技术资料

The contribution of mast cells in the microenvironment of solid malignancies remains controversial. Here we functionally assess the impact of tumor-adjacent,submucosal mast cell accumulation in murine and human intestinal-type gastric cancer. We find that genetic ablation or therapeutic inactivation of mast cells suppresses accumulation of tumor-associated macrophages,reduces tumor cell proliferation and angiogenesis,and diminishes tumor burden. Mast cells are activated by interleukin (IL)-33,an alarmin produced by the tumor epithelium in response to the inflammatory cytokine IL-11,which is required for the growth of gastric cancers in mice. Accordingly,ablation of the cognate IL-33 receptor St2 limits tumor growth,and reduces mast cell-dependent production and release of the macrophage-attracting factors Csf2,Ccl3,and Il6. Conversely,genetic or therapeutic macrophage depletion reduces tumor burden without affecting mast cell abundance. Therefore,tumor-derived IL-33 sustains a mast cell and macrophage-dependent signaling cascade that is amenable for the treatment of gastric cancer. View Publication

In patients with inactivating mutations in myosin Vb (Myo5B),enterocytes show large inclusions lined by microvilli. The origin of inclusions in small-intestinal enterocytes in microvillus inclusion disease is currently unclear. We postulated that inclusions in Myo5b KO mouse enterocytes form through invagination of the apical brush border membrane. 70-kD FITC-dextran added apically to Myo5b KO intestinal explants accumulated in intracellular inclusions. Live imaging of Myo5b KO-derived enteroids confirmed the formation of inclusions from the apical membrane. Treatment of intestinal explants and enteroids with Dyngo resulted in accumulation of inclusions at the apical membrane. Inclusions in Myo5b KO enterocytes contained VAMP4 and Pacsin 2 (Syndapin 2). Myo5b;Pacsin 2 double-KO mice showed a significant decrease in inclusion formation. Our results suggest that apical bulk endocytosis in Myo5b KO enterocytes resembles activity-dependent bulk endocytosis,the primary mechanism for synaptic vesicle uptake during intense neuronal stimulation. Thus,apical bulk endocytosis mediates the formation of inclusions in neonatal Myo5b KO enterocytes. View Publication

Areas of a junction between two types of epithelia are known to be cancer-prone in many organ systems. However,mechanisms for preferential malignant transformation at the junction areas remain insufficiently elucidated. Here we report that inactivation of tumor suppressor genes Trp53 and Rb1 in the gastric squamous-columnar junction (SCJ) epithelium results in preferential formation of metastatic poorly differentiated neoplasms,which are similar to human gastroesophageal carcinoma. Unlike transformation-resistant antral cells,SCJ cells contain a highly proliferative pool of immature Lgr5-CD44+ cells,which are prone to transformation in organoid assays,comprise early dysplastic lesions,and constitute up to 30{\%} of all neoplastic cells. CD44 ligand osteopontin (OPN) is preferentially expressed in and promotes organoid formation ability and transformation of the SCJ glandular epithelium. OPN and CD44 overexpression correlate with the worst prognosis of human gastroesophageal carcinoma. Thus,detection and selective targeting of the active OPN-CD44 pathway may have direct clinical relevance. View Publication

The higher-order architecture observed in biological systems,like viruses,is very effective in nucleic acid transport. The replications of this system has been attempted with both synthetic and naturally occurring polymers with mixed results. Here we describe a peptide/siRNA quaternary complex that functions as an siRNA delivery system. The rational design of a peptide assembly is inspired by the viral capsids,but not derived from them. We selected the collagen peptide (COL) to provide the structural stability and the folding framework,and hybridize it with the cell penetrating peptide (CPP) that allows for effective penetration of biological barriers. The peptide/siRNA quaternary complex forms stoichiometric,10 nm nanoparticles,that show fast cellular uptake ({\textless}30 min),effective siRNA release,and gene silencing. The complex provides capsid-like protection for siRNA against nucleases without being immunostimulatory,or cytotoxic. Our data suggests that delivery vehicles based on synthetic quaternary structures that exhibit higher-order architecture may be effective in improving delivery and release of nucleic acid cargo. View Publication

Parasitic helminths cause significant damage as they migrate through host tissues to complete their life cycle. While chronic helminth infections are characterized by a well-described Type 2 immune response,the early,tissue-invasive stages are not well understood. Here we investigate the immune pathways activated during the early stages of Heligmosomoides polygyrus bakeri (Hpb),a natural parasitic roundworm of mice. In contrast to the Type 2 immune response present at later stages of infection,a robust Type 1 immune signature including IFNg production was dominant at the time of parasite invasion and granuloma formation. This early response was associated with an accumulation of activated Natural Killer (NK) cells,with no increase of other innate lymphoid cell populations. Parabiosis and confocal microscopy studies indicated that NK cells were recruited from circulation to the small intestine,where they surrounded parasitic larvae. NK cell recruitment required IFN$\gamma$ receptor signaling,but was independent of CXCR3 expression. The depletion of tissue-infiltrating NK cells altered neither worm burden nor parasite fitness,but increased vascular injury,suggesting a role for NK cells in mediating tissue protection. Together,these data identify an unexpected role for NK cells in promoting disease tolerance during the invasive stage of an enteric helminth infection. View Publication

