMT) in the IEOs but mainly disrupted them. Macrophage released cytokines synergistically induced mesenchymal phenotypic changes in TGF-β1 stimulated intestinal organoids. TNF-α and TGF-β1 synergistically induced proliferation of mesenchymal cells as well as EMT in the IEOs. We generated a novel OEMT model based on our finding that TNF-α and TGF-β synergistically induce type 2 EMT in IEOs. This 3D EMT model with in vivo physiology could be used to study EMT associated intestinal fibrosis. View Publication

Irregular mitochondria structure and reduced ATP in some patients with IBD suggest that metabolic stress contributes to disease. Loss-of-function mutation in the nucleotide-binding oligomerization domain (NOD)-2 gene is a major susceptibility trait for IBD. Hence,we assessed if loss of NOD2 further impairs the epithelial barrier function instigated by disruption of mitochondrial ATP synthesis via the hydrogen ionophore dinitrophenol (DNP). NOD2 protein (virtually undetectable in epithelia under basal conditions) was increased in T84 (human colon cell line) cells treated with noninvasive Escherichia coli + DNP (16 h). Increased intracellular bacteria in wild-type (WT) and NOD2 knockdown (KD) cells and colonoids from NOD2(-/-) mice were mediated by reactive oxygen species (ROS) and the MAPK ERK1/2 pathways as determined by cotreatment with the antioxidant mitoTEMPO and the ERK inhibitor U0126: ROS was upstream of ERK1/2 activation. Despite increased E. coli in DNP-treated NOD2 KD compared with WT cells,there were no differences in the internalization of fluorescent inert beads or dead E. coli particles. This suggests that lack of killing in the NOD2 KD cells was responsible for the increased numbers of viable intracellular bacteria; a conclusion supported by evidence of reduced autophagy in NOD2 KD T84 epithelia. Thus,in a two-hit hypothesis,decreased barrier function due to dysfunctional mitochondrial is amplified by lack of NOD2 in transporting enterocytes: subsequently,greater numbers of bacteria entering the mucosa would be a significant inflammatory threat especially since individuals with NOD2 mutations have compromised macrophage and Paneth cell responses to bacteria.NEW & NOTEWORTHY Increased internalization of bacteria by epithelia with dysfunctional mitochondria (reduced ATP) is potentiated if the cells lack nucleotide-binding oligomerization domain 2 (NOD2),mutations in which are inflammatory bowel disease-susceptibility traits. Uptake of bacteria was dependent on reactive oxygen species and MAP-kinase activity,and the increased viable intracellular bacteria in NOD2(-/-) cells likely reflect a reduced ability to recognize and kill bacteria. Thus a significant barrier defect occurs with NOD2 deficiency in conjunction with metabolic stress that could contribute to inflammation. View Publication

Many natural prion diseases of humans and animals are considered to be acquired through oral consumption of contaminated food or pasture. Determining the route by which prions establish host infection will identify the important factors that influence oral prion disease susceptibility and to which intervention strategies can be developed. After exposure,the early accumulation and replication of prions within small intestinal Peyer's patches is essential for the efficient spread of disease to the brain. To replicate within Peyer's patches,the prions must first cross the gut epithelium. M cells are specialised epithelial cells within the epithelia covering Peyer's patches that transcytose particulate antigens and microorganisms. M cell-development is dependent upon RANKL-RANK-signalling,and mice in which RANK is deleted only in the gut epithelium completely lack M cells. In the specific absence of M cells in these mice,the accumulation of prions within Peyer's patches and the spread of disease to the brain was blocked,demonstrating a critical role for M cells in the initial transfer of prions across the gut epithelium in order to establish host infection. Since pathogens,inflammatory stimuli and aging can modify M cell-density in the gut,these factors may also influence oral prion disease susceptibility. Mice were therefore treated with RANKL to enhance M cell density in the gut. We show that prion uptake from the gut lumen was enhanced in RANKL-treated mice,resulting in shortened survival times and increased disease susceptibility,equivalent to a 10-fold higher infectious titre of prions. Together these data demonstrate that M cells are the critical gatekeepers of oral prion infection,whose density in the gut epithelium directly limits or enhances disease susceptibility. Our data suggest that factors which alter M cell-density in the gut epithelium may be important risk factors which influence host susceptibility to orally acquired prion diseases. View Publication

Incorporation of thymidine analogues in replicating DNA,coupled with antibody and fluorophore staining,allows analysis of cell proliferation,but is currently limited to monolayer cultures,fixed cells and end-point assays. We describe a simple microscopy imaging method for live real-time analysis of cell proliferation,S phase progression over several division cycles,effects of anti-proliferative drugs and other applications. It is based on the prominent (˜ 1.7-fold) quenching of fluorescence lifetime of a common cell-permeable nuclear stain,Hoechst 33342 upon the incorporation of 5-bromo-2'-deoxyuridine (BrdU) in genomic DNA and detection by fluorescence lifetime imaging microscopy (FLIM). We show that quantitative and accurate FLIM technique allows high-content,multi-parametric dynamic analyses,far superior to the intensity-based imaging. We demonstrate its uses with monolayer cell cultures,complex 3D tissue models of tumor cell spheroids and intestinal organoids,and in physiological study with metformin treatment. View Publication

NLRs (nucleotide-binding domain and leucine-rich repeats) belong to a large family of cytoplasmic sensors that regulate an extraordinarily diverse range of biological functions. One of these functions is to contribute to immunity against infectious diseases,but dysregulation of their functional activity leads to the development of inflammatory and autoimmune diseases. Cytoplasmic innate immune sensors,including NLRs,are central regulators of intestinal homeostasis. NLRC3 (also known as CLR16.2 or NOD3) is a poorly characterized member of the NLR family and was identified in a genomic screen for genes encoding proteins bearing leucine-rich repeats (LRRs) and nucleotide-binding domains. Expression of NLRC3 is drastically reduced in the tumour tissue of patients with colorectal cancer compared to healthy tissues,highlighting an undefined potential function for this sensor in the development of cancer. Here we show that mice lacking NLRC3 are hyper-susceptible to colitis and colorectal tumorigenesis. The effect of NLRC3 is most dominant in enterocytes,in which it suppresses activation of the mTOR signalling pathways and inhibits cellular proliferation and stem-cell-derived organoid formation. NLRC3 associates with PI3Ks and blocks activation of the PI3K-dependent kinase AKT following binding of growth factor receptors or Toll-like receptor 4. These findings reveal a key role for NLRC3 as an inhibitor of the mTOR pathways,mediating protection against colorectal cancer. View Publication