技术资料

Glucocorticoids (GCs) play an important role in controlling acute graft-versus-host disease (aGvHD),a frequent complication of allogeneic hematopoietic stem cell transplantation. The anti-inflammatory activity of GCs is mainly ascribed to the modulation of T cells and macrophages,for which reason a genetically induced GC resistance of either of these cell types causes aggravated aGvHD. Since only a few genes are currently known that are differentially regulated under these conditions,we analyzed the expression of 54 candidate genes in the inflamed small intestine of mice suffering from aGvHD when either allogeneic T cells or host myeloid cells were GC resistant using a microfluidic dynamic array platform for high-throughput quantitative PCR. The majority of genes categorized as cytokines (e.g. Il2,Il6),chemokines (e.g. Ccl2,Cxcl1),cell surface receptors (e.g. Fasl,Ctla4) and intracellular molecules (e.g. Dusp1,Arg1) were upregulated in mice transplanted with GC resistant allogeneic T cells. Moreover,the expression of several genes linked to energy metabolism (e.g. Glut1) was altered. Surprisingly,mice harboring GC resistant myeloid cells showed almost no changes in gene expression despite their fatal disease course after aGvHD induction. To identify additional genes in the inflamed small intestine that were affected by a GC resistance of allogeneic T cells,we performed an RNAseq analysis,which uncovered more than 500 differentially expressed transcripts (e.g. Cxcr6,Glut3,Otc,Aoc1,Il1r1,Sphk1) that were enriched for biological processes associated with inflammation and tissue disassembly. The changes in gene expression could be confirmed during full-blown disease but hardly any of them in the preclinical phase using high-throughput quantitative PCR. Further analysis of some of these genes revealed a highly selective expression pattern in T cells,intestinal epithelial cells and macrophages,which correlated with their regulation during disease progression. Collectively,we identified an altered gene expression profile caused by GC resistance of transplanted allogeneic T cells,which could help to define new targets for aGvHD therapy. View Publication

BACKGROUND Vitamin D deficiency is associated with intestinal barrier dysfunction,which contributes to pathogenesis of acute intestinal injury in children. We aim to investigate the effects of vitamin D on intestinal injury in intestinal epithelial cells and organoids. METHODS Lipopolysaccharide (LPS) was used to induce injury in intestinal epithelial cells (IEC-18) and organoids,and the effect of vitamin D was assessed. Cell viability was measured and inflammation cytokines TNF$\alpha$ and IL-8 were quantified. FITC-dextran 4 kDa (FD4) permeability was measured using Transwell while tight junction markers were assessed by immunofluorescence staining in IEC-18 and intestinal organoids. Data were compared using one-way ANOVA with Bonferroni post-test. RESULTS IEC-18 viability was decreased by LPS treatment,but was prevented by vitamin D. The upregulation of inflammation was inhibited by vitamin D,which also decreased epithelium permeability. Vitamin D restored tight junction ZO-1 and claudin 2. In addition,vitamin D decreased TNF$\alpha$ expression and prevented the disruption of ZO-1 in injured organoids. CONCLUSIONS Vitamin D rescued epithelial barrier function by improving permeability and restoring tight junctions,leading to decrease inflammation. This study confirms the protective effects of vitamin D,which could be used as a treatment strategy for infants at risk of developing intestinal injury. View Publication

Epitope-specific CD4+ T cells can be labeled in complex cell mixtures from secondary lymphoid organs with fluorophore-labeled peptide:major histocompatibility complex class II (p:MHCII) tetramers and then detected by flow cytometry. Magnetic enrichment of tetramer-bound cells before flow cytometry increases the sensitivity of detection to the point where epitope-specific cells can be studied even when very rare at early and late times after the host has been exposed to the epitope. This method is very useful for studying polyclonal epitope-specific CD4+ T cells under physiological conditions. {\textcopyright} 2019 by John Wiley {\&} Sons,Inc. View Publication

