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

Three-dimensional (3D) culture systems have been developed that can re-capitulate organ level responses,simulate compound diffusion through complex structures,and assess cellular heterogeneity of tissues,making them attractive models for advanced in vitro research and discovery. Organoids are a unique subtype of 3D cell culture that are grown from stem cells,are self-organizing,and closely replicate in vivo pathophysiology. Organoids have been used to understand tissue development,model diseases,test drug sensitivity and toxicity,and advance regenerative medicine. However,traditional organoid culture methods are inadequate because they are low throughput and ill-suited for single organoid imaging,phenotypic assessment,and isolation from heterogenous organoid populations. To address these bottlenecks,we have adapted our tissue culture consumable and instrumentation to enable automated imaging,identification,and isolation of individual organoids. Organoids grown on the 3D CytoSort? Array can be reliably tracked,imaged,and phenotypically analyzed using brightfield and fluorescent microscopy as they grow over time,then released and transferred fully intact for use in downstream applications. Using mouse hepatic and pancreatic organoids,we have demonstrated the use of this technology for single-organoid imaging,clonal organoid generation,parent organoid subcloning,and single-organoid RNA extraction for downstream gene expression or transcriptomic analysis. The results validate the ability of the CellRaft AIR? System to facilitate efficient,user-friendly,and automated workflows broadly applicable to organoid research by overcoming several pain points: 1) single organoid time-course imaging and phenotypic assessment,2) establishment of single cell-derived organoids,and 3) isolation and retrieval of single organoids for downstream applications. View Publication

Zika virus (ZIKV) infects fetal and adult human brain and is associated with serious neurological complications. To date,no therapeutic treatment is available to treat ZIKV-infected patients. We performed a high-content chemical screen using human pluripotent stem cell-derived cortical neural progenitor cells (hNPCs) and found that hippeastrine hydrobromide (HH) and amodiaquine dihydrochloride dihydrate (AQ) can inhibit ZIKV infection in hNPCs. Further validation showed that HH also rescues ZIKV-induced growth and differentiation defects in hNPCs and human fetal-like forebrain organoids. Finally,HH and AQ inhibit ZIKV infection in adult mouse brain in vivo. Strikingly,HH suppresses viral propagation when administered to adult mice with active ZIKV infection,highlighting its therapeutic potential. Our approach highlights the power of stem cell-based screens and validation in human forebrain organoids and mouse models in identifying drug candidates for treating ZIKV infection and related neurological complications in fetal and adult patients. View Publication

Whipworms are parasitic nematodes that live in the gut of more than 500 million people worldwide. Owing to the difficulty in obtaining parasite material,the mouse whipwormTrichuris murishas been extensively used as a model to study human whipworm infections. These nematodes secrete a multitude of compounds that interact with host tissues where they orchestrate a parasitic existence. Herein we provide the first comprehensive characterization of the excretory/secretory products ofT. muris. We identify 148 proteins secreted byT. murisand show for the first time that the mouse whipworm secretes exosome-like extracellular vesicles (EVs) that can interact with host cells. We use an Optiprep{\textregistered} gradient to purify the EVs,highlighting the suitability of this method for purifying EVs secreted by a parasitic nematode. We also characterize the proteomic and genomic content of the EVs,identifying {\textgreater}350 proteins,56 miRNAs (22 novel) and 475 full-length mRNA transcripts mapping toT. murisgene models. Many of the miRNAs putatively mapped to mouse genes are involved in regulation of inflammation,implying a role in parasite-driven immunomodulation. In addition,for the first time to our knowledge,colonic organoids have been used to demonstrate the internalization of parasite EVs by host cells. Understanding how parasites interact with their host is crucial to develop new control measures. This first characterization of the proteins and EVs secreted byT. murisprovides important information on whipworm-host communication and forms the basis for future studies. View Publication

