技术资料

The widely varying therapeutic response of patients with inflammatory bowel disease (IBD) continues to raise questions regarding the unclarified heterogeneity of pathological mechanisms promoting disease progression. While biomarkers for the differentiation of Crohn's disease (CD) versus ulcerative colitis (UC) have been suggested,specific markers for a CD subclassification in ileal CD versus colonic CD are still rare. Since an altered signature of the tryptophan metabolism is associated with chronic inflammatory disease,we sought to characterize potential biomarkers by focusing on the downstream enzymes and metabolites of kynurenine metabolism. Using immunohistochemical stainings,we analyzed and compared the mucosal tryptophan immune metabolism in bioptic samples from patients with active inflammation due to UC or CD versus healthy controls. Localization-specific quantification of immune cell infiltration,tryptophan-metabolizing enzyme expression and mucosal tryptophan downstream metabolite levels was performed. We found generally increased immune cell infiltrates in the tissue of all patients with IBD. However,in patients with CD,significant differences were found between regulatory T cell and neutrophil granulocyte infiltration in the ileum compared with the colon. Furthermore,we observed decreased kynurenine levels as well as strong kynureninase (KYNU) expression specifically in patients with ileal CD. Correspondingly,significantly elevated levels of the kynurenine metabolite 3-hydroxyanthranilic acid were detected in the ileal CD samples. Highlighting the heterogeneity of the different phenotypes of CD,we identified KYNU as a potential mucosal biomarker allowing the localization-specific differentiation of ileal CD versus colonic CD. View Publication

3-dimensional (3D) bioprinting technology provides promising strategy in the fabrication of artificial tissues and organs. As the fundamental element in bioprinting process,preparation of bioink with ideal mechanical properties without sacrifice of biocompatibility is a great challenge. In this study,a supramolecular hydrogel-based bioink is prepared by polyethylene glycol (PEG) grafted chitosan,$\alpha$-cyclodextrin ($\alpha$-CD) and gelatin. It has a primary crosslinking structure through the aggregation of the pseudo-polyrotaxane-like side chains,which are formed from the host-guest interactions between $\alpha$-CD and PEG side chain. Apparent viscosity measurement shows the shear-shinning property of this bioink,which might be due to the reversibility of the physical crosslinking. Moreover,with $\beta$-glycerophosphate at different concentrations as the secondary crosslinking agent,the printed constructs demonstrate different Young's modulus (p {\textless} 0.001). They could also maintain the Young's modulus in cell culture condition for at least 21 days (p {\textless} 0.05). By co-culturing each component with fibroblasts,CCK-8 assay demonstrate cellular viability is higher than 80{\%}. After bioprinting and culturing,immunofluorescence staining with quantification indicate the expression of Ki-67,Paxillin,and N-cadherin is higher in day 14 than those in day 3 (p {\textless} 0.05). Oil red O and Nissl body specific staining reflect strength tunable bioink may have impact on the cell fate of mesenchymal stem cells (p {\textless} 0.05). This work might provide new idea for advanced bioink in the application of re-establishing complicated tissues and organs. View Publication

Overcoming the global health threat of HIV infection requires continuous pipelines of novel drug candidates. We identified the $\gamma$-pyrone polyketides Aureothin/Neoaureothin as potent hits by anti-HIV screening of an extensive natural compound collection. Total synthesis of a structurally diverse group of Aureothin-derivatives successfully identified a lead compound ({\#}7) superior to Aureothin that combines strong anti-HIV activity (IC90{\textless}45 nM),photostability and improved cell safety. Compound {\#}7 inhibited de novo virus production from integrated proviruses by blocking the accumulation of HIV RNAs that encode the structural components of virions and include viral genomic RNAs. Thus,the mode-of-action displayed by compound {\#}7 is different from those of all current clinical drugs. Proteomic analysis indicated that compound {\#}7 does not affect global protein expression in primary blood cells and may modulate cellular pathways linked to HIV infection. Compound {\#}7 inhibited multiple HIV genotypes,including HIV-type 1 and 2 and synergistically inhibited HIV in combination with clinical reverse transcriptase and integrase inhibitors. We conclude that compound {\#}7 represents a promising new class of HIV inhibitors that will facilitate the identification of new virus-host interactions exploitable for antiviral attack and holds promise for further drug development. View Publication

