技术资料

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

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

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

We inhale respiratory pathogens continuously,and the subsequent signaling events between host and microbe are complex,ultimately resulting in clearance of the microbe,stable colonization of the host,or active disease. Traditional in vitro methods are ill-equipped to study these critical events in the context of the lung microenvironment. Here we introduce a microscale organotypic model of the human bronchiole for studying pulmonary infection. By leveraging microscale techniques,the model is designed to approximate the structure of the human bronchiole,containing airway,vascular,and extracellular matrix compartments. To complement direct infection of the organotypic bronchiole,we present a clickable extension that facilitates volatile compound communication between microbial populations and the host model. Using Aspergillus fumigatus,a respiratory pathogen,we characterize the inflammatory response of the organotypic bronchiole to infection. Finally,we demonstrate multikingdom,volatile-mediated communication between the organotypic bronchiole and cultures of Aspergillus fumigatus and Pseudomonas aeruginosa. 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

Severe asthma represents a major unmet clinical need; understanding the pathophysiology is essential for the development of new therapies. Using microarray analysis,we previously found three immunological clusters in asthma: Th2-high,Th17-high,and Th2/17-low. Although new therapies are emerging for Th2-high disease,identifying molecular pathways in Th2-low disease remains an important goal. Further interrogation of our previously described microarray dataset revealed upregulation of gene expression for carcinoembryonic Ag cell adhesion molecule (CEACAM) family members in the bronchi of patients with severe asthma. Our aim was therefore to explore the distribution and cellular localization of CEACAM6 using immunohistochemistry on bronchial biopsy tissue obtained from patients with mild-to-severe asthma and healthy control subjects. Human bronchial epithelial cells were used to investigate cytokine and corticosteroid in vitro regulation of CEACAM6 gene expression. CEACAM6 protein expression in bronchial biopsies was increased in airway epithelial cells and lamina propria inflammatory cells in severe asthma compared with healthy control subjects. CEACAM6 in the lamina propria was localized to neutrophils predominantly. Neutrophil density in the bronchial mucosa was similar across health and the spectrum of asthma severity,but the percentage of neutrophils expressing CEACAM6 was significantly increased in severe asthma,suggesting the presence of an altered neutrophil phenotype. CEACAM6 gene expression in cultured epithelial cells was upregulated by wounding and neutrophil elastase. In summary,CEACAM6 expression is increased in severe asthma and primarily associated with airway epithelial cells and tissue neutrophils. CEACAM6 may contribute to the pathology of treatment-resistant asthma via neutrophil and airway epithelial cell-dependent pathways. View Publication

While readily achieved in cell lines,the application of CRISPR-Cas9 gene editing in human-derived organoids suffers from limited efficacy and complex protocols. Here,we describe a multi-guide RNA CRISPR-Cas9 gene-editing protocol which efficiently achieves complete gene knockout in adult human colonic organoids. This protocol also describes crucial steps including how to harvest patient tissue to maximize gene-editing efficacy and a technique to validate gene knockout following editing with immunofluorescent staining of the organoids against the target protein. View Publication

In cancer microenvironment,aberrant glycosylation events of ECM proteins and cell surface receptors occur. We developed a protocol to generate 3D bioprinted models of colorectal cancer (CRC) crosslinking hyaluronic acid and gelatin functionalized with three signalling glycans characterized in CRC,3'-Sialylgalactose,6'-Sialylgalactose and 2'-Fucosylgalactose. The crosslinking,performed exploiting azide functionalized gelatin and hyaluronic acid and 4arm-PEG-dibenzocyclooctyne,resulted in biocompatible hydrogels that were 3D bioprinted with commercial CRC cells HT-29 and patient derived CRC tumoroids. The glycosylated hydrogels showed good 3D printability,biocompatibility and stability over the time. SEM and synchrotron radiation SAXS/WAXS analysis revealed the influence of glycosylation in the construct morphology,whereas MALDI-MS imaging showed that protein profiles of tumoroid cells vary with glycosylation,indicating that sialylation and fucosylation of ECM proteins induce diverse alterations to the proteome of the tumoroid and surrounding cells. View Publication

Age-related gastrointestinal decline contributes to whole-organism frailty and mortality. Genistein is known to have beneficial effects on age-related diseases,but its precise role in homeostasis of the aging gut remains to be elucidated. Here,wild-type aging mice and Zmpste24-/- progeroid mice were used to investigate the role of genistein in lifespan and homeostasis of the aging gut in mammals. A series of longitudinal,clinically relevant measurements were performed to evaluate the effect of genistein on healthspan. It was found that dietary genistein promoted a healthier and longer life and was associated with a decrease in the levels of systemic inflammatory cytokines in aging mice. Furthermore,dietary genistein ameliorated gut dysfunctions,such as intestinal inflammation,leaky gut,and impaired epithelial regeneration. A distinct genistein-mediated alteration in gut microbiota was observed by increasing Lachnospira abundance and short-chain fatty acid (SCFA) production. Further fecal microbiota transplantation and dirty cage sharing experiments indicated that the gut microbiota from genistein-fed mice rejuvenated the aging gut and extended the lifespan of progeroid mice. It was demonstrated that genistein-associated SCFAs alleviated tumor necrosis factor alpha-induced intestinal organoid damage. Moreover,genistein-associated propionate promoted regulatory T cell-derived interleukin 10 production,which alleviated macrophage-derived inflammation. This study provided the first data,to the authors' knowledge,indicating that dietary genistein modulates homeostasis in the aging gut and extends the healthspan and lifespan of aging mammals. Moreover,the existence of a link between genistein and the gut microbiota provides a rationale for dietary interventions against age-associated frailty. View Publication