BackgroundThere is substantial evidence that signaling through Toll-like receptor 4 (TLR4) contributes to the pathogenesis of necrotizing enterocolitis (NEC). Pregnane X receptor (PXR),a xenobiotic sensor and signaling intermediate for certain host-bacterial metabolites,has been shown to negatively regulate TLR4 signaling. Here we investigated the relationship between PXR and TLR4 in the developing murine intestine and explored the capacity of PXR to modulate inflammatory pathways involved in experimental NEC.MethodsWild-type and PXR-/- mice were studied at various time points of development in an experimental model of NEC. In addition,we studied the ability of the secondary bile acid lithocholic acid (LCA),a known PXR agonist in liver,to activate intestinal PXR and reduce NEC-related intestinal inflammation.ResultsWe found a reciprocal relationship between the developmental expression of PXR and TLR4 in wild-type murine intestine,with PXR acting to reduce TLR4 expression by decreasing TLR4 mRNA stability. In addition,PXR-/- mice exhibited a remarkably heightened severity of disease in experimental NEC. Moreover,LCA attenuated intestinal proinflammatory responses in the early stages of experimental NEC.ConclusionThese findings provide proactive insights into the regulation of TLR4 in the developing intestine. Targeting PXR may be a novel approach for NEC prevention. View Publication

Background and Aims Disturbance of intestinal homeostasis is associated with the development of inflammatory bowel disease (IBD),and TGF-beta$ signaling impairment in mononuclear phagocytes (MPs) causes murine colitis with goblet cell depletion. Here,we examined an organoid-MP co-culture system to study the role of MPs in intestinal epithelial differentiation and homeostasis. Methods Intestinal organoids were co-cultured with lamina propria leukocytes and bone marrow-derived dendritic cells (BMDCs) from CD11c-cre Tgfbr2fl/fl mice. Organoid-MP adhesive interactions were evaluated by microscopy,RT-PCR,and flow cytometry. Murine colitis models (dextran sodium sulphate (DSS),CD11c-cre Tgfbr2fl/fl,T-cell-transfer) were used for histological and immunohistochemical analysis. Anti-E-cadherin antibody treatment or CD11c+-cell-specific CDH1 gene deletion were performed for E-cadherin neutralization or knockout. Colonic biopsies from patients with ulcerative colitis were analyzed by flow cytometry. Results Intestinal organoids co-cultured with CD11c+ lamina propria leukocytes or BMDCs from CD11c-cre Tgfbr2fl/fl mice showed morphological changes and goblet cell depletion with Notch signal activation,analogous to CD11c-cre Tgfbr2fl/fl colitis. E-cadherin was upregulated in CD11c+ MPs,especially CX3CR1+CCR2+ monocytes,of CD11c-cre Tgfbr2fl/fl mice. E-cadherin-mediated BMDC adhesion promoted Notch activation and cystic changes in organoids. Anti-E-cadherin antibody treatment attenuated colitis in CD11c-cre Tgfbr2fl/fl and T-cell-transferred mice. In addition,E-cadherin deletion in CD11c+ cells attenuated colitis in both CD11c-cre Tgfbr2fl/fl and DSS-treated mice. In patients with ulcerative colitis,E-cadherin expressed by intestinal CD11c+ leukocytes was enhanced compared with that in healthy controls. Conclusions E-cadherin-mediated MP-epithelium adhesion is associated with the development of colitis,and blocking these adhesions may have therapeutic potential for IBD. View Publication

Colony-stimulating factor 1 (CSF1) controls the growth and differentiation of macrophages.CSF1R signaling has been implicated in the maintenance of the intestinal stem cell niche and differentiation of Paneth cells,but evidence of expression of CSF1R within the crypt is equivocal. Here we show that CSF1R-dependent macrophages influence intestinal epithelial differentiation and homeostasis. In the intestinal lamina propria CSF1R mRNA expression is restricted to macrophages which are intimately associated with the crypt epithelium,and is undetectable in Paneth cells. Macrophage ablation following CSF1R blockade affects Paneth cell differentiation and leads to a reduction of Lgr5+ intestinal stem cells. The disturbances to the crypt caused by macrophage depletion adversely affect the subsequent differentiation of intestinal epithelial cell lineages. Goblet cell density is enhanced,whereas the development of M cells in Peyer's patches is impeded. We suggest that modification of the phenotype or abundance of macrophages in the gut wall alters the development of the intestinal epithelium and the ability to sample gut antigens. View Publication