CD47 deficient mice are resistant to dextran sulfate sodium (DSS)-induced experimental colitis. The underlying mechanism,however,remains incompletely understood. In this study,we characterized the role of CD47 in modulating homeostasis of gastrointestinal tract. We found that CD47 expression in both human and mouse intestinal epithelium was upregulated in colitic condition compared to that under normal condition. In line with this,CD47 deficiency protected mice from DSS-induced colitis. Analysis based on both intestinal organoid and cultured cell assays showed that CD47 deficiency accelerated intestinal epithelial cell proliferation and migration. Mechanistically,western blot and functional assays indicated that CD47 deficiency promoting mouse intestinal epithelial cell proliferation and migration follow cell injury is likely through upregulating expression of four Yamanaka transcriptional factors Oct4,Sox2,Klf4 and c-Myc (OSKM in abbreviation). Our studies thus reveal CD47 as a negative regulator in intestinal epithelial cell renewal during colitis through downregulating OSKM transcriptional factors. View Publication

Intestinal epithelial organoids established from gut tissue have become a widely used research tool. However,it remains unclear how environmental cues,divergent microbiota composition and other sources of variation before,during and after establishment confound organoid properties,and how these properties relate to the original tissue. While environmental influences cannot be easily addressed in human organoids,mice offer a controlled assay-system. Here,we probed the effect of donor microbiota differences,previously identified as a confounding factor in murine in vivo studies,on organoids. We analysed the proteomes and transcriptomes of primary organoid cultures established from two colonised and one germ-free mouse colony of C57BL/6J genetic background,and compared them to their tissue of origin and commonly used cell lines. While an imprint of microbiota-exposure was observed on the proteome of epithelial samples,the long-term global impact of donor microbiota on organoid expression patterns was negligible. Instead,stochastic culture-to-culture differences accounted for a moderate variability between independently established organoids. Integration of transcriptome and proteome datasets revealed an organoid-typic expression signature comprising 14 transcripts and 10 proteins that distinguished organoids across all donors from murine epithelial cell lines and fibroblasts and closely mimicked expression patterns in the gut epithelium. This included the inflammasome components ASC,Naip1-6,Nlrc4 and Caspase-1,which were highly expressed in all organoids compared to the reference cell line m-ICc12 or mouse embryonic fibroblasts. Taken together,these results reveal that the donor microbiota has little effect on the organoid phenotype and suggest that organoids represent a more suitable culture model than immortalised cell lines,in particular for studies of intestinal epithelial inflammasomes. View Publication

Acute graft-versus-host disease causes significant mortality in patients undergoing allogeneic hematopoietic cell transplantation. Immunosuppressive treatment for graft-versus-host disease can impair the beneficial graft-versus-leukemia effect and facilitate malignancy relapse. Therefore,novel approaches that protect and regenerate injured tissues without impeding the donor immune system are needed. Bile acids regulate multiple cellular processes and are in close contact with the intestinal epithelium,a major target of acute graft-versus-host disease. Here,we found that the bile acid pool is reduced following graft-versus-host disease induction in a preclinical model. We evaluated the efficacy of bile acids to protect the intestinal epithelium without reducing anti-tumor immunity. We observed that application of bile acids decreased cytokine-induced cell death in intestinal organoids and cell lines. Systemic prophylactic administration of tauroursodeoxycholic acid,the most potent compound in our in vitro studies,reduced graft-versus-host disease severity in three different murine transplantation models. This effect was mediated by decreased activity of the antigen presentation machinery and subsequent prevention of apoptosis of the intestinal epithelium. Moreover,bile acid administration did not alter the bacterial composition in the intestine suggesting that its effects are cell-specific and independent of the microbiome. Treatment of human and murine leukemic cell lines with tauroursodeoxycholic acid did not interfere with the expression of antigen presentation-related molecules. Systemic T cell expansion and especially their cytotoxic capacity against leukemic cells remained intact. This study establishes a role for bile acids in the prevention of acute graft-versus-host disease without impairing the graft-versus-leukemia effect. In particular,we provide a scientific rationale for the systematic use of tauroursodeoxycholic acid in patients undergoing allogeneic hematopoietic cell transplantation. View Publication

Human noroviruses (HuNoVs) are the leading cause of nonbacterial gastroenteritis worldwide. Histo-blood group antigen (HBGA) expression is an important susceptibility factor for HuNoV infection based on controlled human infection models and epidemiologic studies that show an association of secretor status with infection caused by several genotypes. The fucosyltransferase 2 gene (FUT2) affects HBGA expression in intestinal epithelial cells; secretors express a functional FUT2 enzyme,while nonsecretors lack this enzyme and are highly resistant to infection and gastroenteritis caused by many HuNoV strains. These epidemiologic associations are confirmed by infections in stem cell-derived human intestinal enteroid (HIE) cultures. GII.4 HuNoV does not replicate in HIE cultures derived from nonsecretor individuals,while HIEs from secretors are permissive to infection. However,whether FUT2 expression alone is critical for infection remains unproven,since routinely used secretor-positive transformed cell lines are resistant to HuNoV replication. To evaluate the role of FUT2 in HuNoV replication,we used CRISPR or overexpression to genetically manipulate FUT2 gene function to produce isogenic HIE lines with or without FUT2 expression. We show that FUT2 expression alone affects both HuNoV binding to the HIE cell surface and susceptibility to HuNoV infection. These findings indicate that initial binding to a molecule(s) glycosylated by FUT2 is critical for HuNoV infection and that the HuNoV receptor is present in nonsecretor HIEs. In addition to HuNoV studies,these isogenic HIE lines will be useful tools to study other enteric microbes where infection and/or disease outcome is associated with secretor status.IMPORTANCE Several studies have demonstrated that secretor status is associated with susceptibility to human norovirus (HuNoV) infection; however,previous reports found that FUT2 expression is not sufficient to allow infection with HuNoV in a variety of continuous laboratory cell lines. Which cellular factor(s) regulates susceptibility to HuNoV infection remains unknown. We used genetic manipulation of HIE cultures to show that secretor status determined by FUT2 gene expression is necessary and sufficient to support HuNoV replication based on analyses of isogenic lines that lack or express FUT2. Fucosylation of HBGAs is critical for initial binding and for modification of another putative receptor(s) in HIEs needed for virus uptake or uncoating and necessary for successful infection by GI.1 and several GII HuNoV strains. View Publication

Background: Differences in DNA methylation have been reported in B and T lymphocyte populations,including CD4+ T cells,isolated from rheumatoid arthritis (RA) patients when compared to healthy controls. CD4+ T cells are a heterogeneous cell type with subpopulations displaying distinct DNA methylation patterns. In this study,we investigated DNA methylation using reduced representation bisulfite sequencing in two CD4+ T cell populations (CD4+ memory and na{\{i}}ve cells) in three groups: newly diagnosed disease modifying antirheumatic drugs (DMARD) na{\"{i}}ve RA patients (N = 11) methotrexate (MTX) treated RA patients (N = 18) and healthy controls (N = 9) matched for age gender and smoking status. Results: Analyses of these data revealed significantly more differentially methylated positions (DMPs) in CD4+ memory than in CD4+ na{\""{i}}ve T cells (904 vs. 19 DMPs) in RA patients compared to controls. The majority of DMPs (72{\%}) identified in newly diagnosed and DMARD na{\""{i}}ve RA patients with active disease showed increased DNA methylation (39 DMPs) whereas most DMPs (80{\%}) identified in the MTX treated RA patients in remission displayed decreased DNA methylation (694 DMPs). Interestingly we also found that about one third of the 101 known RA risk loci overlapped (±500 kb) with the DMPs. Notably introns of the UBASH3A gene harbor both the lead RA risk SNP and two DMPs in CD4+ memory T cells. Conclusion: Our results suggest that RA associated DNA methylation differences vary between the two T cell subsets but are also influenced by RA characteristics such as disease activity disease duration and/or MTX treatment.""" View Publication

