技术资料

The microbes in the gastrointestinal tract are separated from the host by a single layer of intestinal epithelial cells (IECs) that plays pivotal roles in maintaining homeostasis by absorbing nutrients and providing a physical and immunological barrier to potential pathogens. Preservation of homeostasis requires the crosstalk between the epithelium and the microbial environment. One epithelial-driven innate immune mechanism that participates in host-microbe communication involves the release of reactive oxygen species (ROS),such as hydrogen peroxide (H2O2),toward the lumen. Phagocytes produce high amounts of ROS which is critical for microbicidal functions; the functional contribution of epithelial ROS,however,has been hindered by the lack of methodologies to reliably quantify extracellular release of ROS. Here,we used a modified Amplex Red assay to investigate the inflammatory and microbial regulation of IEC-generated H2O2 and the potential role of Duox2,a NADPH oxidase that is an important source of H2O2. We found that colonoids respond to interferon-$\gamma$ and flagellin by enhancing production of H2O2 in a Duox2-mediated fashion. To extend these findings,we analyzed ex vivo production of H2O2 by IECs after acute and chronic inflammation,as well as after exposure to dysbiotic microbiota. While acute inflammation did not induce a significant increase in epithelial-driven H2O2,chronic inflammation caused IECs to release higher levels of H2O2. Furthermore,colonization of germ-free mice with dysbiotic microbiota from mice or patients with IBD resulted in increased H2O2 production compared with healthy controls. Collectively,these data suggest that IECs are capable of H2O2 production during chronic inflammation and dysbiotic states. Our results provide insight into luminal production of H2O2 by IECs as a read-out of innate defense by the mucosa. View Publication

Umbilical cord blood (UCB) transplants in adults have slower hematopoietic recovery compared to bone marrow (BM) or peripheral blood (PB) stem cells mainly due to low number of total nucleated cells and hematopoietic stem and progenitor cells (HSPC). As such in this study,we aimed to perform ex vivo expansion of UCB HSPC from non-enriched mononucleated cells (MNC) using novel azole-based small molecules. Freshly-thawed UCB-MNC were cultured in expansion medium supplemented with small molecules and basal cytokine cocktail. The effects of the expansion protocol were measured based on in vitro and in vivo assays. The proprietary library of {\textgreater}50 small molecules were developed using structure-activity-relationship studies of SB203580,a known p38-MAPK inhibitor. A particular analog,C7,resulted in 1,554.1 ± 27.8-fold increase of absolute viable CD45+ CD34+ CD38- CD45RA- progenitors which was at least 3.7-fold higher than control cultures (p {\textless} .001). In depth phenotypic analysis revealed {\textgreater}600-fold expansion of CD34+ /CD90+ /CD49f+ rare HSPCs coupled with significant (p {\textless} .01) increase of functional colonies from C7 treated cells. Transplantation of C7 expanded UCB grafts to immunodeficient mice resulted in significantly (p {\textless} .001) higher engraftment of human CD45+ and CD45+ CD34+ cells in the PB and BM by day 21 compared to non-expanded and cytokine expanded grafts. The C7 expanded grafts maintained long-term human multilineage chimerism in the BM of primary recipients with sustained human CD45 cell engraftment in secondary recipients. In conclusion,a small molecule,C7,could allow for clinical development of expanded UCB grafts without pre-culture stem cell enrichment that maintains in vitro and in vivo functionality. Stem Cells Translational Medicine 2018;7:376-393. View Publication

The aberrant regulation of the epithelial barrier integrity is involved in many diseases of the digestive tract,including inflammatory bowel diseases and colorectal cancer. Intestinal epithelial cell organoid cultures provide new perspectives for analyses of the intestinal barrier in vitro. However,established methods of barrier function analyses from two dimensional cultures have to be adjusted to the analysis of three dimensional organoid structures. Here we describe the methodology for analysis of epithelial barrier function and molecular regulation in intestinal organoids. Barrier responses to interferon-$\gamma$ of intestinal organoids with and without epithelial cell-specific deletion of the interferon-$\gamma$-receptor 2 gene were used as a model system. The established method allowed monitoring of the kinetics of interferon-$\gamma$-induced permeability changes in living organoids. Proteolytic degradation and altered localization of the tight junction proteins claudin-2,-7,and - 15 was detected using confocal spinning disc microscopy with 3D reconstruction. Hessian analysis was used for quantification of re-localization of claudins. In summary,we provide a novel methodologic approach for quantitative analyses of intestinal epithelial barrier functions in the 3D organoid model. View Publication

Ischemia/reperfusion (I/R) injury is mediated in large part by opening of the mitochondrial permeability transition pore (PTP). Consequently,inhibitors of the PTP hold great promise for the treatment of a variety of cardiovascular disorders. At present,PTP inhibition is obtained only through the use of drugs (e.g. cyclosporine A,CsA) targeting cyclophilin D (CyPD) which is a key modulator,but not a structural component of the PTP. This limitation might explain controversial findings in clinical studies. Therefore,we investigated the protective effects against I/R injury of small-molecule inhibitors of the PTP (63 and TR002) that do not target CyPD. Both compounds exhibited a dose-dependent inhibition of PTP opening in isolated mitochondria and were more potent than CsA. Notably,PTP inhibition was observed also in mitochondria devoid of CyPD. Compounds 63 and TR002 prevented PTP opening and mitochondrial depolarization induced by Ca2+ overload and by reactive oxygen species in neonatal rat ventricular myocytes (NRVMs). Remarkably,both compounds prevented cell death,contractile dysfunction and sarcomeric derangement induced by anoxia/reoxygenation injury in NRVMs at sub-micromolar concentrations,and were more potent than CsA. Cardioprotection was observed also in adult mouse ventricular myocytes and human iPSc-derived cardiomyocytes,as well as ex vivo in perfused hearts. Thus,this study demonstrates that 63 and TR002 represent novel cardioprotective agents that inhibit PTP opening independent of CyPD targeting. View Publication

A coding variant of the inflammatory bowel disease (IBD) risk gene ATG16L1 has been associated with defective autophagy and deregulation of endoplasmic reticulum (ER) function. IL-22 is a barrier protective cytokine by inducing regeneration and antimicrobial responses in the intestinal mucosa. We show that ATG16L1 critically orchestrates IL-22 signaling in the intestinal epithelium. IL-22 stimulation physiologically leads to transient ER stress and subsequent activation of STING-dependent type I interferon (IFN-I) signaling,which is augmented in Atg16l1$\Delta$IEC intestinal organoids. IFN-I signals amplify epithelial TNF production downstream of IL-22 and contribute to necroptotic cell death. In vivo,IL-22 treatment in Atg16l1$\Delta$IEC and Atg16l1$\Delta$IEC/Xbp1$\Delta$IEC mice potentiates endogenous ileal inflammation and causes widespread necroptotic epithelial cell death. Therapeutic blockade of IFN-I signaling ameliorates IL-22-induced ileal inflammation in Atg16l1$\Delta$IEC mice. Our data demonstrate an unexpected role of ATG16L1 in coordinating the outcome of IL-22 signaling in the intestinal epithelium. View Publication