p40,a Lactobacillus rhamnosus GG (LGG)-derived protein,transactivates epidermal growth factor receptor (EGFR) in intestinal epithelial cells,leading to amelioration of intestinal injury and inflammation. To elucidate mechanisms by which p40 regulates mucosal immunity to prevent inflammation,this study aimed to determine the effects and mechanisms of p40 on regulation of a proliferation-inducing ligand (APRIL) expression in intestinal epithelial cells for promoting immunoglobulin A (IgA) production. p40 upregulated April gene expression and protein production in mouse small intestine epithelial (MSIE) cells,which were inhibited by blocking EGFR expression and kinase activity. Enteroids from Egfr(fl/fl),but not Egfr(fl/fl)-Vil-Cre mice with EGFR specifically deleted in intestinal epithelial cells,exhibited increased April gene expression by p40 treatment. p40-conditioned media from MSIE cells increased B-cell class switching to IgA(+) cells and IgA production,which was suppressed by APRIL receptor-neutralizing antibodies. Treatment of B cells with p40 did not show any effects on IgA production. p40 treatment increased April gene expression and protein production in small intestinal epithelial cells,fecal IgA levels,IgA(+)B220(+),IgA(+)CD19(+),and IgA(+) plasma cells in lamina propria of Egfr(fl/fl),but not of Egfr(fl/fl)-Vil-Cre,mice. Thus p40 upregulates EGFR-dependent APRIL production in intestinal epithelial cells,which may contribute to promoting IgA production. View Publication

Group 3 innate lymphoid cells (ILC3) are major regulators of inflammation and infection at mucosal barriers. ILC3 development is thought to be programmed,but how ILC3 perceive,integrate and respond to local environmental signals remains unclear. Here we show that ILC3 in mice sense their environment and control gut defence as part of a glial"ILC3"epithelial cell unit orchestrated by neurotrophic factors. We found that enteric ILC3 express the neuroregulatory receptor RET. ILC3-autonomous Ret ablation led to decreased innate interleukin-22 (IL-22),impaired epithelial reactivity,dysbiosis and increased susceptibility to bowel inflammation and infection. Neurotrophic factors directly controlled innate Il22 downstream of the p38 MAPK/ERK-AKT cascade and STAT3 activation. Notably,ILC3 were adjacent to neurotrophic-factor-expressing glial cells that exhibited stellate-shaped projections into ILC3 aggregates. Glial cells sensed microenvironmental cues in a MYD88-dependent manner to control neurotrophic factors and innate IL-22. Accordingly,glial-intrinsic Myd88 deletion led to impaired production of ILC3-derived IL-22 and a pronounced propensity towards gut inflammation and infection. Our work sheds light on a novel multi-tissue defence unit,revealing that glial cells are central hubs of neuron and innate immune regulation by neurotrophic factor signals. 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

Cholangiocytes are the target of a heterogeneous group of liver diseases known as the cholangiopathies. An evolving understanding of the mechanisms driving biliary development provides the theoretical underpinnings for rational development of induced pluripotent stem cell (iPSC)-derived cholangiocytes (iDCs). Therefore,the aims of this study were to develop an approach to generate iDCs and to fully characterize the cells in vitro and in vivo. Human iPSC lines were generated by forced expression of the Yamanaka pluripotency factors. We then pursued a stepwise differentiation strategy toward iDCs,using precise temporal exposure to key biliary morphogens,and we characterized the cells,using a variety of morphologic,molecular,cell biologic,functional,and in vivo approaches. Morphology shows a stepwise phenotypic change toward an epithelial monolayer. Molecular analysis during differentiation shows appropriate enrichment in markers of iPSC,definitive endoderm,hepatic specification,hepatic progenitors,and ultimately cholangiocytes. Immunostaining,western blotting,and flow cytometry demonstrate enrichment of multiple functionally relevant biliary proteins. RNA sequencing reveals that the transcriptome moves progressively toward that of human cholangiocytes. iDCs generate intracellular calcium signaling in response to ATP,form intact primary cilia,and self-assemble into duct-like structures in three-dimensional culture. In vivo,the cells engraft within mouse liver,following retrograde intrabiliary infusion. In summary,we have developed a novel approach to generate mature cholangiocytes from iPSCs. In addition to providing a model of biliary differentiation,iDCs represent a platform for in vitro disease modeling,pharmacologic testing,and individualized,cell-based,regenerative therapies for the cholangiopathies. View Publication