Human induced pluripotent stem cells (hiPSC) provide an attractive tool to study disease mechanisms of neurodevelopmental disorders such as schizophrenia. A pertinent problem is the development of hiPSC-based assays to discriminate schizophrenia (SZ) from autism spectrum disorder (ASD) models. Healthy control individuals as well as patients with SZ and ASD were examined by a panel of diagnostic tests. Subsequently,skin biopsies were taken for the generation,differentiation,and testing of hiPSC-derived neurons from all individuals. SZ and ASD neurons share a reduced capacity for cortical differentiation as shown by quantitative analysis of the synaptic marker PSD95 and neurite outgrowth. By contrast,pattern analysis of calcium signals turned out to discriminate among healthy control,schizophrenia,and autism samples. Schizophrenia neurons displayed decreased peak frequency accompanied by increased peak areas,while autism neurons showed a slight decrease in peak amplitudes. For further analysis of the schizophrenia phenotype,transcriptome analyses revealed a clear discrimination among schizophrenia,autism,and healthy controls based on differentially expressed genes. However,considerable differences were still evident among schizophrenia patients under inspection. For one individual with schizophrenia,expression analysis revealed deregulation of genes associated with the major histocompatibility complex class II (MHC class II) presentation pathway. Interestingly,antipsychotic treatment of healthy control neurons also increased MHC class II expression. In conclusion,transcriptome analysis combined with pattern analysis of calcium signals appeared as a tool to discriminate between SZ and ASD phenotypes in vitro. View Publication

The use of antiretroviral therapy (ART) has remarkably decreased the morbidity associated with HIV-1 infection,however,the prevalence of HIV-1-associated neurocognitive disorders (HAND) is still increasing. The blood-brain barrier (BBB) is the major impediment for penetration of antiretroviral drugs,causing therapeutics to reach only suboptimal level to the brain. Conventional antiretroviral drug regimens are not sufficient to improve the treatment outcomes of HAND. In our recent report,we have developed a poloxamer-PLGA nanoformulation loaded with elvitegravir (EVG),a commonly used antiretroviral drug. The nanoformulated EVG is capable of elevating intracellular drug uptake and simultaneously enhance viral suppression in HIV-1-infected macrophages. In this work,we identified the clinical parameters including stability,biocompatibility,protein corona,cellular internalization pathway of EVG nanoformulation for its potential clinical translation. We further assessed the ability of this EVG nanoformulation to cross the in vitro BBB model and suppress the HIV-1 in macrophage cells. Compared with EVG native drug,our EVG nanoformulation demonstrated an improved BBB model penetration cross the in vitro BBB model and an enhanced HIV-1 suppression in HIV-1-infected human monocyte-derived macrophages after crossing the BBB model without altering the BBB model integrity. Overall,this is an innovative and optimized treatment strategy that has a potential for therapeutic interventions in reducing HAND. View Publication

The intestine is a highly dynamic environment that requires tight control of the various inputs to maintain homeostasis and allow for proper responses to injury. It was recently found that the stem cell niche and epithelium is regenerated after injury by de-differentiated adult cells,through a process that gives rise to Sca1+ fetal-like cells and is driven by a transient population of Clu+ revival stem cells (revSCs). However,the molecular mechanisms that regulate this dynamic process have not been fully defined. Here we show that TNFAIP8 (also known as TIPE0) is a regulator of intestinal homeostasis that is vital for proper regeneration. TIPE0 functions through inhibiting basal Akt activation by the commensal microbiota via modulating membrane phospholipid abundance. Loss of TIPE0 in mice results in injury-resistant enterocytes,that are hyperproliferative,yet have regenerative deficits and are shifted towards a de-differentiated state. Tipe0-/- enterocytes show basal induction of the Clu+ regenerative program and a fetal gene expression signature marked by Sca1,but upon injury are unable to generate Sca-1+/Clu+ revSCs and could not regenerate the epithelium. This work demonstrates the role of TIPE0 in regulating the dynamic signaling that determines the injury response and enables intestinal epithelial cell regenerative plasticity. View Publication

Human Alzheimer's disease (AD) brains and transgenic AD mouse models manifest hyperexcitability. This aberrant electrical activity is caused by synaptic dysfunction that represents the major pathophysiological correlate of cognitive decline. However,the underlying mechanism for this excessive excitability remains incompletely understood. To investigate the basis for the hyperactivity,we performed electrophysiological and immunofluorescence studies on hiPSC-derived cerebrocortical neuronal cultures and cerebral organoids bearing AD-related mutations in presenilin-1 or amyloid precursor protein vs. isogenic gene corrected controls. In the AD hiPSC-derived neurons/organoids,we found increased excitatory bursting activity,which could be explained in part by a decrease in neurite length. AD hiPSC-derived neurons also displayed increased sodium current density and increased excitatory and decreased inhibitory synaptic activity. Our findings establish hiPSC-derived AD neuronal cultures and organoids as a relevant model of early AD pathophysiology and provide mechanistic insight into the observed hyperexcitability. View Publication