Myeloid-derived suppressor cells (MDSCs) can subdue inflammation. In mice with acute graft-versus-host disease (GVHD),donor MDSC infusion enhances survival that is only partial and transient because of MDSC inflammasome activation early posttransfer,resulting in differentiation and loss of suppressor function. Here we demonstrate that conditioning regimen-induced adenosine triphosphate (ATP) release is a primary driver of MDSC dysfunction through ATP receptor (P2x7R) engagement and NLR pyrin family domain 3 (NLRP3) inflammasome activation. P2x7R or NLRP3 knockout (KO) donor MDSCs provided significantly higher survival than wild-type (WT) MDSCs. Although in vivo pharmacologic targeting of NLRP3 or P2x7R promoted recipient survival,indicating in vivo biologic effects,no synergistic survival advantage was seen when combined with MDSCs. Because activated inflammasomes release mature interleukin-1$\beta$ (IL-1$\beta$),we expected that IL-1$\beta$ KO donor MDSCs would be superior in subverting GVHD,but such MDSCs proved inferior relative to WT. IL-1$\beta$ release and IL-1 receptor expression was required for optimal MDSC function,and exogenous IL-1$\beta$ added to suppression assays that included MDSCs increased suppressor potency. These data indicate that prolonged systemic NLRP3 inflammasome inhibition and decreased IL-1$\beta$ could diminish survival in GVHD. However,loss of inflammasome activation and IL-1$\beta$ release restricted to MDSCs rather than systemic inhibition allowed non-MDSC IL-1$\beta$ signaling,improving survival. Extracellular ATP catalysis with peritransplant apyrase administered into the peritoneum,the ATP release site,synergized with WT MDSCs,as did regulatory T-cell infusion,which we showed reduced but did not eliminate MDSC inflammasome activation,as assessed with a novel inflammasome reporter strain. These findings will inform future clinical using MDSCs to decrease alloresponses in inflammatory environments. View Publication

Microfold (M) cells residing in the follicle-associated epithelium (FAE) of the gut-associated lymphoid tissue are specialized for antigen uptake to initiate mucosal immune responses. The molecular machinery and biological significance of M cell differentiation,however,remain to be fully elucidated. Here,we demonstrate that Sox8,a member of the SRY-related HMG box transcription factor family,is specifically expressed by M cells in the intestinal epithelium. The expression of Sox8 requires activation of RANKL-RelB signaling. Chromatin immunoprecipitation and luciferase assays revealed that Sox8 directly binds the promoter region of Gp2 to increase Gp2 expression,which is the hallmark of functionally mature M cells. Furthermore,genetic deletion of Sox8 causes a marked decrease in the number of mature M cells,resulting in reduced antigen uptake in Peyer's patches. Consequently,juvenile Sox8-deficient mice showed attenuated germinal center reactions and antigen-specific IgA responses. These findings indicate that Sox8 plays an essential role in the development of M cells to establish mucosal immune responses. View Publication

BACKGROUND Human pancreata contain many types of cells,such as endocrine islets,acinar,ductal,fat,and mesenchymal stromal cells (MSCs). MSCs are important and shown to have a promising therapeutic potential to treat various disease conditions. METHODS We investigated intra-pancreatic tissue-derived (IPTD) MSCs isolated from tissue fractions that are routinely discarded during pancreatic islet isolation of human cadaveric donors. Furthermore,whether pro-angiogenic and anti-inflammatory properties of these cells could be enhanced was investigated. RESULTS IPTD-MSCs were expanded in GMP-compatible CMRL-1066 medium supplemented with 5{\%} human platelet lysate (hPL). IPTD-MSCs were found to be highly pure,with {\textgreater} 95{\%} positive for CD90,CD105,and CD73,and negative for CD45,CD34,CD14,and HLA-DR. Immunofluorescence staining of pancreas tissue demonstrated the presence of CD105+ cells in the vicinity of islets. IPTD-MSCs were capable of differentiation into adipocytes,chondrocytes,and osteoblasts in vitro,underscoring their multipotent features. When these cells were cultured in the presence of a low dose of TNF-$\alpha$,gene expression of tumor necrosis factor alpha-stimulated gene-6 (TSG-6) was significantly increased,compared to control. In contrast,treating cells with dimethyloxallyl glycine (DMOG) (a prolyl 4-hydroxylase inhibitor) enhanced mRNA levels of nuclear factor erythroid 2-related factor 2 (NRF2) and vascular endothelial growth factor (VEGF). Interestingly,a combination of TNF-$\alpha$ and DMOG stimulated the optimal expression of all three genes in IPTD-MSCs. Conditioned medium of IPTD-MSCs treated with a combination of DMOG and TNF-$\alpha$ contained higher levels of pro-angiogenic (VEGF,IL-6,and IL-8) compared to controls,promoting angiogenesis of human endothelial cells in vitro. In contrast,levels of MCP-1,a pro-inflammatory cytokine,were reduced in the conditioned medium of IPTD-MSCs treated with a combination of DMOG and TNF-$\alpha$. CONCLUSIONS The results demonstrate that IPTD-MSCs reside within the pancreas and can be separated as part of a standard islet-isolation protocol. These IPTD-MSCs can be expanded and potentiated ex vivo to enhance their anti-inflammatory and pro-angiogenic profiles. The fact that IPTD-MSCs are generated in a GMP-compatible procedure implicates a direct clinical application. View Publication