Hematopoietic stem cells (HSCs) reside in the bone marrow within niches that provide microenvironmental signals in the form of biophysical cues,bound and diffusible biomolecules,and heterotypic cell-cell interactions that influence HSC fate decisions. This study seeks to inform the development of a synthetic culture platform that promotes ex vivo HSC expansion without exhaustion. A library of methacrylamide-functionalized gelatin (GelMA) hydrogels is used to explore remodeling and crosstalk from mesenchymal stromal cells (MSCs) on the expansion and quiescence of murine HSCs. The use of a degradable GelMA hydrogel enables MSC-mediated remodeling,yielding dynamic shifts in the matrix environment over time. An initially low-diffusivity hydrogel for co-culture of hematopoietic stem and progenitor cells to MSCs facilitates maintenance of an early progenitor cell population over 7 days. Excitingly,this platform promotes retention of a quiescent HSC population compared to HSC monocultures. These studies reveal MSC-density-dependent upregulation of MMP-9 and changes in hydrogel mechanical properties ($\Delta$E = 2.61 ± 0.72) suggesting MSC-mediated matrix remodeling may contribute to a dynamic culture environment. Herein,a 3D hydrogel is reported for ex vivo HSC culture,in which HSC expansion and quiescence is sensitive to hydrogel properties,MSC co-culture,and MSC-mediated hydrogel remodeling. View Publication

Exosomes are small extracellular vesicles (sEVs),playing a crucial role in the intercellular communication in physiological as well as pathological processes. Here,we aimed to study whether the melanoma-derived sEV-mediated communication could adapt to microenvironmental stresses. We compared B16F1 cell-derived sEVs released under normal and stress conditions,including cytostatic,heat and oxidative stress. The miRNome and proteome showed substantial differences across the sEV groups and bioinformatics analysis of the obtained data by the Ingenuity Pathway Analysis also revealed significant functional differences. The in silico predicted functional alterations of sEVs were validated by in vitro assays. For instance,melanoma-derived sEVs elicited by oxidative stress increased Ki-67 expression of mesenchymal stem cells (MSCs); cytostatic stress-resulted sEVs facilitated melanoma cell migration; all sEV groups supported microtissue generation of MSC-B16F1 co-cultures in a 3D tumour matrix model. Based on this study,we concluded that (i) molecular patterns of tumour-derived sEVs,dictated by the microenvironmental conditions,resulted in specific response patterns in the recipient cells; (ii) in silico analyses could be useful tools to predict different stress responses; (iii) alteration of the sEV-mediated communication of tumour cells might be a therapy-induced host response,with a potential influence on treatment efficacy. 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

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

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

Bone loss in postmenopausal osteoporosis is induced chiefly by an imbalance of bone-forming osteoblasts and bone-resorbing osteoclasts. Salubrinal is a synthetic compound that inhibits de-phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2$\alpha$). Phosphorylation of eIF2$\alpha$ alleviates endoplasmic reticulum (ER) stress,which may activate autophagy. We hypothesized that eIF2$\alpha$ signaling regulates bone homeostasis by promoting autophagy in osteoblasts and inhibiting osteoclast development. To test the hypothesis,we employed salubrinal to elevate the phosphorylation of eIF2$\alpha$ in an ovariectomized (OVX) mouse model and cell cultures. In the OVX model,salubrinal prevented abnormal expansion of rough ER and decreased the number of acidic vesiculars. It regulated ER stress-associated signaling molecules such as Bip,p-eIF2$\alpha$,ATF4 and CHOP,and promoted autophagy of osteoblasts via regulation of eIF2$\alpha$,Atg7,LC3,and p62. Salubrinal markedly alleviated OVX-induced symptoms such as reduction of bone mineral density and bone volume fraction. In primary bone-marrow-derived cells,salubrinal increased the differentiation of osteoblasts,and decreased the formation of osteoclasts by inhibiting nuclear factor of activated T-cells cytoplasmic 1 (NFATc1). Live cell imaging and RNA interference demonstrated that suppression of osteoclastogenesis is in part mediated by Rac1 GTPase. Collectively,this study demonstrates that ER stress-autophagy axis plays an important role in OVX mice. Bone-forming osteoblasts are restored by maintaining phosphorylation of eIF2$\alpha$,and bone-resorbing osteoclasts are regulated by inhibiting NFATc1 and Rac1 GTPase. View Publication

The gut microbiome contributes to inflammatory bowel disease (IBD),in which bacteria can be present within the epithelium. Epithelial barrier function is decreased in IBD,and dysfunctional epithelial mitochondria and endoplasmic reticulum (ER) stress have been individually associated with IBD. We therefore hypothesized that the combination of ER and mitochondrial stresses significantly disrupt epithelial barrier function. Here,we treated human colonic biopsies,epithelial colonoids,and epithelial cells with an uncoupler of oxidative phosphorylation,dinitrophenol (DNP),with or without the ER stressor tunicamycin and assessed epithelial barrier function by monitoring internalization and translocation of commensal bacteria. We also examined barrier function and colitis in mice exposed to dextran sodium sulfate (DSS) or DNP and co-treated with DAPK6,an inhibitor of death-associated protein kinase 1 (DAPK1). Contrary to our hypothesis,induction of ER stress (i.e. the unfolded protein response) protected against decreased barrier function caused by the disruption of mitochondrial function. ER stress did not prevent DNP-driven uptake of bacteria; rather,specific mobilization of the ATF6 arm of ER stress and recruitment of DAPK1 resulted in enhanced autophagic killing (xenophagy) of bacteria. Of note,epithelia with a Crohn's disease-susceptibility mutation in the autophagy gene ATG16L1 exhibited less xenophagy. Systemic delivery of the DAPK1 inhibitor DAPK6 increased bacterial translocation in DSS- or DNP-treated mice. We conclude that promoting ER stress-ATF6-DAPK1 signaling in transporting enterocytes counters the transcellular passage of bacteria evoked by dysfunctional mitochondria,thereby reducing the potential for metabolic stress to reactivate or perpetuate inflammation. View Publication