Adherens junctions (AJs),together with tight junctions (TJs),form an apical junctional complex that regulates intestinal epithelial cell-to-cell adherence and barrier homeostasis. Within the AJ,membrane-bound E-cadherin binds $\beta$-catenin,which functions as an essential intracellular signaling molecule. We have previously identified a novel protein in the region of the apical junction complex,chloride channel protein-2 (ClC-2),that we have used to study TJ regulation. In this study,we investigated the possible effects of ClC-2 on the regulation of AJs in intestinal mucosal epithelial homeostasis and tumorigenicity. Mucosal homeostasis and junctional proteins were examined in wild-type (WT) and ClC-2 knockout (KO) mice as well as associated colonoids. Tumorigenicity and AJ-associated signaling were evaluated in a murine colitis-associated tumor model and in a colorectal cancer cell line (HT-29). Colonic tissues from ClC-2 KO mice had altered ultrastructural morphology of intercellular junctions with reduced colonocyte differentiation,whereas jejunal tissues had minimal changes. Colonic crypts from ClC-2 KO mice had significantly higher numbers of less-differentiated forms of colonoids compared with WT. Furthermore,the absence of ClC-2 resulted in redistribution of AJ proteins and increased $\beta$-catenin activity. Downregulation of ClC-2 in colorectal cells resulted in significant increases in proliferation associated with disruption of AJs. Colitis-associated tumors in ClC-2 KO mice were significantly increased,associated with $\beta$-catenin transcription factor activation. The absence of ClC-2 results in less differentiated colonic crypts and increased tumorigenicity associated with colitis via dysregulation of AJ proteins and activation of $\beta$-catenin-associated signaling. NEW {\&} NOTEWORTHY Disruption of adherens junctions in the absence of chloride channel protein-2 revealed critical functions of these junctional structures,including maintenance of colonic homeostasis and differentiation as well as driving tumorigenicity by regulating $\beta$-catenin signaling. View Publication

We study the effect of antifreeze glycoproteins (AFGPs) on the survival of organoids under hypothermic conditions. We find that the survival of organoids in cold conditions depends on their developmental stage. Mature organoids die within 24 h when being stored at 4 °C,while cystic organoids can survive up to 48 h. We find that in the presence of AFGPs,the organoid survival is prolonged up to 72 h,irrespective of their developmental stage. Fluorescence microscopy experiments reveal that the AFGPs predominately localize at the cell surface and cover the cell membranes. Our findings support a mechanism in which the positive effect of AFGPs on cell survival during hypothermic storage involves the direct interaction of AFGPs with the cell membrane. Our research highlights organoids as an attractive multicellular model system for studying the action of AFGPs that bridges the gap between single-cell and whole-organ studies. View Publication

Human neutrophilic granulocytes form the largest pool of innate immune cells for host defense against bacterial and fungal pathogens. The dynamic changes that accompany the metamorphosis from a proliferating myeloid progenitor cell in the bone marrow into a mature non-dividing polymorphonuclear blood cell have remained poorly defined. Using mass spectrometry-based quantitative proteomics combined with transcriptomic data,we report on the dynamic changes of five developmental stages in the bone marrow and blood. Integration of transcriptomes and proteome unveils highly dynamic and differential interactions between RNA and protein kinetics during human neutrophil development,which can be linked to functional maturation of typical end-stage blood neutrophil killing activities. View Publication

Early studies in exercise immunology suggested acute bouts of exercise had an immunosuppressive effect in human subjects. However,recent data,show acute bouts of combined aerobic and resistance training increase both lymphocyte activation and proliferation. We quantified resistance exercise-induced changes in the activation state of CD4+ T lymphocytes via surface protein expression and using a medically relevant model of infection (HIV-1). Using a randomized cross-over design,10 untrained subjects completed a control and exercise session. The control session consisted of 30-min seated rest while the exercise session entailed 3 sets × 10 repetitions of back squat,leg press,and leg extensions at 70{\%} 1-RM with 2-min rest between each set. Venous blood samples were obtained pre/post each session. CD4+ T lymphocytes were isolated from whole blood by negative selection. Expression of activation markers (CD69 {\&} CD25) in both nonstimulated and stimulated (costimulation through CD3+ CD28) cells were assessed by flow cytometry. Resistance exercised-induced effects on intracellular activation was further evaluated via in vitro infection with HIV-1. Nonstimulated CD4+ T lymphocytes obtained postexercise exhibited elevated CD25 expression following 24 h in culture. Enhanced HIV-1 replication was observed in cells obtained postexercise. Our results demonstrate that an acute bout of resistance exercise increases the activation state of CD4+ T lymphocytes and results in a greater susceptibility to HIV-1 infection in vitro. These findings offer further evidence that exercise induces activation of T lymphocytes and provides a foundation for the use of medically relevant pathogens as indirect measures of intracellular activation. View Publication

Induced pluripotent stem cells (iPSC) derived from healthy individuals are important controls for disease-modeling studies. Here we apply precision health to create a high-quality resource of control iPSCs. Footprint-free lines were reprogrammed from four volunteers of the Personal Genome Project Canada (PGPC). Multilineage-directed differentiation efficiently produced functional cortical neurons,cardiomyocytes and hepatocytes. Pilot users demonstrated versatility by generating kidney organoids,T lymphocytes,and sensory neurons. A frameshift knockout was introduced into MYBPC3 and these cardiomyocytes exhibited the expected hypertrophic phenotype. Whole-genome sequencing-based annotation of PGPC lines revealed on average 20 coding variants. Importantly,nearly all annotated PGPC and HipSci lines harbored at least one pre-existing or acquired variant with cardiac,neurological,or other disease associations. Overall,PGPC lines were efficiently differentiated by multiple users into cells from six tissues for disease modeling,and variant-preferred healthy control lines were identified for specific disease settings. View Publication

A rapidly increasing number of reports on dysregulated long intergenic non-coding RNA (lincRNA) expression across numerous types of cancers indicates that aberrant lincRNA expression may be a major contributor to tumorigenesis. Marek's disease (MD) is a T cell lymphoma of chickens induced by Marek's disease virus (MDV). Although we have investigated the roles of lincRNAs in bursa tissue of MDV-infected chickens in previous studies,the molecular mechanisms of lincRNA functions in T cells remain poorly understood. In the present study,Linc-GALMD1 was identified from CD4+ T cells and MSB1 cells,and its expression was significantly downregulated in MD-resistant line of birds in response to MDV challenge. Furthermore,loss-of-function experiments indicated that linc-GALMD1 significantly affected the expression of 290 genes in trans. Through integrated analysis of differentially expressed genes (DEGs) induced by MDV and linc-GALMD1,we found that IGLL1 gene expression levels had a positive correlation with the degree of MD infection and could potentially serve as an indicator for clinical diagnosis of MD. Moreover,an interaction between MDV and linc-GALMD1 was also observed. Accordingly,chicken embryonic fibroblast cells were inoculated with MDV with and without the linc-GALMD1 knockdown,and the data showed that linc-GALMD1 could repress MDV gene expression during the course of MDV infection. These findings uncovered a role of linc-GALMD1 as a viral gene regulator and suggested a function of linc-GALMD1 contributing to tumor suppression by coordinating expression of MDV genes and tumor-related genes and regulating immune responses to MDV infection